Structured Review

NeuroMab anti kv1 3
Cytosolic HSP70 and HSP90 chaperones assist with <t>Kv1.3</t> mitochondrial import. HEK 293 cells were transfected with Kv1.3YFP and subsequently stained with antibodies against HSP70 and HSP90. (A–G) Representative confocal images showing colocalization between Kv1.3YFP (green) (A, E) and HSP70 (B) or HSP90 (F) (red). (C, G) The merged image shows colocalization in yellow. (D, H) Histogram showing the pixel-by-pixel analysis of the section indicated by the line in the merged Panels (C, G) . (I, J) Electron micrographs of HEK 293 cells transfected with Kv1.3YFP. Immunogold particles with sizes of 12 and 18 nm show chaperones (white arrowheads) and Kv1.3 (black arrowheads), respectively. (I) HSP70; (J) HSP90. Scale bars represent 200 nm. (K) HEK 293 cells transfected with Kv1.3YFP were treated with DMSO (vehicle), 20 μM VER-155008 (HSP70 inhibitor) or 1 μM 17-DMAG (HSP90 inhibitor) for 18 h Next, mitochondria were purified, and the YFP intensity was analyzed using flow cytometry. Media (DMEM) and untransfected mitochondria were used as negative controls. (L) Quantification of relative mitochondrial Kv1.3YFP expression upon the indicated treatments. Data are presented as the means ± SE of 3 independent experiments. *p
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Images

1) Product Images from "The Mitochondrial Routing of the Kv1.3 Channel"

Article Title: The Mitochondrial Routing of the Kv1.3 Channel

Journal: Frontiers in Oncology

doi: 10.3389/fonc.2022.865686

Cytosolic HSP70 and HSP90 chaperones assist with Kv1.3 mitochondrial import. HEK 293 cells were transfected with Kv1.3YFP and subsequently stained with antibodies against HSP70 and HSP90. (A–G) Representative confocal images showing colocalization between Kv1.3YFP (green) (A, E) and HSP70 (B) or HSP90 (F) (red). (C, G) The merged image shows colocalization in yellow. (D, H) Histogram showing the pixel-by-pixel analysis of the section indicated by the line in the merged Panels (C, G) . (I, J) Electron micrographs of HEK 293 cells transfected with Kv1.3YFP. Immunogold particles with sizes of 12 and 18 nm show chaperones (white arrowheads) and Kv1.3 (black arrowheads), respectively. (I) HSP70; (J) HSP90. Scale bars represent 200 nm. (K) HEK 293 cells transfected with Kv1.3YFP were treated with DMSO (vehicle), 20 μM VER-155008 (HSP70 inhibitor) or 1 μM 17-DMAG (HSP90 inhibitor) for 18 h Next, mitochondria were purified, and the YFP intensity was analyzed using flow cytometry. Media (DMEM) and untransfected mitochondria were used as negative controls. (L) Quantification of relative mitochondrial Kv1.3YFP expression upon the indicated treatments. Data are presented as the means ± SE of 3 independent experiments. *p
Figure Legend Snippet: Cytosolic HSP70 and HSP90 chaperones assist with Kv1.3 mitochondrial import. HEK 293 cells were transfected with Kv1.3YFP and subsequently stained with antibodies against HSP70 and HSP90. (A–G) Representative confocal images showing colocalization between Kv1.3YFP (green) (A, E) and HSP70 (B) or HSP90 (F) (red). (C, G) The merged image shows colocalization in yellow. (D, H) Histogram showing the pixel-by-pixel analysis of the section indicated by the line in the merged Panels (C, G) . (I, J) Electron micrographs of HEK 293 cells transfected with Kv1.3YFP. Immunogold particles with sizes of 12 and 18 nm show chaperones (white arrowheads) and Kv1.3 (black arrowheads), respectively. (I) HSP70; (J) HSP90. Scale bars represent 200 nm. (K) HEK 293 cells transfected with Kv1.3YFP were treated with DMSO (vehicle), 20 μM VER-155008 (HSP70 inhibitor) or 1 μM 17-DMAG (HSP90 inhibitor) for 18 h Next, mitochondria were purified, and the YFP intensity was analyzed using flow cytometry. Media (DMEM) and untransfected mitochondria were used as negative controls. (L) Quantification of relative mitochondrial Kv1.3YFP expression upon the indicated treatments. Data are presented as the means ± SE of 3 independent experiments. *p

Techniques Used: Transfection, Staining, Purification, Flow Cytometry, Expressing

Schematic representation of the mitochondrial import pathway for Kv1.3. The Kv1.3 channel is cotranslated by ER-linked and cytosolic ribosomes. ER ribosomes synthesize the plasma membrane Kv1.3, which interacts early with ancillary proteins, such as Kvβ subunits and caveolin, and is routed to the cell surface via COPII-dependent anterograde trafficking. The yellow circle restrains anterograde trafficking to the plasma membrane. Alternatively, (1) the Kv1.3 mRNA, which is translated by cytosolic ribosomes, follows the mitochondrial route. (2) By interacting with channel hydrophobic domains, cytosolic chaperones and cochaperones provide Kv1.3 with a partially folded state competent for mitochondrial translocation. Sequential transmembrane domains from the N- to C-terminus cooperate to achieve the proper folded state. (3) TOM receptors recognize cytosolic Kv1.3. (4) The TOM40 channel at the OMM translocates Kv1.3 to the TIM complex at the IMM. Finally, (5) the TIM50 receptor facilitates Kv1.3 translocation through the TIM23/17 channel into the IMM (6) . Spatial and temporal mechanisms defining Kv1.3 membrane topology and tetramerization at the IMM remain unknown.
Figure Legend Snippet: Schematic representation of the mitochondrial import pathway for Kv1.3. The Kv1.3 channel is cotranslated by ER-linked and cytosolic ribosomes. ER ribosomes synthesize the plasma membrane Kv1.3, which interacts early with ancillary proteins, such as Kvβ subunits and caveolin, and is routed to the cell surface via COPII-dependent anterograde trafficking. The yellow circle restrains anterograde trafficking to the plasma membrane. Alternatively, (1) the Kv1.3 mRNA, which is translated by cytosolic ribosomes, follows the mitochondrial route. (2) By interacting with channel hydrophobic domains, cytosolic chaperones and cochaperones provide Kv1.3 with a partially folded state competent for mitochondrial translocation. Sequential transmembrane domains from the N- to C-terminus cooperate to achieve the proper folded state. (3) TOM receptors recognize cytosolic Kv1.3. (4) The TOM40 channel at the OMM translocates Kv1.3 to the TIM complex at the IMM. Finally, (5) the TIM50 receptor facilitates Kv1.3 translocation through the TIM23/17 channel into the IMM (6) . Spatial and temporal mechanisms defining Kv1.3 membrane topology and tetramerization at the IMM remain unknown.

Techniques Used: Translocation Assay

The Kv1.3 interactome reveals an unconventional mitochondrial import pathway for Kv1.3. (A) Network showing protein–protein interactions (PPIs) in the mitochondrial Kv1.3 interactome of HEK 293 cells transfected with Kv1.3YFP. The mitochondrial import subnetwork was obtained after a functional enrichment analysis of the Kv1.3 interactome. A force-directed layout was used for network visualization. Protein names are indicated at the node center. The color code indicates functional relations between proteins (purple, chaperones; blue, matrix proteases; red, PAM complex; orange, TIM23 complex; green, TOM complex; yellow, MICOS complex). TOMM34, CDC37 and YWHAE were manually annotated based on published evidence. (B) HEK 293 cells were transfected with Kv1.3YFP. Total cell lysates (SM, starting materials) were immunoprecipitated against Kv1.3 (IP+). IP-, absence of the Kv1.3 antibody. Samples were immunoblotted (IB) with antibodies against Kv1.3 and different TIM/TOM proteins, as indicated.
Figure Legend Snippet: The Kv1.3 interactome reveals an unconventional mitochondrial import pathway for Kv1.3. (A) Network showing protein–protein interactions (PPIs) in the mitochondrial Kv1.3 interactome of HEK 293 cells transfected with Kv1.3YFP. The mitochondrial import subnetwork was obtained after a functional enrichment analysis of the Kv1.3 interactome. A force-directed layout was used for network visualization. Protein names are indicated at the node center. The color code indicates functional relations between proteins (purple, chaperones; blue, matrix proteases; red, PAM complex; orange, TIM23 complex; green, TOM complex; yellow, MICOS complex). TOMM34, CDC37 and YWHAE were manually annotated based on published evidence. (B) HEK 293 cells were transfected with Kv1.3YFP. Total cell lysates (SM, starting materials) were immunoprecipitated against Kv1.3 (IP+). IP-, absence of the Kv1.3 antibody. Samples were immunoblotted (IB) with antibodies against Kv1.3 and different TIM/TOM proteins, as indicated.

Techniques Used: Transfection, Functional Assay, Immunoprecipitation

Mitochondrial targeting of Kv1.3 depends on structural information rather than on discrete motifs. HEK 293 cells were transfected with several Kv1.3-Δx channels, and mitochondrial colocalization was analyzed. (A–I) Representative confocal images of different Kv1.3YFP constructs (a, green) and the mitochondrial marker (b, pmitoRFP in red). Kv1.3-Δx indicates the transmembrane segment deleted from the expressed Kv1.3YFP construct. Kv1.3-ΔN (B) and Kv1.3-ΔC (I) indicate deletion of the N- and C-termini, respectively. Panel c (merge) shows colocalization in yellow. Square inset indicates the region shown at higher magnification. The scale bar represents 10 µm. (J) Quantification of mitochondrial colocalization using Pearson’s correlation coefficient. The dashed line highlights the Kv1.3 WT value. Data are presented as the means ± SE (n > 30). *p
Figure Legend Snippet: Mitochondrial targeting of Kv1.3 depends on structural information rather than on discrete motifs. HEK 293 cells were transfected with several Kv1.3-Δx channels, and mitochondrial colocalization was analyzed. (A–I) Representative confocal images of different Kv1.3YFP constructs (a, green) and the mitochondrial marker (b, pmitoRFP in red). Kv1.3-Δx indicates the transmembrane segment deleted from the expressed Kv1.3YFP construct. Kv1.3-ΔN (B) and Kv1.3-ΔC (I) indicate deletion of the N- and C-termini, respectively. Panel c (merge) shows colocalization in yellow. Square inset indicates the region shown at higher magnification. The scale bar represents 10 µm. (J) Quantification of mitochondrial colocalization using Pearson’s correlation coefficient. The dashed line highlights the Kv1.3 WT value. Data are presented as the means ± SE (n > 30). *p

Techniques Used: Transfection, Construct, Marker

Transmembrane domains are candidate determinants of the mitochondrial import of Shaker channels. HEK 293 cells were transfected with Kv1.1-Kv1.5YFP channels, and mitochondrial expression was analyzed. (A) Membranous (Mb) and mitochondrial (Mit) fractions of HEK 293 cells transfected with the indicated member of the Shaker family. Note a shift in the molecular weights of the Kv1.2, Kv1.3 and Kv1.5 channels between the Mb and Mit fractions. Na + /K + ATPse is used as a plasma membrane marker, and VDAC is used as a mitochondrial marker. (B) Multiple protein sequence alignment of members of the Shaker family of voltage-gated potassium channels performed using Clustal Omega and JalView software. UniprotKB reference numbers are indicated. Hydrophobic residues are indicated in red, and hydrophilic residues are indicated in blue. Conservation among sequences is indicated in yellow. S1-S6 black lines indicate the sequence fragments corresponding to the transmembrane segments.
Figure Legend Snippet: Transmembrane domains are candidate determinants of the mitochondrial import of Shaker channels. HEK 293 cells were transfected with Kv1.1-Kv1.5YFP channels, and mitochondrial expression was analyzed. (A) Membranous (Mb) and mitochondrial (Mit) fractions of HEK 293 cells transfected with the indicated member of the Shaker family. Note a shift in the molecular weights of the Kv1.2, Kv1.3 and Kv1.5 channels between the Mb and Mit fractions. Na + /K + ATPse is used as a plasma membrane marker, and VDAC is used as a mitochondrial marker. (B) Multiple protein sequence alignment of members of the Shaker family of voltage-gated potassium channels performed using Clustal Omega and JalView software. UniprotKB reference numbers are indicated. Hydrophobic residues are indicated in red, and hydrophilic residues are indicated in blue. Conservation among sequences is indicated in yellow. S1-S6 black lines indicate the sequence fragments corresponding to the transmembrane segments.

Techniques Used: Transfection, Expressing, Marker, Sequencing, Software

Sequential deletion of Kv1.3 segments results in differential mitochondrial targeting. HEK 293 cells were transfected with several Kv1.3-Δx channels, and mitochondrial expression was studied. (A) Representative immunoblot showing purified mitochondrial fractions (bottom panels) from total lysates (starting materials) of HEK 293 cells transfected with Kv1.3-Δx. Samples were immunoblotted with antibodies against YFP (Kv1.3), Na + /K + ATPase (membrane marker) and VDAC (mitochondrial marker). (B) Relative mitochondrial Kv1.3-Δx expression. Data are presented as the means ± SE (n=3-5). The linear regression shown in red (y=0.09568x+0.2544) had a Pearson’s correlation coefficient of r=0.8899 (p=0.0125). Kv1.3-ΔC was not included in the calculation because of its substantial mitochondrial-independent ER retention ( 23 ).
Figure Legend Snippet: Sequential deletion of Kv1.3 segments results in differential mitochondrial targeting. HEK 293 cells were transfected with several Kv1.3-Δx channels, and mitochondrial expression was studied. (A) Representative immunoblot showing purified mitochondrial fractions (bottom panels) from total lysates (starting materials) of HEK 293 cells transfected with Kv1.3-Δx. Samples were immunoblotted with antibodies against YFP (Kv1.3), Na + /K + ATPase (membrane marker) and VDAC (mitochondrial marker). (B) Relative mitochondrial Kv1.3-Δx expression. Data are presented as the means ± SE (n=3-5). The linear regression shown in red (y=0.09568x+0.2544) had a Pearson’s correlation coefficient of r=0.8899 (p=0.0125). Kv1.3-ΔC was not included in the calculation because of its substantial mitochondrial-independent ER retention ( 23 ).

Techniques Used: Transfection, Expressing, Purification, Marker

Kv1.3 is targeted to the mitochondria by bypassing the endoplasmic reticulum. (A) Subcellular fractionation to obtain the membranous (Mb), mitochondrial (Mit) and cytosolic (Cyto) fractions of HEK 293 cells transfected with Kv1.3YFP. SM, starting materials. Na + /K + ATPase is used as a plasma membrane (PM) marker, and VDAC is used as a mitochondrial marker. β-actin identified cytosolic-enriched fractions. Note the difference in molecular weight of Kv1.3 bands from the Mb and Mit fractions. (B) Mb and Mit fractions of HEK 293 cells transfected with Kv1.3YFP were obtained. Cells were cotransfected with (+) or without (-) a constitutively active Sar1(H79G) dominant-negative GTPase. (C–H) Electron micrographs of HEK 293 cells transfected with Kv1.3YFP. Kv1.3 was labeled with 18 nm immunogold particles. Arrowheads indicate Kv1.3. (C) Plasma membrane Kv1.3. (D) Kv1.3 in the ER. (E) Kv1.3 in the Golgi (black arrowhead) and in the IMM (white arrowhead). (F–H) Black arrowheads indicate cytosolic Kv1.3, and white arrowheads indicate mitochondrial Kv1.3. Note the presence of Kv1.3 in ribosomes (r) close to a mitochondrion or embedded in the actin cytoskeleton (a) in (F, G) , respectively. The scale bar indicates 500 nm. White square insets in (C, G, H) highlight higher magnification regions with a scale bar representing 200 nm.
Figure Legend Snippet: Kv1.3 is targeted to the mitochondria by bypassing the endoplasmic reticulum. (A) Subcellular fractionation to obtain the membranous (Mb), mitochondrial (Mit) and cytosolic (Cyto) fractions of HEK 293 cells transfected with Kv1.3YFP. SM, starting materials. Na + /K + ATPase is used as a plasma membrane (PM) marker, and VDAC is used as a mitochondrial marker. β-actin identified cytosolic-enriched fractions. Note the difference in molecular weight of Kv1.3 bands from the Mb and Mit fractions. (B) Mb and Mit fractions of HEK 293 cells transfected with Kv1.3YFP were obtained. Cells were cotransfected with (+) or without (-) a constitutively active Sar1(H79G) dominant-negative GTPase. (C–H) Electron micrographs of HEK 293 cells transfected with Kv1.3YFP. Kv1.3 was labeled with 18 nm immunogold particles. Arrowheads indicate Kv1.3. (C) Plasma membrane Kv1.3. (D) Kv1.3 in the ER. (E) Kv1.3 in the Golgi (black arrowhead) and in the IMM (white arrowhead). (F–H) Black arrowheads indicate cytosolic Kv1.3, and white arrowheads indicate mitochondrial Kv1.3. Note the presence of Kv1.3 in ribosomes (r) close to a mitochondrion or embedded in the actin cytoskeleton (a) in (F, G) , respectively. The scale bar indicates 500 nm. White square insets in (C, G, H) highlight higher magnification regions with a scale bar representing 200 nm.

Techniques Used: Fractionation, Transfection, Marker, Molecular Weight, Dominant Negative Mutation, Labeling

Transmembrane segments encode sufficient information to localize Kv1.3 to mitochondria. HEK 293 cells were transfected with several YFP-Sx(1-6) transmembrane peptides, and mitochondrial colocalization was analyzed. (A) Cartoon representing the transmembrane peptide constructs. Sx indicates the transmembrane segment (1-6) inserted in the YFP plasmid. (B–H) Representative confocal images of HEK 293 cells cotransfected with different YFP constructs (a, green) and a mitochondrial marker (b, pmitoRFP in red). The merged image (c) shows colocalization in yellow. The square inset indicates the region shown at higher magnification. The scale bar represents 10 µm. (I) Quantification of colocalization using Pearson’s correlation coefficient. Data are presented as the means ± SE (n > 30). *p
Figure Legend Snippet: Transmembrane segments encode sufficient information to localize Kv1.3 to mitochondria. HEK 293 cells were transfected with several YFP-Sx(1-6) transmembrane peptides, and mitochondrial colocalization was analyzed. (A) Cartoon representing the transmembrane peptide constructs. Sx indicates the transmembrane segment (1-6) inserted in the YFP plasmid. (B–H) Representative confocal images of HEK 293 cells cotransfected with different YFP constructs (a, green) and a mitochondrial marker (b, pmitoRFP in red). The merged image (c) shows colocalization in yellow. The square inset indicates the region shown at higher magnification. The scale bar represents 10 µm. (I) Quantification of colocalization using Pearson’s correlation coefficient. Data are presented as the means ± SE (n > 30). *p

Techniques Used: Transfection, Construct, Plasmid Preparation, Marker

2) Product Images from "Kv1.3 Controls Mitochondrial Dynamics during Cell Cycle Progression"

Article Title: Kv1.3 Controls Mitochondrial Dynamics during Cell Cycle Progression

Journal: Cancers

doi: 10.3390/cancers13174457

Kv1.3 accumulates at perinuclear mitochondria during the G1/S transition. ( A ) Subcellular fractionation of 3T3-L1 wild-type preadipocytes to obtain the membranous (Mb) and mitochondrial (Mit) fractions. The samples were probed for Kv1.3, Na+/K+ ATPase (a membrane marker) and TIMM50 (a mitochondrial marker). ( B ) Electron micrograph showing mitochondria of 3T3-L1 wild-type preadipocytes. Kv1.3 was labeled with 18 nm immunogold particles (black arrowhead) and was located at the inner mitochondrial membrane. The scale bar represents 200 nm. ( C – H ) Cells were either in the G0/G1 or the G1/S phase following serum deprivation or serum readdition for 12 h, respectively. Representative confocal images showing Kv1.3 and mitochondria in wild-type preadipocytes fixed in the G0/G1 ( C – E ) and G1/S ( F – H ) phase. Ea-Eb and Ha-Hb are magnified images of E and H, respectively. Ea and Ha show distal regions, and Eb and Hb show perinuclear regions. Yellow indicates colocalization of Kv1.3 (green) and mitochondria (red). The scale bar represents 20 µm. ( I ) Pearson’s coefficient of colocalization between Kv1.3 and mitochondria. The data are the mean ± SE ( n > 30). *** p
Figure Legend Snippet: Kv1.3 accumulates at perinuclear mitochondria during the G1/S transition. ( A ) Subcellular fractionation of 3T3-L1 wild-type preadipocytes to obtain the membranous (Mb) and mitochondrial (Mit) fractions. The samples were probed for Kv1.3, Na+/K+ ATPase (a membrane marker) and TIMM50 (a mitochondrial marker). ( B ) Electron micrograph showing mitochondria of 3T3-L1 wild-type preadipocytes. Kv1.3 was labeled with 18 nm immunogold particles (black arrowhead) and was located at the inner mitochondrial membrane. The scale bar represents 200 nm. ( C – H ) Cells were either in the G0/G1 or the G1/S phase following serum deprivation or serum readdition for 12 h, respectively. Representative confocal images showing Kv1.3 and mitochondria in wild-type preadipocytes fixed in the G0/G1 ( C – E ) and G1/S ( F – H ) phase. Ea-Eb and Ha-Hb are magnified images of E and H, respectively. Ea and Ha show distal regions, and Eb and Hb show perinuclear regions. Yellow indicates colocalization of Kv1.3 (green) and mitochondria (red). The scale bar represents 20 µm. ( I ) Pearson’s coefficient of colocalization between Kv1.3 and mitochondria. The data are the mean ± SE ( n > 30). *** p

Techniques Used: Fractionation, Marker, Labeling

Kv1.3 regulates the mitochondrial membrane potential during the cell cycle. Ablation of Kv1.3 impairs the mitochondrial membrane potential. ( A ) TMRM intensity in wild-type (black bar) and Kv1.3KD (white bar) 3T3-L1 preadipocytes was analyzed with flow cytometry. The data are the mean ± SE ( n = 3), * p
Figure Legend Snippet: Kv1.3 regulates the mitochondrial membrane potential during the cell cycle. Ablation of Kv1.3 impairs the mitochondrial membrane potential. ( A ) TMRM intensity in wild-type (black bar) and Kv1.3KD (white bar) 3T3-L1 preadipocytes was analyzed with flow cytometry. The data are the mean ± SE ( n = 3), * p

Techniques Used: Flow Cytometry

Kv1.3 facilitates the G1/S transition of the cell cycle in preadipocytes. Serum-starved resting cells were incubated for the indicated time after serum readdition. ( A ) Cell cycle analysis of 3T3-L1 preadipocytes was performed with propidium iodide. Representative histograms at 0, 6, 12, 18 or 24 h after serum readdition. The cells exhibit two blue peaks corresponding to the G0/G1 (left) and G2 (right) phases. The cell population in purple corresponds to cells in the S phase. Left panels, wild-type preadipocytes; right panels, Kv1.3KD preadipocytes. ( B ) The % of cells in the G0/G1 phase, % of cells in the S phase and % of cells in the G2 phase for wild-type (black) and Kv1.3KD (white) preadipocytes. The data are the mean ± SE ( n = 4–10 independent experiments). Two-way ANOVA indicated p
Figure Legend Snippet: Kv1.3 facilitates the G1/S transition of the cell cycle in preadipocytes. Serum-starved resting cells were incubated for the indicated time after serum readdition. ( A ) Cell cycle analysis of 3T3-L1 preadipocytes was performed with propidium iodide. Representative histograms at 0, 6, 12, 18 or 24 h after serum readdition. The cells exhibit two blue peaks corresponding to the G0/G1 (left) and G2 (right) phases. The cell population in purple corresponds to cells in the S phase. Left panels, wild-type preadipocytes; right panels, Kv1.3KD preadipocytes. ( B ) The % of cells in the G0/G1 phase, % of cells in the S phase and % of cells in the G2 phase for wild-type (black) and Kv1.3KD (white) preadipocytes. The data are the mean ± SE ( n = 4–10 independent experiments). Two-way ANOVA indicated p

Techniques Used: Incubation, Cell Cycle Assay

Representative cartoon summarizing the participation of the mitochondrial Kv1.3 (mitoKv1.3) in the proliferation of preadipocytes. Kv1.3 would facilitate the G1/S transition of the cell cycle in preadipocytes accumulating at perinuclear mitochondria. The elucidation of a putative mitochondrial-nuclear communication during this phase of the cell cycle in which Kv1.3 would participate deserves much effort. During the G1/S transition, Kv1.3 would contribute to the mitochondrial fusion/fission equilibrium controlling the mitochondrial membrane potential. Ablation of Kv1.3 (Kv1.3KD) would impair mitochondrial dynamics during cell cycle progression. Kv1.3KD, 3T3-L1 preadipocytes, with a genetic ablation of Kv1.3. Green dots, Kv1.3 channels; magenta, mitochondrial network.
Figure Legend Snippet: Representative cartoon summarizing the participation of the mitochondrial Kv1.3 (mitoKv1.3) in the proliferation of preadipocytes. Kv1.3 would facilitate the G1/S transition of the cell cycle in preadipocytes accumulating at perinuclear mitochondria. The elucidation of a putative mitochondrial-nuclear communication during this phase of the cell cycle in which Kv1.3 would participate deserves much effort. During the G1/S transition, Kv1.3 would contribute to the mitochondrial fusion/fission equilibrium controlling the mitochondrial membrane potential. Ablation of Kv1.3 (Kv1.3KD) would impair mitochondrial dynamics during cell cycle progression. Kv1.3KD, 3T3-L1 preadipocytes, with a genetic ablation of Kv1.3. Green dots, Kv1.3 channels; magenta, mitochondrial network.

Techniques Used:

Kv1.3 participates in the proliferation of preadipocytes. 3T3-L1 preadipocytes express Kv1.3, and genetic ablation of the channel alters cell proliferation. ( A ) Representative immunofluorescence confocal image of Kv1.3 in 3T3-L1 preadipocytes. The scale bar represents 20 µm. ( B ) Kv1.3 silencing in 3T3-L1 preadipocytes. Cells were infected with Kv1.3 shRNA (Kv1.3KD) or scramble shRNA (SCR) lentivirus. β-actin was used as a loading control. Noninfected 3T3-L1 cells were called wild-type (WT) cells. ( C ) Quantification of the efficiency of Kv1.3 silencing. The data are the mean ± SE ( n ≥ 3). * p
Figure Legend Snippet: Kv1.3 participates in the proliferation of preadipocytes. 3T3-L1 preadipocytes express Kv1.3, and genetic ablation of the channel alters cell proliferation. ( A ) Representative immunofluorescence confocal image of Kv1.3 in 3T3-L1 preadipocytes. The scale bar represents 20 µm. ( B ) Kv1.3 silencing in 3T3-L1 preadipocytes. Cells were infected with Kv1.3 shRNA (Kv1.3KD) or scramble shRNA (SCR) lentivirus. β-actin was used as a loading control. Noninfected 3T3-L1 cells were called wild-type (WT) cells. ( C ) Quantification of the efficiency of Kv1.3 silencing. The data are the mean ± SE ( n ≥ 3). * p

Techniques Used: Immunofluorescence, Infection, shRNA

Kv1.3 regulates the mitochondrial fusion/fission equilibrium during the G1/S transition. Confocal images showing mitochondria in cells fixed in the G0/G1 ( A – F ) and G1/S ( G – L ) phase for WT ( A – C , G – I ) and Kv1.3KD preadipocytes ( D – F , J – L ). The scale bar represents 20 µm. Images were processed (tubeness and skeleton) to perform morphometric analysis of mitochondria. ( M ) The length of mitochondrial networks was measured as the average area of the skeletonized binary image. ( N ) Number of mitochondrial particles per µm 2 . ( O ) The form factor describes the particle shape complexity and was computed as the average (perimeter)2/(4π·area). A circle corresponds to a minimum value of 1. The data are the mean ± SE ( n > 30). *, p
Figure Legend Snippet: Kv1.3 regulates the mitochondrial fusion/fission equilibrium during the G1/S transition. Confocal images showing mitochondria in cells fixed in the G0/G1 ( A – F ) and G1/S ( G – L ) phase for WT ( A – C , G – I ) and Kv1.3KD preadipocytes ( D – F , J – L ). The scale bar represents 20 µm. Images were processed (tubeness and skeleton) to perform morphometric analysis of mitochondria. ( M ) The length of mitochondrial networks was measured as the average area of the skeletonized binary image. ( N ) Number of mitochondrial particles per µm 2 . ( O ) The form factor describes the particle shape complexity and was computed as the average (perimeter)2/(4π·area). A circle corresponds to a minimum value of 1. The data are the mean ± SE ( n > 30). *, p

Techniques Used:

3) Product Images from "KCNE4-dependent functional consequences of Kv1.3-related leukocyte physiology"

Article Title: KCNE4-dependent functional consequences of Kv1.3-related leukocyte physiology

Journal: Scientific Reports

doi: 10.1038/s41598-021-94015-9

KCNE4 impaired Kv1.3 accumulation in the IS but did not disrupt IS formation. Human Jurkat T lymphocytes and human Raji B lymphocytes were used to generate cell conjugates. ( A – C ) Activated B-cells (10 µg/mL SEE toxin) were cocultured in the absence ( Ba – Bd ) or presence ( Ca – Cd ) of Jurkat cells, and confocal images were obtained. ( Aa – Ad ) Jurkat T-cells in the absence of B cells. Endogenous Kv1.3 (green), CD3 (marker of T-cells, red), and CD19 (marker of B-cells, blue) were detected. ( Ad , Bd , Cd ) merge panels. Note that triple colocalization (white) in Cd localizes Kv1.3 in the IS, as identified by CD3 staining ( Cb ). Bars are 20 µm. ( D ) Accumulation ratio of mGFP-Kv1.3 at the IS vs. KCNE4-mCherry total intensity (n = 40). ( E ) CD3 recruitment into the IS vs. KCNE4 intensity. The horizontal red line represents the threshold level (1.5) for Kv1.3 and CD3 accumulation in the IS. Values greater or less than 1.5 indicated positive or negative accumulation of proteins at the IS, respectively.
Figure Legend Snippet: KCNE4 impaired Kv1.3 accumulation in the IS but did not disrupt IS formation. Human Jurkat T lymphocytes and human Raji B lymphocytes were used to generate cell conjugates. ( A – C ) Activated B-cells (10 µg/mL SEE toxin) were cocultured in the absence ( Ba – Bd ) or presence ( Ca – Cd ) of Jurkat cells, and confocal images were obtained. ( Aa – Ad ) Jurkat T-cells in the absence of B cells. Endogenous Kv1.3 (green), CD3 (marker of T-cells, red), and CD19 (marker of B-cells, blue) were detected. ( Ad , Bd , Cd ) merge panels. Note that triple colocalization (white) in Cd localizes Kv1.3 in the IS, as identified by CD3 staining ( Cb ). Bars are 20 µm. ( D ) Accumulation ratio of mGFP-Kv1.3 at the IS vs. KCNE4-mCherry total intensity (n = 40). ( E ) CD3 recruitment into the IS vs. KCNE4 intensity. The horizontal red line represents the threshold level (1.5) for Kv1.3 and CD3 accumulation in the IS. Values greater or less than 1.5 indicated positive or negative accumulation of proteins at the IS, respectively.

Techniques Used: Marker, Staining

KCNE4 expression impairs Kv1.3 surface expression and inhibits Kv currents in Jurkat T cells. Confocal imaging of Jurkat T cells transfected with YFP, Kv1.3YFP, KCNE4CFP and KCNE4CFP with YFP-Kv1.3. Nuclei were stained with DAPI (blue). ( Aa – Ad ) Jurkat nontransfected cells (control). ( Ba – Bd ) YFP-transfected cells (YFP). ( Ca – Cd ) Kv1.3YFP transfected cells. ( Da – Dd ) KCNE4CFP transfected cells. ( Ea – Ed ) Kv1.3YFP and KCNE4CFP cotransfected cells. ( Aa , Ba , Ca , Da , Ea ) Kv1.3 in green. ( Ab , Bb , Cb , Db , Eb ) KCNE4 in red. ( Ac , Bc , Cc , Dc , Ec ) DAPI in blue. Merged yellow indicates colocalization between green and red ( Ad , Bd , Cd , Dd , Ed ). Scale bar: 5 µm. ( F ) FRET analysis of the Kv1.3-KCNE4 protein interaction by flow cytometry in Jurkat T lymphocytes. Values are mean ± SE, n = 5–7, *p
Figure Legend Snippet: KCNE4 expression impairs Kv1.3 surface expression and inhibits Kv currents in Jurkat T cells. Confocal imaging of Jurkat T cells transfected with YFP, Kv1.3YFP, KCNE4CFP and KCNE4CFP with YFP-Kv1.3. Nuclei were stained with DAPI (blue). ( Aa – Ad ) Jurkat nontransfected cells (control). ( Ba – Bd ) YFP-transfected cells (YFP). ( Ca – Cd ) Kv1.3YFP transfected cells. ( Da – Dd ) KCNE4CFP transfected cells. ( Ea – Ed ) Kv1.3YFP and KCNE4CFP cotransfected cells. ( Aa , Ba , Ca , Da , Ea ) Kv1.3 in green. ( Ab , Bb , Cb , Db , Eb ) KCNE4 in red. ( Ac , Bc , Cc , Dc , Ec ) DAPI in blue. Merged yellow indicates colocalization between green and red ( Ad , Bd , Cd , Dd , Ed ). Scale bar: 5 µm. ( F ) FRET analysis of the Kv1.3-KCNE4 protein interaction by flow cytometry in Jurkat T lymphocytes. Values are mean ± SE, n = 5–7, *p

Techniques Used: Expressing, Imaging, Transfection, Staining, Flow Cytometry

LPS-dependent activation of CY15 dendritic cells increases the abundance of Kv1.3 at the cell surface. CY15 cells were incubated in the presence (LPS) or the absence (control) of LPS for 24 h. Cells were first stained with WGA (membrane marker) and then immunolabeled against Kv1.3. ( A – D ) Control cells in the absence of LPS. ( E – H ) Cells treated with LPS. Green, Kv1.3; red, WGA; merged panels show colocalization between green and red. ( D , H ) Histogram of the pixel by pixel analysis of the section indicated by the arrow in ( C , G ), respectively. Bars represent 10 μm. ( I ) Mander's overlap coefficient (MOC) quantifying the degree of colocalization between Kv1.3 and membrane surface (WGA) staining. White bar, control; black bar, LPS. Values are mean ± SE of n > 30 cells. ***p
Figure Legend Snippet: LPS-dependent activation of CY15 dendritic cells increases the abundance of Kv1.3 at the cell surface. CY15 cells were incubated in the presence (LPS) or the absence (control) of LPS for 24 h. Cells were first stained with WGA (membrane marker) and then immunolabeled against Kv1.3. ( A – D ) Control cells in the absence of LPS. ( E – H ) Cells treated with LPS. Green, Kv1.3; red, WGA; merged panels show colocalization between green and red. ( D , H ) Histogram of the pixel by pixel analysis of the section indicated by the arrow in ( C , G ), respectively. Bars represent 10 μm. ( I ) Mander's overlap coefficient (MOC) quantifying the degree of colocalization between Kv1.3 and membrane surface (WGA) staining. White bar, control; black bar, LPS. Values are mean ± SE of n > 30 cells. ***p

Techniques Used: Activation Assay, Incubation, Staining, Whole Genome Amplification, Marker, Immunolabeling

KCNE4 overexpression modulates Kv1.3-related physiological events in Jurkat T lymphocytes. Jurkat T cells were electroporated with KCNE4CFP, and positively transfected cells were selected for specific assays. ( A ) Kv1.3 and KCNE4 expression in Jurkat cells. ( B ) Percentage of Jurkat T cell proliferation. Cells were serum starved overnight and cultured for an additional 24 h in the presence of FBS. The alamarBlue dye was used. *p
Figure Legend Snippet: KCNE4 overexpression modulates Kv1.3-related physiological events in Jurkat T lymphocytes. Jurkat T cells were electroporated with KCNE4CFP, and positively transfected cells were selected for specific assays. ( A ) Kv1.3 and KCNE4 expression in Jurkat cells. ( B ) Percentage of Jurkat T cell proliferation. Cells were serum starved overnight and cultured for an additional 24 h in the presence of FBS. The alamarBlue dye was used. *p

Techniques Used: Over Expression, Transfection, Expressing, Cell Culture

Kv1.3 and KCNE4 are differentially expressed in leukocytes. The presence of Kv1.3 and KCNE4 expression was analyzed in human Jurkat T lymphocytes and mouse CY15 dendritic cells. ( A ) Kv1.3 and KCNE4 protein expression in leukocytes. HEK 293 cells were used as a negative control. Although Jurkat and CY15 dendritic cells shared Kv1.3 and KCNE4 expression, the abundance of KCNE4 in T cells was much lower and minimally detected by western blot. In addition, Kv1.5 was abundantly expressed in CY15 cells. Representative cropped blots, clearly separated by vertical white lines, are shown only for qualitative purposes. Voltage-dependent K + currents were elicited in Jurkat ( B ) and CY15 cells ( C ). Cells were held at -60 mV, and 250 ms pulse potentials were applied as indicated. ( D ) Representative confocal images of Kv1.3 ( Da and Dd , in green) and KCNE4 ( Db and De , in red) in Jurkat T lymphocytes ( Da – Dc ) and CY15 dendritic cells ( Dd – Df ). Scale bars: 10 µm. Given the limited expression of KCNE4 in T-cells, IPI was performed in Jurkat cells. ( E ) KCNE4 coimmunoprecipitated with Kv1.3 in dendritic cells. Lysates were immunoprecipitated against Kv1.3 (IP: Kv1.3) and immunoblotted (IB) against Kv1.3 and KCNE4. Upper panel: Kv1.3. Lower panel: KCNE4. SM: starting material. SN+: supernatant from the IP+. SN−: supernatant from the IP−. IP+: Immunoprecipitation in the presence of the anti-Kv1.3 antibody. IP−: Immunoprecipitated in the absence of the anti-Kv1.3 antibody. ( F ) Kv1.3 localized in lipid raft fractions from Jurkat T-cells. ( G ) Kv1.3 and KCNE4 did not localize in lipid rafts from CY15 dendritic cells. Lipid rafts were isolated, and low density (1) to high density (12) sucrose gradient fractions were analyzed by western blot. Flotilin indicated low-buoyancy lipid rafts, whereas clathrin identified nonfloating raft fractions.
Figure Legend Snippet: Kv1.3 and KCNE4 are differentially expressed in leukocytes. The presence of Kv1.3 and KCNE4 expression was analyzed in human Jurkat T lymphocytes and mouse CY15 dendritic cells. ( A ) Kv1.3 and KCNE4 protein expression in leukocytes. HEK 293 cells were used as a negative control. Although Jurkat and CY15 dendritic cells shared Kv1.3 and KCNE4 expression, the abundance of KCNE4 in T cells was much lower and minimally detected by western blot. In addition, Kv1.5 was abundantly expressed in CY15 cells. Representative cropped blots, clearly separated by vertical white lines, are shown only for qualitative purposes. Voltage-dependent K + currents were elicited in Jurkat ( B ) and CY15 cells ( C ). Cells were held at -60 mV, and 250 ms pulse potentials were applied as indicated. ( D ) Representative confocal images of Kv1.3 ( Da and Dd , in green) and KCNE4 ( Db and De , in red) in Jurkat T lymphocytes ( Da – Dc ) and CY15 dendritic cells ( Dd – Df ). Scale bars: 10 µm. Given the limited expression of KCNE4 in T-cells, IPI was performed in Jurkat cells. ( E ) KCNE4 coimmunoprecipitated with Kv1.3 in dendritic cells. Lysates were immunoprecipitated against Kv1.3 (IP: Kv1.3) and immunoblotted (IB) against Kv1.3 and KCNE4. Upper panel: Kv1.3. Lower panel: KCNE4. SM: starting material. SN+: supernatant from the IP+. SN−: supernatant from the IP−. IP+: Immunoprecipitation in the presence of the anti-Kv1.3 antibody. IP−: Immunoprecipitated in the absence of the anti-Kv1.3 antibody. ( F ) Kv1.3 localized in lipid raft fractions from Jurkat T-cells. ( G ) Kv1.3 and KCNE4 did not localize in lipid rafts from CY15 dendritic cells. Lipid rafts were isolated, and low density (1) to high density (12) sucrose gradient fractions were analyzed by western blot. Flotilin indicated low-buoyancy lipid rafts, whereas clathrin identified nonfloating raft fractions.

Techniques Used: Expressing, Negative Control, Western Blot, Immunoprecipitation, Isolation

LPS-dependent activation increases the Kv1.3/KCNE4 ratio in CY15 dendritic cells. Cells were treated for 24 h with LPS (100 ng/ml), and the protein expression of selected K + channel proteins was studied at 0, 6 and 24 h. ( A ) Confocal images of CY15 cells stained with Kv1.3 upon LPS treatment. Scale bars: 20 µm. ( B ) Representative voltage-dependent K+ currents elicited in CY15 cells treated with (LPS 24 h) or without (LPS 0 h) LPS during 24 h. Cells were held at -60 mV and 250 ms pulses to +60 mV were applied. ( C ) Peak current densitiy (pA/pF) of K + currents from CY15 cells in the absence (0 h) or the presence (24 h) of LPS. White bars, LPS 0 h (control); black bars, LPS 24 h. Values are the mean ± SE of 4–6 cells. *p
Figure Legend Snippet: LPS-dependent activation increases the Kv1.3/KCNE4 ratio in CY15 dendritic cells. Cells were treated for 24 h with LPS (100 ng/ml), and the protein expression of selected K + channel proteins was studied at 0, 6 and 24 h. ( A ) Confocal images of CY15 cells stained with Kv1.3 upon LPS treatment. Scale bars: 20 µm. ( B ) Representative voltage-dependent K+ currents elicited in CY15 cells treated with (LPS 24 h) or without (LPS 0 h) LPS during 24 h. Cells were held at -60 mV and 250 ms pulses to +60 mV were applied. ( C ) Peak current densitiy (pA/pF) of K + currents from CY15 cells in the absence (0 h) or the presence (24 h) of LPS. White bars, LPS 0 h (control); black bars, LPS 24 h. Values are the mean ± SE of 4–6 cells. *p

Techniques Used: Activation Assay, Expressing, Staining

4) Product Images from "A novel mitochondrial Kv1.3–caveolin axis controls cell survival and apoptosis"

Article Title: A novel mitochondrial Kv1.3–caveolin axis controls cell survival and apoptosis

Journal: eLife

doi: 10.7554/eLife.69099

Kv1.3 CBD less triggered no ER-stress. HEK-293 cells were transfected with Kv1.3YFP WT and Kv1.3YFP CBD less . ( A ) Representative immunoblots showing the expression of ER-stress markers. Untransfected cells treated or not with 2 µM Thapsigargin (Tg) were used as positive and negative controls of ER-stress, respectively. Total protein extracts were separated by SDS-PAGE and immunoblotted for YFP (Kv1.3), GRP78 (Bip), XBP1, ATF-4, eIF2α, eIF2α pS51, and β-actin (loading control). ( B–E ) Relative expression (arbitrary units, A.U.) of different ER-stress markers. Data are the mean ± SE (n = 3–6). *p
Figure Legend Snippet: Kv1.3 CBD less triggered no ER-stress. HEK-293 cells were transfected with Kv1.3YFP WT and Kv1.3YFP CBD less . ( A ) Representative immunoblots showing the expression of ER-stress markers. Untransfected cells treated or not with 2 µM Thapsigargin (Tg) were used as positive and negative controls of ER-stress, respectively. Total protein extracts were separated by SDS-PAGE and immunoblotted for YFP (Kv1.3), GRP78 (Bip), XBP1, ATF-4, eIF2α, eIF2α pS51, and β-actin (loading control). ( B–E ) Relative expression (arbitrary units, A.U.) of different ER-stress markers. Data are the mean ± SE (n = 3–6). *p

Techniques Used: Transfection, Western Blot, Expressing, SDS Page

The integrity of the CBD domain is involved in the surface expression of Kv1.3. HEK 293 cells were transfected with Kv1.3YFP WT and Kv1.3YFP CBD less . ( A–C ) Representative confocal images show colocalization of Kv1.3YFP WT and Kv1.3YFP CBD less with ( A ) plasma membrane (Mb), ( B ) Golgi, and ( C ) endoplasmic reticulum (ER). Green panels, Kv1.3; red panels, subcellular marker; merge panels show colocalization in yellow. The scale bar is 10 μm. ER (pDsRed-ER) and Mb (Akt-PH-pDsRed) were used as ER and Mb markers, respectively, and were cotransfected with the channel. Golgi was stained with an anti- cis -Golgi antibody (GM130). ( D ) Colocalization analysis (Pearson’s coefficient) between channel and subcellular markers. Gray bars, Kv1.3 WT. White bars, Kv1.3 CBD less . Data are the mean ± SE (n > 30 cells) **p
Figure Legend Snippet: The integrity of the CBD domain is involved in the surface expression of Kv1.3. HEK 293 cells were transfected with Kv1.3YFP WT and Kv1.3YFP CBD less . ( A–C ) Representative confocal images show colocalization of Kv1.3YFP WT and Kv1.3YFP CBD less with ( A ) plasma membrane (Mb), ( B ) Golgi, and ( C ) endoplasmic reticulum (ER). Green panels, Kv1.3; red panels, subcellular marker; merge panels show colocalization in yellow. The scale bar is 10 μm. ER (pDsRed-ER) and Mb (Akt-PH-pDsRed) were used as ER and Mb markers, respectively, and were cotransfected with the channel. Golgi was stained with an anti- cis -Golgi antibody (GM130). ( D ) Colocalization analysis (Pearson’s coefficient) between channel and subcellular markers. Gray bars, Kv1.3 WT. White bars, Kv1.3 CBD less . Data are the mean ± SE (n > 30 cells) **p

Techniques Used: Expressing, Transfection, Marker, Staining

Caveolin modulates the pro-apoptotic activity of Kv1.3. ( A ) Subcellular fractionation was used to isolate mitochondrial (Mit) and plasma membrane (Mb) fractions in HEK-293 cells transfected with Kv1.3YFP WT. Samples were immunoblotted for GFP (Kv1.3), Caveolin, Na + /K + ATPase, and VDAC. ( B, C ) Electron micrographs showing HEK-293 cells transfected with Kv1.3YFP WT. Kv1.3 was immunolabeled with 18 nm gold particles (black arrowheads) and Cav1 with 12 nm gold particles (white arrowhead). The square inset in ( B ) indicates the zoomed in region in ( C ). Scale bars represent 500 nm. ( D ) Regular human Jurkat T lymphocytes express Kv1.3 and a negligible amount of endogenous Cav1 (Cav − ). In addition, a Jurkat cell line with notable expression of Cav1 was selected (Cav+). ( E ) Jurkat cells (Cav − and Cav + ) were electroporated with Kv1.3YFP WT or Kv1.3YFP CBD less . After 24 hr, apoptosis was assessed by Annexin V staining with flow cytometry. Black bar, cells electroporated with YFP; gray bar, Kv1.3 YFP WT; white bar, Kv1.3YFP CBD less . ( F ) Mouse 3T3-L1 and 3T3-L1 Cav - preadipocytes were analyzed for the expression of endogenous Cav1 and Kv1.3. β-actin was used as a loading control. ( G ) Flow cytometric analysis quantifying apoptosis by Annexin V on 3T3-L1 (gray bar) and 3T3-L1 Cav - (white bar) preadipocytes. Note that the amount of Cav1 exerted notable effects on the Kv1.3-related apoptosis in native 3T3-L1 cells. Values are the mean ± SE of 3–6 independent experiments. *p
Figure Legend Snippet: Caveolin modulates the pro-apoptotic activity of Kv1.3. ( A ) Subcellular fractionation was used to isolate mitochondrial (Mit) and plasma membrane (Mb) fractions in HEK-293 cells transfected with Kv1.3YFP WT. Samples were immunoblotted for GFP (Kv1.3), Caveolin, Na + /K + ATPase, and VDAC. ( B, C ) Electron micrographs showing HEK-293 cells transfected with Kv1.3YFP WT. Kv1.3 was immunolabeled with 18 nm gold particles (black arrowheads) and Cav1 with 12 nm gold particles (white arrowhead). The square inset in ( B ) indicates the zoomed in region in ( C ). Scale bars represent 500 nm. ( D ) Regular human Jurkat T lymphocytes express Kv1.3 and a negligible amount of endogenous Cav1 (Cav − ). In addition, a Jurkat cell line with notable expression of Cav1 was selected (Cav+). ( E ) Jurkat cells (Cav − and Cav + ) were electroporated with Kv1.3YFP WT or Kv1.3YFP CBD less . After 24 hr, apoptosis was assessed by Annexin V staining with flow cytometry. Black bar, cells electroporated with YFP; gray bar, Kv1.3 YFP WT; white bar, Kv1.3YFP CBD less . ( F ) Mouse 3T3-L1 and 3T3-L1 Cav - preadipocytes were analyzed for the expression of endogenous Cav1 and Kv1.3. β-actin was used as a loading control. ( G ) Flow cytometric analysis quantifying apoptosis by Annexin V on 3T3-L1 (gray bar) and 3T3-L1 Cav - (white bar) preadipocytes. Note that the amount of Cav1 exerted notable effects on the Kv1.3-related apoptosis in native 3T3-L1 cells. Values are the mean ± SE of 3–6 independent experiments. *p

Techniques Used: Activity Assay, Fractionation, Transfection, Immunolabeling, Expressing, Staining, Flow Cytometry

Functional Kv1.3 CBD less channels exhibit altered biophysical properties and decreased current density at the plasma membrane. Kv1.3 WT and Kv1.3 CBD less were expressed in Xenopus oocytes to perform two-electrode voltage-clamp experiments. Cells were held at −100 mV and 2.5 s depolarizing pulses were applied in 20 mV steps to +40 mV. ( A ) Representative Kv1.3 protein expression of non-injected oocytes (control) and oocytes injected with Kv1.3WT (WT) and Kv1.3 CBD less (CBD less ). Representative currents elicited in a noninjected control oocyte ( B ) and either Kv1.3 WT ( C ) or Kv1.3 CBD less ( D ) expressing oocytes. ( E ) Peak current amplitude at +40 mV. ( F ) Time to peak at +40 mV. ( G ) Steady-state activation (left panel) and kinetic parameters (right panel). Data were fitted to a Boltzman equation. ( H ) Current inactivation at +40 mV and ( I ) τ inactivation values. Cumulative inactivation of Kv1.3 WT ( J ) and Kv1.3 CBD less ( K ) currents. Currents were elicited by a train of 8 depolarizing voltage steps to +40 mV once every second, from a holding potential of −100 mV. ( L ) Percentage of remaining current (I Kv1.3 ) at the end of the last pulse. ( M ) Membrane potential (Em (mV)) of non-injected (control) and injected oocytes with Kv1.3 WT or Kv1.3 CBD less . ( N ) Membrane input resistance (MΩ) of oocytes injected with Kv1.3 WT and Kv1.3 CBD less . Black bars, control (no injection); gray, oocytes injected with Kv1.3 WT; white bars, oocytes expressing Kv1.3 CBD less . Data are the mean ± SE (n: 20–26). *p
Figure Legend Snippet: Functional Kv1.3 CBD less channels exhibit altered biophysical properties and decreased current density at the plasma membrane. Kv1.3 WT and Kv1.3 CBD less were expressed in Xenopus oocytes to perform two-electrode voltage-clamp experiments. Cells were held at −100 mV and 2.5 s depolarizing pulses were applied in 20 mV steps to +40 mV. ( A ) Representative Kv1.3 protein expression of non-injected oocytes (control) and oocytes injected with Kv1.3WT (WT) and Kv1.3 CBD less (CBD less ). Representative currents elicited in a noninjected control oocyte ( B ) and either Kv1.3 WT ( C ) or Kv1.3 CBD less ( D ) expressing oocytes. ( E ) Peak current amplitude at +40 mV. ( F ) Time to peak at +40 mV. ( G ) Steady-state activation (left panel) and kinetic parameters (right panel). Data were fitted to a Boltzman equation. ( H ) Current inactivation at +40 mV and ( I ) τ inactivation values. Cumulative inactivation of Kv1.3 WT ( J ) and Kv1.3 CBD less ( K ) currents. Currents were elicited by a train of 8 depolarizing voltage steps to +40 mV once every second, from a holding potential of −100 mV. ( L ) Percentage of remaining current (I Kv1.3 ) at the end of the last pulse. ( M ) Membrane potential (Em (mV)) of non-injected (control) and injected oocytes with Kv1.3 WT or Kv1.3 CBD less . ( N ) Membrane input resistance (MΩ) of oocytes injected with Kv1.3 WT and Kv1.3 CBD less . Black bars, control (no injection); gray, oocytes injected with Kv1.3 WT; white bars, oocytes expressing Kv1.3 CBD less . Data are the mean ± SE (n: 20–26). *p

Techniques Used: Functional Assay, Expressing, Injection, Activation Assay

Caveolin-1 protects from apoptosis when associated with Kv1.3 in primary human T lymphocytes. Human CD4+ T lymphocytes were transfected with Kv1.3YFP WT or Kv1.3YFP CBD less and the mitochondrial membrane potential (TMRM) and apoptosis were measured. YFP-transfected cells were used as a control. ( A ) Mitochondrial membrane potential was determined by tetramethyl rhodamine methyl ester (TMRM) fluorescence. Cells were incubated with TMRM and analyzed by flow cytometry. A.U, arbitrary units. ( B ) T cells were electroporated with Kv1.3YFP WT or Kv1.3YFP CBD less with (Cav+) or without (Cav−) Cav1 Cerulean. After 24 hr, transfected cells were sorted and apoptosis was assessed by Annexin V staining with flow cytometry. The level of apoptosis in arbitrary units (A.U.) was measured in each group by resting the value of basal apoptosis in cells transfected with YFP in the presence (Cav−) or the absence (Cav−) of Cav1. Black bar, cells electroporated with YFP; gray bar, Kv1.3 YFP WT; white bar, Kv1.3YFP CBD less . Cav−, regular CD4+ cells without Cav 1; Cav+, T cells transfected with Cav1 Cer. Data are the mean ± SE (n = 5–7). *p
Figure Legend Snippet: Caveolin-1 protects from apoptosis when associated with Kv1.3 in primary human T lymphocytes. Human CD4+ T lymphocytes were transfected with Kv1.3YFP WT or Kv1.3YFP CBD less and the mitochondrial membrane potential (TMRM) and apoptosis were measured. YFP-transfected cells were used as a control. ( A ) Mitochondrial membrane potential was determined by tetramethyl rhodamine methyl ester (TMRM) fluorescence. Cells were incubated with TMRM and analyzed by flow cytometry. A.U, arbitrary units. ( B ) T cells were electroporated with Kv1.3YFP WT or Kv1.3YFP CBD less with (Cav+) or without (Cav−) Cav1 Cerulean. After 24 hr, transfected cells were sorted and apoptosis was assessed by Annexin V staining with flow cytometry. The level of apoptosis in arbitrary units (A.U.) was measured in each group by resting the value of basal apoptosis in cells transfected with YFP in the presence (Cav−) or the absence (Cav−) of Cav1. Black bar, cells electroporated with YFP; gray bar, Kv1.3 YFP WT; white bar, Kv1.3YFP CBD less . Cav−, regular CD4+ cells without Cav 1; Cav+, T cells transfected with Cav1 Cer. Data are the mean ± SE (n = 5–7). *p

Techniques Used: Transfection, Fluorescence, Incubation, Flow Cytometry, Staining

Flow cytometry evaluates apoptosis by Annexin V staining. Mouse melanoma B16F10 cells were transfected with Kv1.3YFP WT or Kv1.3YFP CBD less and treated for an additional 24 hr with different pro-apoptotic agents. Transfected cells were sorted and % of Annexin V-positive cells was obtained. Gray bars, Kv1.3 WT. White bars, Kv1.3 CBD less . Data are the mean ± SE (n = 3). *p
Figure Legend Snippet: Flow cytometry evaluates apoptosis by Annexin V staining. Mouse melanoma B16F10 cells were transfected with Kv1.3YFP WT or Kv1.3YFP CBD less and treated for an additional 24 hr with different pro-apoptotic agents. Transfected cells were sorted and % of Annexin V-positive cells was obtained. Gray bars, Kv1.3 WT. White bars, Kv1.3 CBD less . Data are the mean ± SE (n = 3). *p

Techniques Used: Flow Cytometry, Staining, Transfection

Kv1.3 CBD less severely impairs mitochondrial function. HEK-293 cells were transfected with Kv1.3YFP WT and Kv1.3YFP CBD less . Non-transfected cells were used as a control. ( A ) Mitochondrial membrane potential was determined by tetramethyl rhodamine methyl ester (TMRM) fluorescence. Positive transfected cells (separated by sorting) were incubated with TMRM and analyzed by confocal microscopy. Data are the mean ± SE (n = 3). ***p
Figure Legend Snippet: Kv1.3 CBD less severely impairs mitochondrial function. HEK-293 cells were transfected with Kv1.3YFP WT and Kv1.3YFP CBD less . Non-transfected cells were used as a control. ( A ) Mitochondrial membrane potential was determined by tetramethyl rhodamine methyl ester (TMRM) fluorescence. Positive transfected cells (separated by sorting) were incubated with TMRM and analyzed by confocal microscopy. Data are the mean ± SE (n = 3). ***p

Techniques Used: Transfection, Fluorescence, Incubation, Confocal Microscopy

Kv1.3 CBD less targets mitochondria altering mitochondrial morphology. HEK 293 and B16F10 cells were transfected with Kv1.3YFP WT or Kv1.3YFP CBD less . ( A ) Representative confocal images of HEK-293 cells cotransfected with (Aa–Ad) Kv1.3YFP WT, (Ae–Ah) Kv1.3YFP CBD less (green) and pmitoRFP (red). Images were processed (tubeness (Ac, Ag) and skeleton (Ad, Ah)) to perform morphometric analysis ( B–D ) of mitochondria in Kv1.3 positive cells. Scale bar represents 10 μm. ( B ) The form factor (arbitrary units, A.U.) describes the particle shape complexity and is computed as the average (perimeter) 2 /(4π·area). A circle corresponds to a minimum value of 1. ( C ) Average area of particles detected on the binary image. ( D ) The length of mitochondrial networks was measured as the average area of the skeletonized binary image. Data are the mean ± SE (n > 30). ***p
Figure Legend Snippet: Kv1.3 CBD less targets mitochondria altering mitochondrial morphology. HEK 293 and B16F10 cells were transfected with Kv1.3YFP WT or Kv1.3YFP CBD less . ( A ) Representative confocal images of HEK-293 cells cotransfected with (Aa–Ad) Kv1.3YFP WT, (Ae–Ah) Kv1.3YFP CBD less (green) and pmitoRFP (red). Images were processed (tubeness (Ac, Ag) and skeleton (Ad, Ah)) to perform morphometric analysis ( B–D ) of mitochondria in Kv1.3 positive cells. Scale bar represents 10 μm. ( B ) The form factor (arbitrary units, A.U.) describes the particle shape complexity and is computed as the average (perimeter) 2 /(4π·area). A circle corresponds to a minimum value of 1. ( C ) Average area of particles detected on the binary image. ( D ) The length of mitochondrial networks was measured as the average area of the skeletonized binary image. Data are the mean ± SE (n > 30). ***p

Techniques Used: Transfection

Kv1.3 CBD less does not localize to the plasma membrane in B16F10 melanoma cells. B16F10 melanoma cells were transfected with Kv1.3YFP WT and Kv1.3YFP CBD less . ( A ) Colocalization of Kv1.3YFP WT (Aa-Ac) and Kv1.3YFP CBD less (Ad-Af) at the plasma membrane. (Aa, Ad) Kv1.3YFP in green. (Ab, Ae) Plasma membrane staining with FM-464 dye in red. (Ac, Af) Merge shows colocalization in yellow. The scale bar represents 10 μm. ( B ) Quantification of colocalization was performed by Pearson’s coefficient. Data are the mean ± SE (n = 18). *p
Figure Legend Snippet: Kv1.3 CBD less does not localize to the plasma membrane in B16F10 melanoma cells. B16F10 melanoma cells were transfected with Kv1.3YFP WT and Kv1.3YFP CBD less . ( A ) Colocalization of Kv1.3YFP WT (Aa-Ac) and Kv1.3YFP CBD less (Ad-Af) at the plasma membrane. (Aa, Ad) Kv1.3YFP in green. (Ab, Ae) Plasma membrane staining with FM-464 dye in red. (Ac, Af) Merge shows colocalization in yellow. The scale bar represents 10 μm. ( B ) Quantification of colocalization was performed by Pearson’s coefficient. Data are the mean ± SE (n = 18). *p

Techniques Used: Transfection, Staining

Disruption of the CBD of Kv1.3 impairs caveolin colocalization, as well as association with the channel. HEK 293 Cav − cells were cotransfected with Kv1.3YFP WT and Kv1.3YFP mutants (F166A, W171G/F174A, and CBD less ) and Cav1. ( A ) Representative confocal images from an experiment on cell unroofing preparations (CUPs). HEK 293 Cav − cells were cotransfected with Kv1.3YFP WT+Cav1 or Kv1.3YFP mutants (F166A, W171G/F174A, and CBD less )+Cav1. (Aa, Ae, Ai) Kv1.3YFP WT; (Ab, Af, Aj) Kv1.3YFP (F166A); (Ac, Ag, Ak) Kv1.3YFP (W171G/F174A); (Ad, Ah, Al) Kv1.3YFP CBD less . Green, Kv1.3 channels; Red, Cav1; Merge, colocalization between Kv1.3 and Cav1 in yellow. The scale bar represents 10 μm. ( B ) Consensus sequence of the CBD. The amino acid sequence shows the CBD of wild type (WT) Kv1.3. The Kv1.3 CBD mutants contain amino acid substitutions (in red) to disrupt the CBD. ( C ) Pixel by pixel colocalization analysis of Kv1.3 channels and Cav1. Dark gray bar, Kv1.3 WT; light gray bar, Kv1.3 F166A mutant; black bar, Kv1.3 W171G/F174A; white bar, Kv1.3 CBD less . Values are the mean ± SE (n > 25). **p
Figure Legend Snippet: Disruption of the CBD of Kv1.3 impairs caveolin colocalization, as well as association with the channel. HEK 293 Cav − cells were cotransfected with Kv1.3YFP WT and Kv1.3YFP mutants (F166A, W171G/F174A, and CBD less ) and Cav1. ( A ) Representative confocal images from an experiment on cell unroofing preparations (CUPs). HEK 293 Cav − cells were cotransfected with Kv1.3YFP WT+Cav1 or Kv1.3YFP mutants (F166A, W171G/F174A, and CBD less )+Cav1. (Aa, Ae, Ai) Kv1.3YFP WT; (Ab, Af, Aj) Kv1.3YFP (F166A); (Ac, Ag, Ak) Kv1.3YFP (W171G/F174A); (Ad, Ah, Al) Kv1.3YFP CBD less . Green, Kv1.3 channels; Red, Cav1; Merge, colocalization between Kv1.3 and Cav1 in yellow. The scale bar represents 10 μm. ( B ) Consensus sequence of the CBD. The amino acid sequence shows the CBD of wild type (WT) Kv1.3. The Kv1.3 CBD mutants contain amino acid substitutions (in red) to disrupt the CBD. ( C ) Pixel by pixel colocalization analysis of Kv1.3 channels and Cav1. Dark gray bar, Kv1.3 WT; light gray bar, Kv1.3 F166A mutant; black bar, Kv1.3 W171G/F174A; white bar, Kv1.3 CBD less . Values are the mean ± SE (n > 25). **p

Techniques Used: Sequencing, Mutagenesis

The accumulation of Kv1.3 in mitochondria is not responsible for the increase in apoptosis. HEK 293 cells were transfected with Kv1.3 WT, Kv1.3 CBD less , or the Kv1.3 YMVIii mutant. See Materials and methods for details. The pmitoRFP mitochondrial marker was also cotransfected to stain mitochondria. ( A ) Representative confocal images from WT (Aa–Ac), CBD less (Ad–Af), and YMVIii (Ag–Ai) channels. Green, Kv1.3 channels; red, mitochondria; merge, colocalization of channel and mitochondria in yellow. Bars represent 20 μm. ( B ) Quantification of Kv1.3/mitochondria colocalization was performed by Pearson’s coefficient. Data are the mean ± SE (n > 20 cells). ( C ) Coimmunoprecipitation of Kv1.3 channels and Cav1. HEK 293 lysates were immunoprecipitated against Cav1 (IP: Cav1) and blots were immunoblotted against YFP-tagged channels (IB: Kv1.3) and Cav1 (IB: Cav1). IP−, absence of anti-Cav1 antibody. SM, starting material. ( D ) Kv1.3-transfected cells were sorted and % of Annexin V-positive cells was obtained. Gray bars, Kv1.3 WT. White bars, Kv1.3 CBD less . Black bars, Kv1.3 YMVIii. Data are the mean ± SE (n > 4). *p
Figure Legend Snippet: The accumulation of Kv1.3 in mitochondria is not responsible for the increase in apoptosis. HEK 293 cells were transfected with Kv1.3 WT, Kv1.3 CBD less , or the Kv1.3 YMVIii mutant. See Materials and methods for details. The pmitoRFP mitochondrial marker was also cotransfected to stain mitochondria. ( A ) Representative confocal images from WT (Aa–Ac), CBD less (Ad–Af), and YMVIii (Ag–Ai) channels. Green, Kv1.3 channels; red, mitochondria; merge, colocalization of channel and mitochondria in yellow. Bars represent 20 μm. ( B ) Quantification of Kv1.3/mitochondria colocalization was performed by Pearson’s coefficient. Data are the mean ± SE (n > 20 cells). ( C ) Coimmunoprecipitation of Kv1.3 channels and Cav1. HEK 293 lysates were immunoprecipitated against Cav1 (IP: Cav1) and blots were immunoblotted against YFP-tagged channels (IB: Kv1.3) and Cav1 (IB: Cav1). IP−, absence of anti-Cav1 antibody. SM, starting material. ( D ) Kv1.3-transfected cells were sorted and % of Annexin V-positive cells was obtained. Gray bars, Kv1.3 WT. White bars, Kv1.3 CBD less . Black bars, Kv1.3 YMVIii. Data are the mean ± SE (n > 4). *p

Techniques Used: Transfection, Mutagenesis, Marker, Staining, Immunoprecipitation

Kv1.3 colocalizes with plasma membrane and mitochondria in primary human T lymphocytes. CD4+ lymphocytes were isolated from human blood as indicated in Materials and methods. ( A ) Representative western blot from HEK 293 cells and T lymphocytes samples from two independent human donors showing differential protein expression of Kv1.3, Flotillin, and Cav1. β-Actin was a loading control. ( B ) Representative confocal images of Kv1.3 colocalization in plasma membrane (Mb) from Kv1.3YFP WT and Kv1.3YFP CBD less -transfected cells. WGA stained plasma membrane. ( C ) Representative confocal images of Kv1.3 colocalization in mitochondria (mito) from Kv1.3YFP WT and Kv1.3YFP CBD less expressing cells. MitoTracker was used for mitochondrial staining. Scale bar represents 10 μm. Quantification of Kv1.3/Mb ( D ) and Kv1.3/mito ( E ) colocalization was performed by Pearson’s coefficient. Data are the mean ± SE (n > 20), Student’s t-test. Gray bars, Kv1.3YFP WT cells; white bars, Kv1.3YFP CBD less cells.
Figure Legend Snippet: Kv1.3 colocalizes with plasma membrane and mitochondria in primary human T lymphocytes. CD4+ lymphocytes were isolated from human blood as indicated in Materials and methods. ( A ) Representative western blot from HEK 293 cells and T lymphocytes samples from two independent human donors showing differential protein expression of Kv1.3, Flotillin, and Cav1. β-Actin was a loading control. ( B ) Representative confocal images of Kv1.3 colocalization in plasma membrane (Mb) from Kv1.3YFP WT and Kv1.3YFP CBD less -transfected cells. WGA stained plasma membrane. ( C ) Representative confocal images of Kv1.3 colocalization in mitochondria (mito) from Kv1.3YFP WT and Kv1.3YFP CBD less expressing cells. MitoTracker was used for mitochondrial staining. Scale bar represents 10 μm. Quantification of Kv1.3/Mb ( D ) and Kv1.3/mito ( E ) colocalization was performed by Pearson’s coefficient. Data are the mean ± SE (n > 20), Student’s t-test. Gray bars, Kv1.3YFP WT cells; white bars, Kv1.3YFP CBD less cells.

Techniques Used: Isolation, Western Blot, Expressing, Transfection, Whole Genome Amplification, Staining

Kv1.3 CBD less forms tetramers. ( A ) Representative FRET experiment of CUPs from HEK 293 cells cotransfected with Kv1.3YFP WT+Kv1.3 Cer WT (left panels) or Kv1.3YFP CBD less +Kv1.3 Cer CBD less (right panels). Top to bottom: acceptor (Kv1.3-YFP) and donor (Kv1.3-Cer) prebleach and postbleach images. Square insets indicate the bleached zone, and the red area indicates the analyzed ROI. Line graphs show changes in donor (cerulean) and acceptor (yellow) fluorescence after bleaching. The scale bar represents 10 μm. ( B ) FRET efficiency (%) of YFP +Cer (negative control), Kv1.3YFP WT +Kv1.3 Cer WT (positive control), and Kv1.3YFP CBD less +Kv1.3 Cer CBD less . Values are mean ± SE (n > 20). ***p
Figure Legend Snippet: Kv1.3 CBD less forms tetramers. ( A ) Representative FRET experiment of CUPs from HEK 293 cells cotransfected with Kv1.3YFP WT+Kv1.3 Cer WT (left panels) or Kv1.3YFP CBD less +Kv1.3 Cer CBD less (right panels). Top to bottom: acceptor (Kv1.3-YFP) and donor (Kv1.3-Cer) prebleach and postbleach images. Square insets indicate the bleached zone, and the red area indicates the analyzed ROI. Line graphs show changes in donor (cerulean) and acceptor (yellow) fluorescence after bleaching. The scale bar represents 10 μm. ( B ) FRET efficiency (%) of YFP +Cer (negative control), Kv1.3YFP WT +Kv1.3 Cer WT (positive control), and Kv1.3YFP CBD less +Kv1.3 Cer CBD less . Values are mean ± SE (n > 20). ***p

Techniques Used: Fluorescence, Negative Control, Positive Control

5) Product Images from "The C-terminal domain of Kv1.3 regulates functional interactions with the KCNE4 subunit"

Article Title: The C-terminal domain of Kv1.3 regulates functional interactions with the KCNE4 subunit

Journal: Journal of Cell Science

doi: 10.1242/jcs.191650

Kv1.3 and KCNE4 form functional channels in leukocytes. Endogenous expression of Kv1.3 and KCNE4 was analyzed in human Jurkat T-lymphocytes and mouse CY15 dendritic cells. Endogenous voltage-dependent K + currents were elicited in Jurkat (A) and CY15 cells (B). Cells were held at −60 mV, and pulse potentials were applied as indicated. Cumulative inactivation of K + currents was elicited in Jurkat (C) and CY15 cells (D) by a train of 15 depolarizing 250 ms pulses ranging from −80 mV to +60 mV once every 1 s. (E) K + currents elicited in Jurkat (left axis) and CY15 cells (right axis) at the peak current density (+80 mV) in the presence or the absence of 1 and 10 nM MgTx. Black bars, Jurkat T-cells; white bars, CY15 dendritic cells. Values are shown as the mean±s.e.m. ( n =6–8 cells/group). (F) Steady-state activation of outward K + currents. Gray circles, HEK-293 cells transfected with Kv1.3; black circles, Jurkat cells; white circles, CY15 cells. Values are shown as the mean±s.e.m. ( n =4–6 independent cells). (G) Protein expression of Kv1.3, Kv1.5 and KCNE4 in leukocytes as determined by western blotting. HEK-293 cells were used as a negative control. Notably, although Jurkat and CY15 dendritic cells express both Kv1.3 and KCNE4, the abundance of KCNE4 and Kv1.5 is much lower in T-cells and was barely detected. (H) Representative confocal images of Kv1.3 and KCNE4 in Jurkat T-lymphocytes and CY15 dendritic cells. Scale bars: 10 µm. (I) KCNE4 co-immunoprecipitates with Kv1.3 in dendritic cells. Western blots from Kv1.3 and KCNE4 co-immunoprecipitation. Lysates were immunoprecipitated for Kv1.3 (IP: Kv1.3). Upper panel: Kv1.3 immunoblot (IB: Kv1.3). Lower panel: KCNE4 immunoblot (IB: KCNE4). SM, starting material (input); IP+, immunoprecipitation in the presence of the anti-Kv1.3 antibody; IP−, immunoprecipitation in the absence of the anti-Kv1.3 antibody; SN+, supernatant from the IP+; SN−, supernatant from the IP−.
Figure Legend Snippet: Kv1.3 and KCNE4 form functional channels in leukocytes. Endogenous expression of Kv1.3 and KCNE4 was analyzed in human Jurkat T-lymphocytes and mouse CY15 dendritic cells. Endogenous voltage-dependent K + currents were elicited in Jurkat (A) and CY15 cells (B). Cells were held at −60 mV, and pulse potentials were applied as indicated. Cumulative inactivation of K + currents was elicited in Jurkat (C) and CY15 cells (D) by a train of 15 depolarizing 250 ms pulses ranging from −80 mV to +60 mV once every 1 s. (E) K + currents elicited in Jurkat (left axis) and CY15 cells (right axis) at the peak current density (+80 mV) in the presence or the absence of 1 and 10 nM MgTx. Black bars, Jurkat T-cells; white bars, CY15 dendritic cells. Values are shown as the mean±s.e.m. ( n =6–8 cells/group). (F) Steady-state activation of outward K + currents. Gray circles, HEK-293 cells transfected with Kv1.3; black circles, Jurkat cells; white circles, CY15 cells. Values are shown as the mean±s.e.m. ( n =4–6 independent cells). (G) Protein expression of Kv1.3, Kv1.5 and KCNE4 in leukocytes as determined by western blotting. HEK-293 cells were used as a negative control. Notably, although Jurkat and CY15 dendritic cells express both Kv1.3 and KCNE4, the abundance of KCNE4 and Kv1.5 is much lower in T-cells and was barely detected. (H) Representative confocal images of Kv1.3 and KCNE4 in Jurkat T-lymphocytes and CY15 dendritic cells. Scale bars: 10 µm. (I) KCNE4 co-immunoprecipitates with Kv1.3 in dendritic cells. Western blots from Kv1.3 and KCNE4 co-immunoprecipitation. Lysates were immunoprecipitated for Kv1.3 (IP: Kv1.3). Upper panel: Kv1.3 immunoblot (IB: Kv1.3). Lower panel: KCNE4 immunoblot (IB: KCNE4). SM, starting material (input); IP+, immunoprecipitation in the presence of the anti-Kv1.3 antibody; IP−, immunoprecipitation in the absence of the anti-Kv1.3 antibody; SN+, supernatant from the IP+; SN−, supernatant from the IP−.

Techniques Used: Functional Assay, Expressing, Mass Spectrometry, Activation Assay, Transfection, Western Blot, Negative Control, Immunoprecipitation

Kv1.3, but not Kv1.5, associates with KCNE4 in HEK-293 cells. KCNE4 modulates Kv1.3 trafficking and activity. HEK-293 cells were transfected with Kv1.3–YFP or Kv1.5–YFP in the presence or absence of KCNE4–CFP. Confocal images of (A) Kv1.3–YFP, (B) Kv1.5–YFP and (C) KCNE4–CFP. (D) HEK-293 cells co-transfected with Kv1.3 and KCNE4. (E) Kv1.5 and KCNE4. Color code: green, channels; red, KCNE4; yellow in merge panels shows colocalization. Scale bars: 10 µm. Voltage-dependent K + currents were elicited in HEK-293 cells transfected with Kv1.3 (F,G) and Kv1.5 (H,I) in the absence (F,H) or the presence (G,I) of KCNE4. Cells were held at −80 mV, and pulse potentials were applied as indicated. (J) Current density versus voltage plot of outward K + currents. White circles, Kv1.3; black circles, Kv1.3+KCNE4; light gray circles, Kv1.5; dark gray circles, Kv1.5+KCNE4. Values are shown as the mean±s.e.m. ( n =6–10 independent cells). (K) Molecular association of Kv1.3 and Kv1.5 with KCNE4 as measured by FRET efficiency (%). HEK-293 cells were transfected with Kv1.3–YFP and Kv1.5–YFP in the presence of KCNE4–CFP. YFP–CFP and Kv1.3–YFP or Kv1.3–CFP were used as negative and positive controls, respectively. Values are shown as the mean±s.e.m. ( n > 25 independent cells). ** P
Figure Legend Snippet: Kv1.3, but not Kv1.5, associates with KCNE4 in HEK-293 cells. KCNE4 modulates Kv1.3 trafficking and activity. HEK-293 cells were transfected with Kv1.3–YFP or Kv1.5–YFP in the presence or absence of KCNE4–CFP. Confocal images of (A) Kv1.3–YFP, (B) Kv1.5–YFP and (C) KCNE4–CFP. (D) HEK-293 cells co-transfected with Kv1.3 and KCNE4. (E) Kv1.5 and KCNE4. Color code: green, channels; red, KCNE4; yellow in merge panels shows colocalization. Scale bars: 10 µm. Voltage-dependent K + currents were elicited in HEK-293 cells transfected with Kv1.3 (F,G) and Kv1.5 (H,I) in the absence (F,H) or the presence (G,I) of KCNE4. Cells were held at −80 mV, and pulse potentials were applied as indicated. (J) Current density versus voltage plot of outward K + currents. White circles, Kv1.3; black circles, Kv1.3+KCNE4; light gray circles, Kv1.5; dark gray circles, Kv1.5+KCNE4. Values are shown as the mean±s.e.m. ( n =6–10 independent cells). (K) Molecular association of Kv1.3 and Kv1.5 with KCNE4 as measured by FRET efficiency (%). HEK-293 cells were transfected with Kv1.3–YFP and Kv1.5–YFP in the presence of KCNE4–CFP. YFP–CFP and Kv1.3–YFP or Kv1.3–CFP were used as negative and positive controls, respectively. Values are shown as the mean±s.e.m. ( n > 25 independent cells). ** P

Techniques Used: Activity Assay, Transfection

The structure of the Kv1.3 C-terminal domain mediates the association with KCNE4. HEK-293 cells were transfected with Kv1.3 wt, various Kv1.3 truncated mutants and KCNE4. (A) Scheme representing Kv1.3 wt and the different C-terminus truncation mutants. Bars indicate the N-terminus in white (Nt), the S1–S6 transmembrane domains in black and the C-terminal domain in gray (Ct). (B) Schematic diagram of the Kv1.3 C-terminal domain highlighting some important forward trafficking signatures and the distal PDZ motif. The residues in black indicate the truncations. A unique change (A to S) between human and rodents is indicated in gray. The S6 transmembrane domain is represented as a gray barrel. The HRETE, ExExE, YMVIEE and the PDZ motifs are indicated by gray boxes. The SwissProt number, as well as the number of residues in the C-terminus, for rat, mouse and humans is also indicated. (C) Cell lysates. Immunoprecipitation of Kv1.3–YFP C-terminus mutants with anti-GFP antibody. Top panels show the immunoblot against GFP (Kv1.3). Bottom panels show the immunoblot against KCNE4 (arrow). (D) Co-immunoprecipitation (Co-IP). Cell lysates (as in C) were immunoprecipitated with anti-GFP antibody (IP Kv1.3) and immunoblotted against Kv1.3 (IB: Kv1.3, top panel) and KCNE4 (IP: KCNE4, bottom panel). Note that the anti-GFP antibody used recognizes YFP. (E) Plot of the remaining number of C-terminal residues in the Kv1.3 C-terminus truncated mutants versus the co-immunoprecipitation of KCNE4. Values (mean±s.e.m., n =4) are calculated as the logarithm (log) of co-immunoprecipitation in arbitrary units (AU) standardized to the maximum level (scored as 1) of the co-immunoprecipitation of KCNE4 with Kv1.3 wt from D. Values followed a notable linear correlation ( r 2 =0.8175) which yielded a statistical significant ( P =0.0133) Pearson’s coefficient (0.9041).
Figure Legend Snippet: The structure of the Kv1.3 C-terminal domain mediates the association with KCNE4. HEK-293 cells were transfected with Kv1.3 wt, various Kv1.3 truncated mutants and KCNE4. (A) Scheme representing Kv1.3 wt and the different C-terminus truncation mutants. Bars indicate the N-terminus in white (Nt), the S1–S6 transmembrane domains in black and the C-terminal domain in gray (Ct). (B) Schematic diagram of the Kv1.3 C-terminal domain highlighting some important forward trafficking signatures and the distal PDZ motif. The residues in black indicate the truncations. A unique change (A to S) between human and rodents is indicated in gray. The S6 transmembrane domain is represented as a gray barrel. The HRETE, ExExE, YMVIEE and the PDZ motifs are indicated by gray boxes. The SwissProt number, as well as the number of residues in the C-terminus, for rat, mouse and humans is also indicated. (C) Cell lysates. Immunoprecipitation of Kv1.3–YFP C-terminus mutants with anti-GFP antibody. Top panels show the immunoblot against GFP (Kv1.3). Bottom panels show the immunoblot against KCNE4 (arrow). (D) Co-immunoprecipitation (Co-IP). Cell lysates (as in C) were immunoprecipitated with anti-GFP antibody (IP Kv1.3) and immunoblotted against Kv1.3 (IB: Kv1.3, top panel) and KCNE4 (IP: KCNE4, bottom panel). Note that the anti-GFP antibody used recognizes YFP. (E) Plot of the remaining number of C-terminal residues in the Kv1.3 C-terminus truncated mutants versus the co-immunoprecipitation of KCNE4. Values (mean±s.e.m., n =4) are calculated as the logarithm (log) of co-immunoprecipitation in arbitrary units (AU) standardized to the maximum level (scored as 1) of the co-immunoprecipitation of KCNE4 with Kv1.3 wt from D. Values followed a notable linear correlation ( r 2 =0.8175) which yielded a statistical significant ( P =0.0133) Pearson’s coefficient (0.9041).

Techniques Used: Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay

KCNE4 interacts with the C-terminal domain of Kv1.3, thereby impairing the association with the COPII anterograde trafficking mechanism. HEK-293 cells were transfected with Kv1.3 in the presence or the absence of KCNE4, Sec24D–YFP and Sar1(H79G)–HA. (A,B) Co-immunoprecipitation of Kv1.3 and Sec24D in the presence or the absence of KCNE4. (A) Expression of Sec24D, Kv1.3 and KCNE4 in starting materials (cell lysates). (B) Co-immunoprecipitations. Samples were immunoprecipitated (+IP) against Sec24D–YFP and immunoblotted (IB) against GFP (Sec24D), Kv1.3 and KCNE4. Sar1(H79G) and KCNE4 expression led to the retention of Kv1.3 in the ER. Note that KCNE4 notably impaired the association between Kv1.3 and Sec24D. In addition, KCNE4 showed a slight co-immunoprecipitation with Sec24. Note that the anti-GFP antibody used recognizes YFP. (C–H) Molecular interaction between Kv1.3 and Sec24D in the presence or the absence of KCNE4. (C–F) A representative FRET experiment for Kv1.3 and Sec24D in the presence of KCNE4. (C) Sec24D–YFP (red) in the acceptor panel. (D) Kv1.3–CFP (green) in the donor panel. (E) Colocalization is shown in yellow. (F) Magnification of the boxed area in E. Circles highlight some ROIs. Scale bar: 10 µm. (G) FRET efficiency of the Kv1.3–Sec24D interaction in the presence or the absence of Sar1(H79G) and KCNE4. YFP and CFP, and Kv1.3–CFP and Kv1.3–YFP pairs were used as negative and positive controls, respectively. Note that although FRET values between Kv1.3 and Sec24D were positive, the presence of KCNE4 triggered a larger variability. (H) Relative KCNE4 expression in arbitrary units (AU) in FRET-positive or FRET-negative ROIs. Considering the FRET between Kv1.3 and Sec24D, two types of ROI were observed. When ROI triggered clearly positive FRET, KCNE4 expression was low. However, in ROIs with no relevant FRET values, KCNE4 expression was higher. Values are shown as the mean±s.e.m. of n > 30 cells. * P
Figure Legend Snippet: KCNE4 interacts with the C-terminal domain of Kv1.3, thereby impairing the association with the COPII anterograde trafficking mechanism. HEK-293 cells were transfected with Kv1.3 in the presence or the absence of KCNE4, Sec24D–YFP and Sar1(H79G)–HA. (A,B) Co-immunoprecipitation of Kv1.3 and Sec24D in the presence or the absence of KCNE4. (A) Expression of Sec24D, Kv1.3 and KCNE4 in starting materials (cell lysates). (B) Co-immunoprecipitations. Samples were immunoprecipitated (+IP) against Sec24D–YFP and immunoblotted (IB) against GFP (Sec24D), Kv1.3 and KCNE4. Sar1(H79G) and KCNE4 expression led to the retention of Kv1.3 in the ER. Note that KCNE4 notably impaired the association between Kv1.3 and Sec24D. In addition, KCNE4 showed a slight co-immunoprecipitation with Sec24. Note that the anti-GFP antibody used recognizes YFP. (C–H) Molecular interaction between Kv1.3 and Sec24D in the presence or the absence of KCNE4. (C–F) A representative FRET experiment for Kv1.3 and Sec24D in the presence of KCNE4. (C) Sec24D–YFP (red) in the acceptor panel. (D) Kv1.3–CFP (green) in the donor panel. (E) Colocalization is shown in yellow. (F) Magnification of the boxed area in E. Circles highlight some ROIs. Scale bar: 10 µm. (G) FRET efficiency of the Kv1.3–Sec24D interaction in the presence or the absence of Sar1(H79G) and KCNE4. YFP and CFP, and Kv1.3–CFP and Kv1.3–YFP pairs were used as negative and positive controls, respectively. Note that although FRET values between Kv1.3 and Sec24D were positive, the presence of KCNE4 triggered a larger variability. (H) Relative KCNE4 expression in arbitrary units (AU) in FRET-positive or FRET-negative ROIs. Considering the FRET between Kv1.3 and Sec24D, two types of ROI were observed. When ROI triggered clearly positive FRET, KCNE4 expression was low. However, in ROIs with no relevant FRET values, KCNE4 expression was higher. Values are shown as the mean±s.e.m. of n > 30 cells. * P

Techniques Used: Transfection, Immunoprecipitation, Expressing

The KCNE4 interaction further aggravated the intracellular retention of the Kv1.3(E 483/484 I) mutant. HEK-293 cells were transfected with Kv1.3wt, Kv1.3(E 483/484 I) and KCNE4. Membrane surface labeling was performed using WGA as described in the Materials and Methods. (A–G) Representative confocal images of Kv1.3 wt in the absence (A–C) or the presence (D–G) of KCNE4. (H–N) Representative confocal images of Kv1.3(E 483/484 I) in the absence (H–J) or the presence (K–N) of KCNE4. (A,D,H,K) Kv1.3 channels in green. (E,L) KCNE4 in red. (B,F,I,M) WGA membrane staining in blue. (C,G,J,N) Color code in merged panels: Kv1.3 channels and KCNE4 colocalization, yellow; channels and membrane, cyan; triple colocalization, white. Scale bars: 10 µm. (O) Kv1.3 membrane surface expression in arbitrary units (AU) in a pixel-by-pixel analysis. Values are shown as the mean±s.e.m. of n > 25 cells. * P
Figure Legend Snippet: The KCNE4 interaction further aggravated the intracellular retention of the Kv1.3(E 483/484 I) mutant. HEK-293 cells were transfected with Kv1.3wt, Kv1.3(E 483/484 I) and KCNE4. Membrane surface labeling was performed using WGA as described in the Materials and Methods. (A–G) Representative confocal images of Kv1.3 wt in the absence (A–C) or the presence (D–G) of KCNE4. (H–N) Representative confocal images of Kv1.3(E 483/484 I) in the absence (H–J) or the presence (K–N) of KCNE4. (A,D,H,K) Kv1.3 channels in green. (E,L) KCNE4 in red. (B,F,I,M) WGA membrane staining in blue. (C,G,J,N) Color code in merged panels: Kv1.3 channels and KCNE4 colocalization, yellow; channels and membrane, cyan; triple colocalization, white. Scale bars: 10 µm. (O) Kv1.3 membrane surface expression in arbitrary units (AU) in a pixel-by-pixel analysis. Values are shown as the mean±s.e.m. of n > 25 cells. * P

Techniques Used: Mutagenesis, Transfection, Labeling, Whole Genome Amplification, Staining, Expressing

KCNE4 associates with the Kv1.3 C-terminal domain. HEK-293 cells were transfected with Kv1.3–YFP channels and KCNE4. (A–I) Confocal images from HEK-293 cells transfected with wt and mutant Kv1.3–YFP channels and KCNE4–CFP. (A,D,G) Kv1.3–YFP, Kv1.3ΔN–YFP and Kv1.3ΔC–YFP channels in green. (B,E,H) Cellular distribution of KCNE4–CFP in red. (C,F,I) Merged images show colocalization in yellow. Scale bars: 10 µm. Note that in all cases, Kv1.3 is mostly intracellular. Representative cartoons of different Kv1.3 channels are shown at the left of their respective images. The red dotted ellipse highlights the deleted domain. (J) Histogram representing the relative colocalization between Kv1.3 channels and KCNE4. Results in arbitrary units (A.U.) are the mean±s.e.m. of a pixel-by-pixel analysis on 25–40 cells. *** P
Figure Legend Snippet: KCNE4 associates with the Kv1.3 C-terminal domain. HEK-293 cells were transfected with Kv1.3–YFP channels and KCNE4. (A–I) Confocal images from HEK-293 cells transfected with wt and mutant Kv1.3–YFP channels and KCNE4–CFP. (A,D,G) Kv1.3–YFP, Kv1.3ΔN–YFP and Kv1.3ΔC–YFP channels in green. (B,E,H) Cellular distribution of KCNE4–CFP in red. (C,F,I) Merged images show colocalization in yellow. Scale bars: 10 µm. Note that in all cases, Kv1.3 is mostly intracellular. Representative cartoons of different Kv1.3 channels are shown at the left of their respective images. The red dotted ellipse highlights the deleted domain. (J) Histogram representing the relative colocalization between Kv1.3 channels and KCNE4. Results in arbitrary units (A.U.) are the mean±s.e.m. of a pixel-by-pixel analysis on 25–40 cells. *** P

Techniques Used: Transfection, Mutagenesis

The disruption of the ERRM of KCNE4 only partially counteracted the intracellular retention of Kv1.3. HEK-293 cells were transfected with Kv1.3–YFP, KCNE4–CFP and the KCNE4(ERRM)–CFP mutant. (A) Cartoon illustrating the ERRM in the C-terminal domain of KCNE4. Positive residues (R and K) in the wild-type ERRM (brown box) were mutated to alanine residues to disrupt the motif. N-term, N-terminal extracellular domain; C-term, C-terminal intracellular domain; TMD, transmembrane domain. (B–G) Confocal images of KCNE4 wt (B–D) and the KCNE4(ERRM) mutant (E–G). (B,E) KCNE4. (C,F) Membrane staining with WGA. (D,G) Merge channel. (H–O) Representative images of Kv1.3 in the presence of KCNE4 (H–K) and KCNE4(ERRM) (L–O). (H,L) Kv1.3 in green; (I,M) KCNE4 in red; (J,N) membrane in blue; (K,O) merge. Note that although Kv1.3 colocalization with KCNE4 showed marked intracellular retention in all cases, the channel reached the surface better in the presence of KCNE4(ERRM). Scale bars: 10 µm. (P) Pixel-by-pixel analysis of the relative membrane surface expression, in arbitrary units (AU), of KCNE4 (black bars) and Kv1.3 (white bars). Note that KCNE4(ERRM) showed double the KCNE4 membrane surface expression. Furthermore, Kv1.3 reached the cell surface more efficiently in the presence of KCNE4(ERRM) than with KCNE4. Values are shown as the mean±s.e.m. ( n > 25 cells). * P
Figure Legend Snippet: The disruption of the ERRM of KCNE4 only partially counteracted the intracellular retention of Kv1.3. HEK-293 cells were transfected with Kv1.3–YFP, KCNE4–CFP and the KCNE4(ERRM)–CFP mutant. (A) Cartoon illustrating the ERRM in the C-terminal domain of KCNE4. Positive residues (R and K) in the wild-type ERRM (brown box) were mutated to alanine residues to disrupt the motif. N-term, N-terminal extracellular domain; C-term, C-terminal intracellular domain; TMD, transmembrane domain. (B–G) Confocal images of KCNE4 wt (B–D) and the KCNE4(ERRM) mutant (E–G). (B,E) KCNE4. (C,F) Membrane staining with WGA. (D,G) Merge channel. (H–O) Representative images of Kv1.3 in the presence of KCNE4 (H–K) and KCNE4(ERRM) (L–O). (H,L) Kv1.3 in green; (I,M) KCNE4 in red; (J,N) membrane in blue; (K,O) merge. Note that although Kv1.3 colocalization with KCNE4 showed marked intracellular retention in all cases, the channel reached the surface better in the presence of KCNE4(ERRM). Scale bars: 10 µm. (P) Pixel-by-pixel analysis of the relative membrane surface expression, in arbitrary units (AU), of KCNE4 (black bars) and Kv1.3 (white bars). Note that KCNE4(ERRM) showed double the KCNE4 membrane surface expression. Furthermore, Kv1.3 reached the cell surface more efficiently in the presence of KCNE4(ERRM) than with KCNE4. Values are shown as the mean±s.e.m. ( n > 25 cells). * P

Techniques Used: Transfection, Mutagenesis, Staining, Whole Genome Amplification, Expressing

The C-terminus of Kv1.3, but not Kv1.5, is necessary and sufficient for the association with KCNE4. Confocal images from HEK-293 cells transfected with Kv1.3–YFP (A–C), Kv1.3NKv1.5–YFP (D–F) and Kv1.3CKv1.5–YFP (G–I) in the presence of KCNE4–CFP. All Kv1.3 channels presented an intracellular distribution, but the colocalization of Kv1.3CKv1.5 with KCNE4–CFP is 40% lower than that with Kv1.3 or Kv1.3NKv1.5. HEK-293 cells were also transfected with Kv1.5–YFP (J–L), Kv1.5NKv1.3 (M–O) and Kv1.5CKv1.3 (P–R) in the presence of KCNE4. Although all Kv1.5 channels were distributed intracellularly, notable colocalization was only observed between KCNE4 and Kv1.5CKv1.3. Cartoons on top of panels represent chimeric channels with the N- and C-terminal domains, and six transmembrane domains (boxes). Blue, Kv1.3 domains; green, Kv1.5 domains. Color code in confocal images: green, channels; red, KCNE4; yellow, colocalization in merge panels. Scale bars: 10 μm. (S) Histogram representing the relative colocalization between Kv1.3 and Kv1.5 channels and chimeras and KCNE4. Results are mean±s.e.m. ( n =25–30 independent cells). Black columns represent Kv1.3 and Kv1.3–Kv1.5 chimeras (1.3N1.5 and 1.3C1.5). White columns represent Kv1.5 and Kv1.5–Kv1.3 chimeras (1.5N1.3 and 1.5C1.3). *** P
Figure Legend Snippet: The C-terminus of Kv1.3, but not Kv1.5, is necessary and sufficient for the association with KCNE4. Confocal images from HEK-293 cells transfected with Kv1.3–YFP (A–C), Kv1.3NKv1.5–YFP (D–F) and Kv1.3CKv1.5–YFP (G–I) in the presence of KCNE4–CFP. All Kv1.3 channels presented an intracellular distribution, but the colocalization of Kv1.3CKv1.5 with KCNE4–CFP is 40% lower than that with Kv1.3 or Kv1.3NKv1.5. HEK-293 cells were also transfected with Kv1.5–YFP (J–L), Kv1.5NKv1.3 (M–O) and Kv1.5CKv1.3 (P–R) in the presence of KCNE4. Although all Kv1.5 channels were distributed intracellularly, notable colocalization was only observed between KCNE4 and Kv1.5CKv1.3. Cartoons on top of panels represent chimeric channels with the N- and C-terminal domains, and six transmembrane domains (boxes). Blue, Kv1.3 domains; green, Kv1.5 domains. Color code in confocal images: green, channels; red, KCNE4; yellow, colocalization in merge panels. Scale bars: 10 μm. (S) Histogram representing the relative colocalization between Kv1.3 and Kv1.5 channels and chimeras and KCNE4. Results are mean±s.e.m. ( n =25–30 independent cells). Black columns represent Kv1.3 and Kv1.3–Kv1.5 chimeras (1.3N1.5 and 1.3C1.5). White columns represent Kv1.5 and Kv1.5–Kv1.3 chimeras (1.5N1.3 and 1.5C1.3). *** P

Techniques Used: Transfection

6) Product Images from "The C-terminal domain of Kv1.3 regulates functional interactions with the KCNE4 subunit"

Article Title: The C-terminal domain of Kv1.3 regulates functional interactions with the KCNE4 subunit

Journal: Journal of Cell Science

doi: 10.1242/jcs.191650

Kv1.3 and KCNE4 form functional channels in leukocytes. Endogenous expression of Kv1.3 and KCNE4 was analyzed in human Jurkat T-lymphocytes and mouse CY15 dendritic cells. Endogenous voltage-dependent K + currents were elicited in Jurkat (A) and CY15 cells (B). Cells were held at −60 mV, and pulse potentials were applied as indicated. Cumulative inactivation of K + currents was elicited in Jurkat (C) and CY15 cells (D) by a train of 15 depolarizing 250 ms pulses ranging from −80 mV to +60 mV once every 1 s. (E) K + currents elicited in Jurkat (left axis) and CY15 cells (right axis) at the peak current density (+80 mV) in the presence or the absence of 1 and 10 nM MgTx. Black bars, Jurkat T-cells; white bars, CY15 dendritic cells. Values are shown as the mean±s.e.m. ( n =6–8 cells/group). (F) Steady-state activation of outward K + currents. Gray circles, HEK-293 cells transfected with Kv1.3; black circles, Jurkat cells; white circles, CY15 cells. Values are shown as the mean±s.e.m. ( n =4–6 independent cells). (G) Protein expression of Kv1.3, Kv1.5 and KCNE4 in leukocytes as determined by western blotting. HEK-293 cells were used as a negative control. Notably, although Jurkat and CY15 dendritic cells express both Kv1.3 and KCNE4, the abundance of KCNE4 and Kv1.5 is much lower in T-cells and was barely detected. (H) Representative confocal images of Kv1.3 and KCNE4 in Jurkat T-lymphocytes and CY15 dendritic cells. Scale bars: 10 µm. (I) KCNE4 co-immunoprecipitates with Kv1.3 in dendritic cells. Western blots from Kv1.3 and KCNE4 co-immunoprecipitation. Lysates were immunoprecipitated for Kv1.3 (IP: Kv1.3). Upper panel: Kv1.3 immunoblot (IB: Kv1.3). Lower panel: KCNE4 immunoblot (IB: KCNE4). SM, starting material (input); IP+, immunoprecipitation in the presence of the anti-Kv1.3 antibody; IP−, immunoprecipitation in the absence of the anti-Kv1.3 antibody; SN+, supernatant from the IP+; SN−, supernatant from the IP−.
Figure Legend Snippet: Kv1.3 and KCNE4 form functional channels in leukocytes. Endogenous expression of Kv1.3 and KCNE4 was analyzed in human Jurkat T-lymphocytes and mouse CY15 dendritic cells. Endogenous voltage-dependent K + currents were elicited in Jurkat (A) and CY15 cells (B). Cells were held at −60 mV, and pulse potentials were applied as indicated. Cumulative inactivation of K + currents was elicited in Jurkat (C) and CY15 cells (D) by a train of 15 depolarizing 250 ms pulses ranging from −80 mV to +60 mV once every 1 s. (E) K + currents elicited in Jurkat (left axis) and CY15 cells (right axis) at the peak current density (+80 mV) in the presence or the absence of 1 and 10 nM MgTx. Black bars, Jurkat T-cells; white bars, CY15 dendritic cells. Values are shown as the mean±s.e.m. ( n =6–8 cells/group). (F) Steady-state activation of outward K + currents. Gray circles, HEK-293 cells transfected with Kv1.3; black circles, Jurkat cells; white circles, CY15 cells. Values are shown as the mean±s.e.m. ( n =4–6 independent cells). (G) Protein expression of Kv1.3, Kv1.5 and KCNE4 in leukocytes as determined by western blotting. HEK-293 cells were used as a negative control. Notably, although Jurkat and CY15 dendritic cells express both Kv1.3 and KCNE4, the abundance of KCNE4 and Kv1.5 is much lower in T-cells and was barely detected. (H) Representative confocal images of Kv1.3 and KCNE4 in Jurkat T-lymphocytes and CY15 dendritic cells. Scale bars: 10 µm. (I) KCNE4 co-immunoprecipitates with Kv1.3 in dendritic cells. Western blots from Kv1.3 and KCNE4 co-immunoprecipitation. Lysates were immunoprecipitated for Kv1.3 (IP: Kv1.3). Upper panel: Kv1.3 immunoblot (IB: Kv1.3). Lower panel: KCNE4 immunoblot (IB: KCNE4). SM, starting material (input); IP+, immunoprecipitation in the presence of the anti-Kv1.3 antibody; IP−, immunoprecipitation in the absence of the anti-Kv1.3 antibody; SN+, supernatant from the IP+; SN−, supernatant from the IP−.

Techniques Used: Functional Assay, Expressing, Activation Assay, Transfection, Western Blot, Negative Control, Immunoprecipitation

Kv1.3, but not Kv1.5, associates with KCNE4 in HEK-293 cells. KCNE4 modulates Kv1.3 trafficking and activity. HEK-293 cells were transfected with Kv1.3–YFP or Kv1.5–YFP in the presence or absence of KCNE4–CFP. Confocal images of (A) Kv1.3–YFP, (B) Kv1.5–YFP and (C) KCNE4–CFP. (D) HEK-293 cells co-transfected with Kv1.3 and KCNE4. (E) Kv1.5 and KCNE4. Color code: green, channels; red, KCNE4; yellow in merge panels shows colocalization. Scale bars: 10 µm. Voltage-dependent K + currents were elicited in HEK-293 cells transfected with Kv1.3 (F,G) and Kv1.5 (H,I) in the absence (F,H) or the presence (G,I) of KCNE4. Cells were held at −80 mV, and pulse potentials were applied as indicated. (J) Current density versus voltage plot of outward K + currents. White circles, Kv1.3; black circles, Kv1.3+KCNE4; light gray circles, Kv1.5; dark gray circles, Kv1.5+KCNE4. Values are shown as the mean±s.e.m. ( n =6–10 independent cells). (K) Molecular association of Kv1.3 and Kv1.5 with KCNE4 as measured by FRET efficiency (%). HEK-293 cells were transfected with Kv1.3–YFP and Kv1.5–YFP in the presence of KCNE4–CFP. YFP–CFP and Kv1.3–YFP or Kv1.3–CFP were used as negative and positive controls, respectively. Values are shown as the mean±s.e.m. ( n > 25 independent cells). ** P
Figure Legend Snippet: Kv1.3, but not Kv1.5, associates with KCNE4 in HEK-293 cells. KCNE4 modulates Kv1.3 trafficking and activity. HEK-293 cells were transfected with Kv1.3–YFP or Kv1.5–YFP in the presence or absence of KCNE4–CFP. Confocal images of (A) Kv1.3–YFP, (B) Kv1.5–YFP and (C) KCNE4–CFP. (D) HEK-293 cells co-transfected with Kv1.3 and KCNE4. (E) Kv1.5 and KCNE4. Color code: green, channels; red, KCNE4; yellow in merge panels shows colocalization. Scale bars: 10 µm. Voltage-dependent K + currents were elicited in HEK-293 cells transfected with Kv1.3 (F,G) and Kv1.5 (H,I) in the absence (F,H) or the presence (G,I) of KCNE4. Cells were held at −80 mV, and pulse potentials were applied as indicated. (J) Current density versus voltage plot of outward K + currents. White circles, Kv1.3; black circles, Kv1.3+KCNE4; light gray circles, Kv1.5; dark gray circles, Kv1.5+KCNE4. Values are shown as the mean±s.e.m. ( n =6–10 independent cells). (K) Molecular association of Kv1.3 and Kv1.5 with KCNE4 as measured by FRET efficiency (%). HEK-293 cells were transfected with Kv1.3–YFP and Kv1.5–YFP in the presence of KCNE4–CFP. YFP–CFP and Kv1.3–YFP or Kv1.3–CFP were used as negative and positive controls, respectively. Values are shown as the mean±s.e.m. ( n > 25 independent cells). ** P

Techniques Used: Activity Assay, Transfection

The structure of the Kv1.3 C-terminal domain mediates the association with KCNE4. HEK-293 cells were transfected with Kv1.3 wt, various Kv1.3 truncated mutants and KCNE4. (A) Scheme representing Kv1.3 wt and the different C-terminus truncation mutants. Bars indicate the N-terminus in white (Nt), the S1–S6 transmembrane domains in black and the C-terminal domain in gray (Ct). (B) Schematic diagram of the Kv1.3 C-terminal domain highlighting some important forward trafficking signatures and the distal PDZ motif. The residues in black indicate the truncations. A unique change (A to S) between human and rodents is indicated in gray. The S6 transmembrane domain is represented as a gray barrel. The HRETE, ExExE, YMVIEE and the PDZ motifs are indicated by gray boxes. The SwissProt number, as well as the number of residues in the C-terminus, for rat, mouse and humans is also indicated. (C) Cell lysates. Immunoprecipitation of Kv1.3–YFP C-terminus mutants with anti-GFP antibody. Top panels show the immunoblot against GFP (Kv1.3). Bottom panels show the immunoblot against KCNE4 (arrow). (D) Co-immunoprecipitation (Co-IP). Cell lysates (as in C) were immunoprecipitated with anti-GFP antibody (IP Kv1.3) and immunoblotted against Kv1.3 (IB: Kv1.3, top panel) and KCNE4 (IP: KCNE4, bottom panel). Note that the anti-GFP antibody used recognizes YFP. (E) Plot of the remaining number of C-terminal residues in the Kv1.3 C-terminus truncated mutants versus the co-immunoprecipitation of KCNE4. Values (mean±s.e.m., n =4) are calculated as the logarithm (log) of co-immunoprecipitation in arbitrary units (AU) standardized to the maximum level (scored as 1) of the co-immunoprecipitation of KCNE4 with Kv1.3 wt from D. Values followed a notable linear correlation ( r 2 =0.8175) which yielded a statistical significant ( P =0.0133) Pearson’s coefficient (0.9041).
Figure Legend Snippet: The structure of the Kv1.3 C-terminal domain mediates the association with KCNE4. HEK-293 cells were transfected with Kv1.3 wt, various Kv1.3 truncated mutants and KCNE4. (A) Scheme representing Kv1.3 wt and the different C-terminus truncation mutants. Bars indicate the N-terminus in white (Nt), the S1–S6 transmembrane domains in black and the C-terminal domain in gray (Ct). (B) Schematic diagram of the Kv1.3 C-terminal domain highlighting some important forward trafficking signatures and the distal PDZ motif. The residues in black indicate the truncations. A unique change (A to S) between human and rodents is indicated in gray. The S6 transmembrane domain is represented as a gray barrel. The HRETE, ExExE, YMVIEE and the PDZ motifs are indicated by gray boxes. The SwissProt number, as well as the number of residues in the C-terminus, for rat, mouse and humans is also indicated. (C) Cell lysates. Immunoprecipitation of Kv1.3–YFP C-terminus mutants with anti-GFP antibody. Top panels show the immunoblot against GFP (Kv1.3). Bottom panels show the immunoblot against KCNE4 (arrow). (D) Co-immunoprecipitation (Co-IP). Cell lysates (as in C) were immunoprecipitated with anti-GFP antibody (IP Kv1.3) and immunoblotted against Kv1.3 (IB: Kv1.3, top panel) and KCNE4 (IP: KCNE4, bottom panel). Note that the anti-GFP antibody used recognizes YFP. (E) Plot of the remaining number of C-terminal residues in the Kv1.3 C-terminus truncated mutants versus the co-immunoprecipitation of KCNE4. Values (mean±s.e.m., n =4) are calculated as the logarithm (log) of co-immunoprecipitation in arbitrary units (AU) standardized to the maximum level (scored as 1) of the co-immunoprecipitation of KCNE4 with Kv1.3 wt from D. Values followed a notable linear correlation ( r 2 =0.8175) which yielded a statistical significant ( P =0.0133) Pearson’s coefficient (0.9041).

Techniques Used: Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay

KCNE4 interacts with the C-terminal domain of Kv1.3, thereby impairing the association with the COPII anterograde trafficking mechanism. HEK-293 cells were transfected with Kv1.3 in the presence or the absence of KCNE4, Sec24D–YFP and Sar1(H79G)–HA. (A,B) Co-immunoprecipitation of Kv1.3 and Sec24D in the presence or the absence of KCNE4. (A) Expression of Sec24D, Kv1.3 and KCNE4 in starting materials (cell lysates). (B) Co-immunoprecipitations. Samples were immunoprecipitated (+IP) against Sec24D–YFP and immunoblotted (IB) against GFP (Sec24D), Kv1.3 and KCNE4. Sar1(H79G) and KCNE4 expression led to the retention of Kv1.3 in the ER. Note that KCNE4 notably impaired the association between Kv1.3 and Sec24D. In addition, KCNE4 showed a slight co-immunoprecipitation with Sec24. Note that the anti-GFP antibody used recognizes YFP. (C–H) Molecular interaction between Kv1.3 and Sec24D in the presence or the absence of KCNE4. (C–F) A representative FRET experiment for Kv1.3 and Sec24D in the presence of KCNE4. (C) Sec24D–YFP (red) in the acceptor panel. (D) Kv1.3–CFP (green) in the donor panel. (E) Colocalization is shown in yellow. (F) Magnification of the boxed area in E. Circles highlight some ROIs. Scale bar: 10 µm. (G) FRET efficiency of the Kv1.3–Sec24D interaction in the presence or the absence of Sar1(H79G) and KCNE4. YFP and CFP, and Kv1.3–CFP and Kv1.3–YFP pairs were used as negative and positive controls, respectively. Note that although FRET values between Kv1.3 and Sec24D were positive, the presence of KCNE4 triggered a larger variability. (H) Relative KCNE4 expression in arbitrary units (AU) in FRET-positive or FRET-negative ROIs. Considering the FRET between Kv1.3 and Sec24D, two types of ROI were observed. When ROI triggered clearly positive FRET, KCNE4 expression was low. However, in ROIs with no relevant FRET values, KCNE4 expression was higher. Values are shown as the mean±s.e.m. of n > 30 cells. * P
Figure Legend Snippet: KCNE4 interacts with the C-terminal domain of Kv1.3, thereby impairing the association with the COPII anterograde trafficking mechanism. HEK-293 cells were transfected with Kv1.3 in the presence or the absence of KCNE4, Sec24D–YFP and Sar1(H79G)–HA. (A,B) Co-immunoprecipitation of Kv1.3 and Sec24D in the presence or the absence of KCNE4. (A) Expression of Sec24D, Kv1.3 and KCNE4 in starting materials (cell lysates). (B) Co-immunoprecipitations. Samples were immunoprecipitated (+IP) against Sec24D–YFP and immunoblotted (IB) against GFP (Sec24D), Kv1.3 and KCNE4. Sar1(H79G) and KCNE4 expression led to the retention of Kv1.3 in the ER. Note that KCNE4 notably impaired the association between Kv1.3 and Sec24D. In addition, KCNE4 showed a slight co-immunoprecipitation with Sec24. Note that the anti-GFP antibody used recognizes YFP. (C–H) Molecular interaction between Kv1.3 and Sec24D in the presence or the absence of KCNE4. (C–F) A representative FRET experiment for Kv1.3 and Sec24D in the presence of KCNE4. (C) Sec24D–YFP (red) in the acceptor panel. (D) Kv1.3–CFP (green) in the donor panel. (E) Colocalization is shown in yellow. (F) Magnification of the boxed area in E. Circles highlight some ROIs. Scale bar: 10 µm. (G) FRET efficiency of the Kv1.3–Sec24D interaction in the presence or the absence of Sar1(H79G) and KCNE4. YFP and CFP, and Kv1.3–CFP and Kv1.3–YFP pairs were used as negative and positive controls, respectively. Note that although FRET values between Kv1.3 and Sec24D were positive, the presence of KCNE4 triggered a larger variability. (H) Relative KCNE4 expression in arbitrary units (AU) in FRET-positive or FRET-negative ROIs. Considering the FRET between Kv1.3 and Sec24D, two types of ROI were observed. When ROI triggered clearly positive FRET, KCNE4 expression was low. However, in ROIs with no relevant FRET values, KCNE4 expression was higher. Values are shown as the mean±s.e.m. of n > 30 cells. * P

Techniques Used: Transfection, Immunoprecipitation, Expressing

The KCNE4 interaction further aggravated the intracellular retention of the Kv1.3(E 483/484 I) mutant. HEK-293 cells were transfected with Kv1.3wt, Kv1.3(E 483/484 I) and KCNE4. Membrane surface labeling was performed using WGA as described in the Materials and Methods. (A–G) Representative confocal images of Kv1.3 wt in the absence (A–C) or the presence (D–G) of KCNE4. (H–N) Representative confocal images of Kv1.3(E 483/484 I) in the absence (H–J) or the presence (K–N) of KCNE4. (A,D,H,K) Kv1.3 channels in green. (E,L) KCNE4 in red. (B,F,I,M) WGA membrane staining in blue. (C,G,J,N) Color code in merged panels: Kv1.3 channels and KCNE4 colocalization, yellow; channels and membrane, cyan; triple colocalization, white. Scale bars: 10 µm. (O) Kv1.3 membrane surface expression in arbitrary units (AU) in a pixel-by-pixel analysis. Values are shown as the mean±s.e.m. of n > 25 cells. * P
Figure Legend Snippet: The KCNE4 interaction further aggravated the intracellular retention of the Kv1.3(E 483/484 I) mutant. HEK-293 cells were transfected with Kv1.3wt, Kv1.3(E 483/484 I) and KCNE4. Membrane surface labeling was performed using WGA as described in the Materials and Methods. (A–G) Representative confocal images of Kv1.3 wt in the absence (A–C) or the presence (D–G) of KCNE4. (H–N) Representative confocal images of Kv1.3(E 483/484 I) in the absence (H–J) or the presence (K–N) of KCNE4. (A,D,H,K) Kv1.3 channels in green. (E,L) KCNE4 in red. (B,F,I,M) WGA membrane staining in blue. (C,G,J,N) Color code in merged panels: Kv1.3 channels and KCNE4 colocalization, yellow; channels and membrane, cyan; triple colocalization, white. Scale bars: 10 µm. (O) Kv1.3 membrane surface expression in arbitrary units (AU) in a pixel-by-pixel analysis. Values are shown as the mean±s.e.m. of n > 25 cells. * P

Techniques Used: Mutagenesis, Transfection, Labeling, Whole Genome Amplification, Staining, Expressing

KCNE4 associates with the Kv1.3 C-terminal domain. HEK-293 cells were transfected with Kv1.3–YFP channels and KCNE4. (A–I) Confocal images from HEK-293 cells transfected with wt and mutant Kv1.3–YFP channels and KCNE4–CFP. (A,D,G) Kv1.3–YFP, Kv1.3ΔN–YFP and Kv1.3ΔC–YFP channels in green. (B,E,H) Cellular distribution of KCNE4–CFP in red. (C,F,I) Merged images show colocalization in yellow. Scale bars: 10 µm. Note that in all cases, Kv1.3 is mostly intracellular. Representative cartoons of different Kv1.3 channels are shown at the left of their respective images. The red dotted ellipse highlights the deleted domain. (J) Histogram representing the relative colocalization between Kv1.3 channels and KCNE4. Results in arbitrary units (A.U.) are the mean±s.e.m. of a pixel-by-pixel analysis on 25–40 cells. *** P
Figure Legend Snippet: KCNE4 associates with the Kv1.3 C-terminal domain. HEK-293 cells were transfected with Kv1.3–YFP channels and KCNE4. (A–I) Confocal images from HEK-293 cells transfected with wt and mutant Kv1.3–YFP channels and KCNE4–CFP. (A,D,G) Kv1.3–YFP, Kv1.3ΔN–YFP and Kv1.3ΔC–YFP channels in green. (B,E,H) Cellular distribution of KCNE4–CFP in red. (C,F,I) Merged images show colocalization in yellow. Scale bars: 10 µm. Note that in all cases, Kv1.3 is mostly intracellular. Representative cartoons of different Kv1.3 channels are shown at the left of their respective images. The red dotted ellipse highlights the deleted domain. (J) Histogram representing the relative colocalization between Kv1.3 channels and KCNE4. Results in arbitrary units (A.U.) are the mean±s.e.m. of a pixel-by-pixel analysis on 25–40 cells. *** P

Techniques Used: Transfection, Mutagenesis

The disruption of the ERRM of KCNE4 only partially counteracted the intracellular retention of Kv1.3. HEK-293 cells were transfected with Kv1.3–YFP, KCNE4–CFP and the KCNE4(ERRM)–CFP mutant. (A) Cartoon illustrating the ERRM in the C-terminal domain of KCNE4. Positive residues (R and K) in the wild-type ERRM (brown box) were mutated to alanine residues to disrupt the motif. N-term, N-terminal extracellular domain; C-term, C-terminal intracellular domain; TMD, transmembrane domain. (B–G) Confocal images of KCNE4 wt (B–D) and the KCNE4(ERRM) mutant (E–G). (B,E) KCNE4. (C,F) Membrane staining with WGA. (D,G) Merge channel. (H–O) Representative images of Kv1.3 in the presence of KCNE4 (H–K) and KCNE4(ERRM) (L–O). (H,L) Kv1.3 in green; (I,M) KCNE4 in red; (J,N) membrane in blue; (K,O) merge. Note that although Kv1.3 colocalization with KCNE4 showed marked intracellular retention in all cases, the channel reached the surface better in the presence of KCNE4(ERRM). Scale bars: 10 µm. (P) Pixel-by-pixel analysis of the relative membrane surface expression, in arbitrary units (AU), of KCNE4 (black bars) and Kv1.3 (white bars). Note that KCNE4(ERRM) showed double the KCNE4 membrane surface expression. Furthermore, Kv1.3 reached the cell surface more efficiently in the presence of KCNE4(ERRM) than with KCNE4. Values are shown as the mean±s.e.m. ( n > 25 cells). * P
Figure Legend Snippet: The disruption of the ERRM of KCNE4 only partially counteracted the intracellular retention of Kv1.3. HEK-293 cells were transfected with Kv1.3–YFP, KCNE4–CFP and the KCNE4(ERRM)–CFP mutant. (A) Cartoon illustrating the ERRM in the C-terminal domain of KCNE4. Positive residues (R and K) in the wild-type ERRM (brown box) were mutated to alanine residues to disrupt the motif. N-term, N-terminal extracellular domain; C-term, C-terminal intracellular domain; TMD, transmembrane domain. (B–G) Confocal images of KCNE4 wt (B–D) and the KCNE4(ERRM) mutant (E–G). (B,E) KCNE4. (C,F) Membrane staining with WGA. (D,G) Merge channel. (H–O) Representative images of Kv1.3 in the presence of KCNE4 (H–K) and KCNE4(ERRM) (L–O). (H,L) Kv1.3 in green; (I,M) KCNE4 in red; (J,N) membrane in blue; (K,O) merge. Note that although Kv1.3 colocalization with KCNE4 showed marked intracellular retention in all cases, the channel reached the surface better in the presence of KCNE4(ERRM). Scale bars: 10 µm. (P) Pixel-by-pixel analysis of the relative membrane surface expression, in arbitrary units (AU), of KCNE4 (black bars) and Kv1.3 (white bars). Note that KCNE4(ERRM) showed double the KCNE4 membrane surface expression. Furthermore, Kv1.3 reached the cell surface more efficiently in the presence of KCNE4(ERRM) than with KCNE4. Values are shown as the mean±s.e.m. ( n > 25 cells). * P

Techniques Used: Transfection, Mutagenesis, Staining, Whole Genome Amplification, Expressing

The C-terminus of Kv1.3, but not Kv1.5, is necessary and sufficient for the association with KCNE4. Confocal images from HEK-293 cells transfected with Kv1.3–YFP (A–C), Kv1.3NKv1.5–YFP (D–F) and Kv1.3CKv1.5–YFP (G–I) in the presence of KCNE4–CFP. All Kv1.3 channels presented an intracellular distribution, but the colocalization of Kv1.3CKv1.5 with KCNE4–CFP is 40% lower than that with Kv1.3 or Kv1.3NKv1.5. HEK-293 cells were also transfected with Kv1.5–YFP (J–L), Kv1.5NKv1.3 (M–O) and Kv1.5CKv1.3 (P–R) in the presence of KCNE4. Although all Kv1.5 channels were distributed intracellularly, notable colocalization was only observed between KCNE4 and Kv1.5CKv1.3. Cartoons on top of panels represent chimeric channels with the N- and C-terminal domains, and six transmembrane domains (boxes). Blue, Kv1.3 domains; green, Kv1.5 domains. Color code in confocal images: green, channels; red, KCNE4; yellow, colocalization in merge panels. Scale bars: 10 μm. (S) Histogram representing the relative colocalization between Kv1.3 and Kv1.5 channels and chimeras and KCNE4. Results are mean±s.e.m. ( n =25–30 independent cells). Black columns represent Kv1.3 and Kv1.3–Kv1.5 chimeras (1.3N1.5 and 1.3C1.5). White columns represent Kv1.5 and Kv1.5–Kv1.3 chimeras (1.5N1.3 and 1.5C1.3). *** P
Figure Legend Snippet: The C-terminus of Kv1.3, but not Kv1.5, is necessary and sufficient for the association with KCNE4. Confocal images from HEK-293 cells transfected with Kv1.3–YFP (A–C), Kv1.3NKv1.5–YFP (D–F) and Kv1.3CKv1.5–YFP (G–I) in the presence of KCNE4–CFP. All Kv1.3 channels presented an intracellular distribution, but the colocalization of Kv1.3CKv1.5 with KCNE4–CFP is 40% lower than that with Kv1.3 or Kv1.3NKv1.5. HEK-293 cells were also transfected with Kv1.5–YFP (J–L), Kv1.5NKv1.3 (M–O) and Kv1.5CKv1.3 (P–R) in the presence of KCNE4. Although all Kv1.5 channels were distributed intracellularly, notable colocalization was only observed between KCNE4 and Kv1.5CKv1.3. Cartoons on top of panels represent chimeric channels with the N- and C-terminal domains, and six transmembrane domains (boxes). Blue, Kv1.3 domains; green, Kv1.5 domains. Color code in confocal images: green, channels; red, KCNE4; yellow, colocalization in merge panels. Scale bars: 10 μm. (S) Histogram representing the relative colocalization between Kv1.3 and Kv1.5 channels and chimeras and KCNE4. Results are mean±s.e.m. ( n =25–30 independent cells). Black columns represent Kv1.3 and Kv1.3–Kv1.5 chimeras (1.3N1.5 and 1.3C1.5). White columns represent Kv1.5 and Kv1.5–Kv1.3 chimeras (1.5N1.3 and 1.5C1.3). *** P

Techniques Used: Transfection

7) Product Images from "Caveolin interaction governs Kv1.3 lipid raft targeting"

Article Title: Caveolin interaction governs Kv1.3 lipid raft targeting

Journal: Scientific Reports

doi: 10.1038/srep22453

Caveolin expression induces aggregation and slows the lateral diffusion of Kv1.3. ( A,B ) Electronic micrographs of PML from HEK Cav- ( A ) and HEK Cav1 ( B ) expressing Kv1.3. Cav1 and Kv1.3 were immunolocalized with anti-Kv1.3 monoclonal and anti-caveolin polyclonal antibodies and a mixture of 10 nm and 15 nm colloidal gold conjugated secondary antibodies tagging Kv1.3 and caveolin, respectively. Bar Scale 500 nm. White arrows point at microtubules. White arrowhead highlights caveolae. Black arrowhead points at caveolin outside caveolae like structures. ( C–G ) FRAP performed 24 h after Kv1.3YFP transfection in HEK Cav- ( C ) and HEK Cav1 ( D ). Representative images are before (prebleach), during (bleach) and 24 or 38 s after bleach (postbleach). White circles highlight the bleached membrane. ( E ) Fluorescence recovery. ( F ) Mobile fraction and ( G ) half live recovery (the mean ± SE, n = 10). ( H,I ) SPT analysis of HEK Cav- and HEK Cav1 expressing Kv1.3LoopBAD together with BirA for 24 h and tagged for 5 min with Qdots-Streptavidin. ( H ) Qdots were classified as single or multiple units. Histogram displays the relative abundance of both in HEK Cav- and HEK Cav1 (the mean ± SE, n ≥ 15). ( I ) Single Qdots were further classified according to its MSD shape as being free: Brownian movement (linear plot); confined: region restricted movement (decreasing slope plot); or stationary: motionless (diffusion coefficient bellow 0.001 μm 2 /s). Histogram displaying the diffusion coefficient of the different types of single Qdots. Closed bars, HEK Cav-; open bars, HEK Cav1. Data are the mean ± SE (n ≥ 9). *p
Figure Legend Snippet: Caveolin expression induces aggregation and slows the lateral diffusion of Kv1.3. ( A,B ) Electronic micrographs of PML from HEK Cav- ( A ) and HEK Cav1 ( B ) expressing Kv1.3. Cav1 and Kv1.3 were immunolocalized with anti-Kv1.3 monoclonal and anti-caveolin polyclonal antibodies and a mixture of 10 nm and 15 nm colloidal gold conjugated secondary antibodies tagging Kv1.3 and caveolin, respectively. Bar Scale 500 nm. White arrows point at microtubules. White arrowhead highlights caveolae. Black arrowhead points at caveolin outside caveolae like structures. ( C–G ) FRAP performed 24 h after Kv1.3YFP transfection in HEK Cav- ( C ) and HEK Cav1 ( D ). Representative images are before (prebleach), during (bleach) and 24 or 38 s after bleach (postbleach). White circles highlight the bleached membrane. ( E ) Fluorescence recovery. ( F ) Mobile fraction and ( G ) half live recovery (the mean ± SE, n = 10). ( H,I ) SPT analysis of HEK Cav- and HEK Cav1 expressing Kv1.3LoopBAD together with BirA for 24 h and tagged for 5 min with Qdots-Streptavidin. ( H ) Qdots were classified as single or multiple units. Histogram displays the relative abundance of both in HEK Cav- and HEK Cav1 (the mean ± SE, n ≥ 15). ( I ) Single Qdots were further classified according to its MSD shape as being free: Brownian movement (linear plot); confined: region restricted movement (decreasing slope plot); or stationary: motionless (diffusion coefficient bellow 0.001 μm 2 /s). Histogram displaying the diffusion coefficient of the different types of single Qdots. Closed bars, HEK Cav-; open bars, HEK Cav1. Data are the mean ± SE (n ≥ 9). *p

Techniques Used: Expressing, Diffusion-based Assay, Transfection, Fluorescence, Single-particle Tracking

Molecular association of Kv1.3 with caveolin. HEK Cav1 cells were transfected with Kv1.3 ( A ) and Kv1.5 ( B ) and, after lipid raft isolation, floating fractions were subjected to caveolin immunoprecipitation (IP: Cav) in the presence (+) and the absence (−) of caveolin antibody. Blots were analyzed for the presence of Kv1.3 (IB: Kv1.3), Kv1.5 (IB: Kv1.5) and Cav1 (IB: Cav). SM: starting material; SN: Supernatant; IP: immunoprecipitate. ( C ) Representative FRET images of HEK Cav- cotranfected with Cav1-Cerulean and Kv1.3YFP (top panels) or Kv1.5YFP (bottom panels). Images from left to right show acceptor (Channel-YFP) prebleach and postbleach and donor (Cav1-Cerulean) prebleach and postbleach images. Squares insets indicate the bleached zone containing the quantified red limited areas. The line graphs at the right show changes in donor (cyan) and acceptor (yellow) fluorescence after bleach ( D ) Histogram with the FRETeff quantification (%) of YFP-Cer (negative control), Kv1.3YFP-Kv1.3Cer (positive control), and samples from ( C ). Data are the mean ± SE (n > 35). *p
Figure Legend Snippet: Molecular association of Kv1.3 with caveolin. HEK Cav1 cells were transfected with Kv1.3 ( A ) and Kv1.5 ( B ) and, after lipid raft isolation, floating fractions were subjected to caveolin immunoprecipitation (IP: Cav) in the presence (+) and the absence (−) of caveolin antibody. Blots were analyzed for the presence of Kv1.3 (IB: Kv1.3), Kv1.5 (IB: Kv1.5) and Cav1 (IB: Cav). SM: starting material; SN: Supernatant; IP: immunoprecipitate. ( C ) Representative FRET images of HEK Cav- cotranfected with Cav1-Cerulean and Kv1.3YFP (top panels) or Kv1.5YFP (bottom panels). Images from left to right show acceptor (Channel-YFP) prebleach and postbleach and donor (Cav1-Cerulean) prebleach and postbleach images. Squares insets indicate the bleached zone containing the quantified red limited areas. The line graphs at the right show changes in donor (cyan) and acceptor (yellow) fluorescence after bleach ( D ) Histogram with the FRETeff quantification (%) of YFP-Cer (negative control), Kv1.3YFP-Kv1.3Cer (positive control), and samples from ( C ). Data are the mean ± SE (n > 35). *p

Techniques Used: Transfection, Isolation, Immunoprecipitation, Fluorescence, Negative Control, Positive Control

The N-terminal domain of Kv1.3 is essential for the interaction with caveolin. Total protein lysates of HEK Cav1 obtained 24 h after transfection with the indicated channel (tagged with YFP) were subjected to immunoprecipitation against caveolin. Samples separated by SDS-PAGE and immunoblotted (IB) against GFP (channel) and caveolin antibodies. ( A ) Schematic diagrams of Kv1.3 truncated channels and Kv1.3-Kv1.5 chimeras. Kv1.3 domains: white barrels and black lines. Kv1.5 domains: gray barrels and gray lines. ( B ) Starting materials. Top panel, filters were immunobloted against GFP (Channels). Bottom panel, filters were probed against Cav to demonstrate Cav immunoprecipitation. ( C ) Immunoprecipitates. No immunoprecipitation was observed in the absence of antibody (IP-).
Figure Legend Snippet: The N-terminal domain of Kv1.3 is essential for the interaction with caveolin. Total protein lysates of HEK Cav1 obtained 24 h after transfection with the indicated channel (tagged with YFP) were subjected to immunoprecipitation against caveolin. Samples separated by SDS-PAGE and immunoblotted (IB) against GFP (channel) and caveolin antibodies. ( A ) Schematic diagrams of Kv1.3 truncated channels and Kv1.3-Kv1.5 chimeras. Kv1.3 domains: white barrels and black lines. Kv1.5 domains: gray barrels and gray lines. ( B ) Starting materials. Top panel, filters were immunobloted against GFP (Channels). Bottom panel, filters were probed against Cav to demonstrate Cav immunoprecipitation. ( C ) Immunoprecipitates. No immunoprecipitation was observed in the absence of antibody (IP-).

Techniques Used: Transfection, Immunoprecipitation, SDS Page

Caveolin 1 stabilized Kv1.3 and modulated current density and inactivation. HEK Cav- and HEK Cav1 cells were transfected with Kv1.3YFP. ( A ) Time course experiments were performed extracting proteins 3, 6, 12, 24 and 48 hours after transfection. ( A ) Representative experiment is shown. Total protein extracts were separated by SDS-PAGE and immunoblotted using Kv1.3, β-actin, and caveolin antibodies. Kv1.3 expression values were corrected by β-actin levels and normalized with the maximum. (●) HEK Cav- cells; (○) HEK Cav1 cells. ( B ) Results are the mean ± SE of two independent experiments. ( C–E ) HEK Cav- cells were transfected with Kv1.3 in the absence (HEK Cav-) or the presence (HEK Cav- +Cav1) of Cav1. ( C ) K + currents were elicited by 250 ms voltage sweeps ranging from −60 mV to +80 mV in 10 mV increments. ( D ) Current density-voltage relationship of HEK Cav- (○) and HEK Cav- +Cav1 (●) cells (mean ± SE of n = 15 and 9 cells, respectively). ( E,F ) C-type inactivation. ( E ) Representative traces evoked by a 5 s pulse from −60 mV to +60 mV. ( F ) Remaining current density (%) at the end of the pulse shown as the mean ± SE of six cells both in the HEK Cav- (Cav1 -) and the HEK Cav- +Cav1 (Cav1+). *p
Figure Legend Snippet: Caveolin 1 stabilized Kv1.3 and modulated current density and inactivation. HEK Cav- and HEK Cav1 cells were transfected with Kv1.3YFP. ( A ) Time course experiments were performed extracting proteins 3, 6, 12, 24 and 48 hours after transfection. ( A ) Representative experiment is shown. Total protein extracts were separated by SDS-PAGE and immunoblotted using Kv1.3, β-actin, and caveolin antibodies. Kv1.3 expression values were corrected by β-actin levels and normalized with the maximum. (●) HEK Cav- cells; (○) HEK Cav1 cells. ( B ) Results are the mean ± SE of two independent experiments. ( C–E ) HEK Cav- cells were transfected with Kv1.3 in the absence (HEK Cav-) or the presence (HEK Cav- +Cav1) of Cav1. ( C ) K + currents were elicited by 250 ms voltage sweeps ranging from −60 mV to +80 mV in 10 mV increments. ( D ) Current density-voltage relationship of HEK Cav- (○) and HEK Cav- +Cav1 (●) cells (mean ± SE of n = 15 and 9 cells, respectively). ( E,F ) C-type inactivation. ( E ) Representative traces evoked by a 5 s pulse from −60 mV to +60 mV. ( F ) Remaining current density (%) at the end of the pulse shown as the mean ± SE of six cells both in the HEK Cav- (Cav1 -) and the HEK Cav- +Cav1 (Cav1+). *p

Techniques Used: Transfection, SDS Page, Expressing, Mass Spectrometry

The CBD at the N-terminal domain is responsible for the targeting of Kv1.3 to lipid raft and the association with Cav1. Lipid raft isolations performed in HEK Cav- (left panels) and HEK Cav1 (right panels) cells expressing Kv1.3CBD ( A ) and the Kv1.5CBD ( B ) mutant channels ( 166 FQRQVWLLF 174 to AQRQVGLLA and 232 FQRQVWLIF 240 to AQRQVGLIA, respectively). ( C ) HEK Cav1 expressing either Kv1.3 or Kv1.3CBD were subjected to immunoprecipitation against caveolin (IP: Cav1) in the presence (+) and the absence (−) of caveolin antibody. Samples were separated by SDS-PAGE and immunoblotted against Kv1.3 (IB:Kv1.3) and caveolin (IB:Caveolin) SM: starting material; SN: Supernatant; IP: immunoprecipitate.
Figure Legend Snippet: The CBD at the N-terminal domain is responsible for the targeting of Kv1.3 to lipid raft and the association with Cav1. Lipid raft isolations performed in HEK Cav- (left panels) and HEK Cav1 (right panels) cells expressing Kv1.3CBD ( A ) and the Kv1.5CBD ( B ) mutant channels ( 166 FQRQVWLLF 174 to AQRQVGLLA and 232 FQRQVWLIF 240 to AQRQVGLIA, respectively). ( C ) HEK Cav1 expressing either Kv1.3 or Kv1.3CBD were subjected to immunoprecipitation against caveolin (IP: Cav1) in the presence (+) and the absence (−) of caveolin antibody. Samples were separated by SDS-PAGE and immunoblotted against Kv1.3 (IB:Kv1.3) and caveolin (IB:Caveolin) SM: starting material; SN: Supernatant; IP: immunoprecipitate.

Techniques Used: Expressing, Mutagenesis, Immunoprecipitation, SDS Page

Kv1.3, but not Kv1.5, targeted to lipid raft microdomains in a caveolin-dependent manner. Sucrose-density gradients were performed with total lysates from HEK Cav- ( A ), HEK 293 ( B,D ) and HEK Cav1 ( C,E ) expressing Kv1.3YFP ( A–C ) and Kv1.5YFP ( D,E ). Clathrin and Flotillin were used as non-lipid raft and lipid raft markers, respectively. Note that the overexpression of Cav1 (HEK Cav1) partially targeted caveolin to floating (enriched in lipid raft) and non-floating fractions. Colocalization of Kv1.3 (F_H and M – O ) and Kv1.5 ( J – L , P – R ) with caveolin in whole HEK Cav1 cells ( F–L ) and plasma membrane lawns (PML) ( M – R ). Green: channel; Red: caveolin; Merge: colocalization in yellow. Bar scale 10 μm. ( I ) Histogram of the colocalization (%) between channel and caveolin. Closed bars, Kv1.3; open bars, Kv1.5. Data are the mean ± SE of whole-cell ( > 15 cells) and PML ( > 35 cells). ***p
Figure Legend Snippet: Kv1.3, but not Kv1.5, targeted to lipid raft microdomains in a caveolin-dependent manner. Sucrose-density gradients were performed with total lysates from HEK Cav- ( A ), HEK 293 ( B,D ) and HEK Cav1 ( C,E ) expressing Kv1.3YFP ( A–C ) and Kv1.5YFP ( D,E ). Clathrin and Flotillin were used as non-lipid raft and lipid raft markers, respectively. Note that the overexpression of Cav1 (HEK Cav1) partially targeted caveolin to floating (enriched in lipid raft) and non-floating fractions. Colocalization of Kv1.3 (F_H and M – O ) and Kv1.5 ( J – L , P – R ) with caveolin in whole HEK Cav1 cells ( F–L ) and plasma membrane lawns (PML) ( M – R ). Green: channel; Red: caveolin; Merge: colocalization in yellow. Bar scale 10 μm. ( I ) Histogram of the colocalization (%) between channel and caveolin. Closed bars, Kv1.3; open bars, Kv1.5. Data are the mean ± SE of whole-cell ( > 15 cells) and PML ( > 35 cells). ***p

Techniques Used: Expressing, Over Expression

8) Product Images from "Localization of Kv1.3 channels in presynaptic terminals of brainstem auditory neurons"

Article Title: Localization of Kv1.3 channels in presynaptic terminals of brainstem auditory neurons

Journal: The Journal of comparative neurology

doi: 10.1002/cne.22393

Comparison of localization patterns of Kv1.3 in MNTB with Kv1.1, Kv1.2 and Kv1.6. Top row (A–C) shows double immunofluorescence labeling of Kv1.3 with Kv1.1 (A), A and B show Kv1.3 and Kv1.1 immunolabeling and C shows the merged fluorescence images.
Figure Legend Snippet: Comparison of localization patterns of Kv1.3 in MNTB with Kv1.1, Kv1.2 and Kv1.6. Top row (A–C) shows double immunofluorescence labeling of Kv1.3 with Kv1.1 (A), A and B show Kv1.3 and Kv1.1 immunolabeling and C shows the merged fluorescence images.

Techniques Used: Immunofluorescence, Labeling, Immunolabeling, Fluorescence

Gradients of Kv1.3 immunoreactivity in sagittal and coronal sections of the MNTB. A. Sagittal section of MNTB immunostained for Kv 1.3. (scale bar, 20 μm). B and C. Coronal (B) and sagittal (C) sections were divided into 5 equal zones for quantification
Figure Legend Snippet: Gradients of Kv1.3 immunoreactivity in sagittal and coronal sections of the MNTB. A. Sagittal section of MNTB immunostained for Kv 1.3. (scale bar, 20 μm). B and C. Coronal (B) and sagittal (C) sections were divided into 5 equal zones for quantification

Techniques Used:

Immunostaining for the Kv1.3 potassium channel subunit in the MNTB
Figure Legend Snippet: Immunostaining for the Kv1.3 potassium channel subunit in the MNTB

Techniques Used: Immunostaining

Electron microscopic immunogold labeling of Kv 1.3 in the MNTB of Wild-type and Kv 1.3 −/− mice. A and B. Examples of sections stained for Kv1.3 using 10 nm gold particles. Particles are localized primarily to pre-synaptic structures,
Figure Legend Snippet: Electron microscopic immunogold labeling of Kv 1.3 in the MNTB of Wild-type and Kv 1.3 −/− mice. A and B. Examples of sections stained for Kv1.3 using 10 nm gold particles. Particles are localized primarily to pre-synaptic structures,

Techniques Used: Labeling, Mouse Assay, Staining

Co-localization of Kv 1.3 with syntaxin, synaptophysin and synaptotagmin. Double immunofluorescence labeling of Kv1.3 with syntaxin (top panels A – E ), synaptophysin ( center panels F – J ) and synaptotagmin ( bottom panels K – O ). Kv 1.3
Figure Legend Snippet: Co-localization of Kv 1.3 with syntaxin, synaptophysin and synaptotagmin. Double immunofluorescence labeling of Kv1.3 with syntaxin (top panels A – E ), synaptophysin ( center panels F – J ) and synaptotagmin ( bottom panels K – O ). Kv 1.3

Techniques Used: Immunofluorescence, Labeling

Electron microscopic co-localization of Kv1.3 with synaptophysin in wild-type and Kv1.3−/− animals. Double immunogold labeling was carried out using large (10 nm) gold particles to detect synaptophysin and smaller (5nm) particles to localize
Figure Legend Snippet: Electron microscopic co-localization of Kv1.3 with synaptophysin in wild-type and Kv1.3−/− animals. Double immunogold labeling was carried out using large (10 nm) gold particles to detect synaptophysin and smaller (5nm) particles to localize

Techniques Used: Labeling

Immunolocalization of Kv1.3 potassium channels subunits in the MNTB using diaminobenzidine labeling. A. Low power view of part of a coronal section of brainstem, showing bilateral immunostaining of MNTBs in a wild-type (wt) mouse (white arrows). B. Image
Figure Legend Snippet: Immunolocalization of Kv1.3 potassium channels subunits in the MNTB using diaminobenzidine labeling. A. Low power view of part of a coronal section of brainstem, showing bilateral immunostaining of MNTBs in a wild-type (wt) mouse (white arrows). B. Image

Techniques Used: Labeling, Immunostaining

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    NeuroMab anti kv1 3
    Cytosolic HSP70 and HSP90 chaperones assist with <t>Kv1.3</t> mitochondrial import. HEK 293 cells were transfected with Kv1.3YFP and subsequently stained with antibodies against HSP70 and HSP90. (A–G) Representative confocal images showing colocalization between Kv1.3YFP (green) (A, E) and HSP70 (B) or HSP90 (F) (red). (C, G) The merged image shows colocalization in yellow. (D, H) Histogram showing the pixel-by-pixel analysis of the section indicated by the line in the merged Panels (C, G) . (I, J) Electron micrographs of HEK 293 cells transfected with Kv1.3YFP. Immunogold particles with sizes of 12 and 18 nm show chaperones (white arrowheads) and Kv1.3 (black arrowheads), respectively. (I) HSP70; (J) HSP90. Scale bars represent 200 nm. (K) HEK 293 cells transfected with Kv1.3YFP were treated with DMSO (vehicle), 20 μM VER-155008 (HSP70 inhibitor) or 1 μM 17-DMAG (HSP90 inhibitor) for 18 h Next, mitochondria were purified, and the YFP intensity was analyzed using flow cytometry. Media (DMEM) and untransfected mitochondria were used as negative controls. (L) Quantification of relative mitochondrial Kv1.3YFP expression upon the indicated treatments. Data are presented as the means ± SE of 3 independent experiments. *p
    Anti Kv1 3, supplied by NeuroMab, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    NeuroMab kv1 3
    KCNE4 impaired <t>Kv1.3</t> accumulation in the IS but did not disrupt IS formation. Human Jurkat T lymphocytes and human Raji B lymphocytes were used to generate cell conjugates. ( A – C ) Activated B-cells (10 µg/mL SEE toxin) were cocultured in the absence ( Ba – Bd ) or presence ( Ca – Cd ) of Jurkat cells, and confocal images were obtained. ( Aa – Ad ) Jurkat T-cells in the absence of B cells. Endogenous Kv1.3 (green), CD3 (marker of T-cells, red), and CD19 (marker of B-cells, blue) were detected. ( Ad , Bd , Cd ) merge panels. Note that triple colocalization (white) in Cd localizes Kv1.3 in the IS, as identified by CD3 staining ( Cb ). Bars are 20 µm. ( D ) Accumulation ratio of mGFP-Kv1.3 at the IS vs. KCNE4-mCherry total intensity (n = 40). ( E ) CD3 recruitment into the IS vs. KCNE4 intensity. The horizontal red line represents the threshold level (1.5) for Kv1.3 and CD3 accumulation in the IS. Values greater or less than 1.5 indicated positive or negative accumulation of proteins at the IS, respectively.
    Kv1 3, supplied by NeuroMab, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cytosolic HSP70 and HSP90 chaperones assist with Kv1.3 mitochondrial import. HEK 293 cells were transfected with Kv1.3YFP and subsequently stained with antibodies against HSP70 and HSP90. (A–G) Representative confocal images showing colocalization between Kv1.3YFP (green) (A, E) and HSP70 (B) or HSP90 (F) (red). (C, G) The merged image shows colocalization in yellow. (D, H) Histogram showing the pixel-by-pixel analysis of the section indicated by the line in the merged Panels (C, G) . (I, J) Electron micrographs of HEK 293 cells transfected with Kv1.3YFP. Immunogold particles with sizes of 12 and 18 nm show chaperones (white arrowheads) and Kv1.3 (black arrowheads), respectively. (I) HSP70; (J) HSP90. Scale bars represent 200 nm. (K) HEK 293 cells transfected with Kv1.3YFP were treated with DMSO (vehicle), 20 μM VER-155008 (HSP70 inhibitor) or 1 μM 17-DMAG (HSP90 inhibitor) for 18 h Next, mitochondria were purified, and the YFP intensity was analyzed using flow cytometry. Media (DMEM) and untransfected mitochondria were used as negative controls. (L) Quantification of relative mitochondrial Kv1.3YFP expression upon the indicated treatments. Data are presented as the means ± SE of 3 independent experiments. *p

    Journal: Frontiers in Oncology

    Article Title: The Mitochondrial Routing of the Kv1.3 Channel

    doi: 10.3389/fonc.2022.865686

    Figure Lengend Snippet: Cytosolic HSP70 and HSP90 chaperones assist with Kv1.3 mitochondrial import. HEK 293 cells were transfected with Kv1.3YFP and subsequently stained with antibodies against HSP70 and HSP90. (A–G) Representative confocal images showing colocalization between Kv1.3YFP (green) (A, E) and HSP70 (B) or HSP90 (F) (red). (C, G) The merged image shows colocalization in yellow. (D, H) Histogram showing the pixel-by-pixel analysis of the section indicated by the line in the merged Panels (C, G) . (I, J) Electron micrographs of HEK 293 cells transfected with Kv1.3YFP. Immunogold particles with sizes of 12 and 18 nm show chaperones (white arrowheads) and Kv1.3 (black arrowheads), respectively. (I) HSP70; (J) HSP90. Scale bars represent 200 nm. (K) HEK 293 cells transfected with Kv1.3YFP were treated with DMSO (vehicle), 20 μM VER-155008 (HSP70 inhibitor) or 1 μM 17-DMAG (HSP90 inhibitor) for 18 h Next, mitochondria were purified, and the YFP intensity was analyzed using flow cytometry. Media (DMEM) and untransfected mitochondria were used as negative controls. (L) Quantification of relative mitochondrial Kv1.3YFP expression upon the indicated treatments. Data are presented as the means ± SE of 3 independent experiments. *p

    Article Snippet: Samples were mounted over Formvar-coated grills, and sections were finally stained with 2% uranyl acetate for 15 min. Immunolabeling was performed with primary anti-Kv1.3 (1:30, Neuromab), anti-HSP70 (1:50, Abcam) or anti-HSP90 (1:30, Abcam) antibodies.

    Techniques: Transfection, Staining, Purification, Flow Cytometry, Expressing

    Schematic representation of the mitochondrial import pathway for Kv1.3. The Kv1.3 channel is cotranslated by ER-linked and cytosolic ribosomes. ER ribosomes synthesize the plasma membrane Kv1.3, which interacts early with ancillary proteins, such as Kvβ subunits and caveolin, and is routed to the cell surface via COPII-dependent anterograde trafficking. The yellow circle restrains anterograde trafficking to the plasma membrane. Alternatively, (1) the Kv1.3 mRNA, which is translated by cytosolic ribosomes, follows the mitochondrial route. (2) By interacting with channel hydrophobic domains, cytosolic chaperones and cochaperones provide Kv1.3 with a partially folded state competent for mitochondrial translocation. Sequential transmembrane domains from the N- to C-terminus cooperate to achieve the proper folded state. (3) TOM receptors recognize cytosolic Kv1.3. (4) The TOM40 channel at the OMM translocates Kv1.3 to the TIM complex at the IMM. Finally, (5) the TIM50 receptor facilitates Kv1.3 translocation through the TIM23/17 channel into the IMM (6) . Spatial and temporal mechanisms defining Kv1.3 membrane topology and tetramerization at the IMM remain unknown.

    Journal: Frontiers in Oncology

    Article Title: The Mitochondrial Routing of the Kv1.3 Channel

    doi: 10.3389/fonc.2022.865686

    Figure Lengend Snippet: Schematic representation of the mitochondrial import pathway for Kv1.3. The Kv1.3 channel is cotranslated by ER-linked and cytosolic ribosomes. ER ribosomes synthesize the plasma membrane Kv1.3, which interacts early with ancillary proteins, such as Kvβ subunits and caveolin, and is routed to the cell surface via COPII-dependent anterograde trafficking. The yellow circle restrains anterograde trafficking to the plasma membrane. Alternatively, (1) the Kv1.3 mRNA, which is translated by cytosolic ribosomes, follows the mitochondrial route. (2) By interacting with channel hydrophobic domains, cytosolic chaperones and cochaperones provide Kv1.3 with a partially folded state competent for mitochondrial translocation. Sequential transmembrane domains from the N- to C-terminus cooperate to achieve the proper folded state. (3) TOM receptors recognize cytosolic Kv1.3. (4) The TOM40 channel at the OMM translocates Kv1.3 to the TIM complex at the IMM. Finally, (5) the TIM50 receptor facilitates Kv1.3 translocation through the TIM23/17 channel into the IMM (6) . Spatial and temporal mechanisms defining Kv1.3 membrane topology and tetramerization at the IMM remain unknown.

    Article Snippet: Samples were mounted over Formvar-coated grills, and sections were finally stained with 2% uranyl acetate for 15 min. Immunolabeling was performed with primary anti-Kv1.3 (1:30, Neuromab), anti-HSP70 (1:50, Abcam) or anti-HSP90 (1:30, Abcam) antibodies.

    Techniques: Translocation Assay

    The Kv1.3 interactome reveals an unconventional mitochondrial import pathway for Kv1.3. (A) Network showing protein–protein interactions (PPIs) in the mitochondrial Kv1.3 interactome of HEK 293 cells transfected with Kv1.3YFP. The mitochondrial import subnetwork was obtained after a functional enrichment analysis of the Kv1.3 interactome. A force-directed layout was used for network visualization. Protein names are indicated at the node center. The color code indicates functional relations between proteins (purple, chaperones; blue, matrix proteases; red, PAM complex; orange, TIM23 complex; green, TOM complex; yellow, MICOS complex). TOMM34, CDC37 and YWHAE were manually annotated based on published evidence. (B) HEK 293 cells were transfected with Kv1.3YFP. Total cell lysates (SM, starting materials) were immunoprecipitated against Kv1.3 (IP+). IP-, absence of the Kv1.3 antibody. Samples were immunoblotted (IB) with antibodies against Kv1.3 and different TIM/TOM proteins, as indicated.

    Journal: Frontiers in Oncology

    Article Title: The Mitochondrial Routing of the Kv1.3 Channel

    doi: 10.3389/fonc.2022.865686

    Figure Lengend Snippet: The Kv1.3 interactome reveals an unconventional mitochondrial import pathway for Kv1.3. (A) Network showing protein–protein interactions (PPIs) in the mitochondrial Kv1.3 interactome of HEK 293 cells transfected with Kv1.3YFP. The mitochondrial import subnetwork was obtained after a functional enrichment analysis of the Kv1.3 interactome. A force-directed layout was used for network visualization. Protein names are indicated at the node center. The color code indicates functional relations between proteins (purple, chaperones; blue, matrix proteases; red, PAM complex; orange, TIM23 complex; green, TOM complex; yellow, MICOS complex). TOMM34, CDC37 and YWHAE were manually annotated based on published evidence. (B) HEK 293 cells were transfected with Kv1.3YFP. Total cell lysates (SM, starting materials) were immunoprecipitated against Kv1.3 (IP+). IP-, absence of the Kv1.3 antibody. Samples were immunoblotted (IB) with antibodies against Kv1.3 and different TIM/TOM proteins, as indicated.

    Article Snippet: Samples were mounted over Formvar-coated grills, and sections were finally stained with 2% uranyl acetate for 15 min. Immunolabeling was performed with primary anti-Kv1.3 (1:30, Neuromab), anti-HSP70 (1:50, Abcam) or anti-HSP90 (1:30, Abcam) antibodies.

    Techniques: Transfection, Functional Assay, Immunoprecipitation

    Mitochondrial targeting of Kv1.3 depends on structural information rather than on discrete motifs. HEK 293 cells were transfected with several Kv1.3-Δx channels, and mitochondrial colocalization was analyzed. (A–I) Representative confocal images of different Kv1.3YFP constructs (a, green) and the mitochondrial marker (b, pmitoRFP in red). Kv1.3-Δx indicates the transmembrane segment deleted from the expressed Kv1.3YFP construct. Kv1.3-ΔN (B) and Kv1.3-ΔC (I) indicate deletion of the N- and C-termini, respectively. Panel c (merge) shows colocalization in yellow. Square inset indicates the region shown at higher magnification. The scale bar represents 10 µm. (J) Quantification of mitochondrial colocalization using Pearson’s correlation coefficient. The dashed line highlights the Kv1.3 WT value. Data are presented as the means ± SE (n > 30). *p

    Journal: Frontiers in Oncology

    Article Title: The Mitochondrial Routing of the Kv1.3 Channel

    doi: 10.3389/fonc.2022.865686

    Figure Lengend Snippet: Mitochondrial targeting of Kv1.3 depends on structural information rather than on discrete motifs. HEK 293 cells were transfected with several Kv1.3-Δx channels, and mitochondrial colocalization was analyzed. (A–I) Representative confocal images of different Kv1.3YFP constructs (a, green) and the mitochondrial marker (b, pmitoRFP in red). Kv1.3-Δx indicates the transmembrane segment deleted from the expressed Kv1.3YFP construct. Kv1.3-ΔN (B) and Kv1.3-ΔC (I) indicate deletion of the N- and C-termini, respectively. Panel c (merge) shows colocalization in yellow. Square inset indicates the region shown at higher magnification. The scale bar represents 10 µm. (J) Quantification of mitochondrial colocalization using Pearson’s correlation coefficient. The dashed line highlights the Kv1.3 WT value. Data are presented as the means ± SE (n > 30). *p

    Article Snippet: Samples were mounted over Formvar-coated grills, and sections were finally stained with 2% uranyl acetate for 15 min. Immunolabeling was performed with primary anti-Kv1.3 (1:30, Neuromab), anti-HSP70 (1:50, Abcam) or anti-HSP90 (1:30, Abcam) antibodies.

    Techniques: Transfection, Construct, Marker

    Transmembrane domains are candidate determinants of the mitochondrial import of Shaker channels. HEK 293 cells were transfected with Kv1.1-Kv1.5YFP channels, and mitochondrial expression was analyzed. (A) Membranous (Mb) and mitochondrial (Mit) fractions of HEK 293 cells transfected with the indicated member of the Shaker family. Note a shift in the molecular weights of the Kv1.2, Kv1.3 and Kv1.5 channels between the Mb and Mit fractions. Na + /K + ATPse is used as a plasma membrane marker, and VDAC is used as a mitochondrial marker. (B) Multiple protein sequence alignment of members of the Shaker family of voltage-gated potassium channels performed using Clustal Omega and JalView software. UniprotKB reference numbers are indicated. Hydrophobic residues are indicated in red, and hydrophilic residues are indicated in blue. Conservation among sequences is indicated in yellow. S1-S6 black lines indicate the sequence fragments corresponding to the transmembrane segments.

    Journal: Frontiers in Oncology

    Article Title: The Mitochondrial Routing of the Kv1.3 Channel

    doi: 10.3389/fonc.2022.865686

    Figure Lengend Snippet: Transmembrane domains are candidate determinants of the mitochondrial import of Shaker channels. HEK 293 cells were transfected with Kv1.1-Kv1.5YFP channels, and mitochondrial expression was analyzed. (A) Membranous (Mb) and mitochondrial (Mit) fractions of HEK 293 cells transfected with the indicated member of the Shaker family. Note a shift in the molecular weights of the Kv1.2, Kv1.3 and Kv1.5 channels between the Mb and Mit fractions. Na + /K + ATPse is used as a plasma membrane marker, and VDAC is used as a mitochondrial marker. (B) Multiple protein sequence alignment of members of the Shaker family of voltage-gated potassium channels performed using Clustal Omega and JalView software. UniprotKB reference numbers are indicated. Hydrophobic residues are indicated in red, and hydrophilic residues are indicated in blue. Conservation among sequences is indicated in yellow. S1-S6 black lines indicate the sequence fragments corresponding to the transmembrane segments.

    Article Snippet: Samples were mounted over Formvar-coated grills, and sections were finally stained with 2% uranyl acetate for 15 min. Immunolabeling was performed with primary anti-Kv1.3 (1:30, Neuromab), anti-HSP70 (1:50, Abcam) or anti-HSP90 (1:30, Abcam) antibodies.

    Techniques: Transfection, Expressing, Marker, Sequencing, Software

    Sequential deletion of Kv1.3 segments results in differential mitochondrial targeting. HEK 293 cells were transfected with several Kv1.3-Δx channels, and mitochondrial expression was studied. (A) Representative immunoblot showing purified mitochondrial fractions (bottom panels) from total lysates (starting materials) of HEK 293 cells transfected with Kv1.3-Δx. Samples were immunoblotted with antibodies against YFP (Kv1.3), Na + /K + ATPase (membrane marker) and VDAC (mitochondrial marker). (B) Relative mitochondrial Kv1.3-Δx expression. Data are presented as the means ± SE (n=3-5). The linear regression shown in red (y=0.09568x+0.2544) had a Pearson’s correlation coefficient of r=0.8899 (p=0.0125). Kv1.3-ΔC was not included in the calculation because of its substantial mitochondrial-independent ER retention ( 23 ).

    Journal: Frontiers in Oncology

    Article Title: The Mitochondrial Routing of the Kv1.3 Channel

    doi: 10.3389/fonc.2022.865686

    Figure Lengend Snippet: Sequential deletion of Kv1.3 segments results in differential mitochondrial targeting. HEK 293 cells were transfected with several Kv1.3-Δx channels, and mitochondrial expression was studied. (A) Representative immunoblot showing purified mitochondrial fractions (bottom panels) from total lysates (starting materials) of HEK 293 cells transfected with Kv1.3-Δx. Samples were immunoblotted with antibodies against YFP (Kv1.3), Na + /K + ATPase (membrane marker) and VDAC (mitochondrial marker). (B) Relative mitochondrial Kv1.3-Δx expression. Data are presented as the means ± SE (n=3-5). The linear regression shown in red (y=0.09568x+0.2544) had a Pearson’s correlation coefficient of r=0.8899 (p=0.0125). Kv1.3-ΔC was not included in the calculation because of its substantial mitochondrial-independent ER retention ( 23 ).

    Article Snippet: Samples were mounted over Formvar-coated grills, and sections were finally stained with 2% uranyl acetate for 15 min. Immunolabeling was performed with primary anti-Kv1.3 (1:30, Neuromab), anti-HSP70 (1:50, Abcam) or anti-HSP90 (1:30, Abcam) antibodies.

    Techniques: Transfection, Expressing, Purification, Marker

    Kv1.3 is targeted to the mitochondria by bypassing the endoplasmic reticulum. (A) Subcellular fractionation to obtain the membranous (Mb), mitochondrial (Mit) and cytosolic (Cyto) fractions of HEK 293 cells transfected with Kv1.3YFP. SM, starting materials. Na + /K + ATPase is used as a plasma membrane (PM) marker, and VDAC is used as a mitochondrial marker. β-actin identified cytosolic-enriched fractions. Note the difference in molecular weight of Kv1.3 bands from the Mb and Mit fractions. (B) Mb and Mit fractions of HEK 293 cells transfected with Kv1.3YFP were obtained. Cells were cotransfected with (+) or without (-) a constitutively active Sar1(H79G) dominant-negative GTPase. (C–H) Electron micrographs of HEK 293 cells transfected with Kv1.3YFP. Kv1.3 was labeled with 18 nm immunogold particles. Arrowheads indicate Kv1.3. (C) Plasma membrane Kv1.3. (D) Kv1.3 in the ER. (E) Kv1.3 in the Golgi (black arrowhead) and in the IMM (white arrowhead). (F–H) Black arrowheads indicate cytosolic Kv1.3, and white arrowheads indicate mitochondrial Kv1.3. Note the presence of Kv1.3 in ribosomes (r) close to a mitochondrion or embedded in the actin cytoskeleton (a) in (F, G) , respectively. The scale bar indicates 500 nm. White square insets in (C, G, H) highlight higher magnification regions with a scale bar representing 200 nm.

    Journal: Frontiers in Oncology

    Article Title: The Mitochondrial Routing of the Kv1.3 Channel

    doi: 10.3389/fonc.2022.865686

    Figure Lengend Snippet: Kv1.3 is targeted to the mitochondria by bypassing the endoplasmic reticulum. (A) Subcellular fractionation to obtain the membranous (Mb), mitochondrial (Mit) and cytosolic (Cyto) fractions of HEK 293 cells transfected with Kv1.3YFP. SM, starting materials. Na + /K + ATPase is used as a plasma membrane (PM) marker, and VDAC is used as a mitochondrial marker. β-actin identified cytosolic-enriched fractions. Note the difference in molecular weight of Kv1.3 bands from the Mb and Mit fractions. (B) Mb and Mit fractions of HEK 293 cells transfected with Kv1.3YFP were obtained. Cells were cotransfected with (+) or without (-) a constitutively active Sar1(H79G) dominant-negative GTPase. (C–H) Electron micrographs of HEK 293 cells transfected with Kv1.3YFP. Kv1.3 was labeled with 18 nm immunogold particles. Arrowheads indicate Kv1.3. (C) Plasma membrane Kv1.3. (D) Kv1.3 in the ER. (E) Kv1.3 in the Golgi (black arrowhead) and in the IMM (white arrowhead). (F–H) Black arrowheads indicate cytosolic Kv1.3, and white arrowheads indicate mitochondrial Kv1.3. Note the presence of Kv1.3 in ribosomes (r) close to a mitochondrion or embedded in the actin cytoskeleton (a) in (F, G) , respectively. The scale bar indicates 500 nm. White square insets in (C, G, H) highlight higher magnification regions with a scale bar representing 200 nm.

    Article Snippet: Samples were mounted over Formvar-coated grills, and sections were finally stained with 2% uranyl acetate for 15 min. Immunolabeling was performed with primary anti-Kv1.3 (1:30, Neuromab), anti-HSP70 (1:50, Abcam) or anti-HSP90 (1:30, Abcam) antibodies.

    Techniques: Fractionation, Transfection, Marker, Molecular Weight, Dominant Negative Mutation, Labeling

    Transmembrane segments encode sufficient information to localize Kv1.3 to mitochondria. HEK 293 cells were transfected with several YFP-Sx(1-6) transmembrane peptides, and mitochondrial colocalization was analyzed. (A) Cartoon representing the transmembrane peptide constructs. Sx indicates the transmembrane segment (1-6) inserted in the YFP plasmid. (B–H) Representative confocal images of HEK 293 cells cotransfected with different YFP constructs (a, green) and a mitochondrial marker (b, pmitoRFP in red). The merged image (c) shows colocalization in yellow. The square inset indicates the region shown at higher magnification. The scale bar represents 10 µm. (I) Quantification of colocalization using Pearson’s correlation coefficient. Data are presented as the means ± SE (n > 30). *p

    Journal: Frontiers in Oncology

    Article Title: The Mitochondrial Routing of the Kv1.3 Channel

    doi: 10.3389/fonc.2022.865686

    Figure Lengend Snippet: Transmembrane segments encode sufficient information to localize Kv1.3 to mitochondria. HEK 293 cells were transfected with several YFP-Sx(1-6) transmembrane peptides, and mitochondrial colocalization was analyzed. (A) Cartoon representing the transmembrane peptide constructs. Sx indicates the transmembrane segment (1-6) inserted in the YFP plasmid. (B–H) Representative confocal images of HEK 293 cells cotransfected with different YFP constructs (a, green) and a mitochondrial marker (b, pmitoRFP in red). The merged image (c) shows colocalization in yellow. The square inset indicates the region shown at higher magnification. The scale bar represents 10 µm. (I) Quantification of colocalization using Pearson’s correlation coefficient. Data are presented as the means ± SE (n > 30). *p

    Article Snippet: Samples were mounted over Formvar-coated grills, and sections were finally stained with 2% uranyl acetate for 15 min. Immunolabeling was performed with primary anti-Kv1.3 (1:30, Neuromab), anti-HSP70 (1:50, Abcam) or anti-HSP90 (1:30, Abcam) antibodies.

    Techniques: Transfection, Construct, Plasmid Preparation, Marker

    Kv1.3 accumulates at perinuclear mitochondria during the G1/S transition. ( A ) Subcellular fractionation of 3T3-L1 wild-type preadipocytes to obtain the membranous (Mb) and mitochondrial (Mit) fractions. The samples were probed for Kv1.3, Na+/K+ ATPase (a membrane marker) and TIMM50 (a mitochondrial marker). ( B ) Electron micrograph showing mitochondria of 3T3-L1 wild-type preadipocytes. Kv1.3 was labeled with 18 nm immunogold particles (black arrowhead) and was located at the inner mitochondrial membrane. The scale bar represents 200 nm. ( C – H ) Cells were either in the G0/G1 or the G1/S phase following serum deprivation or serum readdition for 12 h, respectively. Representative confocal images showing Kv1.3 and mitochondria in wild-type preadipocytes fixed in the G0/G1 ( C – E ) and G1/S ( F – H ) phase. Ea-Eb and Ha-Hb are magnified images of E and H, respectively. Ea and Ha show distal regions, and Eb and Hb show perinuclear regions. Yellow indicates colocalization of Kv1.3 (green) and mitochondria (red). The scale bar represents 20 µm. ( I ) Pearson’s coefficient of colocalization between Kv1.3 and mitochondria. The data are the mean ± SE ( n > 30). *** p

    Journal: Cancers

    Article Title: Kv1.3 Controls Mitochondrial Dynamics during Cell Cycle Progression

    doi: 10.3390/cancers13174457

    Figure Lengend Snippet: Kv1.3 accumulates at perinuclear mitochondria during the G1/S transition. ( A ) Subcellular fractionation of 3T3-L1 wild-type preadipocytes to obtain the membranous (Mb) and mitochondrial (Mit) fractions. The samples were probed for Kv1.3, Na+/K+ ATPase (a membrane marker) and TIMM50 (a mitochondrial marker). ( B ) Electron micrograph showing mitochondria of 3T3-L1 wild-type preadipocytes. Kv1.3 was labeled with 18 nm immunogold particles (black arrowhead) and was located at the inner mitochondrial membrane. The scale bar represents 200 nm. ( C – H ) Cells were either in the G0/G1 or the G1/S phase following serum deprivation or serum readdition for 12 h, respectively. Representative confocal images showing Kv1.3 and mitochondria in wild-type preadipocytes fixed in the G0/G1 ( C – E ) and G1/S ( F – H ) phase. Ea-Eb and Ha-Hb are magnified images of E and H, respectively. Ea and Ha show distal regions, and Eb and Hb show perinuclear regions. Yellow indicates colocalization of Kv1.3 (green) and mitochondria (red). The scale bar represents 20 µm. ( I ) Pearson’s coefficient of colocalization between Kv1.3 and mitochondria. The data are the mean ± SE ( n > 30). *** p

    Article Snippet: The membranes were immunoblotted with the following specific antibodies: anti-Kv1.3 (1/200, Neuromab, Davis, CA, USA), anti-Cyclin E (1/200, Santa Cruz, Dallas, TX, USA), anti-Cyclin D1 (1/200, Santa Cruz, Dallas, TX, USA), anti-Cyclin A (1/200, Santa Cruz, Dallas, TX, USA), anti-β actin (1/50.000, Sigma, Saint Louis, MO, USA), anti-Glut4 (1/500, OSCRX), anti-TIMM50 (1/100, Abcam, Cambridge, UK), anti-Mtf2 (1/1000, Abcam, Cambridge, UK), anti-Drp1 (1/500, Abcam, Cambridge, UK), and anti-Na+/K+ ATPase (1/1000, Dev Studies Hybridoma Bank, University of Iowa, USA).

    Techniques: Fractionation, Marker, Labeling

    Kv1.3 regulates the mitochondrial membrane potential during the cell cycle. Ablation of Kv1.3 impairs the mitochondrial membrane potential. ( A ) TMRM intensity in wild-type (black bar) and Kv1.3KD (white bar) 3T3-L1 preadipocytes was analyzed with flow cytometry. The data are the mean ± SE ( n = 3), * p

    Journal: Cancers

    Article Title: Kv1.3 Controls Mitochondrial Dynamics during Cell Cycle Progression

    doi: 10.3390/cancers13174457

    Figure Lengend Snippet: Kv1.3 regulates the mitochondrial membrane potential during the cell cycle. Ablation of Kv1.3 impairs the mitochondrial membrane potential. ( A ) TMRM intensity in wild-type (black bar) and Kv1.3KD (white bar) 3T3-L1 preadipocytes was analyzed with flow cytometry. The data are the mean ± SE ( n = 3), * p

    Article Snippet: The membranes were immunoblotted with the following specific antibodies: anti-Kv1.3 (1/200, Neuromab, Davis, CA, USA), anti-Cyclin E (1/200, Santa Cruz, Dallas, TX, USA), anti-Cyclin D1 (1/200, Santa Cruz, Dallas, TX, USA), anti-Cyclin A (1/200, Santa Cruz, Dallas, TX, USA), anti-β actin (1/50.000, Sigma, Saint Louis, MO, USA), anti-Glut4 (1/500, OSCRX), anti-TIMM50 (1/100, Abcam, Cambridge, UK), anti-Mtf2 (1/1000, Abcam, Cambridge, UK), anti-Drp1 (1/500, Abcam, Cambridge, UK), and anti-Na+/K+ ATPase (1/1000, Dev Studies Hybridoma Bank, University of Iowa, USA).

    Techniques: Flow Cytometry

    Kv1.3 facilitates the G1/S transition of the cell cycle in preadipocytes. Serum-starved resting cells were incubated for the indicated time after serum readdition. ( A ) Cell cycle analysis of 3T3-L1 preadipocytes was performed with propidium iodide. Representative histograms at 0, 6, 12, 18 or 24 h after serum readdition. The cells exhibit two blue peaks corresponding to the G0/G1 (left) and G2 (right) phases. The cell population in purple corresponds to cells in the S phase. Left panels, wild-type preadipocytes; right panels, Kv1.3KD preadipocytes. ( B ) The % of cells in the G0/G1 phase, % of cells in the S phase and % of cells in the G2 phase for wild-type (black) and Kv1.3KD (white) preadipocytes. The data are the mean ± SE ( n = 4–10 independent experiments). Two-way ANOVA indicated p

    Journal: Cancers

    Article Title: Kv1.3 Controls Mitochondrial Dynamics during Cell Cycle Progression

    doi: 10.3390/cancers13174457

    Figure Lengend Snippet: Kv1.3 facilitates the G1/S transition of the cell cycle in preadipocytes. Serum-starved resting cells were incubated for the indicated time after serum readdition. ( A ) Cell cycle analysis of 3T3-L1 preadipocytes was performed with propidium iodide. Representative histograms at 0, 6, 12, 18 or 24 h after serum readdition. The cells exhibit two blue peaks corresponding to the G0/G1 (left) and G2 (right) phases. The cell population in purple corresponds to cells in the S phase. Left panels, wild-type preadipocytes; right panels, Kv1.3KD preadipocytes. ( B ) The % of cells in the G0/G1 phase, % of cells in the S phase and % of cells in the G2 phase for wild-type (black) and Kv1.3KD (white) preadipocytes. The data are the mean ± SE ( n = 4–10 independent experiments). Two-way ANOVA indicated p

    Article Snippet: The membranes were immunoblotted with the following specific antibodies: anti-Kv1.3 (1/200, Neuromab, Davis, CA, USA), anti-Cyclin E (1/200, Santa Cruz, Dallas, TX, USA), anti-Cyclin D1 (1/200, Santa Cruz, Dallas, TX, USA), anti-Cyclin A (1/200, Santa Cruz, Dallas, TX, USA), anti-β actin (1/50.000, Sigma, Saint Louis, MO, USA), anti-Glut4 (1/500, OSCRX), anti-TIMM50 (1/100, Abcam, Cambridge, UK), anti-Mtf2 (1/1000, Abcam, Cambridge, UK), anti-Drp1 (1/500, Abcam, Cambridge, UK), and anti-Na+/K+ ATPase (1/1000, Dev Studies Hybridoma Bank, University of Iowa, USA).

    Techniques: Incubation, Cell Cycle Assay

    Representative cartoon summarizing the participation of the mitochondrial Kv1.3 (mitoKv1.3) in the proliferation of preadipocytes. Kv1.3 would facilitate the G1/S transition of the cell cycle in preadipocytes accumulating at perinuclear mitochondria. The elucidation of a putative mitochondrial-nuclear communication during this phase of the cell cycle in which Kv1.3 would participate deserves much effort. During the G1/S transition, Kv1.3 would contribute to the mitochondrial fusion/fission equilibrium controlling the mitochondrial membrane potential. Ablation of Kv1.3 (Kv1.3KD) would impair mitochondrial dynamics during cell cycle progression. Kv1.3KD, 3T3-L1 preadipocytes, with a genetic ablation of Kv1.3. Green dots, Kv1.3 channels; magenta, mitochondrial network.

    Journal: Cancers

    Article Title: Kv1.3 Controls Mitochondrial Dynamics during Cell Cycle Progression

    doi: 10.3390/cancers13174457

    Figure Lengend Snippet: Representative cartoon summarizing the participation of the mitochondrial Kv1.3 (mitoKv1.3) in the proliferation of preadipocytes. Kv1.3 would facilitate the G1/S transition of the cell cycle in preadipocytes accumulating at perinuclear mitochondria. The elucidation of a putative mitochondrial-nuclear communication during this phase of the cell cycle in which Kv1.3 would participate deserves much effort. During the G1/S transition, Kv1.3 would contribute to the mitochondrial fusion/fission equilibrium controlling the mitochondrial membrane potential. Ablation of Kv1.3 (Kv1.3KD) would impair mitochondrial dynamics during cell cycle progression. Kv1.3KD, 3T3-L1 preadipocytes, with a genetic ablation of Kv1.3. Green dots, Kv1.3 channels; magenta, mitochondrial network.

    Article Snippet: The membranes were immunoblotted with the following specific antibodies: anti-Kv1.3 (1/200, Neuromab, Davis, CA, USA), anti-Cyclin E (1/200, Santa Cruz, Dallas, TX, USA), anti-Cyclin D1 (1/200, Santa Cruz, Dallas, TX, USA), anti-Cyclin A (1/200, Santa Cruz, Dallas, TX, USA), anti-β actin (1/50.000, Sigma, Saint Louis, MO, USA), anti-Glut4 (1/500, OSCRX), anti-TIMM50 (1/100, Abcam, Cambridge, UK), anti-Mtf2 (1/1000, Abcam, Cambridge, UK), anti-Drp1 (1/500, Abcam, Cambridge, UK), and anti-Na+/K+ ATPase (1/1000, Dev Studies Hybridoma Bank, University of Iowa, USA).

    Techniques:

    Kv1.3 participates in the proliferation of preadipocytes. 3T3-L1 preadipocytes express Kv1.3, and genetic ablation of the channel alters cell proliferation. ( A ) Representative immunofluorescence confocal image of Kv1.3 in 3T3-L1 preadipocytes. The scale bar represents 20 µm. ( B ) Kv1.3 silencing in 3T3-L1 preadipocytes. Cells were infected with Kv1.3 shRNA (Kv1.3KD) or scramble shRNA (SCR) lentivirus. β-actin was used as a loading control. Noninfected 3T3-L1 cells were called wild-type (WT) cells. ( C ) Quantification of the efficiency of Kv1.3 silencing. The data are the mean ± SE ( n ≥ 3). * p

    Journal: Cancers

    Article Title: Kv1.3 Controls Mitochondrial Dynamics during Cell Cycle Progression

    doi: 10.3390/cancers13174457

    Figure Lengend Snippet: Kv1.3 participates in the proliferation of preadipocytes. 3T3-L1 preadipocytes express Kv1.3, and genetic ablation of the channel alters cell proliferation. ( A ) Representative immunofluorescence confocal image of Kv1.3 in 3T3-L1 preadipocytes. The scale bar represents 20 µm. ( B ) Kv1.3 silencing in 3T3-L1 preadipocytes. Cells were infected with Kv1.3 shRNA (Kv1.3KD) or scramble shRNA (SCR) lentivirus. β-actin was used as a loading control. Noninfected 3T3-L1 cells were called wild-type (WT) cells. ( C ) Quantification of the efficiency of Kv1.3 silencing. The data are the mean ± SE ( n ≥ 3). * p

    Article Snippet: The membranes were immunoblotted with the following specific antibodies: anti-Kv1.3 (1/200, Neuromab, Davis, CA, USA), anti-Cyclin E (1/200, Santa Cruz, Dallas, TX, USA), anti-Cyclin D1 (1/200, Santa Cruz, Dallas, TX, USA), anti-Cyclin A (1/200, Santa Cruz, Dallas, TX, USA), anti-β actin (1/50.000, Sigma, Saint Louis, MO, USA), anti-Glut4 (1/500, OSCRX), anti-TIMM50 (1/100, Abcam, Cambridge, UK), anti-Mtf2 (1/1000, Abcam, Cambridge, UK), anti-Drp1 (1/500, Abcam, Cambridge, UK), and anti-Na+/K+ ATPase (1/1000, Dev Studies Hybridoma Bank, University of Iowa, USA).

    Techniques: Immunofluorescence, Infection, shRNA

    Kv1.3 regulates the mitochondrial fusion/fission equilibrium during the G1/S transition. Confocal images showing mitochondria in cells fixed in the G0/G1 ( A – F ) and G1/S ( G – L ) phase for WT ( A – C , G – I ) and Kv1.3KD preadipocytes ( D – F , J – L ). The scale bar represents 20 µm. Images were processed (tubeness and skeleton) to perform morphometric analysis of mitochondria. ( M ) The length of mitochondrial networks was measured as the average area of the skeletonized binary image. ( N ) Number of mitochondrial particles per µm 2 . ( O ) The form factor describes the particle shape complexity and was computed as the average (perimeter)2/(4π·area). A circle corresponds to a minimum value of 1. The data are the mean ± SE ( n > 30). *, p

    Journal: Cancers

    Article Title: Kv1.3 Controls Mitochondrial Dynamics during Cell Cycle Progression

    doi: 10.3390/cancers13174457

    Figure Lengend Snippet: Kv1.3 regulates the mitochondrial fusion/fission equilibrium during the G1/S transition. Confocal images showing mitochondria in cells fixed in the G0/G1 ( A – F ) and G1/S ( G – L ) phase for WT ( A – C , G – I ) and Kv1.3KD preadipocytes ( D – F , J – L ). The scale bar represents 20 µm. Images were processed (tubeness and skeleton) to perform morphometric analysis of mitochondria. ( M ) The length of mitochondrial networks was measured as the average area of the skeletonized binary image. ( N ) Number of mitochondrial particles per µm 2 . ( O ) The form factor describes the particle shape complexity and was computed as the average (perimeter)2/(4π·area). A circle corresponds to a minimum value of 1. The data are the mean ± SE ( n > 30). *, p

    Article Snippet: The membranes were immunoblotted with the following specific antibodies: anti-Kv1.3 (1/200, Neuromab, Davis, CA, USA), anti-Cyclin E (1/200, Santa Cruz, Dallas, TX, USA), anti-Cyclin D1 (1/200, Santa Cruz, Dallas, TX, USA), anti-Cyclin A (1/200, Santa Cruz, Dallas, TX, USA), anti-β actin (1/50.000, Sigma, Saint Louis, MO, USA), anti-Glut4 (1/500, OSCRX), anti-TIMM50 (1/100, Abcam, Cambridge, UK), anti-Mtf2 (1/1000, Abcam, Cambridge, UK), anti-Drp1 (1/500, Abcam, Cambridge, UK), and anti-Na+/K+ ATPase (1/1000, Dev Studies Hybridoma Bank, University of Iowa, USA).

    Techniques:

    KCNE4 impaired Kv1.3 accumulation in the IS but did not disrupt IS formation. Human Jurkat T lymphocytes and human Raji B lymphocytes were used to generate cell conjugates. ( A – C ) Activated B-cells (10 µg/mL SEE toxin) were cocultured in the absence ( Ba – Bd ) or presence ( Ca – Cd ) of Jurkat cells, and confocal images were obtained. ( Aa – Ad ) Jurkat T-cells in the absence of B cells. Endogenous Kv1.3 (green), CD3 (marker of T-cells, red), and CD19 (marker of B-cells, blue) were detected. ( Ad , Bd , Cd ) merge panels. Note that triple colocalization (white) in Cd localizes Kv1.3 in the IS, as identified by CD3 staining ( Cb ). Bars are 20 µm. ( D ) Accumulation ratio of mGFP-Kv1.3 at the IS vs. KCNE4-mCherry total intensity (n = 40). ( E ) CD3 recruitment into the IS vs. KCNE4 intensity. The horizontal red line represents the threshold level (1.5) for Kv1.3 and CD3 accumulation in the IS. Values greater or less than 1.5 indicated positive or negative accumulation of proteins at the IS, respectively.

    Journal: Scientific Reports

    Article Title: KCNE4-dependent functional consequences of Kv1.3-related leukocyte physiology

    doi: 10.1038/s41598-021-94015-9

    Figure Lengend Snippet: KCNE4 impaired Kv1.3 accumulation in the IS but did not disrupt IS formation. Human Jurkat T lymphocytes and human Raji B lymphocytes were used to generate cell conjugates. ( A – C ) Activated B-cells (10 µg/mL SEE toxin) were cocultured in the absence ( Ba – Bd ) or presence ( Ca – Cd ) of Jurkat cells, and confocal images were obtained. ( Aa – Ad ) Jurkat T-cells in the absence of B cells. Endogenous Kv1.3 (green), CD3 (marker of T-cells, red), and CD19 (marker of B-cells, blue) were detected. ( Ad , Bd , Cd ) merge panels. Note that triple colocalization (white) in Cd localizes Kv1.3 in the IS, as identified by CD3 staining ( Cb ). Bars are 20 µm. ( D ) Accumulation ratio of mGFP-Kv1.3 at the IS vs. KCNE4-mCherry total intensity (n = 40). ( E ) CD3 recruitment into the IS vs. KCNE4 intensity. The horizontal red line represents the threshold level (1.5) for Kv1.3 and CD3 accumulation in the IS. Values greater or less than 1.5 indicated positive or negative accumulation of proteins at the IS, respectively.

    Article Snippet: Cells were incubated with anti-Kv1.3 (1/50, NeuroMab) at 4 °C overnight.

    Techniques: Marker, Staining

    KCNE4 expression impairs Kv1.3 surface expression and inhibits Kv currents in Jurkat T cells. Confocal imaging of Jurkat T cells transfected with YFP, Kv1.3YFP, KCNE4CFP and KCNE4CFP with YFP-Kv1.3. Nuclei were stained with DAPI (blue). ( Aa – Ad ) Jurkat nontransfected cells (control). ( Ba – Bd ) YFP-transfected cells (YFP). ( Ca – Cd ) Kv1.3YFP transfected cells. ( Da – Dd ) KCNE4CFP transfected cells. ( Ea – Ed ) Kv1.3YFP and KCNE4CFP cotransfected cells. ( Aa , Ba , Ca , Da , Ea ) Kv1.3 in green. ( Ab , Bb , Cb , Db , Eb ) KCNE4 in red. ( Ac , Bc , Cc , Dc , Ec ) DAPI in blue. Merged yellow indicates colocalization between green and red ( Ad , Bd , Cd , Dd , Ed ). Scale bar: 5 µm. ( F ) FRET analysis of the Kv1.3-KCNE4 protein interaction by flow cytometry in Jurkat T lymphocytes. Values are mean ± SE, n = 5–7, *p

    Journal: Scientific Reports

    Article Title: KCNE4-dependent functional consequences of Kv1.3-related leukocyte physiology

    doi: 10.1038/s41598-021-94015-9

    Figure Lengend Snippet: KCNE4 expression impairs Kv1.3 surface expression and inhibits Kv currents in Jurkat T cells. Confocal imaging of Jurkat T cells transfected with YFP, Kv1.3YFP, KCNE4CFP and KCNE4CFP with YFP-Kv1.3. Nuclei were stained with DAPI (blue). ( Aa – Ad ) Jurkat nontransfected cells (control). ( Ba – Bd ) YFP-transfected cells (YFP). ( Ca – Cd ) Kv1.3YFP transfected cells. ( Da – Dd ) KCNE4CFP transfected cells. ( Ea – Ed ) Kv1.3YFP and KCNE4CFP cotransfected cells. ( Aa , Ba , Ca , Da , Ea ) Kv1.3 in green. ( Ab , Bb , Cb , Db , Eb ) KCNE4 in red. ( Ac , Bc , Cc , Dc , Ec ) DAPI in blue. Merged yellow indicates colocalization between green and red ( Ad , Bd , Cd , Dd , Ed ). Scale bar: 5 µm. ( F ) FRET analysis of the Kv1.3-KCNE4 protein interaction by flow cytometry in Jurkat T lymphocytes. Values are mean ± SE, n = 5–7, *p

    Article Snippet: Cells were incubated with anti-Kv1.3 (1/50, NeuroMab) at 4 °C overnight.

    Techniques: Expressing, Imaging, Transfection, Staining, Flow Cytometry

    LPS-dependent activation of CY15 dendritic cells increases the abundance of Kv1.3 at the cell surface. CY15 cells were incubated in the presence (LPS) or the absence (control) of LPS for 24 h. Cells were first stained with WGA (membrane marker) and then immunolabeled against Kv1.3. ( A – D ) Control cells in the absence of LPS. ( E – H ) Cells treated with LPS. Green, Kv1.3; red, WGA; merged panels show colocalization between green and red. ( D , H ) Histogram of the pixel by pixel analysis of the section indicated by the arrow in ( C , G ), respectively. Bars represent 10 μm. ( I ) Mander's overlap coefficient (MOC) quantifying the degree of colocalization between Kv1.3 and membrane surface (WGA) staining. White bar, control; black bar, LPS. Values are mean ± SE of n > 30 cells. ***p

    Journal: Scientific Reports

    Article Title: KCNE4-dependent functional consequences of Kv1.3-related leukocyte physiology

    doi: 10.1038/s41598-021-94015-9

    Figure Lengend Snippet: LPS-dependent activation of CY15 dendritic cells increases the abundance of Kv1.3 at the cell surface. CY15 cells were incubated in the presence (LPS) or the absence (control) of LPS for 24 h. Cells were first stained with WGA (membrane marker) and then immunolabeled against Kv1.3. ( A – D ) Control cells in the absence of LPS. ( E – H ) Cells treated with LPS. Green, Kv1.3; red, WGA; merged panels show colocalization between green and red. ( D , H ) Histogram of the pixel by pixel analysis of the section indicated by the arrow in ( C , G ), respectively. Bars represent 10 μm. ( I ) Mander's overlap coefficient (MOC) quantifying the degree of colocalization between Kv1.3 and membrane surface (WGA) staining. White bar, control; black bar, LPS. Values are mean ± SE of n > 30 cells. ***p

    Article Snippet: Cells were incubated with anti-Kv1.3 (1/50, NeuroMab) at 4 °C overnight.

    Techniques: Activation Assay, Incubation, Staining, Whole Genome Amplification, Marker, Immunolabeling

    KCNE4 overexpression modulates Kv1.3-related physiological events in Jurkat T lymphocytes. Jurkat T cells were electroporated with KCNE4CFP, and positively transfected cells were selected for specific assays. ( A ) Kv1.3 and KCNE4 expression in Jurkat cells. ( B ) Percentage of Jurkat T cell proliferation. Cells were serum starved overnight and cultured for an additional 24 h in the presence of FBS. The alamarBlue dye was used. *p

    Journal: Scientific Reports

    Article Title: KCNE4-dependent functional consequences of Kv1.3-related leukocyte physiology

    doi: 10.1038/s41598-021-94015-9

    Figure Lengend Snippet: KCNE4 overexpression modulates Kv1.3-related physiological events in Jurkat T lymphocytes. Jurkat T cells were electroporated with KCNE4CFP, and positively transfected cells were selected for specific assays. ( A ) Kv1.3 and KCNE4 expression in Jurkat cells. ( B ) Percentage of Jurkat T cell proliferation. Cells were serum starved overnight and cultured for an additional 24 h in the presence of FBS. The alamarBlue dye was used. *p

    Article Snippet: Cells were incubated with anti-Kv1.3 (1/50, NeuroMab) at 4 °C overnight.

    Techniques: Over Expression, Transfection, Expressing, Cell Culture

    Kv1.3 and KCNE4 are differentially expressed in leukocytes. The presence of Kv1.3 and KCNE4 expression was analyzed in human Jurkat T lymphocytes and mouse CY15 dendritic cells. ( A ) Kv1.3 and KCNE4 protein expression in leukocytes. HEK 293 cells were used as a negative control. Although Jurkat and CY15 dendritic cells shared Kv1.3 and KCNE4 expression, the abundance of KCNE4 in T cells was much lower and minimally detected by western blot. In addition, Kv1.5 was abundantly expressed in CY15 cells. Representative cropped blots, clearly separated by vertical white lines, are shown only for qualitative purposes. Voltage-dependent K + currents were elicited in Jurkat ( B ) and CY15 cells ( C ). Cells were held at -60 mV, and 250 ms pulse potentials were applied as indicated. ( D ) Representative confocal images of Kv1.3 ( Da and Dd , in green) and KCNE4 ( Db and De , in red) in Jurkat T lymphocytes ( Da – Dc ) and CY15 dendritic cells ( Dd – Df ). Scale bars: 10 µm. Given the limited expression of KCNE4 in T-cells, IPI was performed in Jurkat cells. ( E ) KCNE4 coimmunoprecipitated with Kv1.3 in dendritic cells. Lysates were immunoprecipitated against Kv1.3 (IP: Kv1.3) and immunoblotted (IB) against Kv1.3 and KCNE4. Upper panel: Kv1.3. Lower panel: KCNE4. SM: starting material. SN+: supernatant from the IP+. SN−: supernatant from the IP−. IP+: Immunoprecipitation in the presence of the anti-Kv1.3 antibody. IP−: Immunoprecipitated in the absence of the anti-Kv1.3 antibody. ( F ) Kv1.3 localized in lipid raft fractions from Jurkat T-cells. ( G ) Kv1.3 and KCNE4 did not localize in lipid rafts from CY15 dendritic cells. Lipid rafts were isolated, and low density (1) to high density (12) sucrose gradient fractions were analyzed by western blot. Flotilin indicated low-buoyancy lipid rafts, whereas clathrin identified nonfloating raft fractions.

    Journal: Scientific Reports

    Article Title: KCNE4-dependent functional consequences of Kv1.3-related leukocyte physiology

    doi: 10.1038/s41598-021-94015-9

    Figure Lengend Snippet: Kv1.3 and KCNE4 are differentially expressed in leukocytes. The presence of Kv1.3 and KCNE4 expression was analyzed in human Jurkat T lymphocytes and mouse CY15 dendritic cells. ( A ) Kv1.3 and KCNE4 protein expression in leukocytes. HEK 293 cells were used as a negative control. Although Jurkat and CY15 dendritic cells shared Kv1.3 and KCNE4 expression, the abundance of KCNE4 in T cells was much lower and minimally detected by western blot. In addition, Kv1.5 was abundantly expressed in CY15 cells. Representative cropped blots, clearly separated by vertical white lines, are shown only for qualitative purposes. Voltage-dependent K + currents were elicited in Jurkat ( B ) and CY15 cells ( C ). Cells were held at -60 mV, and 250 ms pulse potentials were applied as indicated. ( D ) Representative confocal images of Kv1.3 ( Da and Dd , in green) and KCNE4 ( Db and De , in red) in Jurkat T lymphocytes ( Da – Dc ) and CY15 dendritic cells ( Dd – Df ). Scale bars: 10 µm. Given the limited expression of KCNE4 in T-cells, IPI was performed in Jurkat cells. ( E ) KCNE4 coimmunoprecipitated with Kv1.3 in dendritic cells. Lysates were immunoprecipitated against Kv1.3 (IP: Kv1.3) and immunoblotted (IB) against Kv1.3 and KCNE4. Upper panel: Kv1.3. Lower panel: KCNE4. SM: starting material. SN+: supernatant from the IP+. SN−: supernatant from the IP−. IP+: Immunoprecipitation in the presence of the anti-Kv1.3 antibody. IP−: Immunoprecipitated in the absence of the anti-Kv1.3 antibody. ( F ) Kv1.3 localized in lipid raft fractions from Jurkat T-cells. ( G ) Kv1.3 and KCNE4 did not localize in lipid rafts from CY15 dendritic cells. Lipid rafts were isolated, and low density (1) to high density (12) sucrose gradient fractions were analyzed by western blot. Flotilin indicated low-buoyancy lipid rafts, whereas clathrin identified nonfloating raft fractions.

    Article Snippet: Cells were incubated with anti-Kv1.3 (1/50, NeuroMab) at 4 °C overnight.

    Techniques: Expressing, Negative Control, Western Blot, Immunoprecipitation, Isolation

    LPS-dependent activation increases the Kv1.3/KCNE4 ratio in CY15 dendritic cells. Cells were treated for 24 h with LPS (100 ng/ml), and the protein expression of selected K + channel proteins was studied at 0, 6 and 24 h. ( A ) Confocal images of CY15 cells stained with Kv1.3 upon LPS treatment. Scale bars: 20 µm. ( B ) Representative voltage-dependent K+ currents elicited in CY15 cells treated with (LPS 24 h) or without (LPS 0 h) LPS during 24 h. Cells were held at -60 mV and 250 ms pulses to +60 mV were applied. ( C ) Peak current densitiy (pA/pF) of K + currents from CY15 cells in the absence (0 h) or the presence (24 h) of LPS. White bars, LPS 0 h (control); black bars, LPS 24 h. Values are the mean ± SE of 4–6 cells. *p

    Journal: Scientific Reports

    Article Title: KCNE4-dependent functional consequences of Kv1.3-related leukocyte physiology

    doi: 10.1038/s41598-021-94015-9

    Figure Lengend Snippet: LPS-dependent activation increases the Kv1.3/KCNE4 ratio in CY15 dendritic cells. Cells were treated for 24 h with LPS (100 ng/ml), and the protein expression of selected K + channel proteins was studied at 0, 6 and 24 h. ( A ) Confocal images of CY15 cells stained with Kv1.3 upon LPS treatment. Scale bars: 20 µm. ( B ) Representative voltage-dependent K+ currents elicited in CY15 cells treated with (LPS 24 h) or without (LPS 0 h) LPS during 24 h. Cells were held at -60 mV and 250 ms pulses to +60 mV were applied. ( C ) Peak current densitiy (pA/pF) of K + currents from CY15 cells in the absence (0 h) or the presence (24 h) of LPS. White bars, LPS 0 h (control); black bars, LPS 24 h. Values are the mean ± SE of 4–6 cells. *p

    Article Snippet: Cells were incubated with anti-Kv1.3 (1/50, NeuroMab) at 4 °C overnight.

    Techniques: Activation Assay, Expressing, Staining

    KCNE4 impaired Kv1.3 accumulation in the IS but did not disrupt IS formation. Human Jurkat T lymphocytes and human Raji B lymphocytes were used to generate cell conjugates. ( A – C ) Activated B-cells (10 µg/mL SEE toxin) were cocultured in the absence ( Ba – Bd ) or presence ( Ca – Cd ) of Jurkat cells, and confocal images were obtained. ( Aa – Ad ) Jurkat T-cells in the absence of B cells. Endogenous Kv1.3 (green), CD3 (marker of T-cells, red), and CD19 (marker of B-cells, blue) were detected. ( Ad , Bd , Cd ) merge panels. Note that triple colocalization (white) in Cd localizes Kv1.3 in the IS, as identified by CD3 staining ( Cb ). Bars are 20 µm. ( D ) Accumulation ratio of mGFP-Kv1.3 at the IS vs. KCNE4-mCherry total intensity (n = 40). ( E ) CD3 recruitment into the IS vs. KCNE4 intensity. The horizontal red line represents the threshold level (1.5) for Kv1.3 and CD3 accumulation in the IS. Values greater or less than 1.5 indicated positive or negative accumulation of proteins at the IS, respectively.

    Journal: Scientific Reports

    Article Title: KCNE4-dependent functional consequences of Kv1.3-related leukocyte physiology

    doi: 10.1038/s41598-021-94015-9

    Figure Lengend Snippet: KCNE4 impaired Kv1.3 accumulation in the IS but did not disrupt IS formation. Human Jurkat T lymphocytes and human Raji B lymphocytes were used to generate cell conjugates. ( A – C ) Activated B-cells (10 µg/mL SEE toxin) were cocultured in the absence ( Ba – Bd ) or presence ( Ca – Cd ) of Jurkat cells, and confocal images were obtained. ( Aa – Ad ) Jurkat T-cells in the absence of B cells. Endogenous Kv1.3 (green), CD3 (marker of T-cells, red), and CD19 (marker of B-cells, blue) were detected. ( Ad , Bd , Cd ) merge panels. Note that triple colocalization (white) in Cd localizes Kv1.3 in the IS, as identified by CD3 staining ( Cb ). Bars are 20 µm. ( D ) Accumulation ratio of mGFP-Kv1.3 at the IS vs. KCNE4-mCherry total intensity (n = 40). ( E ) CD3 recruitment into the IS vs. KCNE4 intensity. The horizontal red line represents the threshold level (1.5) for Kv1.3 and CD3 accumulation in the IS. Values greater or less than 1.5 indicated positive or negative accumulation of proteins at the IS, respectively.

    Article Snippet: In some experiments, T cells were stained against Kv1.3 (anti-Kv1.3 1/200, Neuromab) in combination with wheat germ agglutinin (WGA, plasma membrane marker) and calnexin (ER marker; anti-calnexin 1/100; BD Transduction Laboratories) as described .

    Techniques: Marker, Staining

    KCNE4 expression impairs Kv1.3 surface expression and inhibits Kv currents in Jurkat T cells. Confocal imaging of Jurkat T cells transfected with YFP, Kv1.3YFP, KCNE4CFP and KCNE4CFP with YFP-Kv1.3. Nuclei were stained with DAPI (blue). ( Aa – Ad ) Jurkat nontransfected cells (control). ( Ba – Bd ) YFP-transfected cells (YFP). ( Ca – Cd ) Kv1.3YFP transfected cells. ( Da – Dd ) KCNE4CFP transfected cells. ( Ea – Ed ) Kv1.3YFP and KCNE4CFP cotransfected cells. ( Aa , Ba , Ca , Da , Ea ) Kv1.3 in green. ( Ab , Bb , Cb , Db , Eb ) KCNE4 in red. ( Ac , Bc , Cc , Dc , Ec ) DAPI in blue. Merged yellow indicates colocalization between green and red ( Ad , Bd , Cd , Dd , Ed ). Scale bar: 5 µm. ( F ) FRET analysis of the Kv1.3-KCNE4 protein interaction by flow cytometry in Jurkat T lymphocytes. Values are mean ± SE, n = 5–7, *p

    Journal: Scientific Reports

    Article Title: KCNE4-dependent functional consequences of Kv1.3-related leukocyte physiology

    doi: 10.1038/s41598-021-94015-9

    Figure Lengend Snippet: KCNE4 expression impairs Kv1.3 surface expression and inhibits Kv currents in Jurkat T cells. Confocal imaging of Jurkat T cells transfected with YFP, Kv1.3YFP, KCNE4CFP and KCNE4CFP with YFP-Kv1.3. Nuclei were stained with DAPI (blue). ( Aa – Ad ) Jurkat nontransfected cells (control). ( Ba – Bd ) YFP-transfected cells (YFP). ( Ca – Cd ) Kv1.3YFP transfected cells. ( Da – Dd ) KCNE4CFP transfected cells. ( Ea – Ed ) Kv1.3YFP and KCNE4CFP cotransfected cells. ( Aa , Ba , Ca , Da , Ea ) Kv1.3 in green. ( Ab , Bb , Cb , Db , Eb ) KCNE4 in red. ( Ac , Bc , Cc , Dc , Ec ) DAPI in blue. Merged yellow indicates colocalization between green and red ( Ad , Bd , Cd , Dd , Ed ). Scale bar: 5 µm. ( F ) FRET analysis of the Kv1.3-KCNE4 protein interaction by flow cytometry in Jurkat T lymphocytes. Values are mean ± SE, n = 5–7, *p

    Article Snippet: In some experiments, T cells were stained against Kv1.3 (anti-Kv1.3 1/200, Neuromab) in combination with wheat germ agglutinin (WGA, plasma membrane marker) and calnexin (ER marker; anti-calnexin 1/100; BD Transduction Laboratories) as described .

    Techniques: Expressing, Imaging, Transfection, Staining, Flow Cytometry

    LPS-dependent activation of CY15 dendritic cells increases the abundance of Kv1.3 at the cell surface. CY15 cells were incubated in the presence (LPS) or the absence (control) of LPS for 24 h. Cells were first stained with WGA (membrane marker) and then immunolabeled against Kv1.3. ( A – D ) Control cells in the absence of LPS. ( E – H ) Cells treated with LPS. Green, Kv1.3; red, WGA; merged panels show colocalization between green and red. ( D , H ) Histogram of the pixel by pixel analysis of the section indicated by the arrow in ( C , G ), respectively. Bars represent 10 μm. ( I ) Mander's overlap coefficient (MOC) quantifying the degree of colocalization between Kv1.3 and membrane surface (WGA) staining. White bar, control; black bar, LPS. Values are mean ± SE of n > 30 cells. ***p

    Journal: Scientific Reports

    Article Title: KCNE4-dependent functional consequences of Kv1.3-related leukocyte physiology

    doi: 10.1038/s41598-021-94015-9

    Figure Lengend Snippet: LPS-dependent activation of CY15 dendritic cells increases the abundance of Kv1.3 at the cell surface. CY15 cells were incubated in the presence (LPS) or the absence (control) of LPS for 24 h. Cells were first stained with WGA (membrane marker) and then immunolabeled against Kv1.3. ( A – D ) Control cells in the absence of LPS. ( E – H ) Cells treated with LPS. Green, Kv1.3; red, WGA; merged panels show colocalization between green and red. ( D , H ) Histogram of the pixel by pixel analysis of the section indicated by the arrow in ( C , G ), respectively. Bars represent 10 μm. ( I ) Mander's overlap coefficient (MOC) quantifying the degree of colocalization between Kv1.3 and membrane surface (WGA) staining. White bar, control; black bar, LPS. Values are mean ± SE of n > 30 cells. ***p

    Article Snippet: In some experiments, T cells were stained against Kv1.3 (anti-Kv1.3 1/200, Neuromab) in combination with wheat germ agglutinin (WGA, plasma membrane marker) and calnexin (ER marker; anti-calnexin 1/100; BD Transduction Laboratories) as described .

    Techniques: Activation Assay, Incubation, Staining, Whole Genome Amplification, Marker, Immunolabeling

    KCNE4 overexpression modulates Kv1.3-related physiological events in Jurkat T lymphocytes. Jurkat T cells were electroporated with KCNE4CFP, and positively transfected cells were selected for specific assays. ( A ) Kv1.3 and KCNE4 expression in Jurkat cells. ( B ) Percentage of Jurkat T cell proliferation. Cells were serum starved overnight and cultured for an additional 24 h in the presence of FBS. The alamarBlue dye was used. *p

    Journal: Scientific Reports

    Article Title: KCNE4-dependent functional consequences of Kv1.3-related leukocyte physiology

    doi: 10.1038/s41598-021-94015-9

    Figure Lengend Snippet: KCNE4 overexpression modulates Kv1.3-related physiological events in Jurkat T lymphocytes. Jurkat T cells were electroporated with KCNE4CFP, and positively transfected cells were selected for specific assays. ( A ) Kv1.3 and KCNE4 expression in Jurkat cells. ( B ) Percentage of Jurkat T cell proliferation. Cells were serum starved overnight and cultured for an additional 24 h in the presence of FBS. The alamarBlue dye was used. *p

    Article Snippet: In some experiments, T cells were stained against Kv1.3 (anti-Kv1.3 1/200, Neuromab) in combination with wheat germ agglutinin (WGA, plasma membrane marker) and calnexin (ER marker; anti-calnexin 1/100; BD Transduction Laboratories) as described .

    Techniques: Over Expression, Transfection, Expressing, Cell Culture

    Kv1.3 and KCNE4 are differentially expressed in leukocytes. The presence of Kv1.3 and KCNE4 expression was analyzed in human Jurkat T lymphocytes and mouse CY15 dendritic cells. ( A ) Kv1.3 and KCNE4 protein expression in leukocytes. HEK 293 cells were used as a negative control. Although Jurkat and CY15 dendritic cells shared Kv1.3 and KCNE4 expression, the abundance of KCNE4 in T cells was much lower and minimally detected by western blot. In addition, Kv1.5 was abundantly expressed in CY15 cells. Representative cropped blots, clearly separated by vertical white lines, are shown only for qualitative purposes. Voltage-dependent K + currents were elicited in Jurkat ( B ) and CY15 cells ( C ). Cells were held at -60 mV, and 250 ms pulse potentials were applied as indicated. ( D ) Representative confocal images of Kv1.3 ( Da and Dd , in green) and KCNE4 ( Db and De , in red) in Jurkat T lymphocytes ( Da – Dc ) and CY15 dendritic cells ( Dd – Df ). Scale bars: 10 µm. Given the limited expression of KCNE4 in T-cells, IPI was performed in Jurkat cells. ( E ) KCNE4 coimmunoprecipitated with Kv1.3 in dendritic cells. Lysates were immunoprecipitated against Kv1.3 (IP: Kv1.3) and immunoblotted (IB) against Kv1.3 and KCNE4. Upper panel: Kv1.3. Lower panel: KCNE4. SM: starting material. SN+: supernatant from the IP+. SN−: supernatant from the IP−. IP+: Immunoprecipitation in the presence of the anti-Kv1.3 antibody. IP−: Immunoprecipitated in the absence of the anti-Kv1.3 antibody. ( F ) Kv1.3 localized in lipid raft fractions from Jurkat T-cells. ( G ) Kv1.3 and KCNE4 did not localize in lipid rafts from CY15 dendritic cells. Lipid rafts were isolated, and low density (1) to high density (12) sucrose gradient fractions were analyzed by western blot. Flotilin indicated low-buoyancy lipid rafts, whereas clathrin identified nonfloating raft fractions.

    Journal: Scientific Reports

    Article Title: KCNE4-dependent functional consequences of Kv1.3-related leukocyte physiology

    doi: 10.1038/s41598-021-94015-9

    Figure Lengend Snippet: Kv1.3 and KCNE4 are differentially expressed in leukocytes. The presence of Kv1.3 and KCNE4 expression was analyzed in human Jurkat T lymphocytes and mouse CY15 dendritic cells. ( A ) Kv1.3 and KCNE4 protein expression in leukocytes. HEK 293 cells were used as a negative control. Although Jurkat and CY15 dendritic cells shared Kv1.3 and KCNE4 expression, the abundance of KCNE4 in T cells was much lower and minimally detected by western blot. In addition, Kv1.5 was abundantly expressed in CY15 cells. Representative cropped blots, clearly separated by vertical white lines, are shown only for qualitative purposes. Voltage-dependent K + currents were elicited in Jurkat ( B ) and CY15 cells ( C ). Cells were held at -60 mV, and 250 ms pulse potentials were applied as indicated. ( D ) Representative confocal images of Kv1.3 ( Da and Dd , in green) and KCNE4 ( Db and De , in red) in Jurkat T lymphocytes ( Da – Dc ) and CY15 dendritic cells ( Dd – Df ). Scale bars: 10 µm. Given the limited expression of KCNE4 in T-cells, IPI was performed in Jurkat cells. ( E ) KCNE4 coimmunoprecipitated with Kv1.3 in dendritic cells. Lysates were immunoprecipitated against Kv1.3 (IP: Kv1.3) and immunoblotted (IB) against Kv1.3 and KCNE4. Upper panel: Kv1.3. Lower panel: KCNE4. SM: starting material. SN+: supernatant from the IP+. SN−: supernatant from the IP−. IP+: Immunoprecipitation in the presence of the anti-Kv1.3 antibody. IP−: Immunoprecipitated in the absence of the anti-Kv1.3 antibody. ( F ) Kv1.3 localized in lipid raft fractions from Jurkat T-cells. ( G ) Kv1.3 and KCNE4 did not localize in lipid rafts from CY15 dendritic cells. Lipid rafts were isolated, and low density (1) to high density (12) sucrose gradient fractions were analyzed by western blot. Flotilin indicated low-buoyancy lipid rafts, whereas clathrin identified nonfloating raft fractions.

    Article Snippet: In some experiments, T cells were stained against Kv1.3 (anti-Kv1.3 1/200, Neuromab) in combination with wheat germ agglutinin (WGA, plasma membrane marker) and calnexin (ER marker; anti-calnexin 1/100; BD Transduction Laboratories) as described .

    Techniques: Expressing, Negative Control, Western Blot, Immunoprecipitation, Isolation

    LPS-dependent activation increases the Kv1.3/KCNE4 ratio in CY15 dendritic cells. Cells were treated for 24 h with LPS (100 ng/ml), and the protein expression of selected K + channel proteins was studied at 0, 6 and 24 h. ( A ) Confocal images of CY15 cells stained with Kv1.3 upon LPS treatment. Scale bars: 20 µm. ( B ) Representative voltage-dependent K+ currents elicited in CY15 cells treated with (LPS 24 h) or without (LPS 0 h) LPS during 24 h. Cells were held at -60 mV and 250 ms pulses to +60 mV were applied. ( C ) Peak current densitiy (pA/pF) of K + currents from CY15 cells in the absence (0 h) or the presence (24 h) of LPS. White bars, LPS 0 h (control); black bars, LPS 24 h. Values are the mean ± SE of 4–6 cells. *p

    Journal: Scientific Reports

    Article Title: KCNE4-dependent functional consequences of Kv1.3-related leukocyte physiology

    doi: 10.1038/s41598-021-94015-9

    Figure Lengend Snippet: LPS-dependent activation increases the Kv1.3/KCNE4 ratio in CY15 dendritic cells. Cells were treated for 24 h with LPS (100 ng/ml), and the protein expression of selected K + channel proteins was studied at 0, 6 and 24 h. ( A ) Confocal images of CY15 cells stained with Kv1.3 upon LPS treatment. Scale bars: 20 µm. ( B ) Representative voltage-dependent K+ currents elicited in CY15 cells treated with (LPS 24 h) or without (LPS 0 h) LPS during 24 h. Cells were held at -60 mV and 250 ms pulses to +60 mV were applied. ( C ) Peak current densitiy (pA/pF) of K + currents from CY15 cells in the absence (0 h) or the presence (24 h) of LPS. White bars, LPS 0 h (control); black bars, LPS 24 h. Values are the mean ± SE of 4–6 cells. *p

    Article Snippet: In some experiments, T cells were stained against Kv1.3 (anti-Kv1.3 1/200, Neuromab) in combination with wheat germ agglutinin (WGA, plasma membrane marker) and calnexin (ER marker; anti-calnexin 1/100; BD Transduction Laboratories) as described .

    Techniques: Activation Assay, Expressing, Staining