rabbit anti kv1 5 antibody  (Alomone Labs)


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    Structured Review

    Alomone Labs rabbit anti kv1 5 antibody
    Mutant <t>KCNA5</t> is located in perinuclear packets and not on the cell surface of transfected HEK-293 cells and human (h)PASMC. A : HEK-293 cells transfected with WT KCNA5 ( a and c ) or G182R ( b and d ) were stained with anti-KCNA5 antibody (Ab-KCNA5, red) and
    Rabbit Anti Kv1 5 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit anti kv1 5 antibody - by Bioz Stars, 2022-05
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    Images

    1) Product Images from "Tetramerization domain mutations in KCNA5 affect channel kinetics and cause abnormal trafficking patterns"

    Article Title: Tetramerization domain mutations in KCNA5 affect channel kinetics and cause abnormal trafficking patterns

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00464.2009

    Mutant KCNA5 is located in perinuclear packets and not on the cell surface of transfected HEK-293 cells and human (h)PASMC. A : HEK-293 cells transfected with WT KCNA5 ( a and c ) or G182R ( b and d ) were stained with anti-KCNA5 antibody (Ab-KCNA5, red) and
    Figure Legend Snippet: Mutant KCNA5 is located in perinuclear packets and not on the cell surface of transfected HEK-293 cells and human (h)PASMC. A : HEK-293 cells transfected with WT KCNA5 ( a and c ) or G182R ( b and d ) were stained with anti-KCNA5 antibody (Ab-KCNA5, red) and

    Techniques Used: Mutagenesis, Transfection, Staining

    G182R and E211D mutations cause incomplete processing of KCNA5 in HEK-293 cells. HEK-293 cells were transfected with WT KCNA5, G182R, E211D, or G182R/E211D and subjected to standard immunoblot procedures. A : representative immunoblot from cells transfected
    Figure Legend Snippet: G182R and E211D mutations cause incomplete processing of KCNA5 in HEK-293 cells. HEK-293 cells were transfected with WT KCNA5, G182R, E211D, or G182R/E211D and subjected to standard immunoblot procedures. A : representative immunoblot from cells transfected

    Techniques Used: Transfection

    Mutant KCNA5 forms functional homotetrameric channels. A : COS-1, HEK-293, and human pulmonary artery smooth muscle cells (PASMC) were transiently transfected with either empty vector [green fluorescent protein (GFP)] or WT KCNA5 (WT) as indicated. Whole
    Figure Legend Snippet: Mutant KCNA5 forms functional homotetrameric channels. A : COS-1, HEK-293, and human pulmonary artery smooth muscle cells (PASMC) were transiently transfected with either empty vector [green fluorescent protein (GFP)] or WT KCNA5 (WT) as indicated. Whole

    Techniques Used: Mutagenesis, Functional Assay, Transfection, Plasmid Preparation

    Mutations in KCNA5 at G182 and E211 do not affect the pharmacological effect of 4-aminopyridine (4-AP). A : a standard I-V pulse protocol was delivered to HEK-293 cells transiently transfected with the indicated vector [WT KCNA5 ( a ), G182R ( b ), E211D (
    Figure Legend Snippet: Mutations in KCNA5 at G182 and E211 do not affect the pharmacological effect of 4-aminopyridine (4-AP). A : a standard I-V pulse protocol was delivered to HEK-293 cells transiently transfected with the indicated vector [WT KCNA5 ( a ), G182R ( b ), E211D (

    Techniques Used: Transfection, Plasmid Preparation

    Decreased G182R expression in COS-1 cells cannot be rescued by overexpression of K V β subunits. A , a : HEK-293 cells were transfected with WT KCNA5 or K V β1.3-HA alone or cotransfected with WT KCNA5, G182R, E211D, or G182R/E211D and K V β1.3-HA.
    Figure Legend Snippet: Decreased G182R expression in COS-1 cells cannot be rescued by overexpression of K V β subunits. A , a : HEK-293 cells were transfected with WT KCNA5 or K V β1.3-HA alone or cotransfected with WT KCNA5, G182R, E211D, or G182R/E211D and K V β1.3-HA.

    Techniques Used: Expressing, Over Expression, Transfection

    Two nonsynonymous mutations identified in the KCNA5 gene from idiopathic pulmonary arterial hypertension (IPAH) patients localize to the NH 2 -terminal tetramerization domain (T1 domain). A , left : schematic diagram of voltage-gated K + (K V ) channel subunit
    Figure Legend Snippet: Two nonsynonymous mutations identified in the KCNA5 gene from idiopathic pulmonary arterial hypertension (IPAH) patients localize to the NH 2 -terminal tetramerization domain (T1 domain). A , left : schematic diagram of voltage-gated K + (K V ) channel subunit

    Techniques Used:

    Cotransfection of K V β subunits affects KCNA5 channel kinetics. HEK-293 cells were transfected with WT KCNA5 alone (KCNA5) or in the presence of K V β1.3-hemagglutinin (HA) (KCNA5/K V β1.3). A and B : representative current recordings
    Figure Legend Snippet: Cotransfection of K V β subunits affects KCNA5 channel kinetics. HEK-293 cells were transfected with WT KCNA5 alone (KCNA5) or in the presence of K V β1.3-hemagglutinin (HA) (KCNA5/K V β1.3). A and B : representative current recordings

    Techniques Used: Cotransfection, Transfection

    Voltage-dependent inactivation is accelerated in the G182R mutant KCNA5 channel. A standard 2-pulse inactivation protocol was used to determine channel availability after a 10-s prepulse in HEK-293 cells transiently transfected with WT KCNA5, G182R, E211D,
    Figure Legend Snippet: Voltage-dependent inactivation is accelerated in the G182R mutant KCNA5 channel. A standard 2-pulse inactivation protocol was used to determine channel availability after a 10-s prepulse in HEK-293 cells transiently transfected with WT KCNA5, G182R, E211D,

    Techniques Used: Mutagenesis, Transfection

    G182R protein expression is significantly decreased in COS-1 cells. A : COS-1 cells were transiently transfected with water (Mock), WT-KCNA5, G182R, E211D, or G182R/E211D. Representative images are shown at ×40 magnification. B : transfected cells
    Figure Legend Snippet: G182R protein expression is significantly decreased in COS-1 cells. A : COS-1 cells were transiently transfected with water (Mock), WT-KCNA5, G182R, E211D, or G182R/E211D. Representative images are shown at ×40 magnification. B : transfected cells

    Techniques Used: Expressing, Transfection

    2) Product Images from "Molecular Determinants of Kv1.3 Potassium Channels-induced Proliferation *"

    Article Title: Molecular Determinants of Kv1.3 Potassium Channels-induced Proliferation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.678995

    Characterization of the Kv1.3 and Kv1.5 mutant channels containing the YS segment. A , average normalized activation and inactivation curves are shown as conductance-voltage relationships for Kv1.3, Kv1.5, the truncated Kv1.3-YS channel, and the chimeras Kv1.5-YS 532 and Kv1.5-YS 613 . All datasets were fitted to Boltzmann functions. Each data point is the mean ± S.E. of 6–11 cells. B , confocal images of non-permeabilized cells transfected with Kv1.3-YS-Cherry, Kv1.5-YS 532 -EGFP, and Kv1.5-YS 613 -EGFP. An extracellular anti-Kv1.3 antibody was used to label Kv1.3-YS ( green ), whereas the extracellular anti-Kv1.5 antibody was used for Kv1.5-YS 532 and Kv1.5-YS 613 chimeras ( red ). Nuclei were stained by Hoechst ( blue ). C , proliferation rate of the indicated channels or GFP-transfected cells (control) was determined by measuring EdU incorporation. Significant differences when comparing to Kv1.3 (*) or to control (#) are indicated. Statistical analysis was performed with one-way ANOVA followed by a Tukey's HSD multiple comparison. Each bar is the average of 9–15 determinations from 5 different assays. D , the average peak current amplitude obtained in cell-attached experiments for Kv1.5 channels and all the Kv1.5 chimeras was plotted against the % of the channels expressed at the plasma membrane ( upper graph ) or their normalized effect on proliferation (taking 100% as the proliferation rate of GFP-transfected HEK cells, lower graph ). The correlation between expression and current was fit to a linear regression curve ( y = 18.54 + 0.0066x, R 2 = 0.85, p = 0.008), but there was no correlation between proliferation and current amplitude ( R 2 = 0.23, p = 0.19).
    Figure Legend Snippet: Characterization of the Kv1.3 and Kv1.5 mutant channels containing the YS segment. A , average normalized activation and inactivation curves are shown as conductance-voltage relationships for Kv1.3, Kv1.5, the truncated Kv1.3-YS channel, and the chimeras Kv1.5-YS 532 and Kv1.5-YS 613 . All datasets were fitted to Boltzmann functions. Each data point is the mean ± S.E. of 6–11 cells. B , confocal images of non-permeabilized cells transfected with Kv1.3-YS-Cherry, Kv1.5-YS 532 -EGFP, and Kv1.5-YS 613 -EGFP. An extracellular anti-Kv1.3 antibody was used to label Kv1.3-YS ( green ), whereas the extracellular anti-Kv1.5 antibody was used for Kv1.5-YS 532 and Kv1.5-YS 613 chimeras ( red ). Nuclei were stained by Hoechst ( blue ). C , proliferation rate of the indicated channels or GFP-transfected cells (control) was determined by measuring EdU incorporation. Significant differences when comparing to Kv1.3 (*) or to control (#) are indicated. Statistical analysis was performed with one-way ANOVA followed by a Tukey's HSD multiple comparison. Each bar is the average of 9–15 determinations from 5 different assays. D , the average peak current amplitude obtained in cell-attached experiments for Kv1.5 channels and all the Kv1.5 chimeras was plotted against the % of the channels expressed at the plasma membrane ( upper graph ) or their normalized effect on proliferation (taking 100% as the proliferation rate of GFP-transfected HEK cells, lower graph ). The correlation between expression and current was fit to a linear regression curve ( y = 18.54 + 0.0066x, R 2 = 0.85, p = 0.008), but there was no correlation between proliferation and current amplitude ( R 2 = 0.23, p = 0.19).

    Techniques Used: Mutagenesis, Activation Assay, Transfection, Staining, Expressing

    3) Product Images from "PKC and AMPK regulation of Kv1.5 potassium channels"

    Article Title: PKC and AMPK regulation of Kv1.5 potassium channels

    Journal: Channels

    doi: 10.1080/19336950.2015.1036205

    PKC but not AMPK activation reduces Kv1.5 surface expression in HL-1 cells. ( A and B ) Confocal scans of HL-1 cells transiently transfected with Kv1.5 cDNA and treated with PMA (100 nM), AICAR (1 mM) or PT1 (200 μM). As illustrated, surface expression of Kv1.5 channels was reduced in response to PKC activation ( A ). This response was not observed with either of the 2 AMPK activators ( B ). Cytoskeletal actin staining was used as a marker for the membrane. Scalebar, 10 μm. ( C ) Representative traces of Kv1.5 currents recorded in HL-1 cells before and after treatment with PMA. Protocol shown in the insert. ( D ) Current-voltage relationship of currents recorded in Kv1.5 transfected and untransfected HL-1 cells. PMA significantly down regulated the current level by 74%. Numbers of cells are: Kv1.5 (n=12); Kv1.5+PMC (n=13); untransfected (n=8); untransfected+PMA (n=10).
    Figure Legend Snippet: PKC but not AMPK activation reduces Kv1.5 surface expression in HL-1 cells. ( A and B ) Confocal scans of HL-1 cells transiently transfected with Kv1.5 cDNA and treated with PMA (100 nM), AICAR (1 mM) or PT1 (200 μM). As illustrated, surface expression of Kv1.5 channels was reduced in response to PKC activation ( A ). This response was not observed with either of the 2 AMPK activators ( B ). Cytoskeletal actin staining was used as a marker for the membrane. Scalebar, 10 μm. ( C ) Representative traces of Kv1.5 currents recorded in HL-1 cells before and after treatment with PMA. Protocol shown in the insert. ( D ) Current-voltage relationship of currents recorded in Kv1.5 transfected and untransfected HL-1 cells. PMA significantly down regulated the current level by 74%. Numbers of cells are: Kv1.5 (n=12); Kv1.5+PMC (n=13); untransfected (n=8); untransfected+PMA (n=10).

    Techniques Used: Activation Assay, Expressing, Transfection, Staining, Marker

    Two-electrode voltage clamp recordings of Xenopus laevis oocytes expressing Kv1.5 +/− Nedd4–2 following PKC and AMPK activation. ( A-D ) Representative Kv1.5 current recordings following a step protocol. ( E ) PKC activation by PMA resulted in a drastic Kv1.5 current reduction both in the presence and absence of co-expressed Nedd4–2. ( E ) Similar experiments were conducted using the AMPK activator ZMP (100 nM). Contrary to PKC activation, AMPK activation did not affect Kv1.5 current levels when Nedd4–2 was not co-expressed. However, when Nedd4–2 was co-injected, leading to a down-regulation of Kv1.5 surface current, ZMP induced a further current reduction. The number of cells in each group was (n > 10).
    Figure Legend Snippet: Two-electrode voltage clamp recordings of Xenopus laevis oocytes expressing Kv1.5 +/− Nedd4–2 following PKC and AMPK activation. ( A-D ) Representative Kv1.5 current recordings following a step protocol. ( E ) PKC activation by PMA resulted in a drastic Kv1.5 current reduction both in the presence and absence of co-expressed Nedd4–2. ( E ) Similar experiments were conducted using the AMPK activator ZMP (100 nM). Contrary to PKC activation, AMPK activation did not affect Kv1.5 current levels when Nedd4–2 was not co-expressed. However, when Nedd4–2 was co-injected, leading to a down-regulation of Kv1.5 surface current, ZMP induced a further current reduction. The number of cells in each group was (n > 10).

    Techniques Used: Expressing, Activation Assay, Injection

    Schematic illustration of the PKC and AMPK pathways resulting in Kv1.5 downregulation. The polarization of MDCK cells triggers activation of PKC and AMPK kinases. PKC activation can be mimicked by PMA. We suggest this activation of PKC can impact Kv1.5 channels through 2 pathways, depending on which the cell system used. This might be through AMPK activation (that can be mimicked by PT1, AICAR and ZMP), which will activate Nedd4 ubiquitylating enzymes or by another so far undisclosed mechanism. Activation of both pathways results in a reduced Kv1.5 surface expression.
    Figure Legend Snippet: Schematic illustration of the PKC and AMPK pathways resulting in Kv1.5 downregulation. The polarization of MDCK cells triggers activation of PKC and AMPK kinases. PKC activation can be mimicked by PMA. We suggest this activation of PKC can impact Kv1.5 channels through 2 pathways, depending on which the cell system used. This might be through AMPK activation (that can be mimicked by PT1, AICAR and ZMP), which will activate Nedd4 ubiquitylating enzymes or by another so far undisclosed mechanism. Activation of both pathways results in a reduced Kv1.5 surface expression.

    Techniques Used: Activation Assay, Expressing

    Kv1.5 surface expression is reduced in response to cell polarization, to PKC activation and to AMPK activation in MDCK cells. ( A ) MDCK cells transiently co-transfected with DsRed-ER and Kv1.5 cDNA and subjected to a calcium switch assay (see Materials and Methods). At different time points after initiation of the calcium switch, coverslips were fixed and stained for Kv1.5. Illustrated are confocal horizontal scans of the MDCK cells before the initiation of the calcium switch (t =0h, cells grown in low calcium medium) and 30 min and 3h after the initiation of the polarization process following addition of calcium containing medium. As illustrated, surface expressed Kv1.5 channels disappear from the membrane during the 3 hour calcium switch. ( B and C ) MDCK cells transiently co-transfected with DsRed-ER and Kv1.5 and grown in low calcium media. The cells were treated with the PKC activator PMA (100 nM) ( B ) or the 2 AMPK activators AICAR (500 μM) and PT1 (200 μM) ( C ) for up to 6 hours. As illustrated, both activation of PKC and AMPK lead to disappearance of surface expressed Kv1.5 channels. Cytoskeletal actin staining was used as a membrane marker. Scalebar,10 μm.
    Figure Legend Snippet: Kv1.5 surface expression is reduced in response to cell polarization, to PKC activation and to AMPK activation in MDCK cells. ( A ) MDCK cells transiently co-transfected with DsRed-ER and Kv1.5 cDNA and subjected to a calcium switch assay (see Materials and Methods). At different time points after initiation of the calcium switch, coverslips were fixed and stained for Kv1.5. Illustrated are confocal horizontal scans of the MDCK cells before the initiation of the calcium switch (t =0h, cells grown in low calcium medium) and 30 min and 3h after the initiation of the polarization process following addition of calcium containing medium. As illustrated, surface expressed Kv1.5 channels disappear from the membrane during the 3 hour calcium switch. ( B and C ) MDCK cells transiently co-transfected with DsRed-ER and Kv1.5 and grown in low calcium media. The cells were treated with the PKC activator PMA (100 nM) ( B ) or the 2 AMPK activators AICAR (500 μM) and PT1 (200 μM) ( C ) for up to 6 hours. As illustrated, both activation of PKC and AMPK lead to disappearance of surface expressed Kv1.5 channels. Cytoskeletal actin staining was used as a membrane marker. Scalebar,10 μm.

    Techniques Used: Expressing, Activation Assay, Transfection, Staining, Marker

    4) Product Images from "Increased Expression of MicroRNA‐206 Inhibits Potassium Voltage‐Gated Channel Subfamily A Member 5 in Pulmonary Arterial Smooth Muscle Cells and Is Related to Exaggerated Pulmonary Artery Hypertension Following Intrauterine Growth Retardation in Rats"

    Article Title: Increased Expression of MicroRNA‐206 Inhibits Potassium Voltage‐Gated Channel Subfamily A Member 5 in Pulmonary Arterial Smooth Muscle Cells and Is Related to Exaggerated Pulmonary Artery Hypertension Following Intrauterine Growth Retardation in Rats

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    doi: 10.1161/JAHA.118.010456

    miR‐206 inhibition in vivo increased Kv1.5 channel expression to restore CH ‐ PAH of IUGR rats. A , Fold‐change of miR‐206 expression by qRT ‐ PCR in PA smooth muscle and mesenteric artery smooth muscle of different groups. n=5 per group of CON , IUGR , CON ‐ CH and IUGR ‐ CH ; n=8 per group of CON ‐ CH ‐Anti206, IUGR ‐ CH ‐Anti206, CON ‐Anti206, and IUGR ‐Anti206;* P
    Figure Legend Snippet: miR‐206 inhibition in vivo increased Kv1.5 channel expression to restore CH ‐ PAH of IUGR rats. A , Fold‐change of miR‐206 expression by qRT ‐ PCR in PA smooth muscle and mesenteric artery smooth muscle of different groups. n=5 per group of CON , IUGR , CON ‐ CH and IUGR ‐ CH ; n=8 per group of CON ‐ CH ‐Anti206, IUGR ‐ CH ‐Anti206, CON ‐Anti206, and IUGR ‐Anti206;* P

    Techniques Used: Inhibition, In Vivo, Expressing, Quantitative RT-PCR

    miR‐206 inhibition in primary cultured PASMC s regulates Kv1.5 channel expression and prevents overproliferation of PASMC s from IUGR ‐ CH ‐ PAH rats. A , Fold‐change of miR‐206 in PASMC s of different groups. B , Fold‐change of mRNA of KCNA 5 in PASMC s of different groups. C and D , Representative images of immunoblotting and quantitative analysis of Kv1.5 α‐protein in PASMC s. Data in ( A , B , D ) are presented as means± SEM . * P
    Figure Legend Snippet: miR‐206 inhibition in primary cultured PASMC s regulates Kv1.5 channel expression and prevents overproliferation of PASMC s from IUGR ‐ CH ‐ PAH rats. A , Fold‐change of miR‐206 in PASMC s of different groups. B , Fold‐change of mRNA of KCNA 5 in PASMC s of different groups. C and D , Representative images of immunoblotting and quantitative analysis of Kv1.5 α‐protein in PASMC s. Data in ( A , B , D ) are presented as means± SEM . * P

    Techniques Used: Inhibition, Cell Culture, Expressing

    5) Product Images from "K+ Channel Kv3.4 Is Essential for Axon Growth by Limiting the Influx of Ca2+ into Growth Cones"

    Article Title: K+ Channel Kv3.4 Is Essential for Axon Growth by Limiting the Influx of Ca2+ into Growth Cones

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.1076-16.2017

    Kv3.4 in the axonal growth cones of dorsal spinal commissural neurons. A–F , Transverse sections of the spinal cord of chick embryos were immunostained for Kv3.4. A , Absence of Kv3.4-IR in the dorsal spinal cord at HH17. Kv3.4-IR in precrossing commissural axons ( B–F , arrowheads) is evident during HH19-HH25 but disappears at HH27. D , Arrows indicate postcrossing commissural axons projecting from the other side of spinal cord. FP, Floor plate. G–L , Transverse sections of the spinal cord at HH23 were immunostained as indicated. G , Absence of Kv1.5-IR. H , Kv4.2-IR in the somata and dendrites of motoneurons (MN). I , Kv4.3-IR in the bifurcation zone (BZ). In addition to the BZ, Kv3.1b-IR is strong in postcrossing commissural axons ( J , arrow) but weak in precrossing commissural axons ( J , arrowhead). K , Absence of Kv3.2-IR. L , Kv3.3 in motoneurons. M–M″ , Double staining in transverse sections of the spinal cord at HH21 shows colocalization of Kv3.4 and axonin-1 in the growth cones (arrowheads) of commissural axons. N–N″ , Colocalization of Kv3.4 and axonin-1 in cultured dorsal spinal neurons isolated from HH21-HH23 chick embryos. O–P″ , Red fluorescence-tagged phalloidin colabeling reveals enrichment of Kv3.4 in the growth cone ( O–O″ ) and Kv3.1b in the soma/axon shaft ( P–P″ ) of cultured dorsal spinal neurons. Q–Q″ , Kv3.4 and DiI colabeling. White represents Kv3.4-abundant regions. Blue represents Kv3.4-sparse regions ( Q″ ). R , Ratio of Kv3.4/DiI in the soma, axon shaft, or growth cone of each neuron was obtained by dividing the fluorescence intensity of Kv3.4 by that of DiI. Data are mean ± SEM ( n = 8 neurons, pooled from three independent experiments done on different days). *** p
    Figure Legend Snippet: Kv3.4 in the axonal growth cones of dorsal spinal commissural neurons. A–F , Transverse sections of the spinal cord of chick embryos were immunostained for Kv3.4. A , Absence of Kv3.4-IR in the dorsal spinal cord at HH17. Kv3.4-IR in precrossing commissural axons ( B–F , arrowheads) is evident during HH19-HH25 but disappears at HH27. D , Arrows indicate postcrossing commissural axons projecting from the other side of spinal cord. FP, Floor plate. G–L , Transverse sections of the spinal cord at HH23 were immunostained as indicated. G , Absence of Kv1.5-IR. H , Kv4.2-IR in the somata and dendrites of motoneurons (MN). I , Kv4.3-IR in the bifurcation zone (BZ). In addition to the BZ, Kv3.1b-IR is strong in postcrossing commissural axons ( J , arrow) but weak in precrossing commissural axons ( J , arrowhead). K , Absence of Kv3.2-IR. L , Kv3.3 in motoneurons. M–M″ , Double staining in transverse sections of the spinal cord at HH21 shows colocalization of Kv3.4 and axonin-1 in the growth cones (arrowheads) of commissural axons. N–N″ , Colocalization of Kv3.4 and axonin-1 in cultured dorsal spinal neurons isolated from HH21-HH23 chick embryos. O–P″ , Red fluorescence-tagged phalloidin colabeling reveals enrichment of Kv3.4 in the growth cone ( O–O″ ) and Kv3.1b in the soma/axon shaft ( P–P″ ) of cultured dorsal spinal neurons. Q–Q″ , Kv3.4 and DiI colabeling. White represents Kv3.4-abundant regions. Blue represents Kv3.4-sparse regions ( Q″ ). R , Ratio of Kv3.4/DiI in the soma, axon shaft, or growth cone of each neuron was obtained by dividing the fluorescence intensity of Kv3.4 by that of DiI. Data are mean ± SEM ( n = 8 neurons, pooled from three independent experiments done on different days). *** p

    Techniques Used: Double Staining, Cell Culture, Isolation, Fluorescence

    6) Product Images from "Membrane cholesterol modulates Kv1.5 potassium channel distribution and function in rat cardiomyocytes"

    Article Title: Membrane cholesterol modulates Kv1.5 potassium channel distribution and function in rat cardiomyocytes

    Journal: The Journal of Physiology

    doi: 10.1113/jphysiol.2007.134809

    Some Kv1.5 channel subunits are localized in lipid raft fractions in adult rat atrial myocytes Western blot analysis of the distribution of Kv1.5 subunits, connexin-43 and caveolin-3 on step sucrose gradient of proteins from atrial myocardium.
    Figure Legend Snippet: Some Kv1.5 channel subunits are localized in lipid raft fractions in adult rat atrial myocytes Western blot analysis of the distribution of Kv1.5 subunits, connexin-43 and caveolin-3 on step sucrose gradient of proteins from atrial myocardium.

    Techniques Used: Western Blot

    Kv1.5 do not co-localized with caveolin-3 in adult atrial tissue Immunolocalization of Kv1.5 subunits ( A ) and caveolin-3 ( B ) in cryosections of atrial myocardium. Double immunostaining of connexin-43 (FITC, C ) and caveolin-3 (Texas Red, D ) in cryosections of atrial myocardium. E , merged image of the same area of the section in C and D , showing the lack of overlap between connexin-43 and caveolin-3 stainings.
    Figure Legend Snippet: Kv1.5 do not co-localized with caveolin-3 in adult atrial tissue Immunolocalization of Kv1.5 subunits ( A ) and caveolin-3 ( B ) in cryosections of atrial myocardium. Double immunostaining of connexin-43 (FITC, C ) and caveolin-3 (Texas Red, D ) in cryosections of atrial myocardium. E , merged image of the same area of the section in C and D , showing the lack of overlap between connexin-43 and caveolin-3 stainings.

    Techniques Used: Double Immunostaining

    Surface expression of Kv1.5 subunits in neonatal cardiomyocytes A , in live cardiomyocytes, transfected GFP-tagged Kv1.5 subunits are clustered at the membrane surface adjacent to the bottom of laminin-coated glass support, as shown in the projection of Z sections in the lower panel. In contrast, GFP alone was homogeneously distributed in cardiomyocytes (inset). B , after the application of 2% MCD, clusters increased in size and were redistributed throughout the plasma membrane. C , bar graphs summarizing changes in cluster size upon MCD exposures; data are from 21 cardiomyocytes in control, and following incubation with 2% MCD for 7 min and 1 h 30 min. ** P
    Figure Legend Snippet: Surface expression of Kv1.5 subunits in neonatal cardiomyocytes A , in live cardiomyocytes, transfected GFP-tagged Kv1.5 subunits are clustered at the membrane surface adjacent to the bottom of laminin-coated glass support, as shown in the projection of Z sections in the lower panel. In contrast, GFP alone was homogeneously distributed in cardiomyocytes (inset). B , after the application of 2% MCD, clusters increased in size and were redistributed throughout the plasma membrane. C , bar graphs summarizing changes in cluster size upon MCD exposures; data are from 21 cardiomyocytes in control, and following incubation with 2% MCD for 7 min and 1 h 30 min. ** P

    Techniques Used: Expressing, Transfection, Incubation

    Effect of cholesterol depletion on outward current parameters resulting from Kv1.5 subunit overexpression in neonatal cardiomyocytes Current density–voltage relationships ( A ) and voltage dependence ( B ) of I Kur activation under control conditions (•) and following 7 min MCD application (^). In A and B , each point represents average data from 5 cells.
    Figure Legend Snippet: Effect of cholesterol depletion on outward current parameters resulting from Kv1.5 subunit overexpression in neonatal cardiomyocytes Current density–voltage relationships ( A ) and voltage dependence ( B ) of I Kur activation under control conditions (•) and following 7 min MCD application (^). In A and B , each point represents average data from 5 cells.

    Techniques Used: Over Expression, Activation Assay

    7) Product Images from "Mechanical stretch increases Kv1.5 current through an interaction between the S1–S2 linker and N-terminus of the channel"

    Article Title: Mechanical stretch increases Kv1.5 current through an interaction between the S1–S2 linker and N-terminus of the channel

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA119.011302

    The Kv1.5 S1–S2 linker communicates with the N terminus in a conformational manner. Truncation of N terminus altered the susceptibility of the S1–S2 linker to PK cleavage. WT Kv1.5 displays 75-kDa and 68-kDa bands on Western blot analysis. The 75-kDa band represents the mature, fully glycosylated channel protein in the plasma membrane, whereas the 68-kDa band represents the immature channel protein inside the cell. ΔN209 Kv1.5 also presents as two bands; the 50-kDa band represents mature protein in the plasma membrane, the 43-kDa band represents immature protein inside the cell. Although PK completely cleaved the mature (cell surface) channel proteins of WT Kv1.5, it failed to cleave the mature (cell surface) channel proteins of ΔN209 Kv1.5 ( n = 6).
    Figure Legend Snippet: The Kv1.5 S1–S2 linker communicates with the N terminus in a conformational manner. Truncation of N terminus altered the susceptibility of the S1–S2 linker to PK cleavage. WT Kv1.5 displays 75-kDa and 68-kDa bands on Western blot analysis. The 75-kDa band represents the mature, fully glycosylated channel protein in the plasma membrane, whereas the 68-kDa band represents the immature channel protein inside the cell. ΔN209 Kv1.5 also presents as two bands; the 50-kDa band represents mature protein in the plasma membrane, the 43-kDa band represents immature protein inside the cell. Although PK completely cleaved the mature (cell surface) channel proteins of WT Kv1.5, it failed to cleave the mature (cell surface) channel proteins of ΔN209 Kv1.5 ( n = 6).

    Techniques Used: Western Blot

    Centrifugation increases I Kv1.5 . Kv1.5-HEK cells were centrifuged ( CENTR ) at 70 × g for 5 min. Cells were then re-suspended in normal culture medium for 20 min prior to I Kv1.5 recordings. Kv1.5-HEK cells without centrifugation were used as control (CTL). Representative current traces along with the voltage protocol ( top ) and summarized I-V relationship ( bottom ) are shown. CTL, n = 29; CENTR, n = 38; **, p
    Figure Legend Snippet: Centrifugation increases I Kv1.5 . Kv1.5-HEK cells were centrifuged ( CENTR ) at 70 × g for 5 min. Cells were then re-suspended in normal culture medium for 20 min prior to I Kv1.5 recordings. Kv1.5-HEK cells without centrifugation were used as control (CTL). Representative current traces along with the voltage protocol ( top ) and summarized I-V relationship ( bottom ) are shown. CTL, n = 29; CENTR, n = 38; **, p

    Techniques Used: Centrifugation

    The unique S1–S2 linker of Kv1.5 is involved in LO-mediated increase in I Kv1.5 . A , amino acid sequences of the S1–S2 linker of various Kv channels. Kv1.5 possesses an unusually long S1–S2 linker with 12 nonconserved proline residues (in magenta ). The N -linked glycosylation site is shown in red. B, PK cleavage of the S1–S2 linker abolished LO-induced increase in I Kv1.5 . Schematic illustration of Kv1.5 PK cleavage ( top ) and representative current traces ( middle ) are depicted above the summarized I-V relationships ( bottom ). CTL, n = 14; LO, n = 21. C, mutating all 12 nonconserved prolines (P) to alanines (A) in the S1–S2 linker abolished LO-induced increase in I Kv1.5 . Schematic illustration of the Kv1.5–12PA mutant ( top ) as well as representative current traces ( middle ) are depicted above the summarized I-V relationships ( bottom ). CTL, n = 37; LO, n = 36. D , deletion of amino acid residues 282–300 in the S1–S2 linker abolished LO-induced increase in I Kv1.5 . Schematic illustration of Kv1.5-Δ282–300 mutant ( top ) as well as representative current traces ( middle ) are depicted above the summarized I-V relationships ( bottom ). CTL, n = 15, LO, n = 10. E , inhibition of glycosylation in the S1–S2 linker with tunicamycin ( Tuni ) treatment abolished the LO-induced increase in I Kv1.5 . Schematic illustration of WT Kv1.5 without glycosylation ( top ) as well as representative current traces ( middle ) are depicted above the summarized I-V relationships ( bottom ). CTL, n = 24; LO, n = 24.
    Figure Legend Snippet: The unique S1–S2 linker of Kv1.5 is involved in LO-mediated increase in I Kv1.5 . A , amino acid sequences of the S1–S2 linker of various Kv channels. Kv1.5 possesses an unusually long S1–S2 linker with 12 nonconserved proline residues (in magenta ). The N -linked glycosylation site is shown in red. B, PK cleavage of the S1–S2 linker abolished LO-induced increase in I Kv1.5 . Schematic illustration of Kv1.5 PK cleavage ( top ) and representative current traces ( middle ) are depicted above the summarized I-V relationships ( bottom ). CTL, n = 14; LO, n = 21. C, mutating all 12 nonconserved prolines (P) to alanines (A) in the S1–S2 linker abolished LO-induced increase in I Kv1.5 . Schematic illustration of the Kv1.5–12PA mutant ( top ) as well as representative current traces ( middle ) are depicted above the summarized I-V relationships ( bottom ). CTL, n = 37; LO, n = 36. D , deletion of amino acid residues 282–300 in the S1–S2 linker abolished LO-induced increase in I Kv1.5 . Schematic illustration of Kv1.5-Δ282–300 mutant ( top ) as well as representative current traces ( middle ) are depicted above the summarized I-V relationships ( bottom ). CTL, n = 15, LO, n = 10. E , inhibition of glycosylation in the S1–S2 linker with tunicamycin ( Tuni ) treatment abolished the LO-induced increase in I Kv1.5 . Schematic illustration of WT Kv1.5 without glycosylation ( top ) as well as representative current traces ( middle ) are depicted above the summarized I-V relationships ( bottom ). CTL, n = 24; LO, n = 24.

    Techniques Used: Mutagenesis, Inhibition

    LO medium treatment increases I Kv1.5 in neonatal rat ventricular myocytes transfected with Kv1.5. Representative current traces are depicted above the summarized I-V relationship. CTL, n = 32; LO, n = 32. *, p
    Figure Legend Snippet: LO medium treatment increases I Kv1.5 in neonatal rat ventricular myocytes transfected with Kv1.5. Representative current traces are depicted above the summarized I-V relationship. CTL, n = 32; LO, n = 32. *, p

    Techniques Used: Transfection

    LO medium treatment increases cell size and reversibly increases I Kv1.5 . A, culture of Kv1.5-HEK cells with LO medium for 30 min increased cell size ( n = 13; **, p
    Figure Legend Snippet: LO medium treatment increases cell size and reversibly increases I Kv1.5 . A, culture of Kv1.5-HEK cells with LO medium for 30 min increased cell size ( n = 13; **, p

    Techniques Used:

    Removal of Src-binding sites abolishes LO-mediated increase in I Kv1.5 . A, amino acid sequences showing the two consensus SH3–binding motifs (RPLPPLP, shown in blue ) in the N terminus of Kv1.5 as well as the mutant Kv1.5-ΔPro, in which amino acids 64–82 were removed. B , effects of LO treatment on WT I Kv1.5 . CTL, n = 36; LO, n = 27; **, p
    Figure Legend Snippet: Removal of Src-binding sites abolishes LO-mediated increase in I Kv1.5 . A, amino acid sequences showing the two consensus SH3–binding motifs (RPLPPLP, shown in blue ) in the N terminus of Kv1.5 as well as the mutant Kv1.5-ΔPro, in which amino acids 64–82 were removed. B , effects of LO treatment on WT I Kv1.5 . CTL, n = 36; LO, n = 27; **, p

    Techniques Used: Binding Assay, Mutagenesis

    Effects of LO-treatment and Src inhibitor PP1 on Kv1.5 expression and function. A , LO treatment did not affect the total amount of Kv1.5 proteins but increased the cell surface mature channel expression. The density of the 75-kDa band in LO-treated cells was normalized to that of control cells in the same gel and shown in the scatter plots ( n = 5). B , PP1 treatment did not affect the total amount of Kv1.5 proteins but increased the cell surface mature channel expression. The density of the 75-kDa band in PP1-treated cells was normalized to that of control cells in the same gel and shown in the scatter plot ( n = 5). For A and B , boxes represent interquartile ranges, horizontal lines represent medians, whiskers represent 5–95% ranges, and gray boxes represent means. **, p
    Figure Legend Snippet: Effects of LO-treatment and Src inhibitor PP1 on Kv1.5 expression and function. A , LO treatment did not affect the total amount of Kv1.5 proteins but increased the cell surface mature channel expression. The density of the 75-kDa band in LO-treated cells was normalized to that of control cells in the same gel and shown in the scatter plots ( n = 5). B , PP1 treatment did not affect the total amount of Kv1.5 proteins but increased the cell surface mature channel expression. The density of the 75-kDa band in PP1-treated cells was normalized to that of control cells in the same gel and shown in the scatter plot ( n = 5). For A and B , boxes represent interquartile ranges, horizontal lines represent medians, whiskers represent 5–95% ranges, and gray boxes represent means. **, p

    Techniques Used: Expressing

    8) Product Images from "miR‐1 is increased in pulmonary hypertension and downregulates Kv1.5 channels in rat pulmonary arteries"

    Article Title: miR‐1 is increased in pulmonary hypertension and downregulates Kv1.5 channels in rat pulmonary arteries

    Journal: The Journal of Physiology

    doi: 10.1113/JP276054

    Effects of antagomiR‐1 on hypoxia‐induced ionic remodelling A , current–voltage relationships of Kv currents measured at the end of the pulse and membrane potential values in PASMCs from PAs transfected with scrambled miR or antagomiR‐1. B , expression of miR‐1 was analysed in PA from rats incubated 48 h in Hyp/Su5416 or normoxia. C , representative current traces for 200 ms depolarization pulses from −60 mV to +60 mV in 10 mV increments from a holding potential of −60 mV, current–voltage relationships of Kv currents measured at the end of the pulse and membrane potential in PASMCs isolated from PAs transfected with scrambled miR or antagomiR‐1 maintained in hypoxia (48 h, 3% O 2 ). D , representative Kv current traces measured before (black) and after (grey) the addition of the Kv1.5 channel blocker DPO‐1 (1 μmol L −1 ) and the DPO‐1‐sensitive currents obtained by subtracting the current in the absence and in the presence of the drug in myocytes from PA transfected with scrambled miR or antagomiR1 maintained in hypoxia. E , Kv1.5 protein expression in Hyp/Su5416‐exposed PA transfected with scrambled (Scr) or antagomir‐1 (Ant) analysed by western blotting and normalized by β‐actin expression. Results are the mean ± SEM. The parentheses indicate the number of cells or arteries and the number of animals from which these cells or arteries were obtained, respectively. * P
    Figure Legend Snippet: Effects of antagomiR‐1 on hypoxia‐induced ionic remodelling A , current–voltage relationships of Kv currents measured at the end of the pulse and membrane potential values in PASMCs from PAs transfected with scrambled miR or antagomiR‐1. B , expression of miR‐1 was analysed in PA from rats incubated 48 h in Hyp/Su5416 or normoxia. C , representative current traces for 200 ms depolarization pulses from −60 mV to +60 mV in 10 mV increments from a holding potential of −60 mV, current–voltage relationships of Kv currents measured at the end of the pulse and membrane potential in PASMCs isolated from PAs transfected with scrambled miR or antagomiR‐1 maintained in hypoxia (48 h, 3% O 2 ). D , representative Kv current traces measured before (black) and after (grey) the addition of the Kv1.5 channel blocker DPO‐1 (1 μmol L −1 ) and the DPO‐1‐sensitive currents obtained by subtracting the current in the absence and in the presence of the drug in myocytes from PA transfected with scrambled miR or antagomiR1 maintained in hypoxia. E , Kv1.5 protein expression in Hyp/Su5416‐exposed PA transfected with scrambled (Scr) or antagomir‐1 (Ant) analysed by western blotting and normalized by β‐actin expression. Results are the mean ± SEM. The parentheses indicate the number of cells or arteries and the number of animals from which these cells or arteries were obtained, respectively. * P

    Techniques Used: Transfection, Expressing, Incubation, Isolation, Western Blot

    miR‐1 reduces Kv1.5 currents in PASMCs A , representative Kv currents traces recorded in the absence (control, black) and in the presence of the Kv1.5 channel blocker DPO‐1 (1 μmol L −1 , grey). B , DPO‐1 sensitive current in PASMCs transfected with scrambled miR and miR‐1. C , resting membrane potential values in PASMCs transfected with scrambled or miR‐1 in the presence of the Kv1.5 channel blocker DPO‐1 (1 μmol L −1 ). Results are the mean ± SEM. The parentheses indicate the number of cells and the number of animals from which these cells were obtained, respectively. * P
    Figure Legend Snippet: miR‐1 reduces Kv1.5 currents in PASMCs A , representative Kv currents traces recorded in the absence (control, black) and in the presence of the Kv1.5 channel blocker DPO‐1 (1 μmol L −1 , grey). B , DPO‐1 sensitive current in PASMCs transfected with scrambled miR and miR‐1. C , resting membrane potential values in PASMCs transfected with scrambled or miR‐1 in the presence of the Kv1.5 channel blocker DPO‐1 (1 μmol L −1 ). Results are the mean ± SEM. The parentheses indicate the number of cells and the number of animals from which these cells were obtained, respectively. * P

    Techniques Used: Transfection

    Changes in miR‐1 expression in the PAH model A , predicted sequence alignment between miR‐1 and KCNA5 3′‐UTR mRNA in Homo sapiens from microRNA.org. B , expression of miR‐1 was analysed in lung homogenates from rats with PAH induced by Hyp/Su5416 and controls. C , luciferase activity in Cos7 cells co‐transfected with miR‐1 and the pMirTarget vector containing the 3′‐UTR region of KCNA5 and the luciferase gene. Results are the mean ± SEM. The parentheses indicate the number of experiments. ** P
    Figure Legend Snippet: Changes in miR‐1 expression in the PAH model A , predicted sequence alignment between miR‐1 and KCNA5 3′‐UTR mRNA in Homo sapiens from microRNA.org. B , expression of miR‐1 was analysed in lung homogenates from rats with PAH induced by Hyp/Su5416 and controls. C , luciferase activity in Cos7 cells co‐transfected with miR‐1 and the pMirTarget vector containing the 3′‐UTR region of KCNA5 and the luciferase gene. Results are the mean ± SEM. The parentheses indicate the number of experiments. ** P

    Techniques Used: Expressing, Sequencing, Luciferase, Activity Assay, Transfection, Plasmid Preparation

    Ionic remodelling in Hyp/Su5416 rats A , representative current traces for 200 ms depolarization pulses from –60 mV to +60 mV in 10 mV increments from a holding potential of ‐60 mV in PASMC. B , average current–voltage relationships of K + currents, normalized by cell capacitance, measured at the end of the pulse from experiments as those shown in ( A ). C , representative current traces measured before (black) and after (grey) the addition of the Kv1.5 channel blocker DPO‐1 (1 μmol L −1 ). D , mean DPO‐1‐sensitive currents obtained by subtracting the current in the absence and in the presence of DPO‐1. E , Kv1.5 protein expression in lung homogenates from rats exposed to normoxia (N) or Hyp/Su5416 (H) analysed by western blotting and normalized by β‐actin expression. The parentheses indicate the number of cells or arteries and the number of animals from which these cells or arteries were obtained, respectively. Results are the mean ± SEM. * P
    Figure Legend Snippet: Ionic remodelling in Hyp/Su5416 rats A , representative current traces for 200 ms depolarization pulses from –60 mV to +60 mV in 10 mV increments from a holding potential of ‐60 mV in PASMC. B , average current–voltage relationships of K + currents, normalized by cell capacitance, measured at the end of the pulse from experiments as those shown in ( A ). C , representative current traces measured before (black) and after (grey) the addition of the Kv1.5 channel blocker DPO‐1 (1 μmol L −1 ). D , mean DPO‐1‐sensitive currents obtained by subtracting the current in the absence and in the presence of DPO‐1. E , Kv1.5 protein expression in lung homogenates from rats exposed to normoxia (N) or Hyp/Su5416 (H) analysed by western blotting and normalized by β‐actin expression. The parentheses indicate the number of cells or arteries and the number of animals from which these cells or arteries were obtained, respectively. Results are the mean ± SEM. * P

    Techniques Used: Expressing, Western Blot

    9) Product Images from "Modulation of Kv1.5 Currents by Src Tyrosine Phosphorylation: Potential Role in the Differentiation of Astrocytes"

    Article Title: Modulation of Kv1.5 Currents by Src Tyrosine Phosphorylation: Potential Role in the Differentiation of Astrocytes

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.20-14-05245.2000

    Src inhibitor acutely decreases Kv1.5 currents. A , Representative recording of currents in response to the voltage protocols shown ( insets to the right ). Currents were recorded after achieving whole-cell configuration ( Control ) and at 12 min after dialyzing
    Figure Legend Snippet: Src inhibitor acutely decreases Kv1.5 currents. A , Representative recording of currents in response to the voltage protocols shown ( insets to the right ). Currents were recorded after achieving whole-cell configuration ( Control ) and at 12 min after dialyzing

    Techniques Used:

    Src family-specific inhibitor PP2 decreases phosphorylation of Kv1.5 without affecting channel expression within the membrane. A , Kv1.5 protein levels in cell lysates are unaltered by incubation with the Src-specific inhibitor PP2. B , Tyrosine phosphorylation
    Figure Legend Snippet: Src family-specific inhibitor PP2 decreases phosphorylation of Kv1.5 without affecting channel expression within the membrane. A , Kv1.5 protein levels in cell lysates are unaltered by incubation with the Src-specific inhibitor PP2. B , Tyrosine phosphorylation

    Techniques Used: Expressing, Incubation

    Immunoreactivity for Kv1.5 did not change with proliferative status. A , B , Proliferating astrocytes as well as actively dividing cells ( arrows ) demonstrate diffuse Kv1.5 staining. C , Staining is seen in acutely dissociated spinal cord cells, implying
    Figure Legend Snippet: Immunoreactivity for Kv1.5 did not change with proliferative status. A , B , Proliferating astrocytes as well as actively dividing cells ( arrows ) demonstrate diffuse Kv1.5 staining. C , Staining is seen in acutely dissociated spinal cord cells, implying

    Techniques Used: Staining

    Increased Src activity increases astrocyte proliferation. A , Transfecting Src Y529F, a constitutively active Src ( Src* ), into quiescent astrocytes ( 27 DIV ) restores immunoreactivity of Kv1.5 for phosphotyrosine, as analyzed by Western blot. B , Astrocytes
    Figure Legend Snippet: Increased Src activity increases astrocyte proliferation. A , Transfecting Src Y529F, a constitutively active Src ( Src* ), into quiescent astrocytes ( 27 DIV ) restores immunoreactivity of Kv1.5 for phosphotyrosine, as analyzed by Western blot. B , Astrocytes

    Techniques Used: Activity Assay, Western Blot

    Kv1.5 antisense oligodeoxynucleotides inhibit astrocyte potassium currents and proliferation. A , Whole-cell current traces from a representative nonsense-treated control and antisense-treated cell as elicited by the voltage protocol ( inset above ). Antisense
    Figure Legend Snippet: Kv1.5 antisense oligodeoxynucleotides inhibit astrocyte potassium currents and proliferation. A , Whole-cell current traces from a representative nonsense-treated control and antisense-treated cell as elicited by the voltage protocol ( inset above ). Antisense

    Techniques Used:

    Kv1.5 protein expression is unaltered during astrocyte differentiation. A , A protein band at a molecular weight of 67 kDa was specific for Kv1.5 immunoreactivity and was unaltered neither on cell cycle arrest with RA or TEA nor on differentiation in culture
    Figure Legend Snippet: Kv1.5 protein expression is unaltered during astrocyte differentiation. A , A protein band at a molecular weight of 67 kDa was specific for Kv1.5 immunoreactivity and was unaltered neither on cell cycle arrest with RA or TEA nor on differentiation in culture

    Techniques Used: Expressing, Molecular Weight

    Coprecipitation of Kv1.5 and native Src throughout astrocyte differentiation. A , Immobilized Kv1.5 antibody is able to precipitate Kv1.5 channel protein in actively proliferating cells ( 5 DIV ), throughout differentiation (at 14, 20, and 33 DIV ), in astrocytes
    Figure Legend Snippet: Coprecipitation of Kv1.5 and native Src throughout astrocyte differentiation. A , Immobilized Kv1.5 antibody is able to precipitate Kv1.5 channel protein in actively proliferating cells ( 5 DIV ), throughout differentiation (at 14, 20, and 33 DIV ), in astrocytes

    Techniques Used:

    Increased Src activity increases Kv1.5 currents. A , Representative recording of currents in response to the voltage protocols shown ( insets to the right ). Currents were recorded within 1 min of achieving whole-cell configuration ( Control ) and at 35 min
    Figure Legend Snippet: Increased Src activity increases Kv1.5 currents. A , Representative recording of currents in response to the voltage protocols shown ( insets to the right ). Currents were recorded within 1 min of achieving whole-cell configuration ( Control ) and at 35 min

    Techniques Used: Activity Assay

    10) Product Images from "Membrane cholesterol modulates Kv1.5 potassium channel distribution and function in rat cardiomyocytes"

    Article Title: Membrane cholesterol modulates Kv1.5 potassium channel distribution and function in rat cardiomyocytes

    Journal: The Journal of Physiology

    doi: 10.1113/jphysiol.2007.134809

    Some Kv1.5 channel subunits are localized in lipid raft fractions in adult rat atrial myocytes Western blot analysis of the distribution of Kv1.5 subunits, connexin-43 and caveolin-3 on step sucrose gradient of proteins from atrial myocardium.
    Figure Legend Snippet: Some Kv1.5 channel subunits are localized in lipid raft fractions in adult rat atrial myocytes Western blot analysis of the distribution of Kv1.5 subunits, connexin-43 and caveolin-3 on step sucrose gradient of proteins from atrial myocardium.

    Techniques Used: Western Blot

    Kv1.5 do not co-localized with caveolin-3 in adult atrial tissue Immunolocalization of Kv1.5 subunits ( A ) and caveolin-3 ( B ) in cryosections of atrial myocardium. Double immunostaining of connexin-43 (FITC, C ) and caveolin-3 (Texas Red, D ) in cryosections of atrial myocardium. E , merged image of the same area of the section in C and D , showing the lack of overlap between connexin-43 and caveolin-3 stainings.
    Figure Legend Snippet: Kv1.5 do not co-localized with caveolin-3 in adult atrial tissue Immunolocalization of Kv1.5 subunits ( A ) and caveolin-3 ( B ) in cryosections of atrial myocardium. Double immunostaining of connexin-43 (FITC, C ) and caveolin-3 (Texas Red, D ) in cryosections of atrial myocardium. E , merged image of the same area of the section in C and D , showing the lack of overlap between connexin-43 and caveolin-3 stainings.

    Techniques Used: Double Immunostaining

    Surface expression of Kv1.5 subunits in neonatal cardiomyocytes A , in live cardiomyocytes, transfected GFP-tagged Kv1.5 subunits are clustered at the membrane surface adjacent to the bottom of laminin-coated glass support, as shown in the projection of Z sections in the lower panel. In contrast, GFP alone was homogeneously distributed in cardiomyocytes (inset). B , after the application of 2% MCD, clusters increased in size and were redistributed throughout the plasma membrane. C , bar graphs summarizing changes in cluster size upon MCD exposures; data are from 21 cardiomyocytes in control, and following incubation with 2% MCD for 7 min and 1 h 30 min. ** P
    Figure Legend Snippet: Surface expression of Kv1.5 subunits in neonatal cardiomyocytes A , in live cardiomyocytes, transfected GFP-tagged Kv1.5 subunits are clustered at the membrane surface adjacent to the bottom of laminin-coated glass support, as shown in the projection of Z sections in the lower panel. In contrast, GFP alone was homogeneously distributed in cardiomyocytes (inset). B , after the application of 2% MCD, clusters increased in size and were redistributed throughout the plasma membrane. C , bar graphs summarizing changes in cluster size upon MCD exposures; data are from 21 cardiomyocytes in control, and following incubation with 2% MCD for 7 min and 1 h 30 min. ** P

    Techniques Used: Expressing, Transfection, Incubation

    Effect of cholesterol depletion on outward current parameters resulting from Kv1.5 subunit overexpression in neonatal cardiomyocytes Current density–voltage relationships ( A ) and voltage dependence ( B ) of I Kur activation under control conditions (•) and following 7 min MCD application (^). In A and B , each point represents average data from 5 cells.
    Figure Legend Snippet: Effect of cholesterol depletion on outward current parameters resulting from Kv1.5 subunit overexpression in neonatal cardiomyocytes Current density–voltage relationships ( A ) and voltage dependence ( B ) of I Kur activation under control conditions (•) and following 7 min MCD application (^). In A and B , each point represents average data from 5 cells.

    Techniques Used: Over Expression, Activation Assay

    11) Product Images from "Hesperetin improves diabetic coronary arterial vasomotor responsiveness by upregulating myocyte voltage-gated K+ channels"

    Article Title: Hesperetin improves diabetic coronary arterial vasomotor responsiveness by upregulating myocyte voltage-gated K+ channels

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2020.8670

    Chronic administration of HSP upregulated mRNA and protein expression of Kv1.2 in diabetic RCASMCs. The mRNA levels of myocyte (A) Kv 1.2 and (B) Kv 1.5 channels in the three experimental groups, which were expressed as the percentage relative to NDB. Representative western blot images showing (C) Kv1.2 and (D) Kv1.5 channel protein expression. The β-actin-normalized densitometric values of (E) Kv1.2 and (F) Kv1.5 channel protein expression in rat coronary arteries. Each value was presented as a mean ± SD from 6 measurements of pooled samples from 6-12 animals. * P
    Figure Legend Snippet: Chronic administration of HSP upregulated mRNA and protein expression of Kv1.2 in diabetic RCASMCs. The mRNA levels of myocyte (A) Kv 1.2 and (B) Kv 1.5 channels in the three experimental groups, which were expressed as the percentage relative to NDB. Representative western blot images showing (C) Kv1.2 and (D) Kv1.5 channel protein expression. The β-actin-normalized densitometric values of (E) Kv1.2 and (F) Kv1.5 channel protein expression in rat coronary arteries. Each value was presented as a mean ± SD from 6 measurements of pooled samples from 6-12 animals. * P

    Techniques Used: Expressing, Western Blot

    Co-incubation of RCASMCs with HSP reversed high glucose-induced reductions in Kv1.2 channel expression. (A) Representative western blotting images of Kv1.2 channel protein expression. (B) β-actin-normalized densitometric values of Kv1.2 channel protein expression in RCASMCs. Each value was presented as a mean ± SD from 6 measurements of pooled samples from 6-12 animals. * P
    Figure Legend Snippet: Co-incubation of RCASMCs with HSP reversed high glucose-induced reductions in Kv1.2 channel expression. (A) Representative western blotting images of Kv1.2 channel protein expression. (B) β-actin-normalized densitometric values of Kv1.2 channel protein expression in RCASMCs. Each value was presented as a mean ± SD from 6 measurements of pooled samples from 6-12 animals. * P

    Techniques Used: Incubation, Expressing, Western Blot

    12) Product Images from "The N terminus and transmembrane segment S1 of Kv1.5 can coassemble with the rest of the channel independently of the S1–S2 linkage"

    Article Title: The N terminus and transmembrane segment S1 of Kv1.5 can coassemble with the rest of the channel independently of the S1–S2 linkage

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.004065

    PK cleaves Kv1.5 at the S1–S2 linker and increases I Kv1.5 . A , Western blots depicting Kv1.5 expression following PK treatment (200 μg/ml, 20 min, 37 °C) of Kv1.5-HEK cells cultured with or without Tuni (10 μg/ml) for 48 h ( n = 6). Proteins were detected using anti-N-terminal ( N-Ab ) or anti-C-terminal ( C-Ab ) Kv1.5 antibody. Actin was used as a loading control. Molecular mass marker ( Marker ) is shown in the middle. B , schematic illustration of Kv1.5 PK cleavage. C , co-IP assay showing that the N- ( N-FR ) and C-fragments ( C-FR ) do not associate after PK cleavage. Whole-cell proteins were extracted from PK-treated WT Kv1.5-HEK cells, and an anti-N-terminal Kv1.5 antibody was added to precipitate the N-fragment and associated proteins. Although the N-fragment was detected in the precipitate, the C-fragment was not detected. Western blotting ( WB ) of uncleaved and PK-cleaved Kv1.5 is shown to indicate the fragments. GAPDH was used as the control. IP , immunoprecipitation; IB , immunoblotting. The same results were obtained from five co-IP experiments. D , I Kv1.5 in control ( CTL ) and PK-treated cells. The voltage protocol is shown above the current traces, and the summarized current-voltage ( I-V ) and g-V relationships are shown beneath the current traces ( n = 36 in control; n = 35 in PK cleavage; **, p
    Figure Legend Snippet: PK cleaves Kv1.5 at the S1–S2 linker and increases I Kv1.5 . A , Western blots depicting Kv1.5 expression following PK treatment (200 μg/ml, 20 min, 37 °C) of Kv1.5-HEK cells cultured with or without Tuni (10 μg/ml) for 48 h ( n = 6). Proteins were detected using anti-N-terminal ( N-Ab ) or anti-C-terminal ( C-Ab ) Kv1.5 antibody. Actin was used as a loading control. Molecular mass marker ( Marker ) is shown in the middle. B , schematic illustration of Kv1.5 PK cleavage. C , co-IP assay showing that the N- ( N-FR ) and C-fragments ( C-FR ) do not associate after PK cleavage. Whole-cell proteins were extracted from PK-treated WT Kv1.5-HEK cells, and an anti-N-terminal Kv1.5 antibody was added to precipitate the N-fragment and associated proteins. Although the N-fragment was detected in the precipitate, the C-fragment was not detected. Western blotting ( WB ) of uncleaved and PK-cleaved Kv1.5 is shown to indicate the fragments. GAPDH was used as the control. IP , immunoprecipitation; IB , immunoblotting. The same results were obtained from five co-IP experiments. D , I Kv1.5 in control ( CTL ) and PK-treated cells. The voltage protocol is shown above the current traces, and the summarized current-voltage ( I-V ) and g-V relationships are shown beneath the current traces ( n = 36 in control; n = 35 in PK cleavage; **, p

    Techniques Used: Western Blot, Expressing, Cell Culture, Marker, Co-Immunoprecipitation Assay, Immunoprecipitation, CTL Assay

    Frag(304–613) requires Frag(1–303) to traffic to the plasma membrane. A , Western blots depicting total expression ( left ) and membrane expression ( right ) of Frag(1–303) and Frag(304–613) expressed independently or together ( n = 6). Actin and Na + /K + -ATPase were used as loading controls for total and membrane proteins, respectively. Molecular mass marker ( Marker ) is shown in the middle. B , confocal images portraying the localization of WT Kv1.5, Frag(1–303), or Frag(304–613) relative to the plasma membrane ( n = 4). WT Kv1.5 was detected with the N-terminal antibody and is depicted in red . Frag(1–303) and Frag(304–613), detected with N-terminal ( N-Ab ) and C-terminal ( C-Ab ) antibodies, respectively, are depicted in red . Cell membranes are shown in green . Fluorescence intensities of WT Kv1.5, Frag(1–303), and/or Frag(304–613) and the membrane in the line across the cells were quantified by ImageJ and are shown beneath the representative images.
    Figure Legend Snippet: Frag(304–613) requires Frag(1–303) to traffic to the plasma membrane. A , Western blots depicting total expression ( left ) and membrane expression ( right ) of Frag(1–303) and Frag(304–613) expressed independently or together ( n = 6). Actin and Na + /K + -ATPase were used as loading controls for total and membrane proteins, respectively. Molecular mass marker ( Marker ) is shown in the middle. B , confocal images portraying the localization of WT Kv1.5, Frag(1–303), or Frag(304–613) relative to the plasma membrane ( n = 4). WT Kv1.5 was detected with the N-terminal antibody and is depicted in red . Frag(1–303) and Frag(304–613), detected with N-terminal ( N-Ab ) and C-terminal ( C-Ab ) antibodies, respectively, are depicted in red . Cell membranes are shown in green . Fluorescence intensities of WT Kv1.5, Frag(1–303), and/or Frag(304–613) and the membrane in the line across the cells were quantified by ImageJ and are shown beneath the representative images.

    Techniques Used: Western Blot, Expressing, Marker, Fluorescence

    Expression and function of Kv1.5 N- ( N-FR ) and C-fragments ( C-FR ). A and B , Western blots of Frag(1–303) and Frag(304–613) along with full-length Kv1.5 protein without and with PK cleavage from cells treated without or with Tuni treatment (10 μg/ml, 48 h). Kv1.5 proteins were detected with an N-terminal ( N-Ab ) or C-terminal antibody ( C-Ab ). Actin was used as a loading control ( n = 8). C–E , schematics illustrating Frag(1–303) and Frag(304–613) as well as corresponding current traces upon independent expression or coexpression. The voltage protocol is shown in the inset above the current traces. F , summarized current-voltage relationships of currents generated by Frag(1–303) ( n = 8), Frag(304–613) ( n = 10), and Frag(1–303)+(304–613) ( n = 14). Error bars represent S.E. CTL , control.
    Figure Legend Snippet: Expression and function of Kv1.5 N- ( N-FR ) and C-fragments ( C-FR ). A and B , Western blots of Frag(1–303) and Frag(304–613) along with full-length Kv1.5 protein without and with PK cleavage from cells treated without or with Tuni treatment (10 μg/ml, 48 h). Kv1.5 proteins were detected with an N-terminal ( N-Ab ) or C-terminal antibody ( C-Ab ). Actin was used as a loading control ( n = 8). C–E , schematics illustrating Frag(1–303) and Frag(304–613) as well as corresponding current traces upon independent expression or coexpression. The voltage protocol is shown in the inset above the current traces. F , summarized current-voltage relationships of currents generated by Frag(1–303) ( n = 8), Frag(304–613) ( n = 10), and Frag(1–303)+(304–613) ( n = 14). Error bars represent S.E. CTL , control.

    Techniques Used: Expressing, Western Blot, Generated, CTL Assay

    Voltage-dependent inactivation of WT, ΔN209, and Frag(1–303)+(304–613)-coassembled Kv1.5 channels. A , left , schematic illustration of the channels. Middle , representative current traces recorded using the voltage protocol shown above from cells expressing the channels illustrated. The interpulse interval was 15 s. Right , voltage dependence of inactivation. Currents at 60 mV were normalized to the maximum value and plotted versus the test voltages for WT, ΔN209, and Frag(1–303)+(304–613)-coassembled Kv1.5 channels ( n = 7–11 cells from three independent experiments for each). B , excessive cumulative inactivation of ΔN209 and Frag(1–303)+(304–613)-coassembled Kv1.5 channels compared with WT Kv1.5. The voltage protocol is depicted above plots of normalized current amplitudes against depolarizing steps relative to the value upon the first step ( n = 6–8 cells for each channel). Error bars represent S.E.
    Figure Legend Snippet: Voltage-dependent inactivation of WT, ΔN209, and Frag(1–303)+(304–613)-coassembled Kv1.5 channels. A , left , schematic illustration of the channels. Middle , representative current traces recorded using the voltage protocol shown above from cells expressing the channels illustrated. The interpulse interval was 15 s. Right , voltage dependence of inactivation. Currents at 60 mV were normalized to the maximum value and plotted versus the test voltages for WT, ΔN209, and Frag(1–303)+(304–613)-coassembled Kv1.5 channels ( n = 7–11 cells from three independent experiments for each). B , excessive cumulative inactivation of ΔN209 and Frag(1–303)+(304–613)-coassembled Kv1.5 channels compared with WT Kv1.5. The voltage protocol is depicted above plots of normalized current amplitudes against depolarizing steps relative to the value upon the first step ( n = 6–8 cells for each channel). Error bars represent S.E.

    Techniques Used: Expressing

    Dominant-negative suppression of WT Kv1.5 with Frag(1–303). Shown are the effects of transfecting empty pcDNA3 ( PC3 ; control; n = 27) or plasmids encoding Frag(1–209) ( n = 12), Frag(210–303) ( n = 11), or Frag(1–303) ( n = 17) into Kv1.5-HEK cells on I Kv1.5 . Representative current traces are depicted above the summarized box plots of current amplitudes upon 50-mV depolarization. **, p
    Figure Legend Snippet: Dominant-negative suppression of WT Kv1.5 with Frag(1–303). Shown are the effects of transfecting empty pcDNA3 ( PC3 ; control; n = 27) or plasmids encoding Frag(1–209) ( n = 12), Frag(210–303) ( n = 11), or Frag(1–303) ( n = 17) into Kv1.5-HEK cells on I Kv1.5 . Representative current traces are depicted above the summarized box plots of current amplitudes upon 50-mV depolarization. **, p

    Techniques Used: Dominant Negative Mutation

    13) 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 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

    14) Product Images from "Mechanical stretch increases Kv1.5 current through an interaction between the S1–S2 linker and N-terminus of the channel"

    Article Title: Mechanical stretch increases Kv1.5 current through an interaction between the S1–S2 linker and N-terminus of the channel

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA119.011302

    The Kv1.5 S1–S2 linker communicates with the N terminus in a conformational manner. Truncation of N terminus altered the susceptibility of the S1–S2 linker to PK cleavage. WT Kv1.5 displays 75-kDa and 68-kDa bands on Western blot analysis. The 75-kDa band represents the mature, fully glycosylated channel protein in the plasma membrane, whereas the 68-kDa band represents the immature channel protein inside the cell. ΔN209 Kv1.5 also presents as two bands; the 50-kDa band represents mature protein in the plasma membrane, the 43-kDa band represents immature protein inside the cell. Although PK completely cleaved the mature (cell surface) channel proteins of WT Kv1.5, it failed to cleave the mature (cell surface) channel proteins of ΔN209 Kv1.5 ( n = 6).
    Figure Legend Snippet: The Kv1.5 S1–S2 linker communicates with the N terminus in a conformational manner. Truncation of N terminus altered the susceptibility of the S1–S2 linker to PK cleavage. WT Kv1.5 displays 75-kDa and 68-kDa bands on Western blot analysis. The 75-kDa band represents the mature, fully glycosylated channel protein in the plasma membrane, whereas the 68-kDa band represents the immature channel protein inside the cell. ΔN209 Kv1.5 also presents as two bands; the 50-kDa band represents mature protein in the plasma membrane, the 43-kDa band represents immature protein inside the cell. Although PK completely cleaved the mature (cell surface) channel proteins of WT Kv1.5, it failed to cleave the mature (cell surface) channel proteins of ΔN209 Kv1.5 ( n = 6).

    Techniques Used: Western Blot

    Centrifugation increases I Kv1.5 . Kv1.5-HEK cells were centrifuged ( CENTR ) at 70 × g for 5 min. Cells were then re-suspended in normal culture medium for 20 min prior to I Kv1.5 recordings. Kv1.5-HEK cells without centrifugation were used as control (CTL). Representative current traces along with the voltage protocol ( top ) and summarized I-V relationship ( bottom ) are shown. CTL, n = 29; CENTR, n = 38; **, p
    Figure Legend Snippet: Centrifugation increases I Kv1.5 . Kv1.5-HEK cells were centrifuged ( CENTR ) at 70 × g for 5 min. Cells were then re-suspended in normal culture medium for 20 min prior to I Kv1.5 recordings. Kv1.5-HEK cells without centrifugation were used as control (CTL). Representative current traces along with the voltage protocol ( top ) and summarized I-V relationship ( bottom ) are shown. CTL, n = 29; CENTR, n = 38; **, p

    Techniques Used: Centrifugation

    The unique S1–S2 linker of Kv1.5 is involved in LO-mediated increase in I Kv1.5 . A , amino acid sequences of the S1–S2 linker of various Kv channels. Kv1.5 possesses an unusually long S1–S2 linker with 12 nonconserved proline residues (in magenta ). The N -linked glycosylation site is shown in red. B, PK cleavage of the S1–S2 linker abolished LO-induced increase in I Kv1.5 . Schematic illustration of Kv1.5 PK cleavage ( top ) and representative current traces ( middle ) are depicted above the summarized I-V relationships ( bottom ). CTL, n = 14; LO, n = 21. C, mutating all 12 nonconserved prolines (P) to alanines (A) in the S1–S2 linker abolished LO-induced increase in I Kv1.5 . Schematic illustration of the Kv1.5–12PA mutant ( top ) as well as representative current traces ( middle ) are depicted above the summarized I-V relationships ( bottom ). CTL, n = 37; LO, n = 36. D , deletion of amino acid residues 282–300 in the S1–S2 linker abolished LO-induced increase in I Kv1.5 . Schematic illustration of Kv1.5-Δ282–300 mutant ( top ) as well as representative current traces ( middle ) are depicted above the summarized I-V relationships ( bottom ). CTL, n = 15, LO, n = 10. E , inhibition of glycosylation in the S1–S2 linker with tunicamycin ( Tuni ) treatment abolished the LO-induced increase in I Kv1.5 . Schematic illustration of WT Kv1.5 without glycosylation ( top ) as well as representative current traces ( middle ) are depicted above the summarized I-V relationships ( bottom ). CTL, n = 24; LO, n = 24.
    Figure Legend Snippet: The unique S1–S2 linker of Kv1.5 is involved in LO-mediated increase in I Kv1.5 . A , amino acid sequences of the S1–S2 linker of various Kv channels. Kv1.5 possesses an unusually long S1–S2 linker with 12 nonconserved proline residues (in magenta ). The N -linked glycosylation site is shown in red. B, PK cleavage of the S1–S2 linker abolished LO-induced increase in I Kv1.5 . Schematic illustration of Kv1.5 PK cleavage ( top ) and representative current traces ( middle ) are depicted above the summarized I-V relationships ( bottom ). CTL, n = 14; LO, n = 21. C, mutating all 12 nonconserved prolines (P) to alanines (A) in the S1–S2 linker abolished LO-induced increase in I Kv1.5 . Schematic illustration of the Kv1.5–12PA mutant ( top ) as well as representative current traces ( middle ) are depicted above the summarized I-V relationships ( bottom ). CTL, n = 37; LO, n = 36. D , deletion of amino acid residues 282–300 in the S1–S2 linker abolished LO-induced increase in I Kv1.5 . Schematic illustration of Kv1.5-Δ282–300 mutant ( top ) as well as representative current traces ( middle ) are depicted above the summarized I-V relationships ( bottom ). CTL, n = 15, LO, n = 10. E , inhibition of glycosylation in the S1–S2 linker with tunicamycin ( Tuni ) treatment abolished the LO-induced increase in I Kv1.5 . Schematic illustration of WT Kv1.5 without glycosylation ( top ) as well as representative current traces ( middle ) are depicted above the summarized I-V relationships ( bottom ). CTL, n = 24; LO, n = 24.

    Techniques Used: Mutagenesis, Inhibition

    LO medium treatment increases I Kv1.5 in neonatal rat ventricular myocytes transfected with Kv1.5. Representative current traces are depicted above the summarized I-V relationship. CTL, n = 32; LO, n = 32. *, p
    Figure Legend Snippet: LO medium treatment increases I Kv1.5 in neonatal rat ventricular myocytes transfected with Kv1.5. Representative current traces are depicted above the summarized I-V relationship. CTL, n = 32; LO, n = 32. *, p

    Techniques Used: Transfection

    LO medium treatment increases cell size and reversibly increases I Kv1.5 . A, culture of Kv1.5-HEK cells with LO medium for 30 min increased cell size ( n = 13; **, p
    Figure Legend Snippet: LO medium treatment increases cell size and reversibly increases I Kv1.5 . A, culture of Kv1.5-HEK cells with LO medium for 30 min increased cell size ( n = 13; **, p

    Techniques Used:

    Removal of Src-binding sites abolishes LO-mediated increase in I Kv1.5 . A, amino acid sequences showing the two consensus SH3–binding motifs (RPLPPLP, shown in blue ) in the N terminus of Kv1.5 as well as the mutant Kv1.5-ΔPro, in which amino acids 64–82 were removed. B , effects of LO treatment on WT I Kv1.5 . CTL, n = 36; LO, n = 27; **, p
    Figure Legend Snippet: Removal of Src-binding sites abolishes LO-mediated increase in I Kv1.5 . A, amino acid sequences showing the two consensus SH3–binding motifs (RPLPPLP, shown in blue ) in the N terminus of Kv1.5 as well as the mutant Kv1.5-ΔPro, in which amino acids 64–82 were removed. B , effects of LO treatment on WT I Kv1.5 . CTL, n = 36; LO, n = 27; **, p

    Techniques Used: Binding Assay, Mutagenesis

    Effects of LO-treatment and Src inhibitor PP1 on Kv1.5 expression and function. A , LO treatment did not affect the total amount of Kv1.5 proteins but increased the cell surface mature channel expression. The density of the 75-kDa band in LO-treated cells was normalized to that of control cells in the same gel and shown in the scatter plots ( n = 5). B , PP1 treatment did not affect the total amount of Kv1.5 proteins but increased the cell surface mature channel expression. The density of the 75-kDa band in PP1-treated cells was normalized to that of control cells in the same gel and shown in the scatter plot ( n = 5). For A and B , boxes represent interquartile ranges, horizontal lines represent medians, whiskers represent 5–95% ranges, and gray boxes represent means. **, p
    Figure Legend Snippet: Effects of LO-treatment and Src inhibitor PP1 on Kv1.5 expression and function. A , LO treatment did not affect the total amount of Kv1.5 proteins but increased the cell surface mature channel expression. The density of the 75-kDa band in LO-treated cells was normalized to that of control cells in the same gel and shown in the scatter plots ( n = 5). B , PP1 treatment did not affect the total amount of Kv1.5 proteins but increased the cell surface mature channel expression. The density of the 75-kDa band in PP1-treated cells was normalized to that of control cells in the same gel and shown in the scatter plot ( n = 5). For A and B , boxes represent interquartile ranges, horizontal lines represent medians, whiskers represent 5–95% ranges, and gray boxes represent means. **, p

    Techniques Used: Expressing

    15) Product Images from "Cholesterol modulates the recruitment of Kv1.5 channels from Rab11-associated recycling endosome in native atrial myocytes"

    Article Title: Cholesterol modulates the recruitment of Kv1.5 channels from Rab11-associated recycling endosome in native atrial myocytes

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0902809106

    Cholesterol depletion reduces the mobility and redistributes Kv1.5 channels in atrial myocytes. ( A ) Sequential images from FRAP experiments obtained in control prebleach (prebl.) and at various times postbleach (postbl.) in hKv1.5-EGFP expressing myocytes
    Figure Legend Snippet: Cholesterol depletion reduces the mobility and redistributes Kv1.5 channels in atrial myocytes. ( A ) Sequential images from FRAP experiments obtained in control prebleach (prebl.) and at various times postbleach (postbl.) in hKv1.5-EGFP expressing myocytes

    Techniques Used: Expressing

    16) Product Images from "Kv1.5 channels are regulated by PKC-mediated endocytic degradation"

    Article Title: Kv1.5 channels are regulated by PKC-mediated endocytic degradation

    Journal: The Journal of Biological Chemistry

    doi: 10.1016/j.jbc.2021.100514

    Threonine at amino acid 15 in the N terminus of Kv1.5 is required for PMA-induced reduction in Kv1.5 expression and I Kv1.5 in HEK293 cells. For Kv1.5-WT ( A ), Kv1.5-ΔN209 ( B ), Kv1.5-Δ2-19 ( C ), and Kv1.5-T15A channels ( D ), schematic diagrams of channel structure are shown on the top row , western blot images are shown in the middle row , representative current traces along with summarized I-V relationships are shown in the bottom row . HEK293 cells stably expressing various channels were treated with 10 nM PMA for 3 h, and experiments were then performed. For western blots, n = 5 for WT, n = 3 for ΔN209, n = 3 for Δ2-19, n= 5 for T15A. ∗∗ p
    Figure Legend Snippet: Threonine at amino acid 15 in the N terminus of Kv1.5 is required for PMA-induced reduction in Kv1.5 expression and I Kv1.5 in HEK293 cells. For Kv1.5-WT ( A ), Kv1.5-ΔN209 ( B ), Kv1.5-Δ2-19 ( C ), and Kv1.5-T15A channels ( D ), schematic diagrams of channel structure are shown on the top row , western blot images are shown in the middle row , representative current traces along with summarized I-V relationships are shown in the bottom row . HEK293 cells stably expressing various channels were treated with 10 nM PMA for 3 h, and experiments were then performed. For western blots, n = 5 for WT, n = 3 for ΔN209, n = 3 for Δ2-19, n= 5 for T15A. ∗∗ p

    Techniques Used: Expressing, Western Blot, Stable Transfection

    PKC activation by PMA treatment induces monoubiquitination of Kv1.5 channels in HEK293 cells. A , effects of 10 nM PMA treatment on KCNA5 mRNA levels detected using real-time PCR (n = 4). B , the western blot image showing that PMA treatment (10 nM, 0.5 h) induces an extra band, close to 83 kDa (n = 4). C , overexpression of UbKO has no effect on the PMA-induced reduction in expression of the 75-kDa form of Kv1.5 channels (n = 6). ∗∗ p
    Figure Legend Snippet: PKC activation by PMA treatment induces monoubiquitination of Kv1.5 channels in HEK293 cells. A , effects of 10 nM PMA treatment on KCNA5 mRNA levels detected using real-time PCR (n = 4). B , the western blot image showing that PMA treatment (10 nM, 0.5 h) induces an extra band, close to 83 kDa (n = 4). C , overexpression of UbKO has no effect on the PMA-induced reduction in expression of the 75-kDa form of Kv1.5 channels (n = 6). ∗∗ p

    Techniques Used: Activation Assay, Real-time Polymerase Chain Reaction, Western Blot, Over Expression, Expressing

    PKC inhibitors abolish PMA-induced reduction in Kv1.5 expression and I Kv1.5 in HEK293 cells. A , western blots result showing effects of 10 nM PMA treatment for 3 h on Kv1.5 expression with or without 10 μM BIM-1. Representative western blot images are displayed along with summarized data (n = 4). ∗∗ p
    Figure Legend Snippet: PKC inhibitors abolish PMA-induced reduction in Kv1.5 expression and I Kv1.5 in HEK293 cells. A , western blots result showing effects of 10 nM PMA treatment for 3 h on Kv1.5 expression with or without 10 μM BIM-1. Representative western blot images are displayed along with summarized data (n = 4). ∗∗ p

    Techniques Used: Expressing, Western Blot

    Schematic illustrating the process of PKC activation-induced endocytic degradation of Kv1.5 channel. PKC activation targets T15 at the N terminus of Kv1.5 channel on the surface membrane and induces monoubiquitination of the channel. Ubiquitinated channel undergoes internalization through MVB/Vps24 into lysosome to degradation.
    Figure Legend Snippet: Schematic illustrating the process of PKC activation-induced endocytic degradation of Kv1.5 channel. PKC activation targets T15 at the N terminus of Kv1.5 channel on the surface membrane and induces monoubiquitination of the channel. Ubiquitinated channel undergoes internalization through MVB/Vps24 into lysosome to degradation.

    Techniques Used: Activation Assay

    PKC activation by PMA treatment decreases I Kv1.5 in human iPSC-derived atrial cardiomyocytes. A , representative current traces and summarized current–voltage relationships of I Kv1.5 before and after 0.1 mM 4-AP application. After currents were recorded in control conditions (before 4-AP), 0.1 mM 4-AP was applied to the bath solution for 2 min to achieve stead-state block, and currents were recorded in the same cell in the presence of 4-AP (after 4-AP). n = 5. ∗∗ p
    Figure Legend Snippet: PKC activation by PMA treatment decreases I Kv1.5 in human iPSC-derived atrial cardiomyocytes. A , representative current traces and summarized current–voltage relationships of I Kv1.5 before and after 0.1 mM 4-AP application. After currents were recorded in control conditions (before 4-AP), 0.1 mM 4-AP was applied to the bath solution for 2 min to achieve stead-state block, and currents were recorded in the same cell in the presence of 4-AP (after 4-AP). n = 5. ∗∗ p

    Techniques Used: Activation Assay, Derivative Assay, Blocking Assay

    Lysosomal inhibitor bafilomycin A1, but not proteasomal inhibitor MG132, completely abolishes PMA-induced reduction in Kv1.5 expression and I Kv1.5 in HEK293 cells. A , effects of 10 nM PMA (3 h) on Kv1.5 expression with or without bafilomycin A1 (Baf, 1 μM) or MG132 (10 μM). Representative western blot images are displayed along with summarized data. n = 3. ∗∗ p
    Figure Legend Snippet: Lysosomal inhibitor bafilomycin A1, but not proteasomal inhibitor MG132, completely abolishes PMA-induced reduction in Kv1.5 expression and I Kv1.5 in HEK293 cells. A , effects of 10 nM PMA (3 h) on Kv1.5 expression with or without bafilomycin A1 (Baf, 1 μM) or MG132 (10 μM). Representative western blot images are displayed along with summarized data. n = 3. ∗∗ p

    Techniques Used: Expressing, Western Blot

    PKC activation by PMA treatment decreases currents of Kv1.5 channels expressed in neonatal rat ventricular myocytes. Representative current traces ( A ) and summarized current–voltage relationships ( B ) of I Kv1.5 recorded from Kv1.5-transfected neonatal rat ventricular myocytes treated with PMA (10 nM) for 3 h in the absence and presence of BIM-1 (10 μM) or Baf (1 μM) are shown. ∗∗ p
    Figure Legend Snippet: PKC activation by PMA treatment decreases currents of Kv1.5 channels expressed in neonatal rat ventricular myocytes. Representative current traces ( A ) and summarized current–voltage relationships ( B ) of I Kv1.5 recorded from Kv1.5-transfected neonatal rat ventricular myocytes treated with PMA (10 nM) for 3 h in the absence and presence of BIM-1 (10 μM) or Baf (1 μM) are shown. ∗∗ p

    Techniques Used: Activation Assay, Transfection

    PMA treatment (10 nM) for 3 h has no effect on Kv1.1, Kv1.2, Kv1.3, and Kv1.4 channels in HEK293 cells. A , western blot images showing the effect of PMA (10 nM) for 3 h on expressions of Kv1.1, Kv1.2, Kv1.3, and Kv1.4 channels stably expressed in HEK293 cells (n = 3–4). B , I-V relationships showing the effect of PMA treatment for 3 h on currents of Kv1.1, Kv1.2, Kv1.3, and Kv1.4 channels stably expressed in HEK293 cells (n = 7–18 cells for each channel type). For western blots, data are presented as box plots and mean ± SD. For patch clamp, data are presented as mean + SD. C , sequence alignment of the N terminus of Kv channels. Amino acid residues marked in blue are identical or similar among all channels; amino acid resides marked in light blue are different among channels; amino acid resides marked in black are segments that are absent in some channels. Threonine (T) at amino acid 15 in the N terminus of Kv1.5 is marked as red .
    Figure Legend Snippet: PMA treatment (10 nM) for 3 h has no effect on Kv1.1, Kv1.2, Kv1.3, and Kv1.4 channels in HEK293 cells. A , western blot images showing the effect of PMA (10 nM) for 3 h on expressions of Kv1.1, Kv1.2, Kv1.3, and Kv1.4 channels stably expressed in HEK293 cells (n = 3–4). B , I-V relationships showing the effect of PMA treatment for 3 h on currents of Kv1.1, Kv1.2, Kv1.3, and Kv1.4 channels stably expressed in HEK293 cells (n = 7–18 cells for each channel type). For western blots, data are presented as box plots and mean ± SD. For patch clamp, data are presented as mean + SD. C , sequence alignment of the N terminus of Kv channels. Amino acid residues marked in blue are identical or similar among all channels; amino acid resides marked in light blue are different among channels; amino acid resides marked in black are segments that are absent in some channels. Threonine (T) at amino acid 15 in the N terminus of Kv1.5 is marked as red .

    Techniques Used: Western Blot, Stable Transfection, Patch Clamp, Sequencing

    PMA treatment decreases expression and current of Kv1.5 channels in HEK293 cells. A and B , concentration-dependent effects of PMA treatment for 3 h on the Kv1.5 expression ( A , n = 5) and I Kv1.5 ( B , n = 6–13 cells for each concentration). C and D , time-dependent effects of PMA (10 nM) on the Kv1.5 expression ( C , n = 5) and I Kv1.5 ( D , n = 7–14 cells for each time point). For western blot images, the actual molecular markers (the BLUeye Prestained Protein Ladder) are indicated beside the bands run on the same gels throughout the study. The 75-kDa band represents mature, fully glycosylated plasma membrane-located Kv1.5 channels, whereas the 68-kDa band represents core-glycosylated ER-located Kv1.5 channels. For western blot analysis, ∗ p
    Figure Legend Snippet: PMA treatment decreases expression and current of Kv1.5 channels in HEK293 cells. A and B , concentration-dependent effects of PMA treatment for 3 h on the Kv1.5 expression ( A , n = 5) and I Kv1.5 ( B , n = 6–13 cells for each concentration). C and D , time-dependent effects of PMA (10 nM) on the Kv1.5 expression ( C , n = 5) and I Kv1.5 ( D , n = 7–14 cells for each time point). For western blot images, the actual molecular markers (the BLUeye Prestained Protein Ladder) are indicated beside the bands run on the same gels throughout the study. The 75-kDa band represents mature, fully glycosylated plasma membrane-located Kv1.5 channels, whereas the 68-kDa band represents core-glycosylated ER-located Kv1.5 channels. For western blot analysis, ∗ p

    Techniques Used: Expressing, Concentration Assay, Western Blot

    Vps24 mediates PKC activation-induced Kv1.5 reduction in HEK293 cells. A , overexpression of Vps24 accelerates PMA (10 nM, 1 h)-induced Kv1.5 reduction. ∗ p
    Figure Legend Snippet: Vps24 mediates PKC activation-induced Kv1.5 reduction in HEK293 cells. A , overexpression of Vps24 accelerates PMA (10 nM, 1 h)-induced Kv1.5 reduction. ∗ p

    Techniques Used: Activation Assay, Over Expression

    17) Product Images from "Tetramerization domain mutations in KCNA5 affect channel kinetics and cause abnormal trafficking patterns"

    Article Title: Tetramerization domain mutations in KCNA5 affect channel kinetics and cause abnormal trafficking patterns

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00464.2009

    Mutant KCNA5 is located in perinuclear packets and not on the cell surface of transfected HEK-293 cells and human (h)PASMC. A : HEK-293 cells transfected with WT KCNA5 ( a and c ) or G182R ( b and d ) were stained with anti-KCNA5 antibody (Ab-KCNA5, red) and
    Figure Legend Snippet: Mutant KCNA5 is located in perinuclear packets and not on the cell surface of transfected HEK-293 cells and human (h)PASMC. A : HEK-293 cells transfected with WT KCNA5 ( a and c ) or G182R ( b and d ) were stained with anti-KCNA5 antibody (Ab-KCNA5, red) and

    Techniques Used: Mutagenesis, Transfection, Staining

    G182R and E211D mutations cause incomplete processing of KCNA5 in HEK-293 cells. HEK-293 cells were transfected with WT KCNA5, G182R, E211D, or G182R/E211D and subjected to standard immunoblot procedures. A : representative immunoblot from cells transfected
    Figure Legend Snippet: G182R and E211D mutations cause incomplete processing of KCNA5 in HEK-293 cells. HEK-293 cells were transfected with WT KCNA5, G182R, E211D, or G182R/E211D and subjected to standard immunoblot procedures. A : representative immunoblot from cells transfected

    Techniques Used: Transfection

    Mutant KCNA5 forms functional homotetrameric channels. A : COS-1, HEK-293, and human pulmonary artery smooth muscle cells (PASMC) were transiently transfected with either empty vector [green fluorescent protein (GFP)] or WT KCNA5 (WT) as indicated. Whole
    Figure Legend Snippet: Mutant KCNA5 forms functional homotetrameric channels. A : COS-1, HEK-293, and human pulmonary artery smooth muscle cells (PASMC) were transiently transfected with either empty vector [green fluorescent protein (GFP)] or WT KCNA5 (WT) as indicated. Whole

    Techniques Used: Mutagenesis, Functional Assay, Transfection, Plasmid Preparation

    Mutations in KCNA5 at G182 and E211 do not affect the pharmacological effect of 4-aminopyridine (4-AP). A : a standard I-V pulse protocol was delivered to HEK-293 cells transiently transfected with the indicated vector [WT KCNA5 ( a ), G182R ( b ), E211D (
    Figure Legend Snippet: Mutations in KCNA5 at G182 and E211 do not affect the pharmacological effect of 4-aminopyridine (4-AP). A : a standard I-V pulse protocol was delivered to HEK-293 cells transiently transfected with the indicated vector [WT KCNA5 ( a ), G182R ( b ), E211D (

    Techniques Used: Transfection, Plasmid Preparation

    Decreased G182R expression in COS-1 cells cannot be rescued by overexpression of K V β subunits. A , a : HEK-293 cells were transfected with WT KCNA5 or K V β1.3-HA alone or cotransfected with WT KCNA5, G182R, E211D, or G182R/E211D and K V β1.3-HA.
    Figure Legend Snippet: Decreased G182R expression in COS-1 cells cannot be rescued by overexpression of K V β subunits. A , a : HEK-293 cells were transfected with WT KCNA5 or K V β1.3-HA alone or cotransfected with WT KCNA5, G182R, E211D, or G182R/E211D and K V β1.3-HA.

    Techniques Used: Expressing, Over Expression, Transfection

    Two nonsynonymous mutations identified in the KCNA5 gene from idiopathic pulmonary arterial hypertension (IPAH) patients localize to the NH 2 -terminal tetramerization domain (T1 domain). A , left : schematic diagram of voltage-gated K + (K V ) channel subunit
    Figure Legend Snippet: Two nonsynonymous mutations identified in the KCNA5 gene from idiopathic pulmonary arterial hypertension (IPAH) patients localize to the NH 2 -terminal tetramerization domain (T1 domain). A , left : schematic diagram of voltage-gated K + (K V ) channel subunit

    Techniques Used:

    Cotransfection of K V β subunits affects KCNA5 channel kinetics. HEK-293 cells were transfected with WT KCNA5 alone (KCNA5) or in the presence of K V β1.3-hemagglutinin (HA) (KCNA5/K V β1.3). A and B : representative current recordings
    Figure Legend Snippet: Cotransfection of K V β subunits affects KCNA5 channel kinetics. HEK-293 cells were transfected with WT KCNA5 alone (KCNA5) or in the presence of K V β1.3-hemagglutinin (HA) (KCNA5/K V β1.3). A and B : representative current recordings

    Techniques Used: Cotransfection, Transfection

    Voltage-dependent inactivation is accelerated in the G182R mutant KCNA5 channel. A standard 2-pulse inactivation protocol was used to determine channel availability after a 10-s prepulse in HEK-293 cells transiently transfected with WT KCNA5, G182R, E211D,
    Figure Legend Snippet: Voltage-dependent inactivation is accelerated in the G182R mutant KCNA5 channel. A standard 2-pulse inactivation protocol was used to determine channel availability after a 10-s prepulse in HEK-293 cells transiently transfected with WT KCNA5, G182R, E211D,

    Techniques Used: Mutagenesis, Transfection

    G182R protein expression is significantly decreased in COS-1 cells. A : COS-1 cells were transiently transfected with water (Mock), WT-KCNA5, G182R, E211D, or G182R/E211D. Representative images are shown at ×40 magnification. B : transfected cells
    Figure Legend Snippet: G182R protein expression is significantly decreased in COS-1 cells. A : COS-1 cells were transiently transfected with water (Mock), WT-KCNA5, G182R, E211D, or G182R/E211D. Representative images are shown at ×40 magnification. B : transfected cells

    Techniques Used: Expressing, Transfection

    18) Product Images from "Tetramerization domain mutations in KCNA5 affect channel kinetics and cause abnormal trafficking patterns"

    Article Title: Tetramerization domain mutations in KCNA5 affect channel kinetics and cause abnormal trafficking patterns

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00464.2009

    Mutant KCNA5 is located in perinuclear packets and not on the cell surface of transfected HEK-293 cells and human (h)PASMC. A : HEK-293 cells transfected with WT KCNA5 ( a and c ) or G182R ( b and d ) were stained with anti-KCNA5 antibody (Ab-KCNA5, red) and
    Figure Legend Snippet: Mutant KCNA5 is located in perinuclear packets and not on the cell surface of transfected HEK-293 cells and human (h)PASMC. A : HEK-293 cells transfected with WT KCNA5 ( a and c ) or G182R ( b and d ) were stained with anti-KCNA5 antibody (Ab-KCNA5, red) and

    Techniques Used: Mutagenesis, Transfection, Staining

    G182R and E211D mutations cause incomplete processing of KCNA5 in HEK-293 cells. HEK-293 cells were transfected with WT KCNA5, G182R, E211D, or G182R/E211D and subjected to standard immunoblot procedures. A : representative immunoblot from cells transfected
    Figure Legend Snippet: G182R and E211D mutations cause incomplete processing of KCNA5 in HEK-293 cells. HEK-293 cells were transfected with WT KCNA5, G182R, E211D, or G182R/E211D and subjected to standard immunoblot procedures. A : representative immunoblot from cells transfected

    Techniques Used: Transfection

    Mutant KCNA5 forms functional homotetrameric channels. A : COS-1, HEK-293, and human pulmonary artery smooth muscle cells (PASMC) were transiently transfected with either empty vector [green fluorescent protein (GFP)] or WT KCNA5 (WT) as indicated. Whole
    Figure Legend Snippet: Mutant KCNA5 forms functional homotetrameric channels. A : COS-1, HEK-293, and human pulmonary artery smooth muscle cells (PASMC) were transiently transfected with either empty vector [green fluorescent protein (GFP)] or WT KCNA5 (WT) as indicated. Whole

    Techniques Used: Mutagenesis, Functional Assay, Transfection, Plasmid Preparation

    Mutations in KCNA5 at G182 and E211 do not affect the pharmacological effect of 4-aminopyridine (4-AP). A : a standard I-V pulse protocol was delivered to HEK-293 cells transiently transfected with the indicated vector [WT KCNA5 ( a ), G182R ( b ), E211D (
    Figure Legend Snippet: Mutations in KCNA5 at G182 and E211 do not affect the pharmacological effect of 4-aminopyridine (4-AP). A : a standard I-V pulse protocol was delivered to HEK-293 cells transiently transfected with the indicated vector [WT KCNA5 ( a ), G182R ( b ), E211D (

    Techniques Used: Transfection, Plasmid Preparation

    Decreased G182R expression in COS-1 cells cannot be rescued by overexpression of K V β subunits. A , a : HEK-293 cells were transfected with WT KCNA5 or K V β1.3-HA alone or cotransfected with WT KCNA5, G182R, E211D, or G182R/E211D and K V β1.3-HA.
    Figure Legend Snippet: Decreased G182R expression in COS-1 cells cannot be rescued by overexpression of K V β subunits. A , a : HEK-293 cells were transfected with WT KCNA5 or K V β1.3-HA alone or cotransfected with WT KCNA5, G182R, E211D, or G182R/E211D and K V β1.3-HA.

    Techniques Used: Expressing, Over Expression, Transfection

    Two nonsynonymous mutations identified in the KCNA5 gene from idiopathic pulmonary arterial hypertension (IPAH) patients localize to the NH 2 -terminal tetramerization domain (T1 domain). A , left : schematic diagram of voltage-gated K + (K V ) channel subunit
    Figure Legend Snippet: Two nonsynonymous mutations identified in the KCNA5 gene from idiopathic pulmonary arterial hypertension (IPAH) patients localize to the NH 2 -terminal tetramerization domain (T1 domain). A , left : schematic diagram of voltage-gated K + (K V ) channel subunit

    Techniques Used:

    Cotransfection of K V β subunits affects KCNA5 channel kinetics. HEK-293 cells were transfected with WT KCNA5 alone (KCNA5) or in the presence of K V β1.3-hemagglutinin (HA) (KCNA5/K V β1.3). A and B : representative current recordings
    Figure Legend Snippet: Cotransfection of K V β subunits affects KCNA5 channel kinetics. HEK-293 cells were transfected with WT KCNA5 alone (KCNA5) or in the presence of K V β1.3-hemagglutinin (HA) (KCNA5/K V β1.3). A and B : representative current recordings

    Techniques Used: Cotransfection, Transfection

    Voltage-dependent inactivation is accelerated in the G182R mutant KCNA5 channel. A standard 2-pulse inactivation protocol was used to determine channel availability after a 10-s prepulse in HEK-293 cells transiently transfected with WT KCNA5, G182R, E211D,
    Figure Legend Snippet: Voltage-dependent inactivation is accelerated in the G182R mutant KCNA5 channel. A standard 2-pulse inactivation protocol was used to determine channel availability after a 10-s prepulse in HEK-293 cells transiently transfected with WT KCNA5, G182R, E211D,

    Techniques Used: Mutagenesis, Transfection

    G182R protein expression is significantly decreased in COS-1 cells. A : COS-1 cells were transiently transfected with water (Mock), WT-KCNA5, G182R, E211D, or G182R/E211D. Representative images are shown at ×40 magnification. B : transfected cells
    Figure Legend Snippet: G182R protein expression is significantly decreased in COS-1 cells. A : COS-1 cells were transiently transfected with water (Mock), WT-KCNA5, G182R, E211D, or G182R/E211D. Representative images are shown at ×40 magnification. B : transfected cells

    Techniques Used: Expressing, Transfection

    19) Product Images from "Metabolic regulation of Kv channels and cardiac repolarization by Kvβ2 subunits"

    Article Title: Metabolic regulation of Kv channels and cardiac repolarization by Kvβ2 subunits

    Journal: Journal of molecular and cellular cardiology

    doi: 10.1016/j.yjmcc.2019.09.013

    Myocardial Kvβ2 associates with Kv1 and Kv4 proteins. ( A ) Representative blot images showing immunoreactive bands for Kv1.4, Kv1.5, Kv4.2, Kv4.3 and Kvβ2 in whole heart lysates and Kvβ2 immunoprecipitates. Absence of immunoreactive bands at expected molecular weights for each protein is also shown for mouse IgG immunoprecipitates as a negative control. Representative of 3 independent experiments. ( B ) Representative blot image showing immunoreactivity for Kvβ2 (predicted molecular weight, ~37 kDa) in Kvβ2 immunoprecipitates from heart lysates of wild type and Kvβ2 −/− animals. ( C ) Differential interference contrast (DIC) and proximity ligation assay (PLA)-associated fluorescence images of isolated adult ventricular myocytes from wild type and Kvβ2 −/− animals after PLA targeting of Kvβ2 complexes using mouse and rabbit-derived anti-Kvβ2 primary antibodies. PLA images are shown as flattened 2D maximum intensity z-projections from z-series captured for each cell. ( D ) PLA images of isolated ventricular myocytes treated with anti-Kvβ2 only, or anti-Kvβ2 with anti-Kv1.4, anti-Kv1.5, anti-Kv2.1 anti-Kv4.2, anti-Kv4.3. ( E ) Summary of fluorescent PLA-associated punctae, normalized to cell footprint area, for cells treated with anti-Kvβ2 only, or anti-Kvβ2 with anti-Kv1.4, anti-Kv1.5, anti-Kv2.1, anti-Kv4.2 and anti-Kv4.3 primary antibodies (n = 6–10). *P
    Figure Legend Snippet: Myocardial Kvβ2 associates with Kv1 and Kv4 proteins. ( A ) Representative blot images showing immunoreactive bands for Kv1.4, Kv1.5, Kv4.2, Kv4.3 and Kvβ2 in whole heart lysates and Kvβ2 immunoprecipitates. Absence of immunoreactive bands at expected molecular weights for each protein is also shown for mouse IgG immunoprecipitates as a negative control. Representative of 3 independent experiments. ( B ) Representative blot image showing immunoreactivity for Kvβ2 (predicted molecular weight, ~37 kDa) in Kvβ2 immunoprecipitates from heart lysates of wild type and Kvβ2 −/− animals. ( C ) Differential interference contrast (DIC) and proximity ligation assay (PLA)-associated fluorescence images of isolated adult ventricular myocytes from wild type and Kvβ2 −/− animals after PLA targeting of Kvβ2 complexes using mouse and rabbit-derived anti-Kvβ2 primary antibodies. PLA images are shown as flattened 2D maximum intensity z-projections from z-series captured for each cell. ( D ) PLA images of isolated ventricular myocytes treated with anti-Kvβ2 only, or anti-Kvβ2 with anti-Kv1.4, anti-Kv1.5, anti-Kv2.1 anti-Kv4.2, anti-Kv4.3. ( E ) Summary of fluorescent PLA-associated punctae, normalized to cell footprint area, for cells treated with anti-Kvβ2 only, or anti-Kvβ2 with anti-Kv1.4, anti-Kv1.5, anti-Kv2.1, anti-Kv4.2 and anti-Kv4.3 primary antibodies (n = 6–10). *P

    Techniques Used: Negative Control, Molecular Weight, Proximity Ligation Assay, Fluorescence, Isolation, Derivative Assay

    Kvβ2 promotes Kv1 and Kv4 surface expression in cardiac myocytes. ( A ) Differential interference contrast (DIC) and confocal images showing Kvβ2-associated fluorescence (red) in isolated cardiac myocytes from wild type (wt) and Kvβ2 −/− animals. Nuclei (dapi) are shown in blue. ( B ) Western blots showing immunoreactive bands for Kv pore-forming and auxiliary subunits at respective predicted molecular weights as indicated in heart lysates from wt (n = 3) and Kvβ2 −/− (n = 3) animals. As a representative loading control, immunoreactive bands for GAPDH are shown for each lane of Kv1.5 blot. ( C ) Summarized densitometric data for Kv1.4, Kv1.5, Kv2.1, Kv4.2, Kv4.3, Kvβ1.1, Kvβ1.2, Kvβ2, and KChIP2 proteins in heart lysates of wt animals. Data are normalized to GAPDH (run for each blot) and expressed relative to wt controls. *P
    Figure Legend Snippet: Kvβ2 promotes Kv1 and Kv4 surface expression in cardiac myocytes. ( A ) Differential interference contrast (DIC) and confocal images showing Kvβ2-associated fluorescence (red) in isolated cardiac myocytes from wild type (wt) and Kvβ2 −/− animals. Nuclei (dapi) are shown in blue. ( B ) Western blots showing immunoreactive bands for Kv pore-forming and auxiliary subunits at respective predicted molecular weights as indicated in heart lysates from wt (n = 3) and Kvβ2 −/− (n = 3) animals. As a representative loading control, immunoreactive bands for GAPDH are shown for each lane of Kv1.5 blot. ( C ) Summarized densitometric data for Kv1.4, Kv1.5, Kv2.1, Kv4.2, Kv4.3, Kvβ1.1, Kvβ1.2, Kvβ2, and KChIP2 proteins in heart lysates of wt animals. Data are normalized to GAPDH (run for each blot) and expressed relative to wt controls. *P

    Techniques Used: Expressing, Fluorescence, Isolation, Western Blot

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    Alomone Labs rabbit anti kv1 5 antibody
    Mutant <t>KCNA5</t> is located in perinuclear packets and not on the cell surface of transfected HEK-293 cells and human (h)PASMC. A : HEK-293 cells transfected with WT KCNA5 ( a and c ) or G182R ( b and d ) were stained with anti-KCNA5 antibody (Ab-KCNA5, red) and
    Rabbit Anti Kv1 5 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Mutant KCNA5 is located in perinuclear packets and not on the cell surface of transfected HEK-293 cells and human (h)PASMC. A : HEK-293 cells transfected with WT KCNA5 ( a and c ) or G182R ( b and d ) were stained with anti-KCNA5 antibody (Ab-KCNA5, red) and

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Tetramerization domain mutations in KCNA5 affect channel kinetics and cause abnormal trafficking patterns

    doi: 10.1152/ajpcell.00464.2009

    Figure Lengend Snippet: Mutant KCNA5 is located in perinuclear packets and not on the cell surface of transfected HEK-293 cells and human (h)PASMC. A : HEK-293 cells transfected with WT KCNA5 ( a and c ) or G182R ( b and d ) were stained with anti-KCNA5 antibody (Ab-KCNA5, red) and

    Article Snippet: After cells were fixed in 4% paraformaldehyde-PBS for 15 min at room temperature, coverslips were incubated in blocking solution (2% BSA, 2% FBS, 0.1% Triton X-100 in PBS) for 1 h. Coverslips were then exposed to rabbit anti-KV1.5 antibody (Alomone Labs) at a dilution of 1:100 in blocking solution for 2 h at room temperature.

    Techniques: Mutagenesis, Transfection, Staining

    G182R and E211D mutations cause incomplete processing of KCNA5 in HEK-293 cells. HEK-293 cells were transfected with WT KCNA5, G182R, E211D, or G182R/E211D and subjected to standard immunoblot procedures. A : representative immunoblot from cells transfected

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Tetramerization domain mutations in KCNA5 affect channel kinetics and cause abnormal trafficking patterns

    doi: 10.1152/ajpcell.00464.2009

    Figure Lengend Snippet: G182R and E211D mutations cause incomplete processing of KCNA5 in HEK-293 cells. HEK-293 cells were transfected with WT KCNA5, G182R, E211D, or G182R/E211D and subjected to standard immunoblot procedures. A : representative immunoblot from cells transfected

    Article Snippet: After cells were fixed in 4% paraformaldehyde-PBS for 15 min at room temperature, coverslips were incubated in blocking solution (2% BSA, 2% FBS, 0.1% Triton X-100 in PBS) for 1 h. Coverslips were then exposed to rabbit anti-KV1.5 antibody (Alomone Labs) at a dilution of 1:100 in blocking solution for 2 h at room temperature.

    Techniques: Transfection

    Mutant KCNA5 forms functional homotetrameric channels. A : COS-1, HEK-293, and human pulmonary artery smooth muscle cells (PASMC) were transiently transfected with either empty vector [green fluorescent protein (GFP)] or WT KCNA5 (WT) as indicated. Whole

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Tetramerization domain mutations in KCNA5 affect channel kinetics and cause abnormal trafficking patterns

    doi: 10.1152/ajpcell.00464.2009

    Figure Lengend Snippet: Mutant KCNA5 forms functional homotetrameric channels. A : COS-1, HEK-293, and human pulmonary artery smooth muscle cells (PASMC) were transiently transfected with either empty vector [green fluorescent protein (GFP)] or WT KCNA5 (WT) as indicated. Whole

    Article Snippet: After cells were fixed in 4% paraformaldehyde-PBS for 15 min at room temperature, coverslips were incubated in blocking solution (2% BSA, 2% FBS, 0.1% Triton X-100 in PBS) for 1 h. Coverslips were then exposed to rabbit anti-KV1.5 antibody (Alomone Labs) at a dilution of 1:100 in blocking solution for 2 h at room temperature.

    Techniques: Mutagenesis, Functional Assay, Transfection, Plasmid Preparation

    Mutations in KCNA5 at G182 and E211 do not affect the pharmacological effect of 4-aminopyridine (4-AP). A : a standard I-V pulse protocol was delivered to HEK-293 cells transiently transfected with the indicated vector [WT KCNA5 ( a ), G182R ( b ), E211D (

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Tetramerization domain mutations in KCNA5 affect channel kinetics and cause abnormal trafficking patterns

    doi: 10.1152/ajpcell.00464.2009

    Figure Lengend Snippet: Mutations in KCNA5 at G182 and E211 do not affect the pharmacological effect of 4-aminopyridine (4-AP). A : a standard I-V pulse protocol was delivered to HEK-293 cells transiently transfected with the indicated vector [WT KCNA5 ( a ), G182R ( b ), E211D (

    Article Snippet: After cells were fixed in 4% paraformaldehyde-PBS for 15 min at room temperature, coverslips were incubated in blocking solution (2% BSA, 2% FBS, 0.1% Triton X-100 in PBS) for 1 h. Coverslips were then exposed to rabbit anti-KV1.5 antibody (Alomone Labs) at a dilution of 1:100 in blocking solution for 2 h at room temperature.

    Techniques: Transfection, Plasmid Preparation

    Decreased G182R expression in COS-1 cells cannot be rescued by overexpression of K V β subunits. A , a : HEK-293 cells were transfected with WT KCNA5 or K V β1.3-HA alone or cotransfected with WT KCNA5, G182R, E211D, or G182R/E211D and K V β1.3-HA.

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Tetramerization domain mutations in KCNA5 affect channel kinetics and cause abnormal trafficking patterns

    doi: 10.1152/ajpcell.00464.2009

    Figure Lengend Snippet: Decreased G182R expression in COS-1 cells cannot be rescued by overexpression of K V β subunits. A , a : HEK-293 cells were transfected with WT KCNA5 or K V β1.3-HA alone or cotransfected with WT KCNA5, G182R, E211D, or G182R/E211D and K V β1.3-HA.

    Article Snippet: After cells were fixed in 4% paraformaldehyde-PBS for 15 min at room temperature, coverslips were incubated in blocking solution (2% BSA, 2% FBS, 0.1% Triton X-100 in PBS) for 1 h. Coverslips were then exposed to rabbit anti-KV1.5 antibody (Alomone Labs) at a dilution of 1:100 in blocking solution for 2 h at room temperature.

    Techniques: Expressing, Over Expression, Transfection

    Two nonsynonymous mutations identified in the KCNA5 gene from idiopathic pulmonary arterial hypertension (IPAH) patients localize to the NH 2 -terminal tetramerization domain (T1 domain). A , left : schematic diagram of voltage-gated K + (K V ) channel subunit

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Tetramerization domain mutations in KCNA5 affect channel kinetics and cause abnormal trafficking patterns

    doi: 10.1152/ajpcell.00464.2009

    Figure Lengend Snippet: Two nonsynonymous mutations identified in the KCNA5 gene from idiopathic pulmonary arterial hypertension (IPAH) patients localize to the NH 2 -terminal tetramerization domain (T1 domain). A , left : schematic diagram of voltage-gated K + (K V ) channel subunit

    Article Snippet: After cells were fixed in 4% paraformaldehyde-PBS for 15 min at room temperature, coverslips were incubated in blocking solution (2% BSA, 2% FBS, 0.1% Triton X-100 in PBS) for 1 h. Coverslips were then exposed to rabbit anti-KV1.5 antibody (Alomone Labs) at a dilution of 1:100 in blocking solution for 2 h at room temperature.

    Techniques:

    Cotransfection of K V β subunits affects KCNA5 channel kinetics. HEK-293 cells were transfected with WT KCNA5 alone (KCNA5) or in the presence of K V β1.3-hemagglutinin (HA) (KCNA5/K V β1.3). A and B : representative current recordings

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Tetramerization domain mutations in KCNA5 affect channel kinetics and cause abnormal trafficking patterns

    doi: 10.1152/ajpcell.00464.2009

    Figure Lengend Snippet: Cotransfection of K V β subunits affects KCNA5 channel kinetics. HEK-293 cells were transfected with WT KCNA5 alone (KCNA5) or in the presence of K V β1.3-hemagglutinin (HA) (KCNA5/K V β1.3). A and B : representative current recordings

    Article Snippet: After cells were fixed in 4% paraformaldehyde-PBS for 15 min at room temperature, coverslips were incubated in blocking solution (2% BSA, 2% FBS, 0.1% Triton X-100 in PBS) for 1 h. Coverslips were then exposed to rabbit anti-KV1.5 antibody (Alomone Labs) at a dilution of 1:100 in blocking solution for 2 h at room temperature.

    Techniques: Cotransfection, Transfection

    Voltage-dependent inactivation is accelerated in the G182R mutant KCNA5 channel. A standard 2-pulse inactivation protocol was used to determine channel availability after a 10-s prepulse in HEK-293 cells transiently transfected with WT KCNA5, G182R, E211D,

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Tetramerization domain mutations in KCNA5 affect channel kinetics and cause abnormal trafficking patterns

    doi: 10.1152/ajpcell.00464.2009

    Figure Lengend Snippet: Voltage-dependent inactivation is accelerated in the G182R mutant KCNA5 channel. A standard 2-pulse inactivation protocol was used to determine channel availability after a 10-s prepulse in HEK-293 cells transiently transfected with WT KCNA5, G182R, E211D,

    Article Snippet: After cells were fixed in 4% paraformaldehyde-PBS for 15 min at room temperature, coverslips were incubated in blocking solution (2% BSA, 2% FBS, 0.1% Triton X-100 in PBS) for 1 h. Coverslips were then exposed to rabbit anti-KV1.5 antibody (Alomone Labs) at a dilution of 1:100 in blocking solution for 2 h at room temperature.

    Techniques: Mutagenesis, Transfection

    G182R protein expression is significantly decreased in COS-1 cells. A : COS-1 cells were transiently transfected with water (Mock), WT-KCNA5, G182R, E211D, or G182R/E211D. Representative images are shown at ×40 magnification. B : transfected cells

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Tetramerization domain mutations in KCNA5 affect channel kinetics and cause abnormal trafficking patterns

    doi: 10.1152/ajpcell.00464.2009

    Figure Lengend Snippet: G182R protein expression is significantly decreased in COS-1 cells. A : COS-1 cells were transiently transfected with water (Mock), WT-KCNA5, G182R, E211D, or G182R/E211D. Representative images are shown at ×40 magnification. B : transfected cells

    Article Snippet: After cells were fixed in 4% paraformaldehyde-PBS for 15 min at room temperature, coverslips were incubated in blocking solution (2% BSA, 2% FBS, 0.1% Triton X-100 in PBS) for 1 h. Coverslips were then exposed to rabbit anti-KV1.5 antibody (Alomone Labs) at a dilution of 1:100 in blocking solution for 2 h at room temperature.

    Techniques: Expressing, Transfection

    Characterization of the Kv1.3 and Kv1.5 mutant channels containing the YS segment. A , average normalized activation and inactivation curves are shown as conductance-voltage relationships for Kv1.3, Kv1.5, the truncated Kv1.3-YS channel, and the chimeras Kv1.5-YS 532 and Kv1.5-YS 613 . All datasets were fitted to Boltzmann functions. Each data point is the mean ± S.E. of 6–11 cells. B , confocal images of non-permeabilized cells transfected with Kv1.3-YS-Cherry, Kv1.5-YS 532 -EGFP, and Kv1.5-YS 613 -EGFP. An extracellular anti-Kv1.3 antibody was used to label Kv1.3-YS ( green ), whereas the extracellular anti-Kv1.5 antibody was used for Kv1.5-YS 532 and Kv1.5-YS 613 chimeras ( red ). Nuclei were stained by Hoechst ( blue ). C , proliferation rate of the indicated channels or GFP-transfected cells (control) was determined by measuring EdU incorporation. Significant differences when comparing to Kv1.3 (*) or to control (#) are indicated. Statistical analysis was performed with one-way ANOVA followed by a Tukey's HSD multiple comparison. Each bar is the average of 9–15 determinations from 5 different assays. D , the average peak current amplitude obtained in cell-attached experiments for Kv1.5 channels and all the Kv1.5 chimeras was plotted against the % of the channels expressed at the plasma membrane ( upper graph ) or their normalized effect on proliferation (taking 100% as the proliferation rate of GFP-transfected HEK cells, lower graph ). The correlation between expression and current was fit to a linear regression curve ( y = 18.54 + 0.0066x, R 2 = 0.85, p = 0.008), but there was no correlation between proliferation and current amplitude ( R 2 = 0.23, p = 0.19).

    Journal: The Journal of Biological Chemistry

    Article Title: Molecular Determinants of Kv1.3 Potassium Channels-induced Proliferation *

    doi: 10.1074/jbc.M115.678995

    Figure Lengend Snippet: Characterization of the Kv1.3 and Kv1.5 mutant channels containing the YS segment. A , average normalized activation and inactivation curves are shown as conductance-voltage relationships for Kv1.3, Kv1.5, the truncated Kv1.3-YS channel, and the chimeras Kv1.5-YS 532 and Kv1.5-YS 613 . All datasets were fitted to Boltzmann functions. Each data point is the mean ± S.E. of 6–11 cells. B , confocal images of non-permeabilized cells transfected with Kv1.3-YS-Cherry, Kv1.5-YS 532 -EGFP, and Kv1.5-YS 613 -EGFP. An extracellular anti-Kv1.3 antibody was used to label Kv1.3-YS ( green ), whereas the extracellular anti-Kv1.5 antibody was used for Kv1.5-YS 532 and Kv1.5-YS 613 chimeras ( red ). Nuclei were stained by Hoechst ( blue ). C , proliferation rate of the indicated channels or GFP-transfected cells (control) was determined by measuring EdU incorporation. Significant differences when comparing to Kv1.3 (*) or to control (#) are indicated. Statistical analysis was performed with one-way ANOVA followed by a Tukey's HSD multiple comparison. Each bar is the average of 9–15 determinations from 5 different assays. D , the average peak current amplitude obtained in cell-attached experiments for Kv1.5 channels and all the Kv1.5 chimeras was plotted against the % of the channels expressed at the plasma membrane ( upper graph ) or their normalized effect on proliferation (taking 100% as the proliferation rate of GFP-transfected HEK cells, lower graph ). The correlation between expression and current was fit to a linear regression curve ( y = 18.54 + 0.0066x, R 2 = 0.85, p = 0.008), but there was no correlation between proliferation and current amplitude ( R 2 = 0.23, p = 0.19).

    Article Snippet: Non-permeabilized cells were incubated with anti-Kv1.3 or anti Kv1.5 extracellular primary antibodies (APC101 or APC150, Alomone Labs), whereas permeabilized cells were incubated with anti-Kv1.3 COOH (75-009, NeuroMab) or anti-Kv1.5 COOH (APC004, Alomone Labs), all at a final concentration of 1:50.

    Techniques: Mutagenesis, Activation Assay, Transfection, Staining, Expressing

    PKC but not AMPK activation reduces Kv1.5 surface expression in HL-1 cells. ( A and B ) Confocal scans of HL-1 cells transiently transfected with Kv1.5 cDNA and treated with PMA (100 nM), AICAR (1 mM) or PT1 (200 μM). As illustrated, surface expression of Kv1.5 channels was reduced in response to PKC activation ( A ). This response was not observed with either of the 2 AMPK activators ( B ). Cytoskeletal actin staining was used as a marker for the membrane. Scalebar, 10 μm. ( C ) Representative traces of Kv1.5 currents recorded in HL-1 cells before and after treatment with PMA. Protocol shown in the insert. ( D ) Current-voltage relationship of currents recorded in Kv1.5 transfected and untransfected HL-1 cells. PMA significantly down regulated the current level by 74%. Numbers of cells are: Kv1.5 (n=12); Kv1.5+PMC (n=13); untransfected (n=8); untransfected+PMA (n=10).

    Journal: Channels

    Article Title: PKC and AMPK regulation of Kv1.5 potassium channels

    doi: 10.1080/19336950.2015.1036205

    Figure Lengend Snippet: PKC but not AMPK activation reduces Kv1.5 surface expression in HL-1 cells. ( A and B ) Confocal scans of HL-1 cells transiently transfected with Kv1.5 cDNA and treated with PMA (100 nM), AICAR (1 mM) or PT1 (200 μM). As illustrated, surface expression of Kv1.5 channels was reduced in response to PKC activation ( A ). This response was not observed with either of the 2 AMPK activators ( B ). Cytoskeletal actin staining was used as a marker for the membrane. Scalebar, 10 μm. ( C ) Representative traces of Kv1.5 currents recorded in HL-1 cells before and after treatment with PMA. Protocol shown in the insert. ( D ) Current-voltage relationship of currents recorded in Kv1.5 transfected and untransfected HL-1 cells. PMA significantly down regulated the current level by 74%. Numbers of cells are: Kv1.5 (n=12); Kv1.5+PMC (n=13); untransfected (n=8); untransfected+PMA (n=10).

    Article Snippet: The antibodies used in this study were rabbit polyclonal anti-Kv1.5 (1:50, APC-004, Alomone Labs), Alexa Fluor®488-conjugated donkey anti-rabbit IgG (1:200, Invitrogen), Alexa Flour®647 -conjugated phalloidin (1:200, Invitrogen) and 4′,6-diamidino-2-phenylindole (DAPI, 1:300, Invitrogen).

    Techniques: Activation Assay, Expressing, Transfection, Staining, Marker

    Two-electrode voltage clamp recordings of Xenopus laevis oocytes expressing Kv1.5 +/− Nedd4–2 following PKC and AMPK activation. ( A-D ) Representative Kv1.5 current recordings following a step protocol. ( E ) PKC activation by PMA resulted in a drastic Kv1.5 current reduction both in the presence and absence of co-expressed Nedd4–2. ( E ) Similar experiments were conducted using the AMPK activator ZMP (100 nM). Contrary to PKC activation, AMPK activation did not affect Kv1.5 current levels when Nedd4–2 was not co-expressed. However, when Nedd4–2 was co-injected, leading to a down-regulation of Kv1.5 surface current, ZMP induced a further current reduction. The number of cells in each group was (n > 10).

    Journal: Channels

    Article Title: PKC and AMPK regulation of Kv1.5 potassium channels

    doi: 10.1080/19336950.2015.1036205

    Figure Lengend Snippet: Two-electrode voltage clamp recordings of Xenopus laevis oocytes expressing Kv1.5 +/− Nedd4–2 following PKC and AMPK activation. ( A-D ) Representative Kv1.5 current recordings following a step protocol. ( E ) PKC activation by PMA resulted in a drastic Kv1.5 current reduction both in the presence and absence of co-expressed Nedd4–2. ( E ) Similar experiments were conducted using the AMPK activator ZMP (100 nM). Contrary to PKC activation, AMPK activation did not affect Kv1.5 current levels when Nedd4–2 was not co-expressed. However, when Nedd4–2 was co-injected, leading to a down-regulation of Kv1.5 surface current, ZMP induced a further current reduction. The number of cells in each group was (n > 10).

    Article Snippet: The antibodies used in this study were rabbit polyclonal anti-Kv1.5 (1:50, APC-004, Alomone Labs), Alexa Fluor®488-conjugated donkey anti-rabbit IgG (1:200, Invitrogen), Alexa Flour®647 -conjugated phalloidin (1:200, Invitrogen) and 4′,6-diamidino-2-phenylindole (DAPI, 1:300, Invitrogen).

    Techniques: Expressing, Activation Assay, Injection

    Schematic illustration of the PKC and AMPK pathways resulting in Kv1.5 downregulation. The polarization of MDCK cells triggers activation of PKC and AMPK kinases. PKC activation can be mimicked by PMA. We suggest this activation of PKC can impact Kv1.5 channels through 2 pathways, depending on which the cell system used. This might be through AMPK activation (that can be mimicked by PT1, AICAR and ZMP), which will activate Nedd4 ubiquitylating enzymes or by another so far undisclosed mechanism. Activation of both pathways results in a reduced Kv1.5 surface expression.

    Journal: Channels

    Article Title: PKC and AMPK regulation of Kv1.5 potassium channels

    doi: 10.1080/19336950.2015.1036205

    Figure Lengend Snippet: Schematic illustration of the PKC and AMPK pathways resulting in Kv1.5 downregulation. The polarization of MDCK cells triggers activation of PKC and AMPK kinases. PKC activation can be mimicked by PMA. We suggest this activation of PKC can impact Kv1.5 channels through 2 pathways, depending on which the cell system used. This might be through AMPK activation (that can be mimicked by PT1, AICAR and ZMP), which will activate Nedd4 ubiquitylating enzymes or by another so far undisclosed mechanism. Activation of both pathways results in a reduced Kv1.5 surface expression.

    Article Snippet: The antibodies used in this study were rabbit polyclonal anti-Kv1.5 (1:50, APC-004, Alomone Labs), Alexa Fluor®488-conjugated donkey anti-rabbit IgG (1:200, Invitrogen), Alexa Flour®647 -conjugated phalloidin (1:200, Invitrogen) and 4′,6-diamidino-2-phenylindole (DAPI, 1:300, Invitrogen).

    Techniques: Activation Assay, Expressing

    Kv1.5 surface expression is reduced in response to cell polarization, to PKC activation and to AMPK activation in MDCK cells. ( A ) MDCK cells transiently co-transfected with DsRed-ER and Kv1.5 cDNA and subjected to a calcium switch assay (see Materials and Methods). At different time points after initiation of the calcium switch, coverslips were fixed and stained for Kv1.5. Illustrated are confocal horizontal scans of the MDCK cells before the initiation of the calcium switch (t =0h, cells grown in low calcium medium) and 30 min and 3h after the initiation of the polarization process following addition of calcium containing medium. As illustrated, surface expressed Kv1.5 channels disappear from the membrane during the 3 hour calcium switch. ( B and C ) MDCK cells transiently co-transfected with DsRed-ER and Kv1.5 and grown in low calcium media. The cells were treated with the PKC activator PMA (100 nM) ( B ) or the 2 AMPK activators AICAR (500 μM) and PT1 (200 μM) ( C ) for up to 6 hours. As illustrated, both activation of PKC and AMPK lead to disappearance of surface expressed Kv1.5 channels. Cytoskeletal actin staining was used as a membrane marker. Scalebar,10 μm.

    Journal: Channels

    Article Title: PKC and AMPK regulation of Kv1.5 potassium channels

    doi: 10.1080/19336950.2015.1036205

    Figure Lengend Snippet: Kv1.5 surface expression is reduced in response to cell polarization, to PKC activation and to AMPK activation in MDCK cells. ( A ) MDCK cells transiently co-transfected with DsRed-ER and Kv1.5 cDNA and subjected to a calcium switch assay (see Materials and Methods). At different time points after initiation of the calcium switch, coverslips were fixed and stained for Kv1.5. Illustrated are confocal horizontal scans of the MDCK cells before the initiation of the calcium switch (t =0h, cells grown in low calcium medium) and 30 min and 3h after the initiation of the polarization process following addition of calcium containing medium. As illustrated, surface expressed Kv1.5 channels disappear from the membrane during the 3 hour calcium switch. ( B and C ) MDCK cells transiently co-transfected with DsRed-ER and Kv1.5 and grown in low calcium media. The cells were treated with the PKC activator PMA (100 nM) ( B ) or the 2 AMPK activators AICAR (500 μM) and PT1 (200 μM) ( C ) for up to 6 hours. As illustrated, both activation of PKC and AMPK lead to disappearance of surface expressed Kv1.5 channels. Cytoskeletal actin staining was used as a membrane marker. Scalebar,10 μm.

    Article Snippet: The antibodies used in this study were rabbit polyclonal anti-Kv1.5 (1:50, APC-004, Alomone Labs), Alexa Fluor®488-conjugated donkey anti-rabbit IgG (1:200, Invitrogen), Alexa Flour®647 -conjugated phalloidin (1:200, Invitrogen) and 4′,6-diamidino-2-phenylindole (DAPI, 1:300, Invitrogen).

    Techniques: Expressing, Activation Assay, Transfection, Staining, Marker

    miR‐206 inhibition in vivo increased Kv1.5 channel expression to restore CH ‐ PAH of IUGR rats. A , Fold‐change of miR‐206 expression by qRT ‐ PCR in PA smooth muscle and mesenteric artery smooth muscle of different groups. n=5 per group of CON , IUGR , CON ‐ CH and IUGR ‐ CH ; n=8 per group of CON ‐ CH ‐Anti206, IUGR ‐ CH ‐Anti206, CON ‐Anti206, and IUGR ‐Anti206;* P

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    Article Title: Increased Expression of MicroRNA‐206 Inhibits Potassium Voltage‐Gated Channel Subfamily A Member 5 in Pulmonary Arterial Smooth Muscle Cells and Is Related to Exaggerated Pulmonary Artery Hypertension Following Intrauterine Growth Retardation in Rats

    doi: 10.1161/JAHA.118.010456

    Figure Lengend Snippet: miR‐206 inhibition in vivo increased Kv1.5 channel expression to restore CH ‐ PAH of IUGR rats. A , Fold‐change of miR‐206 expression by qRT ‐ PCR in PA smooth muscle and mesenteric artery smooth muscle of different groups. n=5 per group of CON , IUGR , CON ‐ CH and IUGR ‐ CH ; n=8 per group of CON ‐ CH ‐Anti206, IUGR ‐ CH ‐Anti206, CON ‐Anti206, and IUGR ‐Anti206;* P

    Article Snippet: Total protein extracts and western blotting were performed as previously reported., The membrane proteins were immunoblotted with antibodies to Kv1.5 (APC‐004; Alomone Labs, Jerusalem, Israel).

    Techniques: Inhibition, In Vivo, Expressing, Quantitative RT-PCR

    miR‐206 inhibition in primary cultured PASMC s regulates Kv1.5 channel expression and prevents overproliferation of PASMC s from IUGR ‐ CH ‐ PAH rats. A , Fold‐change of miR‐206 in PASMC s of different groups. B , Fold‐change of mRNA of KCNA 5 in PASMC s of different groups. C and D , Representative images of immunoblotting and quantitative analysis of Kv1.5 α‐protein in PASMC s. Data in ( A , B , D ) are presented as means± SEM . * P

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    Article Title: Increased Expression of MicroRNA‐206 Inhibits Potassium Voltage‐Gated Channel Subfamily A Member 5 in Pulmonary Arterial Smooth Muscle Cells and Is Related to Exaggerated Pulmonary Artery Hypertension Following Intrauterine Growth Retardation in Rats

    doi: 10.1161/JAHA.118.010456

    Figure Lengend Snippet: miR‐206 inhibition in primary cultured PASMC s regulates Kv1.5 channel expression and prevents overproliferation of PASMC s from IUGR ‐ CH ‐ PAH rats. A , Fold‐change of miR‐206 in PASMC s of different groups. B , Fold‐change of mRNA of KCNA 5 in PASMC s of different groups. C and D , Representative images of immunoblotting and quantitative analysis of Kv1.5 α‐protein in PASMC s. Data in ( A , B , D ) are presented as means± SEM . * P

    Article Snippet: Total protein extracts and western blotting were performed as previously reported., The membrane proteins were immunoblotted with antibodies to Kv1.5 (APC‐004; Alomone Labs, Jerusalem, Israel).

    Techniques: Inhibition, Cell Culture, Expressing