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. 2012 Sep 14;111(7):842-53.
doi: 10.1161/CIRCRESAHA.111.263525. Epub 2012 Jul 27.

Redox-sensitive sulfenic acid modification regulates surface expression of the cardiovascular voltage-gated potassium channel Kv1.5

Laurie K Svoboda  1 , Khalilah G Reddie, Lian Zhang, Eileen D Vesely, Elizabeth S Williams, Sarah M Schumacher, Ryan P O'Connell, Robin Shaw, Sharlene M Day, Justus M Anumonwo, Kate S Carroll, Jeffrey R Martens
Affiliations

Redox-sensitive sulfenic acid modification regulates surface expression of the cardiovascular voltage-gated potassium channel Kv1.5

Laurie K Svoboda et al. Circ Res. .

Abstract

Rationale: Kv1.5 (KCNA5) is expressed in the heart, where it underlies the I(Kur) current that controls atrial repolarization, and in the pulmonary vasculature, where it regulates vessel contractility in response to changes in oxygen tension. Atrial fibrillation and hypoxic pulmonary hypertension are characterized by downregulation of Kv1.5 protein expression, as well as with oxidative stress. Formation of sulfenic acid on cysteine residues of proteins is an important, dynamic mechanism for protein regulation under oxidative stress. Kv1.5 is widely reported to be redox-sensitive, and the channel possesses 6 potentially redox-sensitive intracellular cysteines. We therefore hypothesized that sulfenic acid modification of the channel itself may regulate Kv1.5 in response to oxidative stress.

Objective: To investigate how oxidative stress, via redox-sensitive modification of the channel with sulfenic acid, regulates trafficking and expression of Kv1.5.

Methods and results: Labeling studies with the sulfenic acid-specific probe DAz and horseradish peroxidase-streptavidin Western blotting demonstrated a global increase in sulfenic acid-modified proteins in human patients with atrial fibrillation, as well as sulfenic acid modification to Kv1.5 in the heart. Further studies showed that Kv1.5 is modified with sulfenic acid on a single COOH-terminal cysteine (C581), and the level of sulfenic acid increases in response to oxidant exposure. Using live-cell immunofluorescence and whole-cell voltage-clamping, we found that modification of this cysteine is necessary and sufficient to reduce channel surface expression, promote its internalization, and block channel recycling back to the cell surface. Moreover, Western blotting demonstrated that sulfenic acid modification is a trigger for channel degradation under prolonged oxidative stress.

Conclusions: Sulfenic acid modification to proteins, which is elevated in diseased human heart, regulates Kv1.5 channel surface expression and stability under oxidative stress and diverts channel from a recycling pathway to degradation. This provides a molecular mechanism linking oxidative stress and downregulation of channel expression observed in cardiovascular diseases.

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Figures

Figure 1
Figure 1. Increase in global sulfenic acid modification in human heart tissue with AF
A, HRP-streptavidin western blot depicting DAz-labeled sulfenic acid in atrial tissue lysate from healthy and diseased human hearts. B, Global sulfenic acid modification (HRP-streptavidin signal) from patient samples was quantified via densitometry and normalized to GAPDH levels. p=.04, significant.
Figure 2
Figure 2. Sulfenic acid modification of Kv1.5
A, Topology of a single alpha subunit of wild type, human Kv1.5, showing all ten cysteine residues. Aligned vertebrate Kv1.5 sequences centered on human C-terminal cysteines. Conserved cysteines are highlighted, with C581 in magenta. B, LTK cells stably expressing V5-tagged wild-type (WT) were labeled with DAz-1 and conjugated to p-biotin (lane 3). Sulfenic acid modification was detected by HRP-streptavidin western blot. C, Top: LTK cells stably expressing V5-tagged wild-type (WT) Kv1.5, Kv1.5-4CS, or Kv1.5-6CS were labeled with DAz-1. Bottom: averaged quantified densitometry data from three experiments normalized to WT Kv1.5, and analyzed using 1-way ANOVA, followed by Tukey’s post-hoc comparison. *** indicates p<0.0001 relative to WT D, Top: COOH-terminal cysteines were individually re-introduced into the null background of Kv1.5-6CS. Sulfenic acid modifications were detected by labeling with DAz-1. Bottom: Summary of three experiments quantified via densitometry, normalized to WT Kv1.5, and analyzed using 1-way ANOVA, followed by Tukey’s post-hoc comparison. *** indicates p<0.0001 relative to WT.
Figure 3
Figure 3. Redox sensitivity of Kv1.5 sulfenic acid modification
A, Top: HRP-streptavidin western blot depicting LTK cells stably expressing Kv1.5-WT, treated for 30 min with tBOOH or vehicle in the presence or absence of N-acetylcysteine (NAC), followed by labeling with DAz-1. Bottom: Data from three separate experiments were quantified via densitometry. HRP-streptavidin signal was normalized to V5 (Kv1.5) signal, converted to ratios (relative to vehicle) and analyzed using 1-way ANOVA, followed by Tukey’s post-hoc comparison. ** indicates p<0.01, * indicates p<0.05. B-C, Top: LTK cells stably expressing Kv1.5-WT were treated for 30 min with H2O2 (B), diamide (C) or vehicle, followed by labeling with DAz-1, and HRP-streptavidin western blot. Bottom: Data from three separate experiments were quantified via densitometry and analyzed via unpaired t-test with Welch’s correction. * indicates p<0.05.
Figure 4
Figure 4. Redox sensitivity of endogenous Kv1.5 and IKur curents
A, Representative Western blot images depicting sulfenic acid modification on Kv1.5 immunoprecipitated from isolated, perfused rat hearts. Data from 5 experiments were quantified using densitometry and analyzed via unpaired t test with Welch’s correction. p=.03, significant B Rat ventricular myocytes were acutely isolated and currents were recorded following 1 hr. treatment with dimedone, which is specific for sulfenic acid-modified proteins (schematic at top). The protocol used to isolate IKslow (IKur + Iss) and Iss are shown with representative traces. Bar graph depicts quantification of Kv1.5-specific (IKur) current density upon treatment with dimedone. * indicates p < 0.05 as determined by t test. C, HL-1 cells transiently expressing Kv1.5-GFP were treated with vehicle, tBOOH and/or dimedone for 60 min. Cells were then stained live to label surface populations of Kv1.5 (red, collapsed Z-stacks). Surface channel was quantified using NIH ImageJ software, normalized to total GFP fluorescence (green, i.e. total Kv1.5), and analyzed via Kruskal-Wallis test, followed by Dunn’s post test, n=100+ cells per condition, three experiments. * indicates p<0.05, relative to vehicle. Scale bar: 30 microns. D, Results of whole cell voltage clamping experiments in HL-1 cells transiently transfected with Kv1.5-GFP-WT and treated with vehicle (n=6) or tBOOH (n=4) for 60 min. Top: Sample outward Kv channel current traces for vehicle and peroxide-treated cells. Bottom: Current density. Results were analyzed via unpaired t-test. *indicates p<0.05 relative to control.
Figure 5
Figure 5. Functional effect of oxidant induced sulfenic acid modification of Kv1.5 at C581
A, HL-1 cells transiently expressing Kv1.5-C581S-GFP were treated with vehicle, tBOOH, and/or dimedone for 60 min. Cells were then analyzed as described in Figure 3B (n=60+ cells per condition, three experiments). B, Results of whole cell voltage clamping experiments in HL-1 cells transiently transfected with Kv1.5-GFP-C581S and treated with vehicle (n=5) or tBOOH (n=4) for 60 min. Top: Sample outward Kv channel current traces for vehicle and tBOOH treated cells. Bottom: Current density. Results were analyzed via unpaired t-test. C, HL-1 cells transiently expressing Kv1.5-GFP-S581C were treated and analyzed as outlined in (A) (n=60+ cells per condition, three experiments). D, Results of whole cell voltage clamping experiments in HL-1 cells transiently transfected with Kv1.5-GFP-S581C and treated with vehicle (n=5) or tBOOH (n=4) for 60 min. and analyzed as in (B) ** indicates p<0.001. *** indicates p<0.0001.
Figure 6
Figure 6. Effect of sulfenic acid on Kv1.5 internalization and recycling
A, HL-1 cells transiently expressing Kv1.5-WT-GFP were treated with vehicle, tBOOH, or dimedone for 60 min. Internalized channel (cyan) was visualized using confocal microscopy, quantified using NIH ImageJ software, normalized to GFP fluorescence (i.e. total Kv1.5), and analyzed via Kruskal-Wallis test, followed by Dunn’s post test (n= 85+ cells per condition, three experiments). ** indicates p<0.001, *** p<.0001 relative to vehicle. Scale bar: 30 microns. B, Left: In HL-1 cells, recycled channel (gray) at 60 min was measured after vehicle or dimedone treatment for 90 min. Data were quantified using NIH ImageJ software and analyzed via 1-way ANOVA, followed by Tukey’s post-hoc comparison (n= at least 70 cells per condition, three experiments). *** indicates p<0.0001 relative to vehicle control.
Figure 7
Figure 7. Sub-cellular localization of internalized Kv1.5 following oxidative stress
A, HL-1 cells transiently expressing Kv1.5-GFP were treated for 60 min with tBOOH, followed by staining with EEA1 antibody. Figure depicts a cross sectional image. Yellow puncta indicate colocalization between internalized Kv1.5 and EEA1. Bottom: Quantified data (n=25 cells, 3 separate experiments), * indicates p<.05 relative to vehicle control. B, HL-1 cells transiently expressing Kv1.5-GFP were treated for 60 min with tBOOH, followed by staining with HSP70 antibody. Figure depicts a cross sectional image. Yellow puncta indicate colocalization between internalized Kv1.5 and HSP70. Bottom: Quantified data (n=30 cells, 3 separate experiments), * indicates p<.05 relative to vehicle control.
Figure 8
Figure 8. Effect of sulfenic acid on Kv1.5 degradation
A, HL-1 cells stably expressing Kv1.5-WT were treated with vehicle or tBOOH, alone or concurrently with the proteasome inhibitor, MG132 in the presence of cycloheximide (5 μM) to block the synthesis of new proteins. Cell lysates were generated and analyzed by western blotting with anti-V5 antibody. B, HL-1 cells stably expressing Kv1.5-C581S were treated and analyzed as in (A) (quantified data based on three separate experiments). C, HL-1 cells stably expressing Kv1.5-WT were treated with tBOOH as outlined in (A), alone or concurrently with dimedone. Cell lysates were generated and analyzed by western blotting with anti-V5 antibody (quantified data based on three separate experiments). D, Hypothetical model for sulfenic acid modulation of Kv1.5 trafficking and stability.
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