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Millipore kv1 3
Fyn modulates the posttranslational modification of <t>Kv1.3.</t> (A) Western blot analysis of postmortem human PD and age-matched control brains showing increased phosphorylation of Kv1.3. (B) Immunoprecipitation of Fyn and Kv1.3 showing direct Fyn-Kv1.3 interaction after αSyn Agg treatment. ( C ) Duolink PLA showing αSyn Agg -induced interaction between Kv1.3 and Fyn. Scale bar: 25 μm. ( D ) Western blot of Fyn WT and KO PMCs revealed that Kv1.3 phosphorylation at residue 135 was Fyn dependent. ( E ) IHC analysis of substantia nigra from Fyn +/+ and Fyn –/– mice showing reduced phosphorylation of Kv1.3 after αSyn PFF injection. Scale bars: 100 μm; 60 μm (insets). ( F ) IHC of substantia nigra from MitoPark mice and their littermate controls showing that pharmacological inhibition of Fyn by saracatinib reduced Kv1.3 phosphorylation. Scale bar: 100 μm. ( G – J ) Immortalized MMCs were either transfected with WT Kv1.3 or aY135A Kv1.3 plasmid. ( G ) qRT-PCR analysis and ( H ) Griess assay showing reduced levels of inducible NOS (iNOS) and nitrite release, respectively, in Y135A Kv1.3-transfected cells compared with WT cells. ( I ) qRT-PCR analysis showing reduced IL-1β production in Y135A Kv1.3–transfected versus WT Kv1.3–transfected MMCs. ( J ) Luminex assay showing reduced IL-1β secretion in Y135A Kv1.3–transfected compared with WT Kv1.3–transfected MMCs. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. A 2-tailed Student’s t test was used to compare 2 groups in A . Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–4 biological replicates from 2–3 independent experiments. * P ≤ 0.05, ** P
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1) Product Images from "Kv1.3 modulates neuroinflammation and neurodegeneration in Parkinson’s disease"

Article Title: Kv1.3 modulates neuroinflammation and neurodegeneration in Parkinson’s disease

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI136174

Fyn modulates the posttranslational modification of Kv1.3. (A) Western blot analysis of postmortem human PD and age-matched control brains showing increased phosphorylation of Kv1.3. (B) Immunoprecipitation of Fyn and Kv1.3 showing direct Fyn-Kv1.3 interaction after αSyn Agg treatment. ( C ) Duolink PLA showing αSyn Agg -induced interaction between Kv1.3 and Fyn. Scale bar: 25 μm. ( D ) Western blot of Fyn WT and KO PMCs revealed that Kv1.3 phosphorylation at residue 135 was Fyn dependent. ( E ) IHC analysis of substantia nigra from Fyn +/+ and Fyn –/– mice showing reduced phosphorylation of Kv1.3 after αSyn PFF injection. Scale bars: 100 μm; 60 μm (insets). ( F ) IHC of substantia nigra from MitoPark mice and their littermate controls showing that pharmacological inhibition of Fyn by saracatinib reduced Kv1.3 phosphorylation. Scale bar: 100 μm. ( G – J ) Immortalized MMCs were either transfected with WT Kv1.3 or aY135A Kv1.3 plasmid. ( G ) qRT-PCR analysis and ( H ) Griess assay showing reduced levels of inducible NOS (iNOS) and nitrite release, respectively, in Y135A Kv1.3-transfected cells compared with WT cells. ( I ) qRT-PCR analysis showing reduced IL-1β production in Y135A Kv1.3–transfected versus WT Kv1.3–transfected MMCs. ( J ) Luminex assay showing reduced IL-1β secretion in Y135A Kv1.3–transfected compared with WT Kv1.3–transfected MMCs. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. A 2-tailed Student’s t test was used to compare 2 groups in A . Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–4 biological replicates from 2–3 independent experiments. * P ≤ 0.05, ** P
Figure Legend Snippet: Fyn modulates the posttranslational modification of Kv1.3. (A) Western blot analysis of postmortem human PD and age-matched control brains showing increased phosphorylation of Kv1.3. (B) Immunoprecipitation of Fyn and Kv1.3 showing direct Fyn-Kv1.3 interaction after αSyn Agg treatment. ( C ) Duolink PLA showing αSyn Agg -induced interaction between Kv1.3 and Fyn. Scale bar: 25 μm. ( D ) Western blot of Fyn WT and KO PMCs revealed that Kv1.3 phosphorylation at residue 135 was Fyn dependent. ( E ) IHC analysis of substantia nigra from Fyn +/+ and Fyn –/– mice showing reduced phosphorylation of Kv1.3 after αSyn PFF injection. Scale bars: 100 μm; 60 μm (insets). ( F ) IHC of substantia nigra from MitoPark mice and their littermate controls showing that pharmacological inhibition of Fyn by saracatinib reduced Kv1.3 phosphorylation. Scale bar: 100 μm. ( G – J ) Immortalized MMCs were either transfected with WT Kv1.3 or aY135A Kv1.3 plasmid. ( G ) qRT-PCR analysis and ( H ) Griess assay showing reduced levels of inducible NOS (iNOS) and nitrite release, respectively, in Y135A Kv1.3-transfected cells compared with WT cells. ( I ) qRT-PCR analysis showing reduced IL-1β production in Y135A Kv1.3–transfected versus WT Kv1.3–transfected MMCs. ( J ) Luminex assay showing reduced IL-1β secretion in Y135A Kv1.3–transfected compared with WT Kv1.3–transfected MMCs. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. A 2-tailed Student’s t test was used to compare 2 groups in A . Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–4 biological replicates from 2–3 independent experiments. * P ≤ 0.05, ** P

Techniques Used: Modification, Western Blot, Immunoprecipitation, Proximity Ligation Assay, Immunohistochemistry, Mouse Assay, Injection, Inhibition, Transfection, Plasmid Preparation, Quantitative RT-PCR, Griess Assay, Luminex

Kv1.3 inhibition protects against αSyn PFF -induced behavior deficit and dopaminergic neuronal loss. ( A ) Treatment paradigm corresponding to the αSyn PFF mouse model of PD. ( B ) Representative movement tracks showing that PAP-1 rescued movement deficits induced by αSyn PFF . ( C – E ) A VersaMax open-field test showed decreased ( C ) rest time and increased ( D ) horizontal activity and ( E ) total distance traveled for αSyn PFF mice treated with PAP-1. ( F and G ) HPLC showing that PAP-1 treatment protected against loss of ( F ) dopamine and ( G ) DOPAC induced by αSyn PFF . ( H ) Western blot analysis of TH showing loss of TH induced by αSyn PFF in the SNpc region. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–7 animals per group. * P ≤ 0.05, ** P
Figure Legend Snippet: Kv1.3 inhibition protects against αSyn PFF -induced behavior deficit and dopaminergic neuronal loss. ( A ) Treatment paradigm corresponding to the αSyn PFF mouse model of PD. ( B ) Representative movement tracks showing that PAP-1 rescued movement deficits induced by αSyn PFF . ( C – E ) A VersaMax open-field test showed decreased ( C ) rest time and increased ( D ) horizontal activity and ( E ) total distance traveled for αSyn PFF mice treated with PAP-1. ( F and G ) HPLC showing that PAP-1 treatment protected against loss of ( F ) dopamine and ( G ) DOPAC induced by αSyn PFF . ( H ) Western blot analysis of TH showing loss of TH induced by αSyn PFF in the SNpc region. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–7 animals per group. * P ≤ 0.05, ** P

Techniques Used: Inhibition, Activity Assay, Mouse Assay, High Performance Liquid Chromatography, Western Blot

Upregulated expression of the potassium channel Kv1.3 upon aggregated αSyn stimulation in ex vivo slices and B cells derived from patients with PD. ( A ) Midbrain slice cultures were treated with 1 μM αSyn Agg for 24 hours. qRT-PCR shows upregulated Kv1.3 mRNA expression. ( B ) Western blot shows upregulated Kv1.3 protein level in midbrain slice cultures treated with 1 μM αSyn Agg for 24 hours. ( C ) qRT-PCR of midbrain slice cultures treated with 1 μM αSyn Agg for 24 hours, revealing upregulation of the proinflammatory factors Nos2 , Csf2 , IL-6 , IL-1β , and Tnfa . ( D ) qRT-PCR shows increased Kv1.3 mRNA expression in B cell lymphocytes isolated from patients with PD compared with expression in B cell lymphocytes from age-matched controls. ( E ) Whole-cell patch clamping of B cell lymphocytes isolated from patients with PD showed higher Kv1.3 channel activity compared with that observed in age-matched controls ( n = 3 control and n = 3 PD). A 1-way ANOVA was used to compare multiple groups in C and D . Tukey’s post hoc analysis was applied. A 2-tailed Student’s t test was used to compare 2 groups. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–7 biological replicates from 2–3 independent experiments unless otherwise indicated. * P ≤ 0.05 and ** P
Figure Legend Snippet: Upregulated expression of the potassium channel Kv1.3 upon aggregated αSyn stimulation in ex vivo slices and B cells derived from patients with PD. ( A ) Midbrain slice cultures were treated with 1 μM αSyn Agg for 24 hours. qRT-PCR shows upregulated Kv1.3 mRNA expression. ( B ) Western blot shows upregulated Kv1.3 protein level in midbrain slice cultures treated with 1 μM αSyn Agg for 24 hours. ( C ) qRT-PCR of midbrain slice cultures treated with 1 μM αSyn Agg for 24 hours, revealing upregulation of the proinflammatory factors Nos2 , Csf2 , IL-6 , IL-1β , and Tnfa . ( D ) qRT-PCR shows increased Kv1.3 mRNA expression in B cell lymphocytes isolated from patients with PD compared with expression in B cell lymphocytes from age-matched controls. ( E ) Whole-cell patch clamping of B cell lymphocytes isolated from patients with PD showed higher Kv1.3 channel activity compared with that observed in age-matched controls ( n = 3 control and n = 3 PD). A 1-way ANOVA was used to compare multiple groups in C and D . Tukey’s post hoc analysis was applied. A 2-tailed Student’s t test was used to compare 2 groups. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–7 biological replicates from 2–3 independent experiments unless otherwise indicated. * P ≤ 0.05 and ** P

Techniques Used: Expressing, Ex Vivo, Derivative Assay, Quantitative RT-PCR, Western Blot, Isolation, Activity Assay

Kv1.3 expression is highly induced in microglial cells in experimental models of PD and postmortem PD brains. ( A ) Western blot showing increased Kv1.3 protein levels in the substantia nigra of the Syn-AAV mouse model of PD. ( B ) qRT-PCR analysis of 8- to 24-week-old nigral tissues from the MitoPark mouse model of PD showing Kv1.3 induction compared with age-matched littermate controls. ( C ) Western blot of 24-week-old nigral tissues from the MitoPark mouse model of PD (MP) showing induction of Kv1.3 protein expression compared with age-matched littermate control mice (LM). ( D ) IHC in 24-week-old nigral tissues from the MitoPark mouse model of PD showing higher Kv1.3 protein levels (red) in IBA1-positive microglial cells (green) compared with age-matched controls as revealed by their colocalization (yellow). Scale bar: 20 μm. ( E ) qRT-PCR analysis of nigral tissues from the MPTP mouse model revealing induction of Kv1.3 mRNA expression. ( F ) Western blot showing increased Kv1.3 protein levels in substantia nigra of the MPTP mouse model of PD. ( G ) qRT-PCR analysis of postmortem human PD brains showing elevated Kv1.3 mRNA expression. ( H ) Western blot of the SN region of postmortem human PD brain showing induction of Kv1.3 protein expression compared with age-matched controls. n = 6–8. ( I ) Immunostaining revealing higher Kv1.3 levels in the prefrontal cortex of postmortem human PD brains compared with age-matched controls. Lower panel shows the deconvoluted binary image used for analysis. Three regions per brain were analyzed. Scale bar: 200 μm. ( J ) Dual DAB staining showing induction of Kv1.3 expression in HLA-DR–positive microglial cells in patients with DLBs compared with age-matched controls. Scale bars: 100 μm; 20 μm (enlarged insets). A 1-way ANOVA was used to compare multiple groups. Tukey’s post hoc analysis was applied B . A 2-tailed Student’s t test was used to compare 2 groups. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–9 biological replicates from 2–3 independent experiments unless otherwise indicated. * P ≤ 0.05, ** P
Figure Legend Snippet: Kv1.3 expression is highly induced in microglial cells in experimental models of PD and postmortem PD brains. ( A ) Western blot showing increased Kv1.3 protein levels in the substantia nigra of the Syn-AAV mouse model of PD. ( B ) qRT-PCR analysis of 8- to 24-week-old nigral tissues from the MitoPark mouse model of PD showing Kv1.3 induction compared with age-matched littermate controls. ( C ) Western blot of 24-week-old nigral tissues from the MitoPark mouse model of PD (MP) showing induction of Kv1.3 protein expression compared with age-matched littermate control mice (LM). ( D ) IHC in 24-week-old nigral tissues from the MitoPark mouse model of PD showing higher Kv1.3 protein levels (red) in IBA1-positive microglial cells (green) compared with age-matched controls as revealed by their colocalization (yellow). Scale bar: 20 μm. ( E ) qRT-PCR analysis of nigral tissues from the MPTP mouse model revealing induction of Kv1.3 mRNA expression. ( F ) Western blot showing increased Kv1.3 protein levels in substantia nigra of the MPTP mouse model of PD. ( G ) qRT-PCR analysis of postmortem human PD brains showing elevated Kv1.3 mRNA expression. ( H ) Western blot of the SN region of postmortem human PD brain showing induction of Kv1.3 protein expression compared with age-matched controls. n = 6–8. ( I ) Immunostaining revealing higher Kv1.3 levels in the prefrontal cortex of postmortem human PD brains compared with age-matched controls. Lower panel shows the deconvoluted binary image used for analysis. Three regions per brain were analyzed. Scale bar: 200 μm. ( J ) Dual DAB staining showing induction of Kv1.3 expression in HLA-DR–positive microglial cells in patients with DLBs compared with age-matched controls. Scale bars: 100 μm; 20 μm (enlarged insets). A 1-way ANOVA was used to compare multiple groups. Tukey’s post hoc analysis was applied B . A 2-tailed Student’s t test was used to compare 2 groups. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–9 biological replicates from 2–3 independent experiments unless otherwise indicated. * P ≤ 0.05, ** P

Techniques Used: Expressing, Western Blot, Quantitative RT-PCR, Mouse Assay, Immunohistochemistry, Immunostaining, Staining

Fyn modulates the transcriptional regulation of Kv1.3 in microglial cells through the Fyn/PKCδ kinase signaling cascade. ( A ) In silico analysis of the promoter sequence of Kv1.3 revealed probable Nf-κB– and SP1-binding sites. ( B ) qRT-PCR analysis of immortalized MMCs cotreated with αSyn Agg and either SN50 (100 μg/mL) or SB203580 (1 μM), showing that both compounds attenuated αSyn Agg -induced Kv1.3 expression. ( C ) Western blot of Fyn WT and KO PMCs treated with αSyn Agg , showing that Fyn KO reduced the induction of the p38 MAPK pathway. ( D ) qRT-PCR analysis revealed that Fyn KO reduced αSyn Agg -induced Kv1.3 mRNA levels. ( E ) Whole-cell patch-clamp recording showing that Fyn KO attenuated αSyn Agg - and LPS-induced Kv1.3 activity compared with Fyn WT PMCs (WT control n = 24, WT αSyn Agg n = 12, WT LPS n = 29, Fyn KO αSyn Agg n = 20, Fyn KO LPS n = 15). ( F ) ICC showing that Fyn KO reduced αSyn Agg -induced Kv1.3 protein levels in PMCs. Scale bar: 15 μm. ( G ) ICC of PMCs revealed that αSyn Agg -induced Kv1.3 protein expression was reduced by PKCδ KO. Scale bar: 15 μm. ( H ) qRT-PCR analysis of PMCs showing that PKC KO reduced the expression of αSyn Agg -induced Kv1.3 mRNA. ( I ) Whole-cell patch clam recording of PMCs showing that PKC KO attenuated αSyn Agg - and LPS-induced Kv1.3 activity compared with PKC WT PMCs (WT control n = 24, WT αSyn Agg n = 12, WT LPS n = 20, PKC-KO αSyn Agg n = 29, PKC-KO LPS n = 35). Data are presented as the mean ± SD. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–4 biological replicates from 2–3 independent experiments unless otherwise indicated. * P ≤ 0.05, ** P
Figure Legend Snippet: Fyn modulates the transcriptional regulation of Kv1.3 in microglial cells through the Fyn/PKCδ kinase signaling cascade. ( A ) In silico analysis of the promoter sequence of Kv1.3 revealed probable Nf-κB– and SP1-binding sites. ( B ) qRT-PCR analysis of immortalized MMCs cotreated with αSyn Agg and either SN50 (100 μg/mL) or SB203580 (1 μM), showing that both compounds attenuated αSyn Agg -induced Kv1.3 expression. ( C ) Western blot of Fyn WT and KO PMCs treated with αSyn Agg , showing that Fyn KO reduced the induction of the p38 MAPK pathway. ( D ) qRT-PCR analysis revealed that Fyn KO reduced αSyn Agg -induced Kv1.3 mRNA levels. ( E ) Whole-cell patch-clamp recording showing that Fyn KO attenuated αSyn Agg - and LPS-induced Kv1.3 activity compared with Fyn WT PMCs (WT control n = 24, WT αSyn Agg n = 12, WT LPS n = 29, Fyn KO αSyn Agg n = 20, Fyn KO LPS n = 15). ( F ) ICC showing that Fyn KO reduced αSyn Agg -induced Kv1.3 protein levels in PMCs. Scale bar: 15 μm. ( G ) ICC of PMCs revealed that αSyn Agg -induced Kv1.3 protein expression was reduced by PKCδ KO. Scale bar: 15 μm. ( H ) qRT-PCR analysis of PMCs showing that PKC KO reduced the expression of αSyn Agg -induced Kv1.3 mRNA. ( I ) Whole-cell patch clam recording of PMCs showing that PKC KO attenuated αSyn Agg - and LPS-induced Kv1.3 activity compared with PKC WT PMCs (WT control n = 24, WT αSyn Agg n = 12, WT LPS n = 20, PKC-KO αSyn Agg n = 29, PKC-KO LPS n = 35). Data are presented as the mean ± SD. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–4 biological replicates from 2–3 independent experiments unless otherwise indicated. * P ≤ 0.05, ** P

Techniques Used: In Silico, Sequencing, Binding Assay, Quantitative RT-PCR, Expressing, Western Blot, Patch Clamp, Activity Assay, Immunocytochemistry

Kv1.3 modulates neuroinflammation in a cell culture model of PD. ( A – C ) Kv1.3 WT and KO PMCs were treated with 1 μM αSyn Agg for 24 hours. Luminex analysis shows that Kv1.3 KO reduced the release of the αSyn Agg -induced proinflammatory factors ( A ) TNF-α, ( B ) IL-12, and ( C ) IL-1β. ( D – H ) Immortalized MMCs were transfected with WT a Kv1.3 plasmid, and then 48 hours after transfection, cells were treated with 1 μM αSyn Agg for 24 hours. ( D – F ) qRT-PCR analysis showing that Kv1.3 overexpression aggravated αSyn Agg -induced production of the proinflammatory factors ( D ) Nos2 , ( E ) pro– IL-1β , and ( F ) TNF-α . ( G and H ) Luminex analysis showing that Kv1.3 overexpression potentiated the release of the proinflammatory factors ( G ) IL-6 and ( H ) IL-12. ( I ) Voltage ramp from –120 mV to 40 mV elicited a characteristic outward rectifying current in αSyn Agg -treated microglia that was sensitive to the Kv1.3-selective inhibitor PAP-1. ( J ) LDH assay showing that PAP-1 reduced αSyn Agg -induced LDH release from microglial cells. ( K – M ) Luminex assay revealing that PAP-1 attenuated the αSyn Agg -induced proinflammatory factors ( K ) IL-12, ( L ) TNF-α, and ( M ) IL-6. ( N ) Western blot analysis demonstrating that PAP-1 reduced αSyn Agg -induced NLRP3 expression. ( O ) ICC analysis revealed that PAP-1 reduced NLRP3 expression induced by αSyn Agg . Scale bar: 25 μm. A 1-way ANOVA was performed to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–4 biological replicates from 2–3 independent experiments. * P ≤ 0.05, ** P
Figure Legend Snippet: Kv1.3 modulates neuroinflammation in a cell culture model of PD. ( A – C ) Kv1.3 WT and KO PMCs were treated with 1 μM αSyn Agg for 24 hours. Luminex analysis shows that Kv1.3 KO reduced the release of the αSyn Agg -induced proinflammatory factors ( A ) TNF-α, ( B ) IL-12, and ( C ) IL-1β. ( D – H ) Immortalized MMCs were transfected with WT a Kv1.3 plasmid, and then 48 hours after transfection, cells were treated with 1 μM αSyn Agg for 24 hours. ( D – F ) qRT-PCR analysis showing that Kv1.3 overexpression aggravated αSyn Agg -induced production of the proinflammatory factors ( D ) Nos2 , ( E ) pro– IL-1β , and ( F ) TNF-α . ( G and H ) Luminex analysis showing that Kv1.3 overexpression potentiated the release of the proinflammatory factors ( G ) IL-6 and ( H ) IL-12. ( I ) Voltage ramp from –120 mV to 40 mV elicited a characteristic outward rectifying current in αSyn Agg -treated microglia that was sensitive to the Kv1.3-selective inhibitor PAP-1. ( J ) LDH assay showing that PAP-1 reduced αSyn Agg -induced LDH release from microglial cells. ( K – M ) Luminex assay revealing that PAP-1 attenuated the αSyn Agg -induced proinflammatory factors ( K ) IL-12, ( L ) TNF-α, and ( M ) IL-6. ( N ) Western blot analysis demonstrating that PAP-1 reduced αSyn Agg -induced NLRP3 expression. ( O ) ICC analysis revealed that PAP-1 reduced NLRP3 expression induced by αSyn Agg . Scale bar: 25 μm. A 1-way ANOVA was performed to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–4 biological replicates from 2–3 independent experiments. * P ≤ 0.05, ** P

Techniques Used: Cell Culture, Luminex, Transfection, Plasmid Preparation, Quantitative RT-PCR, Over Expression, Lactate Dehydrogenase Assay, Western Blot, Expressing, Immunocytochemistry

Upregulated expression of the potassium channel Kv1.3 upon aggregated αSyn stimulation in microglial cells in vitro. ( A ) Whole-cell patch-clamp recordings of PMCs treated with 1 μM αSyn Agg for 24–48 hours, showing αSyn Agg -induced increased Kv1.3 activity (control n = 24 and αSyn Agg n = 12). Kv1.3 was identified by its characteristic use dependence, which was revealed when applying a train of ten 200-ms pulses from –80 to 40 mV at 1-second intervals (1 Hz). ( B ) qRT-PCR showing that αSyn Agg induced Kv1.3 mRNA expression without significantly altering other potassium channels. ( C ) Western blot of αSyn Agg -induced Kv1.3 protein expression in PMCs. ( D ) ICC of αSyn Agg -induced Kv1.3 protein expression in PMCs. Scale bar: 100 μm. ( E ) Flow cytometric analysis of immortalized MMCs treated with 1 μM αSyn Agg for 24 hours, showing αSyn Agg -induced Kv1.3 surface expression. ( F ) qRT-PCR of human microglia treated with LPS (1 μg/mL) and IL-4 (20 ng/mL) for 6 hours, showing LPS-induced Kv1.3 expression. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied in B and F . A 2-tailed Student’s t test was used for all other figures when comparing 2 groups. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–5 biological replicates from 2–3 independent experiments unless otherwise noted. * P ≤ 0.05, ** P
Figure Legend Snippet: Upregulated expression of the potassium channel Kv1.3 upon aggregated αSyn stimulation in microglial cells in vitro. ( A ) Whole-cell patch-clamp recordings of PMCs treated with 1 μM αSyn Agg for 24–48 hours, showing αSyn Agg -induced increased Kv1.3 activity (control n = 24 and αSyn Agg n = 12). Kv1.3 was identified by its characteristic use dependence, which was revealed when applying a train of ten 200-ms pulses from –80 to 40 mV at 1-second intervals (1 Hz). ( B ) qRT-PCR showing that αSyn Agg induced Kv1.3 mRNA expression without significantly altering other potassium channels. ( C ) Western blot of αSyn Agg -induced Kv1.3 protein expression in PMCs. ( D ) ICC of αSyn Agg -induced Kv1.3 protein expression in PMCs. Scale bar: 100 μm. ( E ) Flow cytometric analysis of immortalized MMCs treated with 1 μM αSyn Agg for 24 hours, showing αSyn Agg -induced Kv1.3 surface expression. ( F ) qRT-PCR of human microglia treated with LPS (1 μg/mL) and IL-4 (20 ng/mL) for 6 hours, showing LPS-induced Kv1.3 expression. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied in B and F . A 2-tailed Student’s t test was used for all other figures when comparing 2 groups. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–5 biological replicates from 2–3 independent experiments unless otherwise noted. * P ≤ 0.05, ** P

Techniques Used: Expressing, In Vitro, Patch Clamp, Activity Assay, Quantitative RT-PCR, Western Blot, Immunocytochemistry

2) Product Images from "Kv1.3 modulates neuroinflammation and neurodegeneration in Parkinson’s disease"

Article Title: Kv1.3 modulates neuroinflammation and neurodegeneration in Parkinson’s disease

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI136174

Fyn modulates the posttranslational modification of Kv1.3. (A) Western blot analysis of postmortem human PD and age-matched control brains showing increased phosphorylation of Kv1.3. (B) Immunoprecipitation of Fyn and Kv1.3 showing direct Fyn-Kv1.3 interaction after αSyn Agg treatment. ( C ) Duolink PLA showing αSyn Agg -induced interaction between Kv1.3 and Fyn. Scale bar: 25 μm. ( D ) Western blot of Fyn WT and KO PMCs revealed that Kv1.3 phosphorylation at residue 135 was Fyn dependent. ( E ) IHC analysis of substantia nigra from Fyn +/+ and Fyn –/– mice showing reduced phosphorylation of Kv1.3 after αSyn PFF injection. Scale bars: 100 μm; 60 μm (insets). ( F ) IHC of substantia nigra from MitoPark mice and their littermate controls showing that pharmacological inhibition of Fyn by saracatinib reduced Kv1.3 phosphorylation. Scale bar: 100 μm. ( G – J ) Immortalized MMCs were either transfected with WT Kv1.3 or aY135A Kv1.3 plasmid. ( G ) qRT-PCR analysis and ( H ) Griess assay showing reduced levels of inducible NOS (iNOS) and nitrite release, respectively, in Y135A Kv1.3-transfected cells compared with WT cells. ( I ) qRT-PCR analysis showing reduced IL-1β production in Y135A Kv1.3–transfected versus WT Kv1.3–transfected MMCs. ( J ) Luminex assay showing reduced IL-1β secretion in Y135A Kv1.3–transfected compared with WT Kv1.3–transfected MMCs. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. A 2-tailed Student’s t test was used to compare 2 groups in A . Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–4 biological replicates from 2–3 independent experiments. * P ≤ 0.05, ** P
Figure Legend Snippet: Fyn modulates the posttranslational modification of Kv1.3. (A) Western blot analysis of postmortem human PD and age-matched control brains showing increased phosphorylation of Kv1.3. (B) Immunoprecipitation of Fyn and Kv1.3 showing direct Fyn-Kv1.3 interaction after αSyn Agg treatment. ( C ) Duolink PLA showing αSyn Agg -induced interaction between Kv1.3 and Fyn. Scale bar: 25 μm. ( D ) Western blot of Fyn WT and KO PMCs revealed that Kv1.3 phosphorylation at residue 135 was Fyn dependent. ( E ) IHC analysis of substantia nigra from Fyn +/+ and Fyn –/– mice showing reduced phosphorylation of Kv1.3 after αSyn PFF injection. Scale bars: 100 μm; 60 μm (insets). ( F ) IHC of substantia nigra from MitoPark mice and their littermate controls showing that pharmacological inhibition of Fyn by saracatinib reduced Kv1.3 phosphorylation. Scale bar: 100 μm. ( G – J ) Immortalized MMCs were either transfected with WT Kv1.3 or aY135A Kv1.3 plasmid. ( G ) qRT-PCR analysis and ( H ) Griess assay showing reduced levels of inducible NOS (iNOS) and nitrite release, respectively, in Y135A Kv1.3-transfected cells compared with WT cells. ( I ) qRT-PCR analysis showing reduced IL-1β production in Y135A Kv1.3–transfected versus WT Kv1.3–transfected MMCs. ( J ) Luminex assay showing reduced IL-1β secretion in Y135A Kv1.3–transfected compared with WT Kv1.3–transfected MMCs. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. A 2-tailed Student’s t test was used to compare 2 groups in A . Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–4 biological replicates from 2–3 independent experiments. * P ≤ 0.05, ** P

Techniques Used: Modification, Western Blot, Immunoprecipitation, Proximity Ligation Assay, Immunohistochemistry, Mouse Assay, Injection, Inhibition, Transfection, Plasmid Preparation, Quantitative RT-PCR, Griess Assay, Luminex

Kv1.3 inhibition protects against αSyn PFF -induced behavior deficit and dopaminergic neuronal loss. ( A ) Treatment paradigm corresponding to the αSyn PFF mouse model of PD. ( B ) Representative movement tracks showing that PAP-1 rescued movement deficits induced by αSyn PFF . ( C – E ) A VersaMax open-field test showed decreased ( C ) rest time and increased ( D ) horizontal activity and ( E ) total distance traveled for αSyn PFF mice treated with PAP-1. ( F and G ) HPLC showing that PAP-1 treatment protected against loss of ( F ) dopamine and ( G ) DOPAC induced by αSyn PFF . ( H ) Western blot analysis of TH showing loss of TH induced by αSyn PFF in the SNpc region. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–7 animals per group. * P ≤ 0.05, ** P
Figure Legend Snippet: Kv1.3 inhibition protects against αSyn PFF -induced behavior deficit and dopaminergic neuronal loss. ( A ) Treatment paradigm corresponding to the αSyn PFF mouse model of PD. ( B ) Representative movement tracks showing that PAP-1 rescued movement deficits induced by αSyn PFF . ( C – E ) A VersaMax open-field test showed decreased ( C ) rest time and increased ( D ) horizontal activity and ( E ) total distance traveled for αSyn PFF mice treated with PAP-1. ( F and G ) HPLC showing that PAP-1 treatment protected against loss of ( F ) dopamine and ( G ) DOPAC induced by αSyn PFF . ( H ) Western blot analysis of TH showing loss of TH induced by αSyn PFF in the SNpc region. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–7 animals per group. * P ≤ 0.05, ** P

Techniques Used: Inhibition, Activity Assay, Mouse Assay, High Performance Liquid Chromatography, Western Blot

Upregulated expression of the potassium channel Kv1.3 upon aggregated αSyn stimulation in ex vivo slices and B cells derived from patients with PD. ( A ) Midbrain slice cultures were treated with 1 μM αSyn Agg for 24 hours. qRT-PCR shows upregulated Kv1.3 mRNA expression. ( B ) Western blot shows upregulated Kv1.3 protein level in midbrain slice cultures treated with 1 μM αSyn Agg for 24 hours. ( C ) qRT-PCR of midbrain slice cultures treated with 1 μM αSyn Agg for 24 hours, revealing upregulation of the proinflammatory factors Nos2 , Csf2 , IL-6 , IL-1β , and Tnfa . ( D ) qRT-PCR shows increased Kv1.3 mRNA expression in B cell lymphocytes isolated from patients with PD compared with expression in B cell lymphocytes from age-matched controls. ( E ) Whole-cell patch clamping of B cell lymphocytes isolated from patients with PD showed higher Kv1.3 channel activity compared with that observed in age-matched controls ( n = 3 control and n = 3 PD). A 1-way ANOVA was used to compare multiple groups in C and D . Tukey’s post hoc analysis was applied. A 2-tailed Student’s t test was used to compare 2 groups. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–7 biological replicates from 2–3 independent experiments unless otherwise indicated. * P ≤ 0.05 and ** P
Figure Legend Snippet: Upregulated expression of the potassium channel Kv1.3 upon aggregated αSyn stimulation in ex vivo slices and B cells derived from patients with PD. ( A ) Midbrain slice cultures were treated with 1 μM αSyn Agg for 24 hours. qRT-PCR shows upregulated Kv1.3 mRNA expression. ( B ) Western blot shows upregulated Kv1.3 protein level in midbrain slice cultures treated with 1 μM αSyn Agg for 24 hours. ( C ) qRT-PCR of midbrain slice cultures treated with 1 μM αSyn Agg for 24 hours, revealing upregulation of the proinflammatory factors Nos2 , Csf2 , IL-6 , IL-1β , and Tnfa . ( D ) qRT-PCR shows increased Kv1.3 mRNA expression in B cell lymphocytes isolated from patients with PD compared with expression in B cell lymphocytes from age-matched controls. ( E ) Whole-cell patch clamping of B cell lymphocytes isolated from patients with PD showed higher Kv1.3 channel activity compared with that observed in age-matched controls ( n = 3 control and n = 3 PD). A 1-way ANOVA was used to compare multiple groups in C and D . Tukey’s post hoc analysis was applied. A 2-tailed Student’s t test was used to compare 2 groups. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–7 biological replicates from 2–3 independent experiments unless otherwise indicated. * P ≤ 0.05 and ** P

Techniques Used: Expressing, Ex Vivo, Derivative Assay, Quantitative RT-PCR, Western Blot, Isolation, Activity Assay

Kv1.3 expression is highly induced in microglial cells in experimental models of PD and postmortem PD brains. ( A ) Western blot showing increased Kv1.3 protein levels in the substantia nigra of the Syn-AAV mouse model of PD. ( B ) qRT-PCR analysis of 8- to 24-week-old nigral tissues from the MitoPark mouse model of PD showing Kv1.3 induction compared with age-matched littermate controls. ( C ) Western blot of 24-week-old nigral tissues from the MitoPark mouse model of PD (MP) showing induction of Kv1.3 protein expression compared with age-matched littermate control mice (LM). ( D ) IHC in 24-week-old nigral tissues from the MitoPark mouse model of PD showing higher Kv1.3 protein levels (red) in IBA1-positive microglial cells (green) compared with age-matched controls as revealed by their colocalization (yellow). Scale bar: 20 μm. ( E ) qRT-PCR analysis of nigral tissues from the MPTP mouse model revealing induction of Kv1.3 mRNA expression. ( F ) Western blot showing increased Kv1.3 protein levels in substantia nigra of the MPTP mouse model of PD. ( G ) qRT-PCR analysis of postmortem human PD brains showing elevated Kv1.3 mRNA expression. ( H ) Western blot of the SN region of postmortem human PD brain showing induction of Kv1.3 protein expression compared with age-matched controls. n = 6–8. ( I ) Immunostaining revealing higher Kv1.3 levels in the prefrontal cortex of postmortem human PD brains compared with age-matched controls. Lower panel shows the deconvoluted binary image used for analysis. Three regions per brain were analyzed. Scale bar: 200 μm. ( J ) Dual DAB staining showing induction of Kv1.3 expression in HLA-DR–positive microglial cells in patients with DLBs compared with age-matched controls. Scale bars: 100 μm; 20 μm (enlarged insets). A 1-way ANOVA was used to compare multiple groups. Tukey’s post hoc analysis was applied B . A 2-tailed Student’s t test was used to compare 2 groups. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–9 biological replicates from 2–3 independent experiments unless otherwise indicated. * P ≤ 0.05, ** P
Figure Legend Snippet: Kv1.3 expression is highly induced in microglial cells in experimental models of PD and postmortem PD brains. ( A ) Western blot showing increased Kv1.3 protein levels in the substantia nigra of the Syn-AAV mouse model of PD. ( B ) qRT-PCR analysis of 8- to 24-week-old nigral tissues from the MitoPark mouse model of PD showing Kv1.3 induction compared with age-matched littermate controls. ( C ) Western blot of 24-week-old nigral tissues from the MitoPark mouse model of PD (MP) showing induction of Kv1.3 protein expression compared with age-matched littermate control mice (LM). ( D ) IHC in 24-week-old nigral tissues from the MitoPark mouse model of PD showing higher Kv1.3 protein levels (red) in IBA1-positive microglial cells (green) compared with age-matched controls as revealed by their colocalization (yellow). Scale bar: 20 μm. ( E ) qRT-PCR analysis of nigral tissues from the MPTP mouse model revealing induction of Kv1.3 mRNA expression. ( F ) Western blot showing increased Kv1.3 protein levels in substantia nigra of the MPTP mouse model of PD. ( G ) qRT-PCR analysis of postmortem human PD brains showing elevated Kv1.3 mRNA expression. ( H ) Western blot of the SN region of postmortem human PD brain showing induction of Kv1.3 protein expression compared with age-matched controls. n = 6–8. ( I ) Immunostaining revealing higher Kv1.3 levels in the prefrontal cortex of postmortem human PD brains compared with age-matched controls. Lower panel shows the deconvoluted binary image used for analysis. Three regions per brain were analyzed. Scale bar: 200 μm. ( J ) Dual DAB staining showing induction of Kv1.3 expression in HLA-DR–positive microglial cells in patients with DLBs compared with age-matched controls. Scale bars: 100 μm; 20 μm (enlarged insets). A 1-way ANOVA was used to compare multiple groups. Tukey’s post hoc analysis was applied B . A 2-tailed Student’s t test was used to compare 2 groups. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–9 biological replicates from 2–3 independent experiments unless otherwise indicated. * P ≤ 0.05, ** P

Techniques Used: Expressing, Western Blot, Quantitative RT-PCR, Mouse Assay, Immunohistochemistry, Immunostaining, Staining

Fyn modulates the transcriptional regulation of Kv1.3 in microglial cells through the Fyn/PKCδ kinase signaling cascade. ( A ) In silico analysis of the promoter sequence of Kv1.3 revealed probable Nf-κB– and SP1-binding sites. ( B ) qRT-PCR analysis of immortalized MMCs cotreated with αSyn Agg and either SN50 (100 μg/mL) or SB203580 (1 μM), showing that both compounds attenuated αSyn Agg -induced Kv1.3 expression. ( C ) Western blot of Fyn WT and KO PMCs treated with αSyn Agg , showing that Fyn KO reduced the induction of the p38 MAPK pathway. ( D ) qRT-PCR analysis revealed that Fyn KO reduced αSyn Agg -induced Kv1.3 mRNA levels. ( E ) Whole-cell patch-clamp recording showing that Fyn KO attenuated αSyn Agg - and LPS-induced Kv1.3 activity compared with Fyn WT PMCs (WT control n = 24, WT αSyn Agg n = 12, WT LPS n = 29, Fyn KO αSyn Agg n = 20, Fyn KO LPS n = 15). ( F ) ICC showing that Fyn KO reduced αSyn Agg -induced Kv1.3 protein levels in PMCs. Scale bar: 15 μm. ( G ) ICC of PMCs revealed that αSyn Agg -induced Kv1.3 protein expression was reduced by PKCδ KO. Scale bar: 15 μm. ( H ) qRT-PCR analysis of PMCs showing that PKC KO reduced the expression of αSyn Agg -induced Kv1.3 mRNA. ( I ) Whole-cell patch clam recording of PMCs showing that PKC KO attenuated αSyn Agg - and LPS-induced Kv1.3 activity compared with PKC WT PMCs (WT control n = 24, WT αSyn Agg n = 12, WT LPS n = 20, PKC-KO αSyn Agg n = 29, PKC-KO LPS n = 35). Data are presented as the mean ± SD. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–4 biological replicates from 2–3 independent experiments unless otherwise indicated. * P ≤ 0.05, ** P
Figure Legend Snippet: Fyn modulates the transcriptional regulation of Kv1.3 in microglial cells through the Fyn/PKCδ kinase signaling cascade. ( A ) In silico analysis of the promoter sequence of Kv1.3 revealed probable Nf-κB– and SP1-binding sites. ( B ) qRT-PCR analysis of immortalized MMCs cotreated with αSyn Agg and either SN50 (100 μg/mL) or SB203580 (1 μM), showing that both compounds attenuated αSyn Agg -induced Kv1.3 expression. ( C ) Western blot of Fyn WT and KO PMCs treated with αSyn Agg , showing that Fyn KO reduced the induction of the p38 MAPK pathway. ( D ) qRT-PCR analysis revealed that Fyn KO reduced αSyn Agg -induced Kv1.3 mRNA levels. ( E ) Whole-cell patch-clamp recording showing that Fyn KO attenuated αSyn Agg - and LPS-induced Kv1.3 activity compared with Fyn WT PMCs (WT control n = 24, WT αSyn Agg n = 12, WT LPS n = 29, Fyn KO αSyn Agg n = 20, Fyn KO LPS n = 15). ( F ) ICC showing that Fyn KO reduced αSyn Agg -induced Kv1.3 protein levels in PMCs. Scale bar: 15 μm. ( G ) ICC of PMCs revealed that αSyn Agg -induced Kv1.3 protein expression was reduced by PKCδ KO. Scale bar: 15 μm. ( H ) qRT-PCR analysis of PMCs showing that PKC KO reduced the expression of αSyn Agg -induced Kv1.3 mRNA. ( I ) Whole-cell patch clam recording of PMCs showing that PKC KO attenuated αSyn Agg - and LPS-induced Kv1.3 activity compared with PKC WT PMCs (WT control n = 24, WT αSyn Agg n = 12, WT LPS n = 20, PKC-KO αSyn Agg n = 29, PKC-KO LPS n = 35). Data are presented as the mean ± SD. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–4 biological replicates from 2–3 independent experiments unless otherwise indicated. * P ≤ 0.05, ** P

Techniques Used: In Silico, Sequencing, Binding Assay, Quantitative RT-PCR, Expressing, Western Blot, Patch Clamp, Activity Assay, Immunocytochemistry

Kv1.3 modulates neuroinflammation in a cell culture model of PD. ( A – C ) Kv1.3 WT and KO PMCs were treated with 1 μM αSyn Agg for 24 hours. Luminex analysis shows that Kv1.3 KO reduced the release of the αSyn Agg -induced proinflammatory factors ( A ) TNF-α, ( B ) IL-12, and ( C ) IL-1β. ( D – H ) Immortalized MMCs were transfected with WT a Kv1.3 plasmid, and then 48 hours after transfection, cells were treated with 1 μM αSyn Agg for 24 hours. ( D – F ) qRT-PCR analysis showing that Kv1.3 overexpression aggravated αSyn Agg -induced production of the proinflammatory factors ( D ) Nos2 , ( E ) pro– IL-1β , and ( F ) TNF-α . ( G and H ) Luminex analysis showing that Kv1.3 overexpression potentiated the release of the proinflammatory factors ( G ) IL-6 and ( H ) IL-12. ( I ) Voltage ramp from –120 mV to 40 mV elicited a characteristic outward rectifying current in αSyn Agg -treated microglia that was sensitive to the Kv1.3-selective inhibitor PAP-1. ( J ) LDH assay showing that PAP-1 reduced αSyn Agg -induced LDH release from microglial cells. ( K – M ) Luminex assay revealing that PAP-1 attenuated the αSyn Agg -induced proinflammatory factors ( K ) IL-12, ( L ) TNF-α, and ( M ) IL-6. ( N ) Western blot analysis demonstrating that PAP-1 reduced αSyn Agg -induced NLRP3 expression. ( O ) ICC analysis revealed that PAP-1 reduced NLRP3 expression induced by αSyn Agg . Scale bar: 25 μm. A 1-way ANOVA was performed to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–4 biological replicates from 2–3 independent experiments. * P ≤ 0.05, ** P
Figure Legend Snippet: Kv1.3 modulates neuroinflammation in a cell culture model of PD. ( A – C ) Kv1.3 WT and KO PMCs were treated with 1 μM αSyn Agg for 24 hours. Luminex analysis shows that Kv1.3 KO reduced the release of the αSyn Agg -induced proinflammatory factors ( A ) TNF-α, ( B ) IL-12, and ( C ) IL-1β. ( D – H ) Immortalized MMCs were transfected with WT a Kv1.3 plasmid, and then 48 hours after transfection, cells were treated with 1 μM αSyn Agg for 24 hours. ( D – F ) qRT-PCR analysis showing that Kv1.3 overexpression aggravated αSyn Agg -induced production of the proinflammatory factors ( D ) Nos2 , ( E ) pro– IL-1β , and ( F ) TNF-α . ( G and H ) Luminex analysis showing that Kv1.3 overexpression potentiated the release of the proinflammatory factors ( G ) IL-6 and ( H ) IL-12. ( I ) Voltage ramp from –120 mV to 40 mV elicited a characteristic outward rectifying current in αSyn Agg -treated microglia that was sensitive to the Kv1.3-selective inhibitor PAP-1. ( J ) LDH assay showing that PAP-1 reduced αSyn Agg -induced LDH release from microglial cells. ( K – M ) Luminex assay revealing that PAP-1 attenuated the αSyn Agg -induced proinflammatory factors ( K ) IL-12, ( L ) TNF-α, and ( M ) IL-6. ( N ) Western blot analysis demonstrating that PAP-1 reduced αSyn Agg -induced NLRP3 expression. ( O ) ICC analysis revealed that PAP-1 reduced NLRP3 expression induced by αSyn Agg . Scale bar: 25 μm. A 1-way ANOVA was performed to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–4 biological replicates from 2–3 independent experiments. * P ≤ 0.05, ** P

Techniques Used: Cell Culture, Luminex, Transfection, Plasmid Preparation, Quantitative RT-PCR, Over Expression, Lactate Dehydrogenase Assay, Western Blot, Expressing, Immunocytochemistry

Upregulated expression of the potassium channel Kv1.3 upon aggregated αSyn stimulation in microglial cells in vitro. ( A ) Whole-cell patch-clamp recordings of PMCs treated with 1 μM αSyn Agg for 24–48 hours, showing αSyn Agg -induced increased Kv1.3 activity (control n = 24 and αSyn Agg n = 12). Kv1.3 was identified by its characteristic use dependence, which was revealed when applying a train of ten 200-ms pulses from –80 to 40 mV at 1-second intervals (1 Hz). ( B ) qRT-PCR showing that αSyn Agg induced Kv1.3 mRNA expression without significantly altering other potassium channels. ( C ) Western blot of αSyn Agg -induced Kv1.3 protein expression in PMCs. ( D ) ICC of αSyn Agg -induced Kv1.3 protein expression in PMCs. Scale bar: 100 μm. ( E ) Flow cytometric analysis of immortalized MMCs treated with 1 μM αSyn Agg for 24 hours, showing αSyn Agg -induced Kv1.3 surface expression. ( F ) qRT-PCR of human microglia treated with LPS (1 μg/mL) and IL-4 (20 ng/mL) for 6 hours, showing LPS-induced Kv1.3 expression. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied in B and F . A 2-tailed Student’s t test was used for all other figures when comparing 2 groups. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–5 biological replicates from 2–3 independent experiments unless otherwise noted. * P ≤ 0.05, ** P
Figure Legend Snippet: Upregulated expression of the potassium channel Kv1.3 upon aggregated αSyn stimulation in microglial cells in vitro. ( A ) Whole-cell patch-clamp recordings of PMCs treated with 1 μM αSyn Agg for 24–48 hours, showing αSyn Agg -induced increased Kv1.3 activity (control n = 24 and αSyn Agg n = 12). Kv1.3 was identified by its characteristic use dependence, which was revealed when applying a train of ten 200-ms pulses from –80 to 40 mV at 1-second intervals (1 Hz). ( B ) qRT-PCR showing that αSyn Agg induced Kv1.3 mRNA expression without significantly altering other potassium channels. ( C ) Western blot of αSyn Agg -induced Kv1.3 protein expression in PMCs. ( D ) ICC of αSyn Agg -induced Kv1.3 protein expression in PMCs. Scale bar: 100 μm. ( E ) Flow cytometric analysis of immortalized MMCs treated with 1 μM αSyn Agg for 24 hours, showing αSyn Agg -induced Kv1.3 surface expression. ( F ) qRT-PCR of human microglia treated with LPS (1 μg/mL) and IL-4 (20 ng/mL) for 6 hours, showing LPS-induced Kv1.3 expression. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied in B and F . A 2-tailed Student’s t test was used for all other figures when comparing 2 groups. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–5 biological replicates from 2–3 independent experiments unless otherwise noted. * P ≤ 0.05, ** P

Techniques Used: Expressing, In Vitro, Patch Clamp, Activity Assay, Quantitative RT-PCR, Western Blot, Immunocytochemistry

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  • 93
    Millipore antibodies against kv1 3
    Analysis of <t>Kv1.3</t> activation kinetics. HEK-293 cells were transfected with different Kv1.3-KCNE4 chimeras with (+) or without (-) free KCNE4. Different combinations yielded fixed or putative Kv1.3:KCNE4 stoichiometries. Cells were held at −80 mV, and current traces were elicited by 200 ms pulses from −100 mV to +60 mV in 20 mV increments. Peak currents from −20 mV to +60 mV were calculated. ( A ) Time to reach the peak current in the open channels from −20 mV to +60 mV. The time scale is presented in log values for easier viewing. ( B ) Time to peak at 0 mV. Values are the means ± SE of 8–14 cells; * p
    Antibodies Against Kv1 3, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/antibodies against kv1 3/product/Millipore
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    90
    Millipore fitc conjugated kv1 3 ab
    Effector memory T-cell phenotype is not sufficient to confer sensitivity to ShK-mediated inhibition. ( a ) Expression of <t>Kv1.3</t> and KCa3.1 on sorted CD4 + and CD8 + naive, T CM and T EM subsets. CD45RO and CCR7 as markers to define naive, T CM and T EM subsets as shown in dot plots. Expression was determined on ex vivo cell subsets or on sorted T-cell subsets after 4 days stimulation with anti-CD3 and anti-CD28. Kv1.3 and KCa3.1 expression were normalized to RPL19 expression and shown relative to expression in ex vivo naive cells. ( b ) Effect of ShK on naive, T CM and T EM CD4 + subsets. Sorted cells were stimulated with anti-CD3 and either vehicle or 1 μM ShK. Proliferation was determined at day 4 of stimulation; IFN-γ was measured after 3 days. ( c ) Bulk PBMC from healthy donors were stimulated with anti-CD3 and anti-CD28 in the absence or presence of 10 nM ShK or 1 μM TRAM-34. IFN-γ production was measured after 3 days stimulation. Proliferation responses were determined at day 7 by CFSE dilution of CD4 + - or CD8 + -gated cells. Representative dot plots from one donor are shown. % inhibition of proliferation was determined by comparing proliferation in the presence of inhibitor to proliferation of cells in the presence of vehicle control. Data are shown as individual data points with mean±s.d. ( n =15 biological replicates). ( d ) CD4 + T cells repeatedly stimulated with anti-CD3 and anti-CD28. Purified CD4 + T cells from healthy donor were stimulated with 5 μg ml −1 plate-bound anti-CD3 and 2 μg ml −1 soluble anti-CD28 for 4 days. Cells were then harvested, washed, and rested for 3 days. In subsequent stimulations, cells were reactivated with 1 μg ml −1 soluble anti-CD3 and 1 μg ml −1 anti-CD28. In the fourth round of stimulation, cells were cultured with 10-fold increases in ShK concentrations, starting at 10 − 5 nM. Cell supernatants were harvested 3 days after initiation of last round of stimulation for determination of IFN-γ. Proliferation responses were determined at day 4 by 3 H-thymidine incorporation. ( e ) Purified memory CD4 + T cells were stimulated for six rounds with anti-CD3 and anti-CD28, as described in d .
    Fitc Conjugated Kv1 3 Ab, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fitc conjugated kv1 3 ab/product/Millipore
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    93
    Millipore kv1 3
    Fyn modulates the posttranslational modification of <t>Kv1.3.</t> (A) Western blot analysis of postmortem human PD and age-matched control brains showing increased phosphorylation of Kv1.3. (B) Immunoprecipitation of Fyn and Kv1.3 showing direct Fyn-Kv1.3 interaction after αSyn Agg treatment. ( C ) Duolink PLA showing αSyn Agg -induced interaction between Kv1.3 and Fyn. Scale bar: 25 μm. ( D ) Western blot of Fyn WT and KO PMCs revealed that Kv1.3 phosphorylation at residue 135 was Fyn dependent. ( E ) IHC analysis of substantia nigra from Fyn +/+ and Fyn –/– mice showing reduced phosphorylation of Kv1.3 after αSyn PFF injection. Scale bars: 100 μm; 60 μm (insets). ( F ) IHC of substantia nigra from MitoPark mice and their littermate controls showing that pharmacological inhibition of Fyn by saracatinib reduced Kv1.3 phosphorylation. Scale bar: 100 μm. ( G – J ) Immortalized MMCs were either transfected with WT Kv1.3 or aY135A Kv1.3 plasmid. ( G ) qRT-PCR analysis and ( H ) Griess assay showing reduced levels of inducible NOS (iNOS) and nitrite release, respectively, in Y135A Kv1.3-transfected cells compared with WT cells. ( I ) qRT-PCR analysis showing reduced IL-1β production in Y135A Kv1.3–transfected versus WT Kv1.3–transfected MMCs. ( J ) Luminex assay showing reduced IL-1β secretion in Y135A Kv1.3–transfected compared with WT Kv1.3–transfected MMCs. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. A 2-tailed Student’s t test was used to compare 2 groups in A . Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–4 biological replicates from 2–3 independent experiments. * P ≤ 0.05, ** P
    Kv1 3, supplied by Millipore, 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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    Analysis of Kv1.3 activation kinetics. HEK-293 cells were transfected with different Kv1.3-KCNE4 chimeras with (+) or without (-) free KCNE4. Different combinations yielded fixed or putative Kv1.3:KCNE4 stoichiometries. Cells were held at −80 mV, and current traces were elicited by 200 ms pulses from −100 mV to +60 mV in 20 mV increments. Peak currents from −20 mV to +60 mV were calculated. ( A ) Time to reach the peak current in the open channels from −20 mV to +60 mV. The time scale is presented in log values for easier viewing. ( B ) Time to peak at 0 mV. Values are the means ± SE of 8–14 cells; * p

    Journal: Cells

    Article Title: Functional Consequences of the Variable Stoichiometry of the Kv1.3-KCNE4 Complex

    doi: 10.3390/cells9051128

    Figure Lengend Snippet: Analysis of Kv1.3 activation kinetics. HEK-293 cells were transfected with different Kv1.3-KCNE4 chimeras with (+) or without (-) free KCNE4. Different combinations yielded fixed or putative Kv1.3:KCNE4 stoichiometries. Cells were held at −80 mV, and current traces were elicited by 200 ms pulses from −100 mV to +60 mV in 20 mV increments. Peak currents from −20 mV to +60 mV were calculated. ( A ) Time to reach the peak current in the open channels from −20 mV to +60 mV. The time scale is presented in log values for easier viewing. ( B ) Time to peak at 0 mV. Values are the means ± SE of 8–14 cells; * p

    Article Snippet: The membranes were immunoblotted with antibodies against Kv1.3 (1/500, Millipore) and KCNE4 (anti-GFP, 1/500, Roche, Basel, Switzerland).

    Techniques: Activation Assay, Transfection

    KCNE4 specifically modulates Kv1.3. HEK-293 cells were transfected with Kv1.3-YFP in the presence (+KCNE4) or the absence of KCNE4-CFP. Cells were held at −80 mV, and currents were elicited by 1 s depolarizing pulses from −60 mV to +60 mV in 10 mV intervals. ( A ) Representative traces at +60 mV. ( B ) Current density vs. the voltage of K + currents ( p

    Journal: Cells

    Article Title: Functional Consequences of the Variable Stoichiometry of the Kv1.3-KCNE4 Complex

    doi: 10.3390/cells9051128

    Figure Lengend Snippet: KCNE4 specifically modulates Kv1.3. HEK-293 cells were transfected with Kv1.3-YFP in the presence (+KCNE4) or the absence of KCNE4-CFP. Cells were held at −80 mV, and currents were elicited by 1 s depolarizing pulses from −60 mV to +60 mV in 10 mV intervals. ( A ) Representative traces at +60 mV. ( B ) Current density vs. the voltage of K + currents ( p

    Article Snippet: The membranes were immunoblotted with antibodies against Kv1.3 (1/500, Millipore) and KCNE4 (anti-GFP, 1/500, Roche, Basel, Switzerland).

    Techniques: Transfection

    GFP bleaching steps from HEK-293 cells transfected with Kv1.3 in the absence or presence of KCNE4. ( A – C ) Cells were transfected with Kv1.3-loopBAD-GFP. ( A ) Representative 1.5 s and 6.5 s time-lapse snapshots from the TIRFM video (488 nm laser). White arrow heads (a, b) point to the yellow square ROI (6 × 6 pixels) in panel c. (a) Initial fluorescence intensity; (b) fluorescence intensity after the first bleaching step at the same spot; (c) merge image. Yellow square indicates the ROI. The white line delineates the cell shape. Scale bar, 5 μm. ( B ) Representative graph of the bleaching steps for the different spots analyzed. Red arrows point to 4 bleaching steps. Squares, at the right, represent the fluorescence intensity after 4 consecutive bleaching steps at the same ROI. 0 s, initial fluorescence; 43.9 s, background intensity remaining after 4 consecutive bleaching steps. ( C ) Relative frequency of 1–4 bleaching events counted per spot. The black bars correspond to the experimental frequencies observed. Red dashed lines correspond to the theoretical distributions of bleaching steps with p = 0.67. ( D – G ) GFP bleaching steps from HEK-293 cells transfected with KCNE4-loopBAD-GFP and Kv1.3-Apple. ( D ) Representative snapshots from a cropped TIRF microscopy video. Upper panel (561 nm laser): Kv1.3-Apple. Lower panel (488 nm laser): KCNE4-loop-BADGFP. Merge panel zooms squares from previous panels. White squares ROIs (6 × 6 pixels) indicate colocalizing stationary spots. Putative oligomeric composition of the Kv1.3/KCNE4 complex is represented with circles at the left. While 4 white circles indicate the Kv1.3 tetramer, KCNE4 units are represented with gray and black circles. ( E – G ) Representative graph of the GFP bleaching steps from the different spots analyzed. ( E ) Two bleaching steps indicating a 4:2 Kv1.3:KCNE4 stoichiometry. ( F ) Three bleaching steps suggesting a 4:3 Kv1.3:KCNE4 ratio. ( G ) Four bleaching steps highlighting a 4:4 Kv1.3:KCNE4 stoichiometry. ( H ) Histogram with the relative frequency of 1–4 bleaching events counted for spot. Bars correspond to the experimental frequencies observed. ( I ) Histograms representing the theoretical distribution of the bleaching events when KCNE4 is present at the Kv1.3/KCNE4 complex as a monomer (4:1 stoichiometry), dimer (4:2), trimer (4:3) or tetramer (4:4) with p = 0.67. In contrast to the functional Kv1.3 tetramers ( C ), no expected KCNE4 distribution fit with the distribution observed during the analysis of the experimental data.

    Journal: Cells

    Article Title: Functional Consequences of the Variable Stoichiometry of the Kv1.3-KCNE4 Complex

    doi: 10.3390/cells9051128

    Figure Lengend Snippet: GFP bleaching steps from HEK-293 cells transfected with Kv1.3 in the absence or presence of KCNE4. ( A – C ) Cells were transfected with Kv1.3-loopBAD-GFP. ( A ) Representative 1.5 s and 6.5 s time-lapse snapshots from the TIRFM video (488 nm laser). White arrow heads (a, b) point to the yellow square ROI (6 × 6 pixels) in panel c. (a) Initial fluorescence intensity; (b) fluorescence intensity after the first bleaching step at the same spot; (c) merge image. Yellow square indicates the ROI. The white line delineates the cell shape. Scale bar, 5 μm. ( B ) Representative graph of the bleaching steps for the different spots analyzed. Red arrows point to 4 bleaching steps. Squares, at the right, represent the fluorescence intensity after 4 consecutive bleaching steps at the same ROI. 0 s, initial fluorescence; 43.9 s, background intensity remaining after 4 consecutive bleaching steps. ( C ) Relative frequency of 1–4 bleaching events counted per spot. The black bars correspond to the experimental frequencies observed. Red dashed lines correspond to the theoretical distributions of bleaching steps with p = 0.67. ( D – G ) GFP bleaching steps from HEK-293 cells transfected with KCNE4-loopBAD-GFP and Kv1.3-Apple. ( D ) Representative snapshots from a cropped TIRF microscopy video. Upper panel (561 nm laser): Kv1.3-Apple. Lower panel (488 nm laser): KCNE4-loop-BADGFP. Merge panel zooms squares from previous panels. White squares ROIs (6 × 6 pixels) indicate colocalizing stationary spots. Putative oligomeric composition of the Kv1.3/KCNE4 complex is represented with circles at the left. While 4 white circles indicate the Kv1.3 tetramer, KCNE4 units are represented with gray and black circles. ( E – G ) Representative graph of the GFP bleaching steps from the different spots analyzed. ( E ) Two bleaching steps indicating a 4:2 Kv1.3:KCNE4 stoichiometry. ( F ) Three bleaching steps suggesting a 4:3 Kv1.3:KCNE4 ratio. ( G ) Four bleaching steps highlighting a 4:4 Kv1.3:KCNE4 stoichiometry. ( H ) Histogram with the relative frequency of 1–4 bleaching events counted for spot. Bars correspond to the experimental frequencies observed. ( I ) Histograms representing the theoretical distribution of the bleaching events when KCNE4 is present at the Kv1.3/KCNE4 complex as a monomer (4:1 stoichiometry), dimer (4:2), trimer (4:3) or tetramer (4:4) with p = 0.67. In contrast to the functional Kv1.3 tetramers ( C ), no expected KCNE4 distribution fit with the distribution observed during the analysis of the experimental data.

    Article Snippet: The membranes were immunoblotted with antibodies against Kv1.3 (1/500, Millipore) and KCNE4 (anti-GFP, 1/500, Roche, Basel, Switzerland).

    Techniques: Transfection, Fluorescence, Microscopy, Functional Assay

    Analysis of C-type inactivation. HEK-293 cells were transfected with different Kv1.3-KCNE4 chimeras with (+) or without (−) free KCNE4. Different combinations yielded fixed and putative Kv1.3:KCNE4 stoichiometries. ( A ) Representative traces from C-type inactivation recordings. An initial 100 ms pulse at −80 mV was applied prior to a 5 s depolarizing pulse at + 60 mV. ( B ) Relative intensity of voltage-dependent K + currents from all combinations during the first 2 s. ( C ) The constant (τ) of inactivation (in ms) of all groups. Values are the means ± SE, n = 8–14; * p

    Journal: Cells

    Article Title: Functional Consequences of the Variable Stoichiometry of the Kv1.3-KCNE4 Complex

    doi: 10.3390/cells9051128

    Figure Lengend Snippet: Analysis of C-type inactivation. HEK-293 cells were transfected with different Kv1.3-KCNE4 chimeras with (+) or without (−) free KCNE4. Different combinations yielded fixed and putative Kv1.3:KCNE4 stoichiometries. ( A ) Representative traces from C-type inactivation recordings. An initial 100 ms pulse at −80 mV was applied prior to a 5 s depolarizing pulse at + 60 mV. ( B ) Relative intensity of voltage-dependent K + currents from all combinations during the first 2 s. ( C ) The constant (τ) of inactivation (in ms) of all groups. Values are the means ± SE, n = 8–14; * p

    Article Snippet: The membranes were immunoblotted with antibodies against Kv1.3 (1/500, Millipore) and KCNE4 (anti-GFP, 1/500, Roche, Basel, Switzerland).

    Techniques: Transfection

    Analysis of the cumulative inactivation. HEK-293 cells were transfected with different Kv1.3-KCNE4 constructs, and K + currents were analyzed. ( A ) Chimeras with fixed Kv1.3-KCNE4 stoichiometry. Kv1.3 (4:0), Kv1.3T (4:0), KCNE4:Kv1.3T (4:2), KCNE4:Kv1.3 (4:4). ( B ) Functional complexes with putative Kv1.3-KCNE4 stoichiometries due to further addition of extra free KCNE4 units. Kv1.3 + KCNE4: Kv1.3 in the presence of KCNE4 (4:(1–4)). Kv1.3T + KCNE4: Kv1.3T in the presence of KCNE4 (4:(1–4)). KCNE4-Kv1.3T + KCNE4: KCNE4-Kv1.3T in the presence of KCNE4 (4:(2–4)). Cells were held at −80 mV, and a train of 25 depolarizing pulses to +60 mV for 200 ms was applied. ( C ) I/Imax vs. the pulse number. The ratio of the peak current amplitude during each pulse relative to that during the 1st pulse (I/Imax) was plotted against every pulse. ( D ) I/Imax reached at the last pulse train of depolarization. * p

    Journal: Cells

    Article Title: Functional Consequences of the Variable Stoichiometry of the Kv1.3-KCNE4 Complex

    doi: 10.3390/cells9051128

    Figure Lengend Snippet: Analysis of the cumulative inactivation. HEK-293 cells were transfected with different Kv1.3-KCNE4 constructs, and K + currents were analyzed. ( A ) Chimeras with fixed Kv1.3-KCNE4 stoichiometry. Kv1.3 (4:0), Kv1.3T (4:0), KCNE4:Kv1.3T (4:2), KCNE4:Kv1.3 (4:4). ( B ) Functional complexes with putative Kv1.3-KCNE4 stoichiometries due to further addition of extra free KCNE4 units. Kv1.3 + KCNE4: Kv1.3 in the presence of KCNE4 (4:(1–4)). Kv1.3T + KCNE4: Kv1.3T in the presence of KCNE4 (4:(1–4)). KCNE4-Kv1.3T + KCNE4: KCNE4-Kv1.3T in the presence of KCNE4 (4:(2–4)). Cells were held at −80 mV, and a train of 25 depolarizing pulses to +60 mV for 200 ms was applied. ( C ) I/Imax vs. the pulse number. The ratio of the peak current amplitude during each pulse relative to that during the 1st pulse (I/Imax) was plotted against every pulse. ( D ) I/Imax reached at the last pulse train of depolarization. * p

    Article Snippet: The membranes were immunoblotted with antibodies against Kv1.3 (1/500, Millipore) and KCNE4 (anti-GFP, 1/500, Roche, Basel, Switzerland).

    Techniques: Transfection, Construct, Functional Assay

    Chimeric constructs, protein expression and putative oligomeric formations. ( A ) Representative cartoon of the fusion proteins. All chimeras were tagged with either YFP or CFP. White and black barrels represent Kv1.3 peptides. Dark and light gray correspond to KCNE4 structures. In KCNE4-Kv1.3 and KCNE4-Kv1.3T, the 18 aa link is also indicated. ( B ) Western blot of the protein lysates of the nontransfected HEK-293 cells and HEK-293 cells transfected with KCNE4 and Kv1.3. ( C ) Protein levels of cells expressing Kv1.3T, KCNE4-Kv1.3T and KCNE4-Kv1.3. ( D ) Putative oligomerization of Kv1.3 and KCNE4 complexes according to the construct combination. Basic channels formed by chimeras exhibited fixed stoichiometries. The addition of free KCNE4 units yielded forced channels with putative stoichiometries. 1–4, the number of KCNE units by complex, which varied from 1 to 4. 2–4, the number of KCNE units by complex, which varied from 2 to 4. White and black circles represent Kv1.3 peptides. Light gray corresponds to KCNE4 chimeras linked to Kv1.3. Dark gray highlights excess KCNE4 units.

    Journal: Cells

    Article Title: Functional Consequences of the Variable Stoichiometry of the Kv1.3-KCNE4 Complex

    doi: 10.3390/cells9051128

    Figure Lengend Snippet: Chimeric constructs, protein expression and putative oligomeric formations. ( A ) Representative cartoon of the fusion proteins. All chimeras were tagged with either YFP or CFP. White and black barrels represent Kv1.3 peptides. Dark and light gray correspond to KCNE4 structures. In KCNE4-Kv1.3 and KCNE4-Kv1.3T, the 18 aa link is also indicated. ( B ) Western blot of the protein lysates of the nontransfected HEK-293 cells and HEK-293 cells transfected with KCNE4 and Kv1.3. ( C ) Protein levels of cells expressing Kv1.3T, KCNE4-Kv1.3T and KCNE4-Kv1.3. ( D ) Putative oligomerization of Kv1.3 and KCNE4 complexes according to the construct combination. Basic channels formed by chimeras exhibited fixed stoichiometries. The addition of free KCNE4 units yielded forced channels with putative stoichiometries. 1–4, the number of KCNE units by complex, which varied from 1 to 4. 2–4, the number of KCNE units by complex, which varied from 2 to 4. White and black circles represent Kv1.3 peptides. Light gray corresponds to KCNE4 chimeras linked to Kv1.3. Dark gray highlights excess KCNE4 units.

    Article Snippet: The membranes were immunoblotted with antibodies against Kv1.3 (1/500, Millipore) and KCNE4 (anti-GFP, 1/500, Roche, Basel, Switzerland).

    Techniques: Construct, Expressing, Western Blot, Transfection

    Current density versus voltage of K + currents. HEK-293 cells were transfected with different Kv1.3-KCNE4 constructs, and the K + currents were analyzed. Cells were clamped at −80 mV, and current traces were elicited by 200 ms pulses from −100 mV to +60 mV in 20 mV increments. ( A ) Representative traces from chimeras with fixed Kv1.3-KCNE4 stoichiometry. Kv1.3 (4:0), Kv1.3T (4:0), KCNE4:Kv1.3T (4:2), and KCNE4:Kv1.3 (4:4). ( B ) Representative traces from functional complexes with putative Kv1.3-KCNE4 stoichiometries due to the addition of excess free KCNE4 units to Kv1.3-KCNE4 chimeras. Kv1.3 + KCNE4: Kv1.3 in the presence of KCNE4 (4:(1–4)). Kv1.3T + KCNE4: Kv1.3T in the presence of KCNE4 (4:(1–4)). KCNE4-Kv1.3T + KCNE4: KCNE4-Kv1.3T in the presence of KCNE4 (4:(2–4)). ( C ) Current density (pA/pF) plotted against voltage (mV). ( D ) Peak current densities, at +60 mV, of different combinations without or with + KCNE4 and free KCNE4 added. Values are the means ± SE of 8–14 cells; ** p

    Journal: Cells

    Article Title: Functional Consequences of the Variable Stoichiometry of the Kv1.3-KCNE4 Complex

    doi: 10.3390/cells9051128

    Figure Lengend Snippet: Current density versus voltage of K + currents. HEK-293 cells were transfected with different Kv1.3-KCNE4 constructs, and the K + currents were analyzed. Cells were clamped at −80 mV, and current traces were elicited by 200 ms pulses from −100 mV to +60 mV in 20 mV increments. ( A ) Representative traces from chimeras with fixed Kv1.3-KCNE4 stoichiometry. Kv1.3 (4:0), Kv1.3T (4:0), KCNE4:Kv1.3T (4:2), and KCNE4:Kv1.3 (4:4). ( B ) Representative traces from functional complexes with putative Kv1.3-KCNE4 stoichiometries due to the addition of excess free KCNE4 units to Kv1.3-KCNE4 chimeras. Kv1.3 + KCNE4: Kv1.3 in the presence of KCNE4 (4:(1–4)). Kv1.3T + KCNE4: Kv1.3T in the presence of KCNE4 (4:(1–4)). KCNE4-Kv1.3T + KCNE4: KCNE4-Kv1.3T in the presence of KCNE4 (4:(2–4)). ( C ) Current density (pA/pF) plotted against voltage (mV). ( D ) Peak current densities, at +60 mV, of different combinations without or with + KCNE4 and free KCNE4 added. Values are the means ± SE of 8–14 cells; ** p

    Article Snippet: The membranes were immunoblotted with antibodies against Kv1.3 (1/500, Millipore) and KCNE4 (anti-GFP, 1/500, Roche, Basel, Switzerland).

    Techniques: Transfection, Construct, Functional Assay

    Multicolour flow cytometry analysis of tumour-associated Kv1.3-expressing T cells after treatment. Percentages of ( A ) Kv1.3 CD4 + T EM cells relative to total CD4 + cells ( B ) CD4 + T EM cells relative to total CD4 + cells MFI ( C ) Kv1.3 CD8 + T EM cells relative to total CD8 + cells MFI ( D ) Kv1.3 CD8 + T EM cells relative to total CD8 + cells MFI between TR and TNR. Data are shown as individual values with the mean ± S.D. and are representative of n = 8–10 mice/ group. ** p

    Journal: Cancers

    Article Title: Imaging Kv1.3 Expressing Memory T Cells as a Marker of Immunotherapy Response

    doi: 10.3390/cancers14051217

    Figure Lengend Snippet: Multicolour flow cytometry analysis of tumour-associated Kv1.3-expressing T cells after treatment. Percentages of ( A ) Kv1.3 CD4 + T EM cells relative to total CD4 + cells ( B ) CD4 + T EM cells relative to total CD4 + cells MFI ( C ) Kv1.3 CD8 + T EM cells relative to total CD8 + cells MFI ( D ) Kv1.3 CD8 + T EM cells relative to total CD8 + cells MFI between TR and TNR. Data are shown as individual values with the mean ± S.D. and are representative of n = 8–10 mice/ group. ** p

    Article Snippet: Cells were stained with antibodies against Kv1.3 (polyclonal FITC; Sigma-Aldrich), CD103 (clone M290 FITC; BD Biosciences, San Jose, CA, USA), CD25 (clone PC61 BB700; BD Biosciences), CD45 (clone 30-F11 BUV395; BD Biosciences), Fixable Live/Dead Blue (Invitrogen, Waltham, MA, USA), CD62L (clone MEL-14 BUV563; BD Biosciences), CD86 (clone GL1 BUV615; BD Biosciences), F4/80 (clone T45-2342; BD Biosciences), NKp46 (clone 29A1.4 BUV737; BD Biosciences), CD3e (clone 500A2 BUV805; BD Biosciences), FoxP3 (clone 150D AlexaFluor647; Biolegend, San Diego, CA, USA), CD44 (clone IM7 APC-R700; BD Biosciences), CD11b (clone M1/70 APC-Cy7; Biolegend, San Diego, CA, USA), Granzyme B (clone QA16A02 PE; Biolegend), CCR7 (clone 4B12 PE-CF594; BD Biosciences), CD19 (clone 6D5 PE-Cy5; Biolegend), CD206 (clone C068C2 PE-Cy7; Biolegend), CD127 (clone SB/199 BV421; BD Biosciences), Ly6G (clone 1A8 BV480; BD Biosciences), CD8 (clone 53-6.7 BV510; BD Biosciences), CD11c (clone N418 BV570; Biolegend), Ly6C (clone HK1.4 BV605; Biolegend), Siglec F (clone E50-2440 BV650, BD Biosciences), CD68 (clone FA-11 BV711; Biolegend), CD4 (clone GK1.5 BV750; BD Biosciences), I-A/I-E (clone M5/114.15.2 BV785; Biolegend).

    Techniques: Flow Cytometry, Expressing, Mouse Assay

    Effector memory T-cell phenotype is not sufficient to confer sensitivity to ShK-mediated inhibition. ( a ) Expression of Kv1.3 and KCa3.1 on sorted CD4 + and CD8 + naive, T CM and T EM subsets. CD45RO and CCR7 as markers to define naive, T CM and T EM subsets as shown in dot plots. Expression was determined on ex vivo cell subsets or on sorted T-cell subsets after 4 days stimulation with anti-CD3 and anti-CD28. Kv1.3 and KCa3.1 expression were normalized to RPL19 expression and shown relative to expression in ex vivo naive cells. ( b ) Effect of ShK on naive, T CM and T EM CD4 + subsets. Sorted cells were stimulated with anti-CD3 and either vehicle or 1 μM ShK. Proliferation was determined at day 4 of stimulation; IFN-γ was measured after 3 days. ( c ) Bulk PBMC from healthy donors were stimulated with anti-CD3 and anti-CD28 in the absence or presence of 10 nM ShK or 1 μM TRAM-34. IFN-γ production was measured after 3 days stimulation. Proliferation responses were determined at day 7 by CFSE dilution of CD4 + - or CD8 + -gated cells. Representative dot plots from one donor are shown. % inhibition of proliferation was determined by comparing proliferation in the presence of inhibitor to proliferation of cells in the presence of vehicle control. Data are shown as individual data points with mean±s.d. ( n =15 biological replicates). ( d ) CD4 + T cells repeatedly stimulated with anti-CD3 and anti-CD28. Purified CD4 + T cells from healthy donor were stimulated with 5 μg ml −1 plate-bound anti-CD3 and 2 μg ml −1 soluble anti-CD28 for 4 days. Cells were then harvested, washed, and rested for 3 days. In subsequent stimulations, cells were reactivated with 1 μg ml −1 soluble anti-CD3 and 1 μg ml −1 anti-CD28. In the fourth round of stimulation, cells were cultured with 10-fold increases in ShK concentrations, starting at 10 − 5 nM. Cell supernatants were harvested 3 days after initiation of last round of stimulation for determination of IFN-γ. Proliferation responses were determined at day 4 by 3 H-thymidine incorporation. ( e ) Purified memory CD4 + T cells were stimulated for six rounds with anti-CD3 and anti-CD28, as described in d .

    Journal: Nature Communications

    Article Title: Potassium channels Kv1.3 and KCa3.1 cooperatively and compensatorily regulate antigen-specific memory T cell functions

    doi: 10.1038/ncomms14644

    Figure Lengend Snippet: Effector memory T-cell phenotype is not sufficient to confer sensitivity to ShK-mediated inhibition. ( a ) Expression of Kv1.3 and KCa3.1 on sorted CD4 + and CD8 + naive, T CM and T EM subsets. CD45RO and CCR7 as markers to define naive, T CM and T EM subsets as shown in dot plots. Expression was determined on ex vivo cell subsets or on sorted T-cell subsets after 4 days stimulation with anti-CD3 and anti-CD28. Kv1.3 and KCa3.1 expression were normalized to RPL19 expression and shown relative to expression in ex vivo naive cells. ( b ) Effect of ShK on naive, T CM and T EM CD4 + subsets. Sorted cells were stimulated with anti-CD3 and either vehicle or 1 μM ShK. Proliferation was determined at day 4 of stimulation; IFN-γ was measured after 3 days. ( c ) Bulk PBMC from healthy donors were stimulated with anti-CD3 and anti-CD28 in the absence or presence of 10 nM ShK or 1 μM TRAM-34. IFN-γ production was measured after 3 days stimulation. Proliferation responses were determined at day 7 by CFSE dilution of CD4 + - or CD8 + -gated cells. Representative dot plots from one donor are shown. % inhibition of proliferation was determined by comparing proliferation in the presence of inhibitor to proliferation of cells in the presence of vehicle control. Data are shown as individual data points with mean±s.d. ( n =15 biological replicates). ( d ) CD4 + T cells repeatedly stimulated with anti-CD3 and anti-CD28. Purified CD4 + T cells from healthy donor were stimulated with 5 μg ml −1 plate-bound anti-CD3 and 2 μg ml −1 soluble anti-CD28 for 4 days. Cells were then harvested, washed, and rested for 3 days. In subsequent stimulations, cells were reactivated with 1 μg ml −1 soluble anti-CD3 and 1 μg ml −1 anti-CD28. In the fourth round of stimulation, cells were cultured with 10-fold increases in ShK concentrations, starting at 10 − 5 nM. Cell supernatants were harvested 3 days after initiation of last round of stimulation for determination of IFN-γ. Proliferation responses were determined at day 4 by 3 H-thymidine incorporation. ( e ) Purified memory CD4 + T cells were stimulated for six rounds with anti-CD3 and anti-CD28, as described in d .

    Article Snippet: FITC-conjugated Kv1.3 Ab was purchased from Sigma-Aldrich.

    Techniques: Inhibition, Expressing, Ex Vivo, Purification, Cell Culture

    AIA and DTH response in Kcna3 − / − rats. ( a ) WT (blue) or Kcna3 − / − (green) rats were given a single injection of CFA to induce AIA (filled symbols, n =5 biological replicates per group) or were untreated (open symbols, naive, n =2 per group) and clinical score assessed at day 21. Individual animals are represented by discrete symbols and mean±s.d. are shown. Delayed-type hypersensitivity. ( b ) WT (blue) or Kcna3 − / − (green) OVA-immunized rats were subsequently challenged with OVA (filled symbols, n =6 biological replicates per group) or PBS (open symbols, n =4 per group) and ear swelling was measured 24 h later. Individual animals are represented by discrete symbols and mean±s.d. are shown. Experiment was performed twice, with each experiment delineated by the dotted line. Statistically significant differences are denoted with P values as determined by Student's t- test. ( c ) Effect of Kv1.3 blockade on DTH inflammatory responses. WT rats were immunized with OVA then subsequently challenged 1 week later with either PBS or OVA. OVA-rechallenged animals were treated with control anti-ragweed (aRGW) antibody (pink), CTLA4-Ig (orange) or ShK (blue). Ear swelling was measured 24 h later. Individual biological replicates (n=6 per group) are shown with mean±s.d. Experiment was performed three times, with each experiment delineated by dotted lines. Statistically significant differences between ShK and anti-ragweed control groups are denoted with P values; CTLA4-Ig inhibition was statistically significant in all experiments ( P

    Journal: Nature Communications

    Article Title: Potassium channels Kv1.3 and KCa3.1 cooperatively and compensatorily regulate antigen-specific memory T cell functions

    doi: 10.1038/ncomms14644

    Figure Lengend Snippet: AIA and DTH response in Kcna3 − / − rats. ( a ) WT (blue) or Kcna3 − / − (green) rats were given a single injection of CFA to induce AIA (filled symbols, n =5 biological replicates per group) or were untreated (open symbols, naive, n =2 per group) and clinical score assessed at day 21. Individual animals are represented by discrete symbols and mean±s.d. are shown. Delayed-type hypersensitivity. ( b ) WT (blue) or Kcna3 − / − (green) OVA-immunized rats were subsequently challenged with OVA (filled symbols, n =6 biological replicates per group) or PBS (open symbols, n =4 per group) and ear swelling was measured 24 h later. Individual animals are represented by discrete symbols and mean±s.d. are shown. Experiment was performed twice, with each experiment delineated by the dotted line. Statistically significant differences are denoted with P values as determined by Student's t- test. ( c ) Effect of Kv1.3 blockade on DTH inflammatory responses. WT rats were immunized with OVA then subsequently challenged 1 week later with either PBS or OVA. OVA-rechallenged animals were treated with control anti-ragweed (aRGW) antibody (pink), CTLA4-Ig (orange) or ShK (blue). Ear swelling was measured 24 h later. Individual biological replicates (n=6 per group) are shown with mean±s.d. Experiment was performed three times, with each experiment delineated by dotted lines. Statistically significant differences between ShK and anti-ragweed control groups are denoted with P values; CTLA4-Ig inhibition was statistically significant in all experiments ( P

    Article Snippet: FITC-conjugated Kv1.3 Ab was purchased from Sigma-Aldrich.

    Techniques: Injection, Inhibition

    Effects of siRNA knockdown of Kv1.3 or KCa3.1 on TT-specific human T-cell sensitivity to inhibitors. ( a ) Kv1.3 (left) and KCa3.1 (right) expression was measured in primary TT-stimulated T cells (1° TT) or T cells that underwent four rounds of TT stimulation (4° TT) that were transfected with Kv1.3 siRNA (blue bars), KCa3.1 siRNA (green bars), a combination of both siRNA (orange bars) or scramble control siRNA (grey bars). Relative expression of targeted genes is shown in comparison with scramble siRNA transfected cells. Data are shown as mean±s.d. of triplicate measurements from one representative experiment. ( b ) Kv1.3 surface protein expression on 1° TT or 4° TT cells following siRNA transfection. Primary (left) or repeatedly stimulated (4°, right) TT cells were transfected with Kv1.3 siRNA (red histograms) or scramble siRNA (blue histograms) and then stained for Kv1.3 48 h later. Grey filled histogram represents isotype control. T-cell responses of 1° TT-stimulated T cells ( c , d ) or 4° TT-stimulated T cells ( e , f ) transfected with Kv1.3 siRNA (blue), KCa3.1 siRNA (green), a combination of both (orange) or scramble control siRNA (black). Cells were then restimulated with anti-CD3 (0.5 μg ml −1 ) in the absence or presence of ShK ( c , e ) or TRAM-34 ( d , f ) at the indicated concentrations. Proliferation responses were determined at day 4 of culture by 3 H-thymidine incorporation; IFN-γ concentration was measured in culture supernatants harvested 3 days after restimulation. Data are shown as mean±s.d. of replicate wells and are representative of at least two independent experiments.

    Journal: Nature Communications

    Article Title: Potassium channels Kv1.3 and KCa3.1 cooperatively and compensatorily regulate antigen-specific memory T cell functions

    doi: 10.1038/ncomms14644

    Figure Lengend Snippet: Effects of siRNA knockdown of Kv1.3 or KCa3.1 on TT-specific human T-cell sensitivity to inhibitors. ( a ) Kv1.3 (left) and KCa3.1 (right) expression was measured in primary TT-stimulated T cells (1° TT) or T cells that underwent four rounds of TT stimulation (4° TT) that were transfected with Kv1.3 siRNA (blue bars), KCa3.1 siRNA (green bars), a combination of both siRNA (orange bars) or scramble control siRNA (grey bars). Relative expression of targeted genes is shown in comparison with scramble siRNA transfected cells. Data are shown as mean±s.d. of triplicate measurements from one representative experiment. ( b ) Kv1.3 surface protein expression on 1° TT or 4° TT cells following siRNA transfection. Primary (left) or repeatedly stimulated (4°, right) TT cells were transfected with Kv1.3 siRNA (red histograms) or scramble siRNA (blue histograms) and then stained for Kv1.3 48 h later. Grey filled histogram represents isotype control. T-cell responses of 1° TT-stimulated T cells ( c , d ) or 4° TT-stimulated T cells ( e , f ) transfected with Kv1.3 siRNA (blue), KCa3.1 siRNA (green), a combination of both (orange) or scramble control siRNA (black). Cells were then restimulated with anti-CD3 (0.5 μg ml −1 ) in the absence or presence of ShK ( c , e ) or TRAM-34 ( d , f ) at the indicated concentrations. Proliferation responses were determined at day 4 of culture by 3 H-thymidine incorporation; IFN-γ concentration was measured in culture supernatants harvested 3 days after restimulation. Data are shown as mean±s.d. of replicate wells and are representative of at least two independent experiments.

    Article Snippet: FITC-conjugated Kv1.3 Ab was purchased from Sigma-Aldrich.

    Techniques: Expressing, Transfection, Staining, Concentration Assay

    Full inhibition of antigen-specific human T cells requires blockade of both Kv1.3 and KCa3.1. ( a , b ) Inhibition of CD4 + ( a ) or CD8 + ( b ) T-cell proliferation response to TT stimulation as determined by CFSE dilution. One representative donor is shown. Percentages indicated in red denote % reduction in proliferation relative to vehicle-treated cells. Primary (1°) T-cell response to TT was determined in the absence or presence of either 10 nM ShK ( n =8 biological replicates) or 1 μM TRAM-34 ( n =9). ( c ) Proliferation responses were determined at day 4; IFN-γ concentrations were measured at day 3. % inhibition was determined by comparing proliferation or IFN-γ concentration in the presence of inhibitor to vehicle control. Data are shown as individual data points with mean±s.d. Statistically significant differences are denoted with P values as determined by Student's t- test. ( d ) Inhibition of both Kv1.3 and KCa3.1 fully abrogates TT-specific T cell responses. PBMC from T1D donors were stimulated with TT in the absence or presence of 10 nM ShK, 1 μM TRAM-34 or a combination of both (combo). ‘No stim' denotes absence of TT. Data are shown as mean±s.d. of replicate wells and are representative of three independent experiments. ( e ) Proliferation and IFN-γ production of TT-specific T cells following four rounds of stimulation (4°). After three rounds of TT stimulation, cells were rested and then restimulated in the presence of irradiated autologous PBMC with TT in the absence or presence of either 10 nM ShK ( n =11 biological replicates) or 1 μM TRAM-34 ( n =4). ( f ) Effect of ShK or TRAM-34 on proliferation or IFN-γ production of TT-specific T cells after increasing rounds of stimulation. Data shown are representative of one donor. ( g ) Gene expression of KCNA3 and KCNN4 in purified T cells after 1° or 4° TT stimulation. Expression of each channel is presented relative to expression in naive T cells. Data shown are representative of three donors. ( h ) Kv1.3 surface protein expression on 1° TT or 4° TT cells. Blue histogram represents Kv1.3 staining; grey filled histogram represents isotype control.

    Journal: Nature Communications

    Article Title: Potassium channels Kv1.3 and KCa3.1 cooperatively and compensatorily regulate antigen-specific memory T cell functions

    doi: 10.1038/ncomms14644

    Figure Lengend Snippet: Full inhibition of antigen-specific human T cells requires blockade of both Kv1.3 and KCa3.1. ( a , b ) Inhibition of CD4 + ( a ) or CD8 + ( b ) T-cell proliferation response to TT stimulation as determined by CFSE dilution. One representative donor is shown. Percentages indicated in red denote % reduction in proliferation relative to vehicle-treated cells. Primary (1°) T-cell response to TT was determined in the absence or presence of either 10 nM ShK ( n =8 biological replicates) or 1 μM TRAM-34 ( n =9). ( c ) Proliferation responses were determined at day 4; IFN-γ concentrations were measured at day 3. % inhibition was determined by comparing proliferation or IFN-γ concentration in the presence of inhibitor to vehicle control. Data are shown as individual data points with mean±s.d. Statistically significant differences are denoted with P values as determined by Student's t- test. ( d ) Inhibition of both Kv1.3 and KCa3.1 fully abrogates TT-specific T cell responses. PBMC from T1D donors were stimulated with TT in the absence or presence of 10 nM ShK, 1 μM TRAM-34 or a combination of both (combo). ‘No stim' denotes absence of TT. Data are shown as mean±s.d. of replicate wells and are representative of three independent experiments. ( e ) Proliferation and IFN-γ production of TT-specific T cells following four rounds of stimulation (4°). After three rounds of TT stimulation, cells were rested and then restimulated in the presence of irradiated autologous PBMC with TT in the absence or presence of either 10 nM ShK ( n =11 biological replicates) or 1 μM TRAM-34 ( n =4). ( f ) Effect of ShK or TRAM-34 on proliferation or IFN-γ production of TT-specific T cells after increasing rounds of stimulation. Data shown are representative of one donor. ( g ) Gene expression of KCNA3 and KCNN4 in purified T cells after 1° or 4° TT stimulation. Expression of each channel is presented relative to expression in naive T cells. Data shown are representative of three donors. ( h ) Kv1.3 surface protein expression on 1° TT or 4° TT cells. Blue histogram represents Kv1.3 staining; grey filled histogram represents isotype control.

    Article Snippet: FITC-conjugated Kv1.3 Ab was purchased from Sigma-Aldrich.

    Techniques: Inhibition, Concentration Assay, Irradiation, Expressing, Purification, Staining

    Kv1.3 and KCa3.1 are both required for human autoreactive T-cell responses. ( a ) PBMC from HLA-DR4 + T1D donors ( n =10 biological replicates) were stimulated with a combination of HLA-DR4-restricted GAD65 peptides in the absence or presence of either 10 nM ShK or 1 μM TRAM-34. ( b ) PBMC from non-HLA- or HLA-typed T1D donors were stimulated with GAD65 protein ( n =13 biological replicates). Proliferation responses (left) were determined at day 4. IFN-γ concentrations (right) were measured 3 days after stimulation. %inhibition was determined by comparing proliferation or IFN-γ concentration in the presence of inhibitor to vehicle control. Data are shown as individual data points with mean±s.d. Statistically significant differences are denoted with P values as determined by Student's t- test. ( c ) Inhibition of both Kv1.3 and KCa3.1 fully abrogates autologous autoreactive T cell responses. PBMC from T1D donors were stimulated with GAD65 protein in the absence or presence of 10 nM ShK, 1 μM TRAM-34 or a combination of both (combo). ‘No stim' denotes unstimulated cell conditions (absence of GAD65 protein). Data are shown as mean±s.d. of replicate wells and are representative of three independent experiments. Gene expression for Kv1.3 ( KCNA3 , d ) and KCa3.1 ( KCNN4, e ) in GAD65-stimulated purified T cells following siRNA transfection with Kv1.3 siRNA (blue), KCa3.1 siRNA (green) or scramble control siRNA (grey). ( f , g ) Effects of siRNA knockdown of Kv1.3 or KCa3.1 on sensitivity to inhibitors. PBMC from T1D donor was stimulated with GAD65 protein for 7 days. After 3 days rest, T cells were purified and transfected with Kv1.3 siRNA (blue), KCa3.1 siRNA (green) or scramble control siRNA (grey) and then restimulated with anti-CD3 (0.5 μg ml −1 ) in the absence or presence of ShK ( f ) or TRAM-34 ( g ) at the indicated concentrations. Data are shown as mean±s.d. of replicate wells and are representative of at least two independent experiments.

    Journal: Nature Communications

    Article Title: Potassium channels Kv1.3 and KCa3.1 cooperatively and compensatorily regulate antigen-specific memory T cell functions

    doi: 10.1038/ncomms14644

    Figure Lengend Snippet: Kv1.3 and KCa3.1 are both required for human autoreactive T-cell responses. ( a ) PBMC from HLA-DR4 + T1D donors ( n =10 biological replicates) were stimulated with a combination of HLA-DR4-restricted GAD65 peptides in the absence or presence of either 10 nM ShK or 1 μM TRAM-34. ( b ) PBMC from non-HLA- or HLA-typed T1D donors were stimulated with GAD65 protein ( n =13 biological replicates). Proliferation responses (left) were determined at day 4. IFN-γ concentrations (right) were measured 3 days after stimulation. %inhibition was determined by comparing proliferation or IFN-γ concentration in the presence of inhibitor to vehicle control. Data are shown as individual data points with mean±s.d. Statistically significant differences are denoted with P values as determined by Student's t- test. ( c ) Inhibition of both Kv1.3 and KCa3.1 fully abrogates autologous autoreactive T cell responses. PBMC from T1D donors were stimulated with GAD65 protein in the absence or presence of 10 nM ShK, 1 μM TRAM-34 or a combination of both (combo). ‘No stim' denotes unstimulated cell conditions (absence of GAD65 protein). Data are shown as mean±s.d. of replicate wells and are representative of three independent experiments. Gene expression for Kv1.3 ( KCNA3 , d ) and KCa3.1 ( KCNN4, e ) in GAD65-stimulated purified T cells following siRNA transfection with Kv1.3 siRNA (blue), KCa3.1 siRNA (green) or scramble control siRNA (grey). ( f , g ) Effects of siRNA knockdown of Kv1.3 or KCa3.1 on sensitivity to inhibitors. PBMC from T1D donor was stimulated with GAD65 protein for 7 days. After 3 days rest, T cells were purified and transfected with Kv1.3 siRNA (blue), KCa3.1 siRNA (green) or scramble control siRNA (grey) and then restimulated with anti-CD3 (0.5 μg ml −1 ) in the absence or presence of ShK ( f ) or TRAM-34 ( g ) at the indicated concentrations. Data are shown as mean±s.d. of replicate wells and are representative of at least two independent experiments.

    Article Snippet: FITC-conjugated Kv1.3 Ab was purchased from Sigma-Aldrich.

    Techniques: Inhibition, Concentration Assay, Expressing, Purification, Transfection

    Characterization of Kcna3 − / − T cells. ( a ) K + -channel expression in human, rat and mouse T cells. Gene expression of Kv1 family members and KCa3.1 in naive CD4 + T cells from human (left), rat (centre) or mouse (right). Relative expression was determined by normalizing to housekeeping gene RPL19. Electrophysiological and pharmacological tests show null Kv1.3 channel in Kcna3 − / − T cells. ( b ) Representative voltage-currents from WT and Kcna3 − / − T cells. Currents were elicited by depolarizing voltage steps from −60 to +40 mV (10 mV increments every 30 s, with −80 mV membrane-holding potential). ( c ) Kv1.3 channel number in WT ( n =40) and Kcna3 − / − ( n =50) T cells after 48 h activation. The mean Kv1.3 channel numbers were 1667±187 in WT and undetectable in Kcna3 − / − T cells. ( d ) Normalized WT and Kcna3 − / − T cell K + currents before and after Shk inhibition. 89% WT T cell K + current was blocked by 1 nM Shk, but no Shk-sensitive current was detected in Kcna3 − / − T cells. Kcna3 − / − T-cell responses to activation. ( e ) Proliferation (left) and IFN-γ (right) responses to anti-CD3 and anti-CD28 stimulation. Spleen cells from WT or Kcna3 − / − rats were stimulated for 3 days. Individual biological replicates ( n =4 per group) are shown with mean±s.d. ( f ) Effects of ShK and TRAM-34 on polyclonal T-cell activation. Data are shown as mean±s.d. ( n =4 biological replicates per group). ( g ) OVA-specific T-cell proliferation responses. Draining lymph node and spleen cells from OVA-immunized WT and Kcna3 − / − rats were plated at a 1:10 lymph node:spleen cell ratio and stimulated in vitro with OVA at various concentrations. Data are shown as mean±s.d. ( n =4 biological replicates per group). ( h , i ) Kcna3 − / − rat dendritic cell competency. CD4 + ( h ) or CD8 + . ( i ) T cells isolated from DLN of OVA-immunized WT (blue) and Kcna3 − / − (green) rats were co-cultured with APCs from WT (filled circles) or Kcna3 − / − (open circles) rats and stimulated with OVA. Proliferation responses were determined at day 3 of culture. Individual biological replicates ( n =4 per group) are shown with mean±s.d.

    Journal: Nature Communications

    Article Title: Potassium channels Kv1.3 and KCa3.1 cooperatively and compensatorily regulate antigen-specific memory T cell functions

    doi: 10.1038/ncomms14644

    Figure Lengend Snippet: Characterization of Kcna3 − / − T cells. ( a ) K + -channel expression in human, rat and mouse T cells. Gene expression of Kv1 family members and KCa3.1 in naive CD4 + T cells from human (left), rat (centre) or mouse (right). Relative expression was determined by normalizing to housekeeping gene RPL19. Electrophysiological and pharmacological tests show null Kv1.3 channel in Kcna3 − / − T cells. ( b ) Representative voltage-currents from WT and Kcna3 − / − T cells. Currents were elicited by depolarizing voltage steps from −60 to +40 mV (10 mV increments every 30 s, with −80 mV membrane-holding potential). ( c ) Kv1.3 channel number in WT ( n =40) and Kcna3 − / − ( n =50) T cells after 48 h activation. The mean Kv1.3 channel numbers were 1667±187 in WT and undetectable in Kcna3 − / − T cells. ( d ) Normalized WT and Kcna3 − / − T cell K + currents before and after Shk inhibition. 89% WT T cell K + current was blocked by 1 nM Shk, but no Shk-sensitive current was detected in Kcna3 − / − T cells. Kcna3 − / − T-cell responses to activation. ( e ) Proliferation (left) and IFN-γ (right) responses to anti-CD3 and anti-CD28 stimulation. Spleen cells from WT or Kcna3 − / − rats were stimulated for 3 days. Individual biological replicates ( n =4 per group) are shown with mean±s.d. ( f ) Effects of ShK and TRAM-34 on polyclonal T-cell activation. Data are shown as mean±s.d. ( n =4 biological replicates per group). ( g ) OVA-specific T-cell proliferation responses. Draining lymph node and spleen cells from OVA-immunized WT and Kcna3 − / − rats were plated at a 1:10 lymph node:spleen cell ratio and stimulated in vitro with OVA at various concentrations. Data are shown as mean±s.d. ( n =4 biological replicates per group). ( h , i ) Kcna3 − / − rat dendritic cell competency. CD4 + ( h ) or CD8 + . ( i ) T cells isolated from DLN of OVA-immunized WT (blue) and Kcna3 − / − (green) rats were co-cultured with APCs from WT (filled circles) or Kcna3 − / − (open circles) rats and stimulated with OVA. Proliferation responses were determined at day 3 of culture. Individual biological replicates ( n =4 per group) are shown with mean±s.d.

    Article Snippet: FITC-conjugated Kv1.3 Ab was purchased from Sigma-Aldrich.

    Techniques: Expressing, Activation Assay, Inhibition, In Vitro, Isolation, Cell Culture

    Autoreactive but not autologous pathogen-specific T cells are sensitive to Kv1.3 blockers. ( a , b ) ShK inhibits GAD65-specific T1D T cells but not autologous TT-specific T cells. PBMC from T1D donors ( n =6 biological replicates) were stimulated with a combination of four HLA-DR4-restricted GAD65 peptides. Proliferation and IFN-γ responses were compared with TT stimulation in the absence or presence of either 10 nM ShK ( a ) or 1 μM TRAM-34 ( b ). Data shown are % inhibition of proliferation response as determined by 3 H-thymidine incorporation after 4 days primary in vitro stimulation and % inhibition of IFN-γ production after 3 days stimulation. Coloured symbols and corresponding lines represent each individual donor.

    Journal: Nature Communications

    Article Title: Potassium channels Kv1.3 and KCa3.1 cooperatively and compensatorily regulate antigen-specific memory T cell functions

    doi: 10.1038/ncomms14644

    Figure Lengend Snippet: Autoreactive but not autologous pathogen-specific T cells are sensitive to Kv1.3 blockers. ( a , b ) ShK inhibits GAD65-specific T1D T cells but not autologous TT-specific T cells. PBMC from T1D donors ( n =6 biological replicates) were stimulated with a combination of four HLA-DR4-restricted GAD65 peptides. Proliferation and IFN-γ responses were compared with TT stimulation in the absence or presence of either 10 nM ShK ( a ) or 1 μM TRAM-34 ( b ). Data shown are % inhibition of proliferation response as determined by 3 H-thymidine incorporation after 4 days primary in vitro stimulation and % inhibition of IFN-γ production after 3 days stimulation. Coloured symbols and corresponding lines represent each individual donor.

    Article Snippet: FITC-conjugated Kv1.3 Ab was purchased from Sigma-Aldrich.

    Techniques: Inhibition, In Vitro

    Fyn modulates the posttranslational modification of Kv1.3. (A) Western blot analysis of postmortem human PD and age-matched control brains showing increased phosphorylation of Kv1.3. (B) Immunoprecipitation of Fyn and Kv1.3 showing direct Fyn-Kv1.3 interaction after αSyn Agg treatment. ( C ) Duolink PLA showing αSyn Agg -induced interaction between Kv1.3 and Fyn. Scale bar: 25 μm. ( D ) Western blot of Fyn WT and KO PMCs revealed that Kv1.3 phosphorylation at residue 135 was Fyn dependent. ( E ) IHC analysis of substantia nigra from Fyn +/+ and Fyn –/– mice showing reduced phosphorylation of Kv1.3 after αSyn PFF injection. Scale bars: 100 μm; 60 μm (insets). ( F ) IHC of substantia nigra from MitoPark mice and their littermate controls showing that pharmacological inhibition of Fyn by saracatinib reduced Kv1.3 phosphorylation. Scale bar: 100 μm. ( G – J ) Immortalized MMCs were either transfected with WT Kv1.3 or aY135A Kv1.3 plasmid. ( G ) qRT-PCR analysis and ( H ) Griess assay showing reduced levels of inducible NOS (iNOS) and nitrite release, respectively, in Y135A Kv1.3-transfected cells compared with WT cells. ( I ) qRT-PCR analysis showing reduced IL-1β production in Y135A Kv1.3–transfected versus WT Kv1.3–transfected MMCs. ( J ) Luminex assay showing reduced IL-1β secretion in Y135A Kv1.3–transfected compared with WT Kv1.3–transfected MMCs. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. A 2-tailed Student’s t test was used to compare 2 groups in A . Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–4 biological replicates from 2–3 independent experiments. * P ≤ 0.05, ** P

    Journal: The Journal of Clinical Investigation

    Article Title: Kv1.3 modulates neuroinflammation and neurodegeneration in Parkinson’s disease

    doi: 10.1172/JCI136174

    Figure Lengend Snippet: Fyn modulates the posttranslational modification of Kv1.3. (A) Western blot analysis of postmortem human PD and age-matched control brains showing increased phosphorylation of Kv1.3. (B) Immunoprecipitation of Fyn and Kv1.3 showing direct Fyn-Kv1.3 interaction after αSyn Agg treatment. ( C ) Duolink PLA showing αSyn Agg -induced interaction between Kv1.3 and Fyn. Scale bar: 25 μm. ( D ) Western blot of Fyn WT and KO PMCs revealed that Kv1.3 phosphorylation at residue 135 was Fyn dependent. ( E ) IHC analysis of substantia nigra from Fyn +/+ and Fyn –/– mice showing reduced phosphorylation of Kv1.3 after αSyn PFF injection. Scale bars: 100 μm; 60 μm (insets). ( F ) IHC of substantia nigra from MitoPark mice and their littermate controls showing that pharmacological inhibition of Fyn by saracatinib reduced Kv1.3 phosphorylation. Scale bar: 100 μm. ( G – J ) Immortalized MMCs were either transfected with WT Kv1.3 or aY135A Kv1.3 plasmid. ( G ) qRT-PCR analysis and ( H ) Griess assay showing reduced levels of inducible NOS (iNOS) and nitrite release, respectively, in Y135A Kv1.3-transfected cells compared with WT cells. ( I ) qRT-PCR analysis showing reduced IL-1β production in Y135A Kv1.3–transfected versus WT Kv1.3–transfected MMCs. ( J ) Luminex assay showing reduced IL-1β secretion in Y135A Kv1.3–transfected compared with WT Kv1.3–transfected MMCs. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. A 2-tailed Student’s t test was used to compare 2 groups in A . Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–4 biological replicates from 2–3 independent experiments. * P ≤ 0.05, ** P

    Article Snippet: The following primary antibodies were used: IBA1 (Wako, 1:1000) (RRID: AB_2314667), IBA1 (Abcam, 1:500) (RRID: AB_870576), Kv1.3 (Alomone Labs, 1:500) (RRID: AB_2040151), Kv1.3 (MilliporeSigma, 1:500)(RRID: AB_11212692), p-Kv1.3 (MilliporeSigma 1:500) (SAB4504254), p38 (Cell Signaling Technology, 1:500) (RRID: AB_330713), and tyrosine hydroxylase (TH) (MilliporeSigma, 1:1000) (RRID: AB_2201526).

    Techniques: Modification, Western Blot, Immunoprecipitation, Proximity Ligation Assay, Immunohistochemistry, Mouse Assay, Injection, Inhibition, Transfection, Plasmid Preparation, Quantitative RT-PCR, Griess Assay, Luminex

    Kv1.3 inhibition protects against αSyn PFF -induced behavior deficit and dopaminergic neuronal loss. ( A ) Treatment paradigm corresponding to the αSyn PFF mouse model of PD. ( B ) Representative movement tracks showing that PAP-1 rescued movement deficits induced by αSyn PFF . ( C – E ) A VersaMax open-field test showed decreased ( C ) rest time and increased ( D ) horizontal activity and ( E ) total distance traveled for αSyn PFF mice treated with PAP-1. ( F and G ) HPLC showing that PAP-1 treatment protected against loss of ( F ) dopamine and ( G ) DOPAC induced by αSyn PFF . ( H ) Western blot analysis of TH showing loss of TH induced by αSyn PFF in the SNpc region. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–7 animals per group. * P ≤ 0.05, ** P

    Journal: The Journal of Clinical Investigation

    Article Title: Kv1.3 modulates neuroinflammation and neurodegeneration in Parkinson’s disease

    doi: 10.1172/JCI136174

    Figure Lengend Snippet: Kv1.3 inhibition protects against αSyn PFF -induced behavior deficit and dopaminergic neuronal loss. ( A ) Treatment paradigm corresponding to the αSyn PFF mouse model of PD. ( B ) Representative movement tracks showing that PAP-1 rescued movement deficits induced by αSyn PFF . ( C – E ) A VersaMax open-field test showed decreased ( C ) rest time and increased ( D ) horizontal activity and ( E ) total distance traveled for αSyn PFF mice treated with PAP-1. ( F and G ) HPLC showing that PAP-1 treatment protected against loss of ( F ) dopamine and ( G ) DOPAC induced by αSyn PFF . ( H ) Western blot analysis of TH showing loss of TH induced by αSyn PFF in the SNpc region. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–7 animals per group. * P ≤ 0.05, ** P

    Article Snippet: The following primary antibodies were used: IBA1 (Wako, 1:1000) (RRID: AB_2314667), IBA1 (Abcam, 1:500) (RRID: AB_870576), Kv1.3 (Alomone Labs, 1:500) (RRID: AB_2040151), Kv1.3 (MilliporeSigma, 1:500)(RRID: AB_11212692), p-Kv1.3 (MilliporeSigma 1:500) (SAB4504254), p38 (Cell Signaling Technology, 1:500) (RRID: AB_330713), and tyrosine hydroxylase (TH) (MilliporeSigma, 1:1000) (RRID: AB_2201526).

    Techniques: Inhibition, Activity Assay, Mouse Assay, High Performance Liquid Chromatography, Western Blot

    Upregulated expression of the potassium channel Kv1.3 upon aggregated αSyn stimulation in ex vivo slices and B cells derived from patients with PD. ( A ) Midbrain slice cultures were treated with 1 μM αSyn Agg for 24 hours. qRT-PCR shows upregulated Kv1.3 mRNA expression. ( B ) Western blot shows upregulated Kv1.3 protein level in midbrain slice cultures treated with 1 μM αSyn Agg for 24 hours. ( C ) qRT-PCR of midbrain slice cultures treated with 1 μM αSyn Agg for 24 hours, revealing upregulation of the proinflammatory factors Nos2 , Csf2 , IL-6 , IL-1β , and Tnfa . ( D ) qRT-PCR shows increased Kv1.3 mRNA expression in B cell lymphocytes isolated from patients with PD compared with expression in B cell lymphocytes from age-matched controls. ( E ) Whole-cell patch clamping of B cell lymphocytes isolated from patients with PD showed higher Kv1.3 channel activity compared with that observed in age-matched controls ( n = 3 control and n = 3 PD). A 1-way ANOVA was used to compare multiple groups in C and D . Tukey’s post hoc analysis was applied. A 2-tailed Student’s t test was used to compare 2 groups. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–7 biological replicates from 2–3 independent experiments unless otherwise indicated. * P ≤ 0.05 and ** P

    Journal: The Journal of Clinical Investigation

    Article Title: Kv1.3 modulates neuroinflammation and neurodegeneration in Parkinson’s disease

    doi: 10.1172/JCI136174

    Figure Lengend Snippet: Upregulated expression of the potassium channel Kv1.3 upon aggregated αSyn stimulation in ex vivo slices and B cells derived from patients with PD. ( A ) Midbrain slice cultures were treated with 1 μM αSyn Agg for 24 hours. qRT-PCR shows upregulated Kv1.3 mRNA expression. ( B ) Western blot shows upregulated Kv1.3 protein level in midbrain slice cultures treated with 1 μM αSyn Agg for 24 hours. ( C ) qRT-PCR of midbrain slice cultures treated with 1 μM αSyn Agg for 24 hours, revealing upregulation of the proinflammatory factors Nos2 , Csf2 , IL-6 , IL-1β , and Tnfa . ( D ) qRT-PCR shows increased Kv1.3 mRNA expression in B cell lymphocytes isolated from patients with PD compared with expression in B cell lymphocytes from age-matched controls. ( E ) Whole-cell patch clamping of B cell lymphocytes isolated from patients with PD showed higher Kv1.3 channel activity compared with that observed in age-matched controls ( n = 3 control and n = 3 PD). A 1-way ANOVA was used to compare multiple groups in C and D . Tukey’s post hoc analysis was applied. A 2-tailed Student’s t test was used to compare 2 groups. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–7 biological replicates from 2–3 independent experiments unless otherwise indicated. * P ≤ 0.05 and ** P

    Article Snippet: The following primary antibodies were used: IBA1 (Wako, 1:1000) (RRID: AB_2314667), IBA1 (Abcam, 1:500) (RRID: AB_870576), Kv1.3 (Alomone Labs, 1:500) (RRID: AB_2040151), Kv1.3 (MilliporeSigma, 1:500)(RRID: AB_11212692), p-Kv1.3 (MilliporeSigma 1:500) (SAB4504254), p38 (Cell Signaling Technology, 1:500) (RRID: AB_330713), and tyrosine hydroxylase (TH) (MilliporeSigma, 1:1000) (RRID: AB_2201526).

    Techniques: Expressing, Ex Vivo, Derivative Assay, Quantitative RT-PCR, Western Blot, Isolation, Activity Assay

    Kv1.3 expression is highly induced in microglial cells in experimental models of PD and postmortem PD brains. ( A ) Western blot showing increased Kv1.3 protein levels in the substantia nigra of the Syn-AAV mouse model of PD. ( B ) qRT-PCR analysis of 8- to 24-week-old nigral tissues from the MitoPark mouse model of PD showing Kv1.3 induction compared with age-matched littermate controls. ( C ) Western blot of 24-week-old nigral tissues from the MitoPark mouse model of PD (MP) showing induction of Kv1.3 protein expression compared with age-matched littermate control mice (LM). ( D ) IHC in 24-week-old nigral tissues from the MitoPark mouse model of PD showing higher Kv1.3 protein levels (red) in IBA1-positive microglial cells (green) compared with age-matched controls as revealed by their colocalization (yellow). Scale bar: 20 μm. ( E ) qRT-PCR analysis of nigral tissues from the MPTP mouse model revealing induction of Kv1.3 mRNA expression. ( F ) Western blot showing increased Kv1.3 protein levels in substantia nigra of the MPTP mouse model of PD. ( G ) qRT-PCR analysis of postmortem human PD brains showing elevated Kv1.3 mRNA expression. ( H ) Western blot of the SN region of postmortem human PD brain showing induction of Kv1.3 protein expression compared with age-matched controls. n = 6–8. ( I ) Immunostaining revealing higher Kv1.3 levels in the prefrontal cortex of postmortem human PD brains compared with age-matched controls. Lower panel shows the deconvoluted binary image used for analysis. Three regions per brain were analyzed. Scale bar: 200 μm. ( J ) Dual DAB staining showing induction of Kv1.3 expression in HLA-DR–positive microglial cells in patients with DLBs compared with age-matched controls. Scale bars: 100 μm; 20 μm (enlarged insets). A 1-way ANOVA was used to compare multiple groups. Tukey’s post hoc analysis was applied B . A 2-tailed Student’s t test was used to compare 2 groups. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–9 biological replicates from 2–3 independent experiments unless otherwise indicated. * P ≤ 0.05, ** P

    Journal: The Journal of Clinical Investigation

    Article Title: Kv1.3 modulates neuroinflammation and neurodegeneration in Parkinson’s disease

    doi: 10.1172/JCI136174

    Figure Lengend Snippet: Kv1.3 expression is highly induced in microglial cells in experimental models of PD and postmortem PD brains. ( A ) Western blot showing increased Kv1.3 protein levels in the substantia nigra of the Syn-AAV mouse model of PD. ( B ) qRT-PCR analysis of 8- to 24-week-old nigral tissues from the MitoPark mouse model of PD showing Kv1.3 induction compared with age-matched littermate controls. ( C ) Western blot of 24-week-old nigral tissues from the MitoPark mouse model of PD (MP) showing induction of Kv1.3 protein expression compared with age-matched littermate control mice (LM). ( D ) IHC in 24-week-old nigral tissues from the MitoPark mouse model of PD showing higher Kv1.3 protein levels (red) in IBA1-positive microglial cells (green) compared with age-matched controls as revealed by their colocalization (yellow). Scale bar: 20 μm. ( E ) qRT-PCR analysis of nigral tissues from the MPTP mouse model revealing induction of Kv1.3 mRNA expression. ( F ) Western blot showing increased Kv1.3 protein levels in substantia nigra of the MPTP mouse model of PD. ( G ) qRT-PCR analysis of postmortem human PD brains showing elevated Kv1.3 mRNA expression. ( H ) Western blot of the SN region of postmortem human PD brain showing induction of Kv1.3 protein expression compared with age-matched controls. n = 6–8. ( I ) Immunostaining revealing higher Kv1.3 levels in the prefrontal cortex of postmortem human PD brains compared with age-matched controls. Lower panel shows the deconvoluted binary image used for analysis. Three regions per brain were analyzed. Scale bar: 200 μm. ( J ) Dual DAB staining showing induction of Kv1.3 expression in HLA-DR–positive microglial cells in patients with DLBs compared with age-matched controls. Scale bars: 100 μm; 20 μm (enlarged insets). A 1-way ANOVA was used to compare multiple groups. Tukey’s post hoc analysis was applied B . A 2-tailed Student’s t test was used to compare 2 groups. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–9 biological replicates from 2–3 independent experiments unless otherwise indicated. * P ≤ 0.05, ** P

    Article Snippet: The following primary antibodies were used: IBA1 (Wako, 1:1000) (RRID: AB_2314667), IBA1 (Abcam, 1:500) (RRID: AB_870576), Kv1.3 (Alomone Labs, 1:500) (RRID: AB_2040151), Kv1.3 (MilliporeSigma, 1:500)(RRID: AB_11212692), p-Kv1.3 (MilliporeSigma 1:500) (SAB4504254), p38 (Cell Signaling Technology, 1:500) (RRID: AB_330713), and tyrosine hydroxylase (TH) (MilliporeSigma, 1:1000) (RRID: AB_2201526).

    Techniques: Expressing, Western Blot, Quantitative RT-PCR, Mouse Assay, Immunohistochemistry, Immunostaining, Staining

    Fyn modulates the transcriptional regulation of Kv1.3 in microglial cells through the Fyn/PKCδ kinase signaling cascade. ( A ) In silico analysis of the promoter sequence of Kv1.3 revealed probable Nf-κB– and SP1-binding sites. ( B ) qRT-PCR analysis of immortalized MMCs cotreated with αSyn Agg and either SN50 (100 μg/mL) or SB203580 (1 μM), showing that both compounds attenuated αSyn Agg -induced Kv1.3 expression. ( C ) Western blot of Fyn WT and KO PMCs treated with αSyn Agg , showing that Fyn KO reduced the induction of the p38 MAPK pathway. ( D ) qRT-PCR analysis revealed that Fyn KO reduced αSyn Agg -induced Kv1.3 mRNA levels. ( E ) Whole-cell patch-clamp recording showing that Fyn KO attenuated αSyn Agg - and LPS-induced Kv1.3 activity compared with Fyn WT PMCs (WT control n = 24, WT αSyn Agg n = 12, WT LPS n = 29, Fyn KO αSyn Agg n = 20, Fyn KO LPS n = 15). ( F ) ICC showing that Fyn KO reduced αSyn Agg -induced Kv1.3 protein levels in PMCs. Scale bar: 15 μm. ( G ) ICC of PMCs revealed that αSyn Agg -induced Kv1.3 protein expression was reduced by PKCδ KO. Scale bar: 15 μm. ( H ) qRT-PCR analysis of PMCs showing that PKC KO reduced the expression of αSyn Agg -induced Kv1.3 mRNA. ( I ) Whole-cell patch clam recording of PMCs showing that PKC KO attenuated αSyn Agg - and LPS-induced Kv1.3 activity compared with PKC WT PMCs (WT control n = 24, WT αSyn Agg n = 12, WT LPS n = 20, PKC-KO αSyn Agg n = 29, PKC-KO LPS n = 35). Data are presented as the mean ± SD. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–4 biological replicates from 2–3 independent experiments unless otherwise indicated. * P ≤ 0.05, ** P

    Journal: The Journal of Clinical Investigation

    Article Title: Kv1.3 modulates neuroinflammation and neurodegeneration in Parkinson’s disease

    doi: 10.1172/JCI136174

    Figure Lengend Snippet: Fyn modulates the transcriptional regulation of Kv1.3 in microglial cells through the Fyn/PKCδ kinase signaling cascade. ( A ) In silico analysis of the promoter sequence of Kv1.3 revealed probable Nf-κB– and SP1-binding sites. ( B ) qRT-PCR analysis of immortalized MMCs cotreated with αSyn Agg and either SN50 (100 μg/mL) or SB203580 (1 μM), showing that both compounds attenuated αSyn Agg -induced Kv1.3 expression. ( C ) Western blot of Fyn WT and KO PMCs treated with αSyn Agg , showing that Fyn KO reduced the induction of the p38 MAPK pathway. ( D ) qRT-PCR analysis revealed that Fyn KO reduced αSyn Agg -induced Kv1.3 mRNA levels. ( E ) Whole-cell patch-clamp recording showing that Fyn KO attenuated αSyn Agg - and LPS-induced Kv1.3 activity compared with Fyn WT PMCs (WT control n = 24, WT αSyn Agg n = 12, WT LPS n = 29, Fyn KO αSyn Agg n = 20, Fyn KO LPS n = 15). ( F ) ICC showing that Fyn KO reduced αSyn Agg -induced Kv1.3 protein levels in PMCs. Scale bar: 15 μm. ( G ) ICC of PMCs revealed that αSyn Agg -induced Kv1.3 protein expression was reduced by PKCδ KO. Scale bar: 15 μm. ( H ) qRT-PCR analysis of PMCs showing that PKC KO reduced the expression of αSyn Agg -induced Kv1.3 mRNA. ( I ) Whole-cell patch clam recording of PMCs showing that PKC KO attenuated αSyn Agg - and LPS-induced Kv1.3 activity compared with PKC WT PMCs (WT control n = 24, WT αSyn Agg n = 12, WT LPS n = 20, PKC-KO αSyn Agg n = 29, PKC-KO LPS n = 35). Data are presented as the mean ± SD. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–4 biological replicates from 2–3 independent experiments unless otherwise indicated. * P ≤ 0.05, ** P

    Article Snippet: The following primary antibodies were used: IBA1 (Wako, 1:1000) (RRID: AB_2314667), IBA1 (Abcam, 1:500) (RRID: AB_870576), Kv1.3 (Alomone Labs, 1:500) (RRID: AB_2040151), Kv1.3 (MilliporeSigma, 1:500)(RRID: AB_11212692), p-Kv1.3 (MilliporeSigma 1:500) (SAB4504254), p38 (Cell Signaling Technology, 1:500) (RRID: AB_330713), and tyrosine hydroxylase (TH) (MilliporeSigma, 1:1000) (RRID: AB_2201526).

    Techniques: In Silico, Sequencing, Binding Assay, Quantitative RT-PCR, Expressing, Western Blot, Patch Clamp, Activity Assay, Immunocytochemistry

    Kv1.3 modulates neuroinflammation in a cell culture model of PD. ( A – C ) Kv1.3 WT and KO PMCs were treated with 1 μM αSyn Agg for 24 hours. Luminex analysis shows that Kv1.3 KO reduced the release of the αSyn Agg -induced proinflammatory factors ( A ) TNF-α, ( B ) IL-12, and ( C ) IL-1β. ( D – H ) Immortalized MMCs were transfected with WT a Kv1.3 plasmid, and then 48 hours after transfection, cells were treated with 1 μM αSyn Agg for 24 hours. ( D – F ) qRT-PCR analysis showing that Kv1.3 overexpression aggravated αSyn Agg -induced production of the proinflammatory factors ( D ) Nos2 , ( E ) pro– IL-1β , and ( F ) TNF-α . ( G and H ) Luminex analysis showing that Kv1.3 overexpression potentiated the release of the proinflammatory factors ( G ) IL-6 and ( H ) IL-12. ( I ) Voltage ramp from –120 mV to 40 mV elicited a characteristic outward rectifying current in αSyn Agg -treated microglia that was sensitive to the Kv1.3-selective inhibitor PAP-1. ( J ) LDH assay showing that PAP-1 reduced αSyn Agg -induced LDH release from microglial cells. ( K – M ) Luminex assay revealing that PAP-1 attenuated the αSyn Agg -induced proinflammatory factors ( K ) IL-12, ( L ) TNF-α, and ( M ) IL-6. ( N ) Western blot analysis demonstrating that PAP-1 reduced αSyn Agg -induced NLRP3 expression. ( O ) ICC analysis revealed that PAP-1 reduced NLRP3 expression induced by αSyn Agg . Scale bar: 25 μm. A 1-way ANOVA was performed to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–4 biological replicates from 2–3 independent experiments. * P ≤ 0.05, ** P

    Journal: The Journal of Clinical Investigation

    Article Title: Kv1.3 modulates neuroinflammation and neurodegeneration in Parkinson’s disease

    doi: 10.1172/JCI136174

    Figure Lengend Snippet: Kv1.3 modulates neuroinflammation in a cell culture model of PD. ( A – C ) Kv1.3 WT and KO PMCs were treated with 1 μM αSyn Agg for 24 hours. Luminex analysis shows that Kv1.3 KO reduced the release of the αSyn Agg -induced proinflammatory factors ( A ) TNF-α, ( B ) IL-12, and ( C ) IL-1β. ( D – H ) Immortalized MMCs were transfected with WT a Kv1.3 plasmid, and then 48 hours after transfection, cells were treated with 1 μM αSyn Agg for 24 hours. ( D – F ) qRT-PCR analysis showing that Kv1.3 overexpression aggravated αSyn Agg -induced production of the proinflammatory factors ( D ) Nos2 , ( E ) pro– IL-1β , and ( F ) TNF-α . ( G and H ) Luminex analysis showing that Kv1.3 overexpression potentiated the release of the proinflammatory factors ( G ) IL-6 and ( H ) IL-12. ( I ) Voltage ramp from –120 mV to 40 mV elicited a characteristic outward rectifying current in αSyn Agg -treated microglia that was sensitive to the Kv1.3-selective inhibitor PAP-1. ( J ) LDH assay showing that PAP-1 reduced αSyn Agg -induced LDH release from microglial cells. ( K – M ) Luminex assay revealing that PAP-1 attenuated the αSyn Agg -induced proinflammatory factors ( K ) IL-12, ( L ) TNF-α, and ( M ) IL-6. ( N ) Western blot analysis demonstrating that PAP-1 reduced αSyn Agg -induced NLRP3 expression. ( O ) ICC analysis revealed that PAP-1 reduced NLRP3 expression induced by αSyn Agg . Scale bar: 25 μm. A 1-way ANOVA was performed to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–4 biological replicates from 2–3 independent experiments. * P ≤ 0.05, ** P

    Article Snippet: The following primary antibodies were used: IBA1 (Wako, 1:1000) (RRID: AB_2314667), IBA1 (Abcam, 1:500) (RRID: AB_870576), Kv1.3 (Alomone Labs, 1:500) (RRID: AB_2040151), Kv1.3 (MilliporeSigma, 1:500)(RRID: AB_11212692), p-Kv1.3 (MilliporeSigma 1:500) (SAB4504254), p38 (Cell Signaling Technology, 1:500) (RRID: AB_330713), and tyrosine hydroxylase (TH) (MilliporeSigma, 1:1000) (RRID: AB_2201526).

    Techniques: Cell Culture, Luminex, Transfection, Plasmid Preparation, Quantitative RT-PCR, Over Expression, Lactate Dehydrogenase Assay, Western Blot, Expressing, Immunocytochemistry

    Upregulated expression of the potassium channel Kv1.3 upon aggregated αSyn stimulation in microglial cells in vitro. ( A ) Whole-cell patch-clamp recordings of PMCs treated with 1 μM αSyn Agg for 24–48 hours, showing αSyn Agg -induced increased Kv1.3 activity (control n = 24 and αSyn Agg n = 12). Kv1.3 was identified by its characteristic use dependence, which was revealed when applying a train of ten 200-ms pulses from –80 to 40 mV at 1-second intervals (1 Hz). ( B ) qRT-PCR showing that αSyn Agg induced Kv1.3 mRNA expression without significantly altering other potassium channels. ( C ) Western blot of αSyn Agg -induced Kv1.3 protein expression in PMCs. ( D ) ICC of αSyn Agg -induced Kv1.3 protein expression in PMCs. Scale bar: 100 μm. ( E ) Flow cytometric analysis of immortalized MMCs treated with 1 μM αSyn Agg for 24 hours, showing αSyn Agg -induced Kv1.3 surface expression. ( F ) qRT-PCR of human microglia treated with LPS (1 μg/mL) and IL-4 (20 ng/mL) for 6 hours, showing LPS-induced Kv1.3 expression. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied in B and F . A 2-tailed Student’s t test was used for all other figures when comparing 2 groups. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–5 biological replicates from 2–3 independent experiments unless otherwise noted. * P ≤ 0.05, ** P

    Journal: The Journal of Clinical Investigation

    Article Title: Kv1.3 modulates neuroinflammation and neurodegeneration in Parkinson’s disease

    doi: 10.1172/JCI136174

    Figure Lengend Snippet: Upregulated expression of the potassium channel Kv1.3 upon aggregated αSyn stimulation in microglial cells in vitro. ( A ) Whole-cell patch-clamp recordings of PMCs treated with 1 μM αSyn Agg for 24–48 hours, showing αSyn Agg -induced increased Kv1.3 activity (control n = 24 and αSyn Agg n = 12). Kv1.3 was identified by its characteristic use dependence, which was revealed when applying a train of ten 200-ms pulses from –80 to 40 mV at 1-second intervals (1 Hz). ( B ) qRT-PCR showing that αSyn Agg induced Kv1.3 mRNA expression without significantly altering other potassium channels. ( C ) Western blot of αSyn Agg -induced Kv1.3 protein expression in PMCs. ( D ) ICC of αSyn Agg -induced Kv1.3 protein expression in PMCs. Scale bar: 100 μm. ( E ) Flow cytometric analysis of immortalized MMCs treated with 1 μM αSyn Agg for 24 hours, showing αSyn Agg -induced Kv1.3 surface expression. ( F ) qRT-PCR of human microglia treated with LPS (1 μg/mL) and IL-4 (20 ng/mL) for 6 hours, showing LPS-induced Kv1.3 expression. A 1-way ANOVA was used to compare multiple groups. In most cases, Tukey’s post hoc analysis was applied in B and F . A 2-tailed Student’s t test was used for all other figures when comparing 2 groups. Each dot on the bar graphs represents a biological replicate. Data are presented as the mean ± SEM, with 3–5 biological replicates from 2–3 independent experiments unless otherwise noted. * P ≤ 0.05, ** P

    Article Snippet: The following primary antibodies were used: IBA1 (Wako, 1:1000) (RRID: AB_2314667), IBA1 (Abcam, 1:500) (RRID: AB_870576), Kv1.3 (Alomone Labs, 1:500) (RRID: AB_2040151), Kv1.3 (MilliporeSigma, 1:500)(RRID: AB_11212692), p-Kv1.3 (MilliporeSigma 1:500) (SAB4504254), p38 (Cell Signaling Technology, 1:500) (RRID: AB_330713), and tyrosine hydroxylase (TH) (MilliporeSigma, 1:1000) (RRID: AB_2201526).

    Techniques: Expressing, In Vitro, Patch Clamp, Activity Assay, Quantitative RT-PCR, Western Blot, Immunocytochemistry