Redox-Sensitive Sulfenic Acid Modification Regulates Surface Expression of the
Cardiovascular Voltage-Gated Potassium Channel Kv1.5
Laurie K. Svoboda, 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 and Jeffrey R. Martens
Circ Res. 2012;111:842-853; originally published online July 27, 2012;
doi: 10.1161/CIRCRESAHA.111.263525
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2012 American Heart Association, Inc. All rights reserved.
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Cellular Biology
Redox-Sensitive Sulfenic Acid Modification Regulates
Surface Expression of the Cardiovascular Voltage-Gated
Potassium Channel Kv1.5
Laurie K. Svoboda, 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
Rationale: Kv1.5 (KCNA5) is expressed in the heart, where it underlies the IKur 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. (Circ Res. 2012;111:842-853.)
Key Words: oxidative stress 䡲 Kv1.5 䡲 sulfenic acid 䡲 atrial fibrillation 䡲 trafficking
䡲 voltage-gated potassium channels 䡲 posttranslational modification
A
trial fibrillation is the most common cardiac arrhythmia
and is predicted to rise dramatically over the next several
years. Treatment includes the use of antiarrhythmic drugs and/or
electric cardioversion. These treatments, although initially successful in managing atrial fibrillation, often become ineffective
over time. One likely contributing factor to this resistance is the
electric and structural remodeling that occurs in the heart with
the persistence of atrial fibrillation. Chronic atrial fibrillation–
induced electrophysiological remodeling of the atria includes
changes to action potential duration and refractory period that
result from alterations in the function and expression of ion
channels in cardiac myocytes.
In the atria, the balance of inward Ca2⫹ and outward K⫹
currents determines action potential duration and refractoriness.
Potassium channel– blocking drugs have been used as antiarrhythmics to treat patients for many years; however, these agents
Original received December 22, 2011; revision received July 25, 2012; accepted July 27, 2012. In June 2012, the average time from submission to first
decision for all original research papers submitted to Circulation Research was 13.35 days.
From Environmental Health Sciences, University of Michigan School of Public Health, Ann Arbor (L.K.S.); Life Sciences Institute, University of
Michigan, Ann Arbor (K.G.R., K.S.C.); the Departments of Pharmacology (L.K.S., L.Z., E.D.V., E.S.W., S.M.S., J.R.M.), Molecular and Integrative
Physiology (R.P.O., J.M.A.), and Internal Medicine (S.M.D., J.M.A.), University of Michigan Medical School, Ann Arbor; the Cardiovascular Research
Institute, University of California, San Francisco (R.S.); and the Department of Chemistry, The Scripps Research Institute, Jupiter, FL (K.S.C.).
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.111.
263525/-/DC1.
Correspondence to Jeffrey R. Martens, PhD, Department of Pharmacology, University of Michigan Medical School, 1301 MSRB III, 1150 W Medical
Center Dr, Ann Arbor, MI 48109-5632. E-mail martensj@umich.edu
© 2012 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.111.263525
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at Scripps Res. Inst./Kresge on February 20, 2013
Svoboda et al
are most effective in treating recent-onset atrial fibrillation and
are less effective in patients with chronic atrial fibrillation. One
of the predominant repolarizing K⫹ currents in the atria is the
ultrarapid delayed-rectifier current IKur. This current is encoded
by the Kv1.5 channel subunit, which in the human heart is
selectively expressed in the atria and is considered an important
therapeutic target for treatment of atrial fibrillation.1,2 Paradoxically, IKur and Kv1.5 protein are reduced in chronic human
atrial fibrillation, which may limit its potential therapeutic
efficacy in patients with persistent or permanent arrhythmias.3
This pathology-specific decrease in Kv1.5 protein level is also
observed in the pulmonary vasculature during chronic hypoxic
pulmonary hypertension (HPH).4 Although it is an important
therapeutic consideration, the precise mechanisms underlying
this ion channel remodeling and decrease in channel protein
remain unknown.
One element common to both HPH and chronic atrial
fibrillation is a metabolic imbalance leading to a pro-oxidant
shift in cellular redox state, which causes cellular oxidative
stress. Oxidative stress, defined as excessive production of
reactive oxygen species (ROS), and/or diminished antioxidant capacity, is a newly recognized hallmark of cardiovascular disease. Rapid activation of the atrium, which occurs
during atrial fibrillation, leads to increased ROS and a
decrease in tissue levels of antioxidants.5,6 Multiple K⫹
channels and currents, including the Kv1.5-encoded IKur, are
regulated by oxidizing agents.4,7–9 Therefore, it is likely that
the altered oxidative state of myocytes contributes to electric
remodeling. However, despite significant evidence linking
conditions of oxidative stress to reduced Kv1.5 expression,
the molecular link(s) between these observations is unclear.
One potential direct mechanism for Kv1.5 regulation is via
redox-sensitive posttranslational modifications to the channel.
The thiol (-SH) group of the amino acid cysteine is a principal
target for ROS in many proteins, including enzymes, signaling
proteins, and transcription factors.10 Oxidation of key cysteine
residues is an important mechanism whereby changes in cellular
redox balance can lead to modification of protein function.
Several different oxidative cysteine modifications (oxoforms)
have been identified in vivo that play a crucial role in protein
stability and function.11 Of these cysteine oxoforms, reversible
formation of cysteine sulfenic acid (Cys-SOH) is emerging as an
Sulfenic Acid and Kv1.5 Expression
843
Non-standard Abbreviations and Acronyms
EEA1
GFP
HPH
HRP
HSP
IK
KHB
MG132
NAC
ROS
SOH
tBOOH
early endosomal antigen 1
green fluorescent protein
hypoxic pulmonary hypertension
horseradish peroxidase
heat shock protein
delayed rectifier potassium current
Krebs-Henseleit buffer
N-(benzyloxycarbonyl)leucinylleucinylleucinal protease
inhibitor
N-acetylcysteine
reactive oxygen species
sulfenic acid
tertiary butyl hydroxperoxide
important mechanism for dynamic regulation of protein function
in response to changes in cellular redox state.10,11 Cys-SOH can
form during conditions of cellular oxidative stress and, depending on the protein microenvironment, afford a metastable modification or represent a transient species leading to a more stable
disulfide or sulfinic acid form.11 Kv1.5 has 6 intracellular
cysteines divided among the NH2 and COOH termini. This,
combined with the widely reported redox sensitivity of the
channel, led to the hypothesis that sulfenic acid modification to
the channel may regulate its function and expression. In the
current study, we show that there is a global increase in the level
of sulfenic acid–modified proteins in the atria of human patients
with chronic atrial fibrillation, compared with healthy control
subjects. We further demonstrate that Kv1.5 is a substrate for
sulfenic acid modification that, under conditions of oxidative
stress, functions as a fate switch to divert the channel from a
recycling to a degradation pathway in myocytes.
Methods
Human Heart Tissue Procurement
Atrial myocardial tissue from patients with chronic atrial fibrillation
was collected at the time of transplantation at the University of
Michigan or the University of California, San Francisco. Atrial myocardial tissue from nonfailing hearts was collected from unmatched
Figure 1. Increase in global sulfenic acid
(SOH) modification in human heart tissue
with atrial fibrillation. A, HRP-streptavidin
Western blot (WB) depicting DAz-labeled sulfenic acid in atrial tissue lysate from healthy
and diseased human. B, Global sulfenic acid
modification (HRP-streptavidin signal) from
patient samples was quantified via densitometry and normalized to GAPDH levels. P⫽0.04,
significant.
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Figure 2. Sulfenic acid modification of Kv1.5. A, Topology of a single ␣-subunit of WT human Kv1.5, showing all 10 cysteine residues. Aligned vertebrate Kv1.5 sequences are centered on human C-terminal cysteines. Conserved cysteines are highlighted, with
C581 in dark gray. B, LTK cells stably expressing V5-tagged WT were labeled with DAz-1 and conjugated to p-biotin (lane 3). Sulfenic
acid modification was detected by HRP-streptavidin Western blot (WB). C, Top, LTK cells stably expressing V5-tagged WT Kv1.5,
Kv1.5 to 4CS, or Kv1.5 to 6CS were labeled with DAz-1. Bottom, Averaged quantified densitometry data from 3 experiments normalized to WT Kv1.5 and analyzed using 1-way ANOVA, followed by Tukey post hoc comparison. ***P⬍0.0001 relative to WT. D, Top,
COOH-terminal cysteines were individually reintroduced into the null background of Kv1.5 6CS. Sulfenic acid modifications were
detected by labeling with DAz. Bottom, Summary of 3 experiments quantified via densitometry, normalized to WT Kv1.5, and analyzed
using 1-way ANOVA, followed by Tukey post hoc comparison. ***P⬍0.0001 relative to WT.
donors from the University of Michigan under approval from the Gift of
Life: Michigan Organ and Tissue Donation Program. Before tissue
retrieval, all hearts were perfused with ice-cold cardioplegia. Samples
from each heart were snap-frozen in liquid N2 in the operating room and
stored at ⫺80°C. Tissue collection from human hearts used in this study
has the approval of the University of Michigan Institutional Review
Board, and subjects gave informed consent.
Stable Cell Lines
Stable cell lines expressing Kv1.5 wild-type (WT) and cysteine
mutant constructs were created in LTK cells (a mouse fibroblast cell
line), using the Retro-X Universal Packaging System from Clontech
(Mountain View, CA), according to the manufacturer’s instructions.
HL-1 cells were a gift from William Claycomb.
DAz Labeling of Sulfenic Acid, Staudinger
Ligation, and Immunoprecipitation of Kv1.5
Rodent or human tissue samples, LTK or HL-1 cells, were harvested
in nondenaturing lysis buffer containing DAz, followed by
Staudinger ligation and horseradish peroxidase (HRP)-streptavidin
Western blotting12 (Online Data Supplement). Blots were stripped
and reprobed with anti-V5 or anti-Kv1.5 antibody to assess immunoprecipitation efficiency.
Perfusion of Isolated Rat Heart
Rat hearts were excised and Langendorff-perfused with KrebsHenseleit buffer (KHB) with or without 200 ␮mol/L diamide for 55
minutes,13 followed by isolation of membranes14 and labeling with
DAz12 (Online Data Supplement).
Immunocytochemistry and Confocal Imaging
For surface, internalization, and recycling assays, immunocytochemistry was performed as described previously, with minor modifications15 (Online Data Supplement).
Electrophysiology
Ionic currents were recorded at room temperature in HL-1 cells
transiently expressing Kv1.5 using the whole-cell configuration of
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Svoboda et al
the patch-clamp technique, as described previously, with minor
modifications16 (Online Data Supplement). For measurement of IKur,
rat cardiac myocytes were isolated sterilely, and currents were
recorded as outlined in the Online Data Supplement.17
Proteasome Activity Assay
An optimized method was used for determining heart tissue
chymotrypsin-like activity.18 Reported values are without ATP and were
averaged from 3 independent experiments. Values for dimedone-treated
cell lysates are expressed as a percentage of values for untreated cells for
each experiment.
Statistics
Statistics were performed using Prism software Version 5 from
Graphpad Prism Software (San Diego, CA). All data are expressed as
mean⫾SEM. Data were analyzed using 1-way ANOVA followed by
Tukey post hoc test or unpaired, 2-tailed t test. A probability value
of ⬍0.05 was considered significant.
Results
Human Chronic Atrial Fibrillation Is Accompanied
by a Global Increase in Sulfenic
Acid–Modified Proteins
Significant evidence indicates that atrial fibrillation is associated
with oxidative stress5,6,19 and that this may play an important role
in the pathological remodeling that occurs in chronic disease5;
however, a role for sulfenic acid modification in this process has
not been investigated. We therefore hypothesized that atrial
fibrillation in human patients is accompanied by an increase in
sulfenic acid–modified proteins. To test this hypothesis, we used
the novel chemical probe DAz. When coupled to a biotinylated
secondary agent, DAz enables detection of sulfenic acid–modified protein in cells and tissue12 (Online Figure IA). We first
used this technique in oxidized mouse heart and detected a
robust increase in sulfenic acid, confirming that DAz labels
sulfenic acid–modified proteins in tissue (Online Figure IB).
Consistent with our hypothesis, the level of sulfenic acid
modification in the human atria from patients with chronic atrial
fibrillation was substantially higher than that of healthy control
patients (Figure 1A and 1B and Online Figure II). This finding,
combined with the evidence that Kv1.5 current is redoxsensitive, led us to explore a mechanistic role for sulfenic acid
regulation of Kv1.5 in atrial fibrillation.
Kv1.5 Is Modified by Sulfenic Acid on a Single
COOH-Terminal Cysteine
Kv1.5 has 6 intracellular cysteines: 2 on the NH2 terminus
and 4 on the COOH terminus, which may be candidates for
oxidation to sulfenic acid (Figure 2A). Sequence comparison
revealed that all 4 COOH-terminal sites are conserved in
mammalian sequences (Figure 2A). Therefore, we hypothesized
that Kv1.5 may be a substrate for sulfenic acid modification and
that this may account for its reported redox sensitivity. Kv1.5
appears as a doublet comprised as a nascent, nonglycosylated
form and a mature, glycosylated form.16 In LTK cells stably
expressing Kv1.5 WT, we found that Kv1.5 is modified with
sulfenic acid, as indicated by a doublet appearing at approximately 80 kDa on the HRP-streptavidin Western blot (Figure
2B, lane 3). Omitting DAz or p-biotin resulted in few nonspecific bands, indicating that DAz is specific for sulfenic acid–
modified proteins and that p-biotin is specific for proteins
Sulfenic Acid and Kv1.5 Expression
845
labeled with DAz (Figure 2B, lanes 1–2). To confirm that the
observed bands were indeed Kv1.5, we performed DAz labeling
and immunoprecipitation, followed by deglycosylation of the
channel with PNGase. Consistent with literature indicating that
Kv1.5 undergoes glycosylation,16 PNGase treatment eliminated
the upper, glycosylated band in both the HRP-streptavidin
Western blot and the anti-V5 Western blots, further indicating
that the sulfenic acid–modified protein is Kv1.5 (Online Figure
IIIA). To test the specificity of this labeling, we used the parent
compound and competitive inhibitor of DAz, 5,5-dimethyl-1,3cyclohexanedione (dimedone).12 Including dimedone with DAz
in the lysis buffer blocked labeling of sulfenic acid–modified
Kv1.5 (Online Figure IIIB), demonstrating the specificity of
DAz for sulfenic acid–modified Kv1.5.
To determine the specific sulfenic acid modification profile
of Kv1.5, we first used LTK cells stably expressing Kv1.5-WT,
as well as 2 additional cysteine mutant proteins: (1) Kv1.5 to
4CS, in which all 4 COOH-terminal cysteines were mutated to
serine, and (2) Kv1.5 to 6CS, in which all 6 intracellular
cysteines were mutated to serine. Importantly, all cysteine
mutant channels undergo proper folding, are able to traffic to the
cell surface, and are fully functional.16 As expected, mutating all
6 intracellular cysteines substantially reduced sulfenic acid
modification of Kv1.5. However, sulfenic acid modification of
Kv1.5 to 4CS was reduced to the same extent (Figure 2C),
indicating that sulfenic acid modification occurs primarily on the
COOH-terminus of the channel. To resolve the specific locus of
this modification, we developed 4 additional stable cell lines,
each expressing Kv1.5 with a different COOH-terminal cysteine
reintroduced individually into the null background of the
6-cysteine mutant (Kv1.5-S564C, S581C, S586C, and S604C).
As shown in Figure 2D, reintroducing C581 restored sulfenic
acid modification of Kv1.5 to a level that was similar to WT.
Taken together, these studies show that C581, located on the
cytoplasmic portion of Kv1.5 near the COOH-terminus, is the
principal site for oxidation to sulfenic acid.
Oxidative Stress Induces Formation of Sulfenic
Acid on Kv1.5
To explore the redox sensitivity of sulfenic acid modification,
we induced oxidative stress in cells stably expressing Kv1.5WT, using the organic hydroperoxide tertiary butyl hydroperoxide (tBOOH). Using DAz labeling, immunoprecipitation of
Kv1.5 and HRP-streptavidin Western blotting, we found that
30-minute treatment with 200 ␮mol/L tBOOH caused a
significant increase in the fraction of sulfenic acid–modifiedKv1.5 (P⬍0.01, Figure 3A). Importantly, concurrent treatment
with the thiol-specific antioxidant N-acetylcysteine (NAC) abolished the tBOOH-induced increase in sulfenic acid, while having
no significant effect alone. We also observed an increase in
sulfenic acid modification to Kv1.5 after treatment with the more
physiologically relevant oxidizing agent, hydrogen peroxide, or
by depletion of reduced intracellular glutathione with diamide20
(Figure 3B and 3C), indicating that the modification is not
specific to a particular oxidant. We also examined the oxidantinduced modification of Kv1.5 in HL-1 atrial myocytes, which
maintain a differentiated, contractile phenotype in vitro.21 Importantly, we observed a similar increase in sulfenic acid
modification to Kv1.5 in myocytes after treatment with either
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Figure 3. Redox sensitivity of Kv1.5 sulfenic acid modification. A, Top, HRP-streptavidin Western blot (WB) depicting LTK cells stably expressing Kv1.5-WT, treated for 30 minutes with tBOOH or vehicle in the presence or absence of N-acetylcysteine (NAC), followed
by labeling with DAz. Bottom, Data from 3 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 post hoc
comparison. **P⬍0.01, *P⬍0.05. B and C, Top, LTK cells stably expressing Kv1.5-WT were treated for 30 minutes with H2O2 (B),
diamide (C), or vehicle, followed by labeling with DAz and HRP-streptavidin Western blot. Bottom, Data from 3 separate experiments
were quantified via densitometry and analyzed via unpaired t test with Welch correction. *P⬍0.05.
tBOOH or diamide (Online Figure IVA and B), indicating that
this modification was not cell type–specific. Together, these data
show that sulfenic acid modification of Kv1.5 reflects changes in
cellular redox state induced by multiple oxidants in varying cell
types, including HL-1 cardiac myocytes.
Endogenous Kv1.5 Is Modified With Sulfenic Acid,
Which Regulates Channel Current and Surface
Levels in Cardiac Myocytes
To further explore a role for sulfenic acid modification in
regulating Kv1.5 in vivo, we perfused freshly isolated, intact
rat hearts with normal KHB solution, or with KHB solution
containing 200 ␮mol/L diamide, which increases intracellular
peroxide levels,22 induces sustained arrhythmia,13 inhibits
other Kv currents in vivo,23 and induced sulfenic acid modification of Kv1.5 in our atrial myocyte cell system (Online Figure
IVB). We immunoprecipitated endogenous Kv1.5 channel and
detected sulfenic acid using DAz labeling. Importantly, we
validated this antibody in experiments that demonstrated that it
recognizes both endogenous and overexpressed channel (Figure
4A and Online Figure VA). Diamide treatment produced a
robust, global increase in sulfenic acid–modified proteins in the
heart (Online Figure VB), and, importantly, increased sulfenic
acid modification to Kv1.5 (Figure 4A). Significant evidence
indicates that oxidative stress attenuates potassium current in the
cardiovascular system. Kv1.5 underlies IKur, an important component of repolarizing current in the heart. Given our discovery
that endogenous Kv1.5 undergoes sulfenic acid modification in
the heart, we next investigated the effect of this modification on
IKur in native cells, using patch-clamp recordings from acutely
dissociated rat cardiac myocytes. Online Figure VI shows that
increased oxidative stress with diamide treatment was accompanied by a significant decrease in IKur current, indicating that
perturbation of cellular redox equilibrium decreases IKur current
in native cardiac myocytes. To determine a specific role for
sulfenic acid modification in modulation of IKur currents, we
used the sulfenic acid–specific alkylating agent dimedone.24 Our
rationale for using dimedone was based on the fact that this
covalent modification of sulfenic acid precludes its reduction
back to the thiol form and prevents further oxidation to sulfinic
or sulfonic acid (Figure 4B, top). As shown in Figure 4B,
treatment with dimedone significantly reduced IKur currents in
dissociated cardiac myocytes. These results collectively show
that Kv1.5 is a substrate for sulfenic acid in the heart, which
regulates endogenous Kv1.5 mediated IKur currents.
Our previous work demonstrated that changes in current
density can result from acute regulation of channel trafficking, leading to altered Kv1.5 levels on the cell surface.15,25
This evidence, combined with the findings shown in Online
Figure VI and Figure 4A and 4B, led us to explore the effects
of sulfenic acid modification on channel trafficking. To this
end, we used HL-1 cells transiently expressing Kv1.5 with an
extracellular green fluorescent protein (GFP) tag inserted
between the S1 and S2 segments (Kv1.5-GFP, Online Figure
VIIA) and measured changes in cell surface expression of
Kv1.5 using live-cell immunofluorescent labeling.15 This
technique utilizes an antibody against the external GFP tag in
live, nonpermeabilized cells, therefore allowing the discrimination between surface and total cellular populations of channel
(total GFP florescence). Our previous work demonstrated that
the GFP tag has no effect on the electrophysiological properties
or glycosylation of the channel.15 In control experiments, we
found that Kv1.5-WT-GFP was modified with sulfenic acid to
the same extent as untagged Kv1.5-WT, suggesting that potential sulfenic acid modification of the GFP tag itself does not
contribute significantly to the signal (Online Figure VIIB). In
live-cell surface labeling experiments, we found that oxidant
treatment resulted in a significant decrease in the surface levels
of Kv1.5 in HL-1 myocytes (Figure 4C). This reduction was
time-dependent (Online Figure VIIC), with no difference in the
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Sulfenic Acid and Kv1.5 Expression
847
Figure 4. Redox sensitivity of endogenous Kv1.5 and IKur currents. A, Representative Western blot (WB) 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 correction. P⫽0.03, significant. B, Rat ventricular myocytes were acutely isolated and currents
were recorded after 1-hour 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 on treatment with dimedone. *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 minutes. 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, ie, total Kv1.5), and analyzed via
Kruskal-Wallis test, followed by Dunn posttest, n⫽100⫹ cells per condition, 3 experiments. *P⬍0.05, relative to vehicle. Scale bar: 30 ␮m. 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 minutes. Top, Sample outward Kv channel current traces for vehicle and peroxide-treated cells. Bottom, Current density. Results were analyzed via unpaired t test. *P⬍0.05 relative to control.
level of GFP fluorescence in oxidant-treated cells compared with
vehicle (Online Figure VIID).
To determine a specific role for sulfenic acid modification
of Kv1.5 in modulation of channel surface expression, we
again used dimedone.24 Treatment with dimedone decreased
surface Kv1.5 (Figure 4C), analogous to treatment with tBOOH,
and combining both agents potentiated this effect. This is
consistent with our biochemical and in vivo data (Figure 2B and
2D and Figure 4A and 4B) showing that there is a basal level of
sulfenic acid modification in nonoxidant treated cells and suggests that hyperoxidation of cysteine to sulfinic or sulfonic acid
does not trigger the decrease in channel surface levels. This
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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 minutes. Cells were then
analyzed as described in Figure 4C (n⫽60⫹ cells per condition, 3 experiments). B,
Results of whole-cell voltageclamping experiments in HL-1
cells transiently transfected
with Kv1.5-GFP-C581S and
treated with vehicle (n⫽5) or
tBOOH (n⫽4) for 60 minutes.
Top, Sample outward Kv channel current traces for vehicleand 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, 3 experiments). D,
Results of whole-cell voltageclamping experiments in HL-1
cells transiently transfected
with Kv1.5-GFP-S581C and
treated with vehicle (n⫽5) or
tBOOH (n⫽4) for 60 minutes
and analyzed as in B.
**P⬍0.001, ***P⬍0.0001.
reduction in channel surface expression was also observed in
cells treated with either hydrogen peroxide or diamide, indicating that the effect is not oxidant-specific (Online Figure VIIIA
and B). As a functional correlate to our immunostaining results,
we performed whole-cell voltage-clamp measurements in HL-1
myocytes expressing Kv1.5-GFP. Treatment with tBOOH
caused a significant reduction in Kv1.5 steady-state current
density (Figure 4D), which paralleled the effects observed in our
immunofluorescence assay. Importantly, we found that oxidant
exposure does not alter the biophysical properties of Kv1.5
(Online Table I), suggesting that a reduction in channel surface
expression underlies the reduction in current. This is the first
report to show that oxidative stress regulates the surface density
of ion channel proteins, providing insight into the molecular
mechanisms underlying the effects of ROS on channel current.
protein expression in these experiments, as indicated by Western
blotting for WT Kv1.5-GFP, as well as total GFP fluorescence in
all experiments (Online Figure IXA through E). Because the
Kv1.5-S581C mutant possesses a single intracellular cysteine,
this finding further implicates sulfenic acid modification of
C581, rather than an intramolecular disulfide bond or other
modification, in modulation of channel surface expression.
Thus, inducing sulfenic acid modification on C581 of Kv1.5
with oxidizing agents, or trapping Kv1.5 in the sulfenic acid–
modified state, causes a reduction in channel surface density and
current. Collectively, these results demonstrate a general mechanism for redox modulation of channel surface expression, in
which acute oxidant exposure induces sulfenic acid modification
of Kv1.5 in atrial myocytes and significantly decreases the levels
of the channel at the plasma membrane.
Oxidation of Kv1.5 C581 Is Necessary and
Sufficient for Reduction in Channel Current and
Surface Density
Oxidation Triggers Internalization of Kv1.5
and Diverts Channel Protein From a
Recycling Pathway
Our data show that C581 is necessary for sulfenic acid modification of Kv1.5 (Figure 2D). Therefore, we tested the requirement of C581 for oxidant-induced changes in channel function.
Mutation of C581 on Kv1.5-GFP (Kv1.5-GFP-C581S) was
sufficient to abrogate the reduction in channel surface expression
and prevent the decrease in Kv1.5 channel current induced by
tBOOH or dimedone in HL-1 atrial myocytes (Figure 5A and
5B). Significantly, reintroduction of this cysteine alone to the
Kv1.5 6-cysteine mutant (Kv1.5-GFP-S581C) restored the effect
on surface levels and channel current (Figure 5C and 5D).
Importantly, there was no significant change in total channel
Several acute mechanisms exist for reduction of Kv1.5
surface expression, including increased channel internalization, impaired recycling of internalized channel back to the
cell surface, and enhanced channel degradation. Our Western
blotting and immunofluorescence data showed no channel
degradation with short-term oxidative stress (Online Figure
IXA and B). Therefore, using a live-cell immunofluorescence-based internalization assay (Online Figure XA),15 we
first measured the effects of tBOOH or dimedone on channel
internalization. To directly measure Kv1.5 internalization,
surface channels were initially labeled at low temperature
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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 minutes. Internalized channel (gray) was visualized using confocal microscopy, quantified using NIH ImageJ software, normalized to GFP fluorescence (ie, total Kv1.5), and analyzed via Kruskal-Wallis test, followed by
Dunn posttest (n⫽85⫹ cells per condition, 3 experiments). **P⬍0.001, ***P⬍0.0001 relative to vehicle. Scale bar: 30 ␮m. B,, In HL-1
cells, recycled channel (gray) at 60 minutes was measured after vehicle or dimedone treatment for 90 minutes. Data were quantified
using NIH ImageJ software and analyzed via 1-way ANOVA, followed by Tukey post hoc comparison (n⫽at least 70 cells per condition,
3 experiments). ***P⬍0.0001 relative to vehicle control.
with a primary antibody directed against the extracellular
GFP epitope tag, followed by treatment at 37°C with tBOOH,
dimedone, or vehicle. Cells were then removed from the
treatment, and channels remaining at the plasma membrane
were labeled at 4°C with a saturated concentration of AlexaFluor 405– conjugated secondary antibody. Cells were subsequently fixed, permeabilized, and incubated with a second
biotinylated secondary antibody, which was detected with
streptavidin Cy5. Using this strategy, only those channels
originally present at the cell surface can be detected. Of these,
only the fraction that remained at the plasma membrane was
labeled with the first AlexaFluor secondary antibody,
whereas the fraction that was internalized was detected with
the second Cy5-labeled secondary antibody. At t⫽0, before
oxidant treatment at 37°C, we observed no internalized
channel, indicating complete saturation of all surface sites,
consistent with our previously published work.15 As expected, increasing sulfenic acid with tBOOH, or trapping this
modification with dimedone, significantly increased internalization of Kv1.5 (Figure 6A). These effects were not due to
differences in overall channel protein expression, as indicated
by total GFP fluorescence (Online Figure IXF).
We previously reported that a population of constitutive
internalized Kv1.5 recycles back to the cell surface.15 Therefore, we determined whether sulfenic acid modification of
Kv1.5 interferes with this dynamic recycling process. Surface
populations of Kv1.5 were first tagged with an anti-GFP
antibody at 4°C. Cells were treated at 37°C for 60 minutes
with dimedone or vehicle, during which time a population of
surface channel was allowed to internalize (Online Figure XB).
Any remaining cell surface channels were then labeled at 4°C
with saturating concentrations of AlexaFluor 405– conjugated
secondary antibody. At t⫽0 minutes, cells were returned to 37°C
for 60 minutes to allow for recycling back to the plasma
membrane. The previously tagged and internalized Kv1.5 channels, which returned to the surface, were then labeled with a
biotin-conjugated anti-rabbit secondary antibody and detected
with Cy5-conjugated streptavidin. The internalized channel population originally labeled on the cell surface is therefore protected against detection with the AlexaFluor 405 secondary
antibody, whereas Kv1.5 that has returned to the plasma membrane, but not newly synthesized channel, is detected with
Cy5-conjugated streptavidin.15 At t⫽0, before returning the cells
to 37°C to permit channel recycling, we observed no recycled
channel, indicating complete saturation of all surface sites
(Online Figure XC). Notably, we observed that trapping sulfenic
acid modification significantly reduced recycling of internalized
Kv1.5 back to the cell surface (Figure 6B). This effect was not
due to changes in total channel protein expression, as total GFP
levels for the treatment groups were not significantly different
(Online Figure IXG). Further, C581 is necessary and sufficient
to mediate this effect (Figure 6B, Kv1.5-C581S and Kv1.5S581C). Therefore, under acute oxidative stress, sulfenic acid
modification triggers internalization of Kv1.5 and significantly
reduces channel recycling back to the cell surface.
Sulfenic Acid Modification of Kv1.5 in
Response to Oxidative Stress Results in
Channel Degradation
On the basis of the preceding results, we sought to determine
the intracellular fate of internalized channel after exposure to
oxidant. Consistent with our previous report,15 intracellularly
retained channel colocalized with early endosomal antigen 1
(EEA1), a marker for early endosomes (Figure 7A). This
interaction was more pronounced after a 60-minute treatment
with tBOOH, an observation that probably reflects the larger
pool of internalized channel (Figure 7A). After performing
coimmunostaining experiments with a number of intracellular
markers, we uncovered a previously unreported, oxidative
stress–induced colocalization of Kv1.5 with the molecular
chaperone heat shock protein (HSP)70 (Figure 7B). HSP70,
which is highly inducible under conditions of cellular stress,
plays a pivotal role in stabilizing proteins, including ion
channels, and in targeting misfolded proteins for ubiquitina-
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Figure 7. Subcellular localization of internalized Kv1.5
after oxidative stress. A, HL-1
cells transiently expressing
Kv1.5-GFP were treated for 60
minutes with tBOOH, followed
by staining with EEA1 antibody. Figure depicts a crosssectional image. Yellow puncta
indicate colocalization between
internalized Kv1.5 and EEA1.
Bottom, Quantified data (n⫽25
cells, 3 separate experiments),
*P⬍0.05 relative to vehicle
control. B, HL-1 cells transiently expressing Kv1.5-GFP
were treated for 60 minutes
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), *P⬍0.05 relative to vehicle control.
tion and proteasomal degradation.26 Consistent with a role for
this protein in cell stress, HSP70 acquires a punctate pattern
after tBOOH treatment and colocalizes with a substantial population of internalized Kv1.5 (Figure 7B). Colocalization of
Kv1.5 with HSP70, combined with data suggesting that overoxidation of proteins may target them for degradation,27–29 led us
to hypothesize that prolonged oxidative stress diverts the channel to a degradation pathway. To investigate this possibility, we
treated HL-1 cells stably expressing WT Kv1.5 with tBOOH,
alone or in the presence of the N-(benzyloxycarbonyl)
leucinylleucinylleucinal protease inhibitor MG132. To prevent
oxidant-induced changes in Kv1.5 transcription,30 we performed
all treatments in the presence of the protein synthesis inhibitor
cycloheximide. Treatment with tBOOH caused degradation of
Kv1.5 in as little as 15 hours, which was rescued by inhibition of
the proteasome (Figure 8A). Importantly, oxidant-induced channel degradation was not observed in the Kv1.5-C581S mutant
protein (Figure 8B), implicating oxidation of this cysteine on the
channel in the degradation. Because sulfenic acid can oxidize
further to sulfinic (Cys-SO2H) or sulfonic (Cys-SO3H) acid,11
we hypothesized that irreversible hyperoxidation of Kv1.5 might
promote degradation. To test this possibility, we used dimedone
to trap the sulfenic acid modification on Kv1.5, thereby preventing further oxidation. Figure 8C shows that dimedone attenuates
Kv1.5 degradation under oxidative stress conditions, as predicted. Although dimedone blocked channel degradation, it
remained possible that these effects were mediated by depletion
of the intracellular pool of ubiquitin, or direct inhibition of the
proteasome. To rule out direct effects of dimedone and oxidant
treatments on the activity of the proteasome, we first treated
HL-1 cells with dimedone and tBOOH, alone and in the
presence of the proteasomal inhibitor MG132, followed by
Western blotting with an antiubiquitin antibody. In contrast with
the positive control samples treated with MG132, treatment of
cells with tBOOH or dimedone alone caused no accumulation of
ubiquitinated proteins in the cells (Online Figure XIA). To
directly assess the effects of dimedone on proteasomal activity,
we measured the chymotrypsin-like activity of the proteasome
using a previously published procedure18 after 15-hour treatments with dimedone. Dimedone treatment alone did not affect
activity of the proteasome (Online Figure XIB). These results
indicate that oxidant and dimedone treatments do not alter
proteasomal activity. Taken together, these findings support the
hypothesis that sulfenic acid diverts Kv1.5 from a recycling to a
degradation pathway (Figure 8D).
Discussion
Numerous reports suggest that Kv1.5, which comprises an
important repolarizing current in the atrium of the human
heart, is sensitive to cellular redox state. Furthermore, disease
states associated with oxidative stress, such as atrial fibrillation and chronic pulmonary hypertension, are characterized
by downregulation of channel protein expression, with no
change in channel transcription.3,4 Despite these observations,
the molecular mechanism(s) underlying this pathological
remodeling have remained elusive. In the present study, we
show that Kv1.5 is modified with sulfenic acid in the heart
and that this increases under oxidative stress. We further
show that this modification occurs on a single, COOHterminal cysteine and that it regulates IKur currents in native
myocytes. To our knowledge, this is the first report demonstrating sulfenic acid modification to an ion channel. We
demonstrate that peroxide-induced stress leads to increased
internalization and impaired recycling of the channel in
cardiac myocytes and that oxidation of COOH-terminal C581
is sufficient to mediate these effects. Importantly, sulfenic
acid modification ultimately leads to channel degradation.
These results therefore provide a molecular mechanism by
which oxidative stress can lead to Kv1.5 degradation in
cardiac myocytes via oxidation of the channel itself.
The finding that sulfenic acid modification occurs on a single
COOH-terminal cysteine emphasizes the fact that, although
formation of sulfenic acid occurs via a nonenzymatic mechanism, it is not a random occurrence. Recent functional site
profiling has revealed that sulfenic acid–modified proteins are
flanked by polar amino acid residues capable of forming
hydrogen bonds.31 It is interesting that the modified cysteine
C581 (Figure 2A, dark gray) is preceded by a polar serine
residue, which is also conserved. Thus, the protein microenvironment flanking reactive cysteines may explain the propensity
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851
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 ␮mol/L) 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 3 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 3 separate experiments). D, Hypothetical model for sulfenic acid
modulation of Kv1.5 trafficking and stability.
for particular cysteines to form sulfenic acid. The conservation
of this cysteine may provide insight into the unique role for
Kv1.5 in oxygen sensing in the heart and probably the pulmonary vasculature. Indeed, our results show that oxidation of this
cysteine to sulfenic acid enables cells to translate acute changes
in redox state into altered cellular excitability.
A link between oxidative stress and downregulation of Kv
channels is currently the subject of intense research.4,9,32
However, studies of the redox sensitivity of Kv channels thus
far have not explored redox-sensitive changes in channel
surface levels. Surface levels of several Kv channels, including HERG1 and Kv1.5, are sensitive to other factors, such
as intracellular signaling molecules33 and pharmacological
agents.25 Importantly, our work provides the first evidence
that oxidative stress, via sulfenic acid modification to the
channel itself, can acutely regulate the level of channel at the
cell surface. Indeed, we observe a significant reduction in
channel surface expression and current after accumulation of
sulfenic acid via 2 distinct means: induction of oxidative
stress with tBOOH, or trapping of the modification with
dimedone, effects that are more pronounced when both agents
are combined (Figure 4B and 4C). The presence of cysteine
581 is necessary and sufficient to mediate these effects
(Figure 5). We observe similar increases in sulfenic acid
modification (Figure 3) and decreased Kv1.5 surface expression (Online Figure VIII) with multiple oxidants, demonstrating that the effects are not specific to one particular oxidant
and probably represent a general cellular mechanism for redox
regulation of channel expression and trafficking. Our whole-cell
voltage-clamping results provide confirmation of our immunofluorescence data via a second, parallel method. Importantly, we
localized these functional effects by using both approaches to a
single COOH-terminal cysteine (Figure 5).
In accordance with our surface labeling experiments, we
find a similar increase in channel internalization after treatment with tBOOH or dimedone. The finding that dimedone
treatment alone causes internalization suggests that there is a
basal level of sulfenic acid–modified channel in cardiac
myocytes. This is supported by data in Figures 2 to 4 and
Online Figure IV, showing a basal level of sulfenic acid–
modified channel in cells and myocytes that have not been
challenged with oxidant. Accordingly, the increase in internalization is accompanied by impairment in channel recycling (Figure 6B), indicating that sulfenic acid modification
diverts channel away from the normal recycling pathway.
These results are in agreement with other studies showing
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that oxidative stress can interfere with trafficking of other
membrane proteins, such as the transferrin receptor,34 or alter
the molecular machinery involved in this trafficking process.35 As the molecular mechanisms governing Kv1.5 endocytosis are elucidated, this may lead to further insight into
how sulfenic acid triggers internalization.
Our results show that human atrial fibrillation is accompanied
by a global increase in sulfenic acid–modified protein in the
atrium and that endogenous Kv1.5 is modified with sulfenic
acid. The findings that Kv1.5 is a substrate for sulfenic acid
modification and that chronic oxidative stress results in channel
degradation suggest that this is at least 1 mechanism linking ion
channel remodeling to persistent atrial fibrillation. The loss of
channel protein associated with atrial fibrillation precludes the
direct measurement of sulfenic acid–modified channel in the
pathophysiological state. However, our results are consistent
with reports showing that chronic heart failure is a substrate for
atrial fibrillation, resulting in the oxidative stress–induced loss of
Kv1.5 channel protein.19 Importantly, it was shown that this ion
channel remodeling was sensitive to the antioxidant NAC,
indicating that redox-sensitive remodeling in the atria may be
prevented or reversed. Targeting sulfenic acid modification to
ion channel proteins may provide a novel therapeutic approach
to treat persistent atrial fibrillation.
In summary, we demonstrate that human chronic atrial
fibrillation is associated with a global increase in sulfenic
acid. We further show that oxidative stress can lead directly
to internalization and protein degradation of Kv1.5 and that
sulfenic acid modification of COOH-terminal C581 alone is
sufficient to trigger these events. Redox modulation of Kv1.5
current is associated with numerous pathophysiological states
that involve loss of channel protein, including atrial fibrillation
and pulmonary hypertension.3,4 We show, for the first time, that
oxidative stress acutely regulates Kv1.5 surface expression. This
is the first report of sulfenic acid modification to an ion channel,
which provides a potential molecular explanation for the pathological remodeling that occurs in cardiovascular diseases.
Although the roles of ROS and modulation of Kv1.5 in the
pathogenesis of disease continue to be intensely studied, it is
clear that other factors peripheral to the channel itself, including
expression of modulatory Kv ␤-subunits, cannot fully account
for channel redox sensitivity.36 Indeed, our results indicate that
oxidative modification of the Kv1.5 channel itself has a profound effect on stability and functional density. By extension,
sulfenic acid modification may also regulate surface levels of
other ion channels, thereby linking oxidative stress to altered
cellular excitability and disease.
Acknowledgments
We would like to thank Dr William Pratt for his advice and critical
review of the manuscript. We thank Dr Yoichi Osawa for providing the
anti-HSP70 antibody and Dr Stephen Ragsdale for providing diamide.
Sources of Funding
This work was funded by an American Heart Association Predoctoral Fellowship 0910001G (to L.K.S.), NIH grant HL0270973 (to
J.R.M.), the NIEHS Toxicology Training Grant T32ES07062, an
AHA Scientist Development Grant 0835419N (to K.S.C.), and
N.I.H. R01 HL093338-01 (to S.M.D.).
Disclosures
None.
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Novelty and Significance
What Is Known?
●
●
●
Multiple potassium channels, which play an essential role in maintaining normal cardiac rhythm and blood vessel contractility, are
regulated by oxidative stress.
Modification of cysteine residues of proteins to sulfenic acids alters the
expression and function of many proteins under oxidative stress.
Diseases such as chronic atrial fibrillation and hypoxic pulmonary
hypertension (HPH) are characterized by both oxidative stress and
reduced expression of the oxygen-sensitive, voltage-gated potassium (Kv) channel Kv1.5.
What New Information Does This Article Contribute?
●
●
●
Chronic atrial fibrillation in human patients is accompanied by a
global increase in sulfenic acid–modified proteins in the heart.
Sulfenic acid containing Kv1.5 protein is present in normal heart, and
the level of this modification increases under oxidative stress.
Sulfenic acid formation in Kv1.5 reduces the level of the channel
protein on the cell surface, disrupts its normal trafficking, and
promotes its degradation in cardiac myocytes.
Oxidative stress, characterized by excessive formation of reactive
oxygen species or insufficient antioxidant capacity, is a common
denominator in a vast array of cardiovascular diseases. Another
common hallmark of cardiovascular disease is a pathological
change in the expression level of ion channel proteins, leading to
impaired vessel contractility and cardiac rhythm. Chronic atrial
fibrillation and HPH are characterized by both oxidative stress and
a reduction in the level of Kv1.5 channel protein, although the
molecular mechanism(s) linking these observations is unclear. In
the present study, we demonstrate a global increase in the level of
sulfenic acid on proteins in the atria of human patients with chronic
atrial fibrillation and that oxidative stress results in formation of
sulfenic acid in Kv1.5 in the rat heart. This modification interrupts
the normal, dynamic trafficking of the channel and promotes its
degradation. This is the first report demonstrating sulfenic acid
modification of an ion channel, and it provides a molecular
mechanism linking oxidative stress to reduced channel expression
levels observed in atrial fibrillation and HPH. Targeting sulfenic acid
modification of ion channel proteins may provide a novel therapeutic approach for treating cardiovascular diseases.
Downloaded from http://circres.ahajournals.org/ at Scripps Res. Inst./Kresge on February 20, 2013
SUPPLEMENTAL MATERIAL
DETAILED METHODS:
Materials
Anti-Kv1.5 antibody (1st extracellular loop, #APC-150) was purchased from Alomone Labs
(Jerusalem, Israel). Mouse anti-V5 antibody (used at 1:5000), polyclonal anti-GFP (used at
1:500), and goat-anti-mouse IgG labeled with Alexa 405, Alexa 594, or Alexa 647 were
purchased from Invitrogen (Carlsbad, CA). Biotin-conjugated goat anti-rabbit secondary
antibody was purchased from Jackson Immunoresearch, Inc. (West Grove, PA). Cy5streptavidin secondary antibody was purchased from GE Healthcare Bio-Sciences Corp.
(Piscataway, NJ). Rabbit anti-alpha tubulin (used at 1:1000) was purchased from Abcam
(Cambridge, MA). HRP-streptavidin conjugate (used at 1:10,000) was purchased from Pierce
(Rockford, IL). Mouse monoclonal HSP70/HSC70 antibody from Abcam, was kindly provided by
Dr. Yoichi Osawa. Mouse monoclonal ubiquitin antibody was from Santa Cruz Biotechnology.
Complete protease inhibitor cocktail tablets were obtained from Roche Applied Science
(Indianapolis, IN). Protein G-agarose beads, tertiary-butyl hydroperoxide (tBOOH), diamide,
hydrogen peroxide (H2O2) and dimedone were purchased from Sigma (St. Louis, MO). HL-1
cells were a generous gift from Dr. William Claycomb (Louisiana State University Health
Sciences Center, New Orleans, LA).
Transient Transfection
HL-1 (mouse cardiomyocyte) cells in 35-mm culture dishes were transfected with human Kv1.51
at 50-60% confluence with 0.65µg of DNA combined with 1.5µL of lipofectamine 2000 reagent
(Invitrogen) in Opti-Mem (Gibco) for 3–5 h, changed to normal medium, and allowed 48 hours
for protein expression.
Stable Cell Lines
Stable cell lines expressing Kv1.5 wild type and cysteine mutant constructs were created in LTK
cells (a mouse fibroblast cell line) using the Retro-X Universal Packaging System from Clontech
(Mountain View, CA), according to manufacturer's instructions. Stable cell lines expressing the
following mutant constructs were generated: Kv1.5 wild type (WT), Kv1.5-6CS, in which the six
NH2-and COOH-terminal cysteines were mutated to serine, and Kv1.5-4CS, in which the COOH
terminal cysteines were mutated to serine. A COOH-terminal point mutant construct was
mutating cysteine 581 to serine: Kv1.5C581S. Four additional Kv1.5 constructs were created
by re-introducing individual cysteines on the COOH terminus into the null background of Kv1.5
6CS: Kv1.5S604C, Kv1.5S586C, Kv1.5S581C and Kv1.5S564C.
Western Blot
LTK or HL-1 cells were harvested in denaturing lysis buffer containing 50 mM Tris-Cl, 10%
glycerol, and 2% SDS containing complete protease inhibitors (Roche Applied Science).
Membranes were isolated and separated by SDS-PAGE on a NuPAGE Novex 4–12% Bis-Tris
gel (Invitrogen). Proteins were transferred to nitrocellulose and probed with the indicated
primary antibody for 1 h at room temperature. Blots were then incubated with secondary
antibody conjugated to horseradish peroxidase (1:5000), and visualized using Western
Lightning enhanced chemiluminescent reagent according to the manufacturer’s protocol
(PerkinElmer Life Sciences). Images were captured using the EpiChemi3 darkroom (UVP, Inc.,
Upland, CA).
1 Downloaded from http://circres.ahajournals.org/ at Scripps Res. Inst./Kresge on February 20, 2013
DAz Labeling of Sulfenic Acid, Staudinger Ligation, and Immunoprecipitation of Kv1.5
For labeling of human and rodent tissue homogenates with DAz, tissue was homogenized in
non-denaturing lysis buffer, pH 7.5, containing 150 mM NaCl, 50 mM Tris-Cl, 5 mM EDTA, 1%
Triton X-100, 1 mM DAz, and protease inhibitors (Roche). The tissue was first cut into small
pieces with a razor blade, followed by mechanical homogenization and passage through a 16gauge needle on ice. Homogenates then underwent shaking at 265 RPM at 37°C for 2 h,
followed by a 10 minute spin at 10,000 g and 4°C. To remove excess DAz, supernatant was
transferred to Amicon Microcon YM-3 spin columns and spun at room temperature, 10,000 g for
90 min. Retentate was resuspended in fresh lysis buffer and spun for another 60 min. For
Staudinger ligation, lysate (at a concentration of 2 mg/mL) was incubated in 100µM p-biotin and
5 mM DTT for 2 hours, nutating at 37°C. Samples were then resuspended in SDS sample
buffer and frozen at -20°C prior to streptavidin western blotting. For labeling sulfenic acidmodified Kv1.5 in cell lysates, LTK or HL-1 cells were harvested in non-denaturing lysis buffer
containing 150 mM NaCl, 50 mM Tris-Cl, 5 mM EDTA, 1% Triton X-100, and 5 mM DAz, pH 7.5
with protease inhibitors (Roche), followed by gentle rocking at 4°C for 20 min. Lysates were
then dounce homogenized, followed by centrifugation at 16,000 g for 4 min at 4°C to remove
cell debris. Supernatant was then incubated at 37°C for 2-2.5h with gentle rocking to allow
labeling of sulfenic acid modified proteins with DAz2. Protein (250 µg) was immunoprecipitated
overnight at 4°C with 1.5 µL of anti-V5 antibody conjugated to 60 µL of Protein G-agarose
beads. The following day, the beads were washed twice with wash buffer containing protease
inhibitors: 150 mM NaCl, 50 mM Tris-Cl, 5 mM EDTA, and .1% Triton X-100. For the ligation
reaction, beads were resuspended in wash buffer with 250 µM p-biotin and incubated 2h at
37°C with gentle rocking, prior to electrophoresis and streptavidin-HRP Western blotting.
Perfusion of Isolated Rat Heart and Immunoprecipitation of Kv1.5
Rat hearts were excised and Langendorff perfused with Krebs-Henseleit buffer (KHB) with or
without 200µM diamide for 55 min., followed by isolation of membranes as previously
described3. Briefly, 200-300 mg tissue was minced with a razor blade and homogenized in
buffer containing 50 mM tris, 10 mM EGTA, 5 mM DAz, and protease inhibitors (pH 7.4).
Homogenate was then centrifuged at 4°C for 10 min. at 2000 RPM. Supernatant was collected
and membranes were isolated via ultracentrifugation at 45000g for 90 min. at 4°C. The
supernatant was then discarded and the pellet resuspended in homogenization buffer (see
above) + 1% NP-40, followed by incubation at 37°C for 2 hours to allow DAz to bind sulfenic
acid-modified proteins. Kv1.5 immunoprecipitation was then performed overnight using 1.5 µL
anti-Kv1.5 antibody (Alomone Labs APC-150), 75 µL protein A-sepharose beads, and 1 mg total
protein. Staudinger ligation and electrophoresis were performed the next day as outlined
above.
N-Glycosidase Treatment of Kv1.5
Vehicle-treated cells were lysed in the presence of DAz, and Kv1.5 was immunoprecipitated and
labeled with p-biotin as outlined in the method. After washing, sample on the beads was
incubated in 1X glycoprotein denaturing buffer for 10 min at 100°C, followed by incubation
overnight in 1 µl of 10x G7 reaction buffer, 1 µl of 10% NP-40, and 1 µl of N-glycosidase F at
37°C. Supernatant from beads was analyzed via western blot.
Immunocytochemistry and Confocal Imaging
For surface, internalization and recycling assays, immunocytochemistry was performed as
described previously4, with minor modifications.
Surface labeling of Kv1.5 in HL-1 cells: 48h post transfection, HL-1 cells transiently expressing
Kv1.5-GFP were administered treatments, followed by live cell staining on ice to stop further
internalization/recycling of Kv1.5. Cells were washed twice with ice-cold PBS, incubated with a
2 Downloaded from http://circres.ahajournals.org/ at Scripps Res. Inst./Kresge on February 20, 2013
polyclonal anti-GFP antibody (1:500) in 2% goat serum for 30 minutes, washed three times with
PBS, incubated with goat anti-rabbit AlexaFluor 594 secondary antibody (1:500) in 2% goat
serum for 30 minutes, washed three times with PBS, fixed with 4% paraformaldehyde and
mounted with ProLong Gold anti-fade reagent (Invitrogen).
Internalization of Kv1.5: HL-1 cells transiently expressing Kv1.5-GFP were live-cell stained with
anti-GFP antibody (1:500) for 30 min on ice. Following this incubation, the cells were
administered treatments at 37°C. Cells were then placed back on ice and any remaining surface
labeled channels were saturated with goat anti-rabbit AlexaFluor 405 secondary antibody
(1:200) for 30 minutes. Cells were then fixed with 4% paraformaldehyde, permeabilized with
0.1% Triton X-100 in 2% goat serum/PBS. Internalized, labeled channel was then detected by
incubating with biotin-conjugated goat anti-rabbit secondary antibody (1:500) 30 minutes,
followed by incubation with Cy5-conjugated streptavidin antibody (1:500) 30 minutes.
Recycling of Kv1.5: HL-1 cells transiently expressing Kv1.5-GFP were live cell stained with an
anti-GFP antibody (1:500) for 30 min on ice. Following this incubation, treatments were
administered for 30 minutes at 37°C, with the remaining treatment duration performed on ice.
Cells were then stained with goat anti-rabbit AlexaFluor 405 secondary antibody (1:200) for 30
min on ice to saturate remaining surface labeled Kv1.5, then returned to 37°C for 60 minutes to
allow recycling of Kv1.5. After this, recycled Kv1.5 was labeled with biotin- conjugated goat
anti-rabbit secondary antibody (1:500) for 30 min on ice, followed by incubation with Cy5conjugated streptavidin antibody (1:500) for 30 min on ice. Cells were then fixed, washed, and
mounted with ProLong Gold.
Staining for colocalization of Kv1.5 with HSP70 or EEA1: For colocalization with HSP70, HL-1
cells transiently expressing Kv1.5-GFP were live-cell stained with anti-GFP antibody (1:500) for
30 min on ice. Following this incubation, the cells were administered treatments at 37°C. Cells
were then placed back on ice and any remaining surface labeled channels were saturated with
goat anti-rabbit AlexaFluor 405 secondary antibody (1:200) for 30 minutes. Cells were then fixed
with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in 2% goat serum/PBS.
Internalized, labeled channel was then detected by incubating with biotin-conjugated goat antirabbit secondary antibody (1:500) 30 minutes, followed by incubation with Cy5-conjugated
streptavidin antibody (1:500) 30 min. Cells were then stained with anti-HSP70 antibody (1:500)
overnight at 4°C, followed by goat anti-mouse AlexaFluor 594 secondary antibody (1:500) for 60
min at room temperature. Cells were then washed and mounted with ProLong Gold. For
colocalization with EEA1, HL-1 cells transiently expressing Kv1.5-GFP were administered
treatments at 37°C. After treatment, cells were fixed with 4% paraformaldehyde, permeabilized
with 0.1% Triton X-100 in 2% donkey serum/PBS, followed by incubation with goat anti-EEA1
antibody (1:100) in 2% donkey serum/PBS. Cells were then incubated with donkey anti-goat
AlexaFluor 594 secondary antibody (1:500) for 30 min at room temperature and mounted with
ProLong Gold. Staining for internalized Kv1.5 was omitted because of cross-reactivity between
donkey anti-goat secondary antibody and the goat antibodies used for the internalization assay.
Imaging: Transfected cells displaying fluorescent signals were acquired on an Olympus
Fluoview 500 confocal microscope using a 60X oil objective. Images were obtained by taking a
series of stacks every 0.5 µm through the cell and combining the images into a composite stack.
To measure oxidant-induced changes in Kv1.5 surface expression, internalization and recycling,
Z-stacks were compressed, and total fluorescence was calculated for total Kv1.5 (eGFP),
surface Kv1.5 (Texas Red or DAPI), and internalized/recycled Kv1.5 (Cy5) using NIH ImageJ
software. Background fluorescence was determined by measuring the background signal for all
channels tested. To determine specific fluorescence, the background signal was subtracted
from the total fluorescent signal. For quantification, Kv1.5 fluorescent signal (surface,
internalized and recycled) was normalized to total Kv1.5-GFP fluorescence in each cell.
3 Downloaded from http://circres.ahajournals.org/ at Scripps Res. Inst./Kresge on February 20, 2013
Electrophysiology
Ionic currents were recorded at room temperature using the whole cell configuration of the patch
clamp technique, as described previously5, with minor modifications. HL-1 cells transiently
expressing WT or cysteine mutant Kv1.5-GFP 48h post-transfection were administered
treatments at 37°C, trypsinized and allowed to settle in the recording chamber. The bath
solution contained (in mmol/L): NaCl 110, KCl 4, MgCl2 1, CaCl2 1.8, HEPES 10, and glucose
10. The pH was adjusted to 7.35 with NaOH. Borosilicate glass pipettes with resistance of 2-4
Mohm were filled with a solution containing (in mmol/L): KCl 20, NaCl 8, HEPES 10, K2BAPTA
10, K2ATP 4, potassium aspartate 110, CaCl2 1, MgCl2 1. The pH was adjusted to 7.2 with
KOH. Transfected cells were identified by GFP fluorescence, and a gigaohm seal was obtained
by gentle suction. To measure Kv channel activation, the membrane voltage was stepped from
-80 to +60 mV in 10-mV increments. Eighty percent of series resistance was compensated.
Steady state currents at the end of each depolarizing pulse were measured and normalized to
cell capacitance to obtain current density. For IKur experiments, rat cardiac myocytes were
isolated and recordings were conducted following treatment with dimedone, diamide or vehicle.
Ca+ current was inhibited by the addition of 200uM CdCl to our bath solution mM (110 NaCl, 4
KCl, 1 MgCl2•H2O, 1.8 CaCl2, 10 HEPES, 1.8 glucose, pH 7.35). Pipette solution consisted of
mM (20 KCl, 8 NaCl2, 1 MgCl2•H2O, 1 CaCl2, 110 L-Aspartic acid potassium salt, 10 HEPES, 10
K4BAPTA, 4 K2ATP, pH to 7.2). IKslow (IKur + Iss) was obtained using a 100ms pre-pulse to -40mV
from a holding potential of -80mV, to inhibit Na+ current and current mediated by Kv4 channels
(Ito), followed by a 500ms pulse to +30mV. Iss, current mediated by Kv2.1 and hERG, was
obtained using the same protocol as above with the addition of 50uM 4-Aminopyridine (4-AP), to
inhibit IKur. Current specific to Kv1.5 (IKur) was then obtained by determining the difference
between IKslow and Iss.
Proteasome Activity Assay
An optimized method was used for determining heart tissue chymotrypsin-like activity6.
Reported values are without ATP and were averaged from 3 independent experiments. Values
for dimedone-treated cell lysates were expressed as a percentage of values for untreated cells
for each experiment.
Statistics
Statistics were performed using Prism software Version 5 from Graphpad Prism Software (San
Diego, CA). All data are expressed as mean +/- SEM. Data were analyzed using one-way
ANOVA followed by Tukey’s post hoc test, or unpaired, two-tailed t-test. A p value of <0.05 was
considered significant.
4 Downloaded from http://circres.ahajournals.org/ at Scripps Res. Inst./Kresge on February 20, 2013
SUPPLEMENTAL FIGURES
Online Figure I: Labeling sulfenic acid-modified proteins with DAz. A, Strategy for
detecting sulfenic acid-modified Kv1.5 using the novel chemical probe, DAz. Cells are lysed in
the presence of DAz, which covalently binds to sulfenic acid-modified proteins. DAz labeled
proteins are then conjugated to a secondary, biotinylated agent, enabling detection of the
sulfenic acid modification via streptavidin western blot. B, HRP-streptavidin western blot
depicting DAz-labeled sulfenic acid in whole tissue lysate from mouse heart treated with tBOOH
or vehicle.
5 Downloaded from http://circres.ahajournals.org/ at Scripps Res. Inst./Kresge on February 20, 2013
Online Figure II: Human atrial fibrillation is accompanied by a global increase in sulfenic
acid-modified proteins. Representative HRP-streptavidin western blot depicting global
sulfenic acid-modified proteins in a human patient with chronic atrial fibrillation, compared to an
unmatched, healthy donor.
6 Downloaded from http://circres.ahajournals.org/ at Scripps Res. Inst./Kresge on February 20, 2013
Online Figure III: DAz recognizes sulfenic acid-modified Kv1.5. A, LTK cells stably
expressing Kv1.5-WT were labeled with DAz. Samples were then treated overnight at 37°C
with reaction buffer (control, lane 1) or PNGase (lane 2) to deglycosylate Kv1.5, which abolished
the doublet, leaving a single, non-glycosylated band. The blot was then stripped and re-probed
with anti-V5 antibody to verify efficient immunoprecipitation. B, LTK cells stably expressing
Kv1.5-WT were labeled with DAz, alone or concurrently with dimedone, the parent compound of
DAz. Dimedone blocks DAz labeling of sulfenic acid-modified Kv1.5.
7 Downloaded from http://circres.ahajournals.org/ at Scripps Res. Inst./Kresge on February 20, 2013
Online Figure IV: Sulfenic acid modification of Kv1.5 is redox-sensitive in cardiac
myocytes. A, HL-1 cells stably expressing Kv1.5-WT were treated for 60 min. with tBOOH,
followed by labeling with DAz. Results from three separate experiments were quantified via
densitometry analyzed via unpaired t-test. *indicates p<.05 relative to control. B, HL-1 cells
stably expressing Kv1.5-WT were treated for 60 min. with diamide, followed by labeling with
DAz. Results were quantified via densitometry and analyzed via unpaired t-test. *indicates
p<.05 relative to control.
8 Downloaded from http://circres.ahajournals.org/ at Scripps Res. Inst./Kresge on February 20, 2013
Online Figure V: Diamide treatment produces a global increase in sulfenic acid-modified
proteins in freshly isolated rat heart. A, Western blot depicting lysates from HEK cells
transfected with Kv1.5-WT or Kv1.5-GFP and probed for Kv1.5 using a commercially available
antibody (Alomone Labs). B, Representative HRP-streptavidin western blot depicting sulfenic
acid-modified proteins in isolated rat hearts perfused with normal KHB solution, or KHB solution
containing diamide.
9 Downloaded from http://circres.ahajournals.org/ at Scripps Res. Inst./Kresge on February 20, 2013
Online Figure VI: IKur, the Kv1.5-encoded current, is redox-sensitive. Rat cardiac
myocytes were isolated sterilely, cultured, and used within 24 hrs. IKur currents were recorded
following 10 min. treatment with M199+ medium alone or with diamide (n=4 cells per treatment).
Data were analyzed using unpaired t-test * denotes p<0.05, ** denotes p<0.01.
10 Downloaded from http://circres.ahajournals.org/ at Scripps Res. Inst./Kresge on February 20, 2013
Online Figure VII: Oxidant treatment causes a significant, time-dependent reduction in
surface levels of Kv1.5 in HL-1 atrial myocytes.
A, Cartoon illustrating the Kv1.5-GFP construct. B, HL-1 cells stably expressing wild type
Kv1.5-GFP were treated with tBOOH or vehicle 60 min., followed by immunoprecipitation and
labeling with DAz and HRP-streptavidin western blot. C, Cells expressing Kv1.5-GFP were
treated with tBOOH or vehicle for 30 or 60 min. Surface channel (red) was quantified using NIH
ImageJ software, normalized to total GFP (i.e. total Kv1.5) fluorescence, and analyzed via oneway ANOVA. n=60+ cells per condition, three experiments. *** indicates p<.0001 relative to
vehicle. D, Levels of total GFP fluorescence (total Kv1.5 expression) were quantified and
analyzed as in (C) Levels are not significantly different (p>.05).
11 Downloaded from http://circres.ahajournals.org/ at Scripps Res. Inst./Kresge on February 20, 2013
Online Figure VIII: The reduction in Kv1.5 surface expression in HL-1 atrial myocytes is
not oxidant-specific. A-B, HL-1 cells transiently expressing Kv1.5-GFP were treated with
H2O2 or vehicle (A) for 60 min. or diamide or vehicle (B) for 30 min., followed by labeling of
surface Kv1.5 (red). Surface channel was quantified using NIH ImageJ software, normalized to
total GFP (i.e. total Kv1.5) fluorescence, and analyzed via unpaired t-test. n=60+ cells per
condition, three experiments. ** indicates p<.01, *** indicates p<.001 relative tovehicle. GFP
levels are not significantly different (p>.05) data not shown. Scale bars: 30 microns.
12 Downloaded from http://circres.ahajournals.org/ at Scripps Res. Inst./Kresge on February 20, 2013
Online Figure IX: Short-term exposure to oxidative stress does not significantly affect
Kv1.5 protein expression. A, Representative western blot image of HL-1 cells stably
expressing V5-tagged Kv1.5-GFP. Cells were treated with vehicle or tBOOH for 60 min. An
additional sample received dimedone alone for 90 min. A final sample was pre-treated with
dimedone for 30 min., followed by 60 min. tBOOH treatment in the continued presence of
dimedone. B, Data from three separate experiments in (A) were quantified via densitometry.
V5 band (Kv1.5-GFP) area density was normalized to alpha tubulin, converted to ratios (relative
to vehicle) and analyzed via 1-way ANOVA, followed by Tukey’s post-hoc test. p>.05, nonsignificant. C-G, GFP fluorescence, used as an internal control for Kv1.5-GFP expression, was
quantified using NIH ImageJ software. Results were analyzed via Kruskal-Wallis test followed
by Dunn's post test, or unpaired t-test (G), p>.05 for all samples.
13 Downloaded from http://circres.ahajournals.org/ at Scripps Res. Inst./Kresge on February 20, 2013
Online Figure X: Internalization and recycling assays. Diagram of live-cell internalization
(A) or recycling (B) assays in HL-1 atrial myocytes. C, HL-1 cells transiently expressing Kv1.5GFP were live cell stained and treated with dimedone or vehicle as outlined in Detailed
Methods. Zero minutes recycling is the time point after dimedone or vehicle treatment, but prior
to returning cells to 37°C to allow recycling of internalized Kv1.5. At this point, as expected,
there is no recycled Kv1.5 (middle panel).
14 Downloaded from http://circres.ahajournals.org/ at Scripps Res. Inst./Kresge on February 20, 2013
Online Figure XI: Oxidant and dimedone treatments do not interfere with function of the
proteasome. A, HL-1 cells were treated with vehicle, tBOOH, dimedone, MG132, or MG132 +
dimedone. After 15 hrs, cell lysates were generated and analyzed via western blotting with
monoclonal anti-ubiquitin antibody. Blot was stripped and re-probed with alpha-tubulin antibody
as a loading control. tBOOH and dimedone treatment do not inhibit the proteasomal
degradation of ubiquitinated proteins. B, HL-1 cells were treated with dimedone or vehicle.
After 15 hrs, cell lysates were generated and an optimized proteasomal activity method was
used for determining heart tissue chymotrypsin-like activity. Reported values are without ATP
and were averaged from three independent experiments. Values for dimedone-treated cell
lysates were expressed as a percentage of values for untreated cells for each experiment and
analyzed via unpaired t-test, p>.05
15 Downloaded from http://circres.ahajournals.org/ at Scripps Res. Inst./Kresge on February 20, 2013
SUPPLEMENTAL TABLE
Online Table I: Oxidant treatment does not significantly alter the biophysical properties
of Kv1.5. Currents were recorded from HL-1atrial myocytes following vehicle or tBOOH
treatment, using the patch clamp technique as described in the detailed methods.
Current Density
V50 Activation
V50 Inactivation
(+60mV)
(+60mV)
(+60mV)
Kv1.5-GFP
428.52 +/- 187.5
- 9.49 +/ - 1.2
-11.71 +/ - 1.6
Kv1.5-GFP + 200uM
171.87 +/-
-10.23 +/ - 0.8
-13.38 +/ - 2.3
47.1
tBOOH
16 Downloaded from http://circres.ahajournals.org/ at Scripps Res. Inst./Kresge on February 20, 2013
1.
2.
3.
4.
5.
6.
Martens, J R, Sakamoto, N, Sullivan, S A, Grobaski, T D, and Tamkun, M M. Isoformspecific localization of voltage-gated K+ channels to distinct lipid raft populations.
Targeting of Kv1.5 to caveolae. J Biol Chem. 2001;276:8409-14.
Reddie, K G, Seo, Y H, Muse Iii, W B, Leonard, S E, and Carroll, K S. A chemical
approach for detecting sulfenic acid-modified proteins in living cells. Mol Biosyst.
2008;4:521-31.
Brackenbury, W J, Davis, T H, Chen, C, Slat, E A, Detrow, M J, Dickendesher, T L,
Ranscht, B, and Isom, L L. Voltage-gated Na+ channel beta1 subunit-mediated neurite
outgrowth requires Fyn kinase and contributes to postnatal CNS development in vivo. J
Neurosci. 2008;28:3246-56.
McEwen, D P, Schumacher, S M, Li, Q, Benson, M D, Iniguez-Lluhi, J A, Van Genderen,
K M, and Martens, J R. Rab-GTPase-dependent endocytic recycling of Kv1.5 in atrial
myocytes. J Biol Chem. 2007;282:29612-20.
Zhang, L, Foster, K, Li, Q, and Martens, J R. S-acylation regulates Kv1.5 channel
surface expression. Am J Physiol Cell Physiol. 2007;293:C152-61.
Predmore, J M, Wang, P, Davis, F, Bartolone, S, Westfall, M V, Dyke, D B, Pagani, F,
Powell, S R, and Day, S M. Ubiquitin proteasome dysfunction in human hypertrophic and
dilated cardiomyopathies. Circulation. 121:997-1004.
17 Downloaded from http://circres.ahajournals.org/ at Scripps Res. Inst./Kresge on February 20, 2013
SUPPLEMENTAL MATERIAL
DETAILED METHODS:
Materials
Anti-Kv1.5 antibody (1st extracellular loop, #APC-150) was purchased from Alomone Labs
(Jerusalem, Israel). Mouse anti-V5 antibody (used at 1:5000), polyclonal anti-GFP (used at
1:500), and goat-anti-mouse IgG labeled with Alexa 405, Alexa 594, or Alexa 647 were
purchased from Invitrogen (Carlsbad, CA). Biotin-conjugated goat anti-rabbit secondary
antibody was purchased from Jackson Immunoresearch, Inc. (West Grove, PA). Cy5streptavidin secondary antibody was purchased from GE Healthcare Bio-Sciences Corp.
(Piscataway, NJ). Rabbit anti-alpha tubulin (used at 1:1000) was purchased from Abcam
(Cambridge, MA). HRP-streptavidin conjugate (used at 1:10,000) was purchased from Pierce
(Rockford, IL). Mouse monoclonal HSP70/HSC70 antibody from Abcam, was kindly provided by
Dr. Yoichi Osawa. Mouse monoclonal ubiquitin antibody was from Santa Cruz Biotechnology.
Complete protease inhibitor cocktail tablets were obtained from Roche Applied Science
(Indianapolis, IN). Protein G-agarose beads, tertiary-butyl hydroperoxide (tBOOH), diamide,
hydrogen peroxide (H2O2) and dimedone were purchased from Sigma (St. Louis, MO). HL-1
cells were a generous gift from Dr. William Claycomb (Louisiana State University Health
Sciences Center, New Orleans, LA).
Transient Transfection
HL-1 (mouse cardiomyocyte) cells in 35-mm culture dishes were transfected with human Kv1.51
at 50-60% confluence with 0.65µg of DNA combined with 1.5µL of lipofectamine 2000 reagent
(Invitrogen) in Opti-Mem (Gibco) for 3–5 h, changed to normal medium, and allowed 48 hours
for protein expression.
Stable Cell Lines
Stable cell lines expressing Kv1.5 wild type and cysteine mutant constructs were created in LTK
cells (a mouse fibroblast cell line) using the Retro-X Universal Packaging System from Clontech
(Mountain View, CA), according to manufacturer's instructions. Stable cell lines expressing the
following mutant constructs were generated: Kv1.5 wild type (WT), Kv1.5-6CS, in which the six
NH2-and COOH-terminal cysteines were mutated to serine, and Kv1.5-4CS, in which the COOH
terminal cysteines were mutated to serine. A COOH-terminal point mutant construct was
mutating cysteine 581 to serine: Kv1.5C581S. Four additional Kv1.5 constructs were created
by re-introducing individual cysteines on the COOH terminus into the null background of Kv1.5
6CS: Kv1.5S604C, Kv1.5S586C, Kv1.5S581C and Kv1.5S564C.
Western Blot
LTK or HL-1 cells were harvested in denaturing lysis buffer containing 50 mM Tris-Cl, 10%
glycerol, and 2% SDS containing complete protease inhibitors (Roche Applied Science).
Membranes were isolated and separated by SDS-PAGE on a NuPAGE Novex 4–12% Bis-Tris
gel (Invitrogen). Proteins were transferred to nitrocellulose and probed with the indicated
primary antibody for 1 h at room temperature. Blots were then incubated with secondary
antibody conjugated to horseradish peroxidase (1:5000), and visualized using Western
Lightning enhanced chemiluminescent reagent according to the manufacturer’s protocol
(PerkinElmer Life Sciences). Images were captured using the EpiChemi3 darkroom (UVP, Inc.,
Upland, CA).
1 DAz Labeling of Sulfenic Acid, Staudinger Ligation, and Immunoprecipitation of Kv1.5
For labeling of human and rodent tissue homogenates with DAz, tissue was homogenized in
non-denaturing lysis buffer, pH 7.5, containing 150 mM NaCl, 50 mM Tris-Cl, 5 mM EDTA, 1%
Triton X-100, 1 mM DAz, and protease inhibitors (Roche). The tissue was first cut into small
pieces with a razor blade, followed by mechanical homogenization and passage through a 16gauge needle on ice. Homogenates then underwent shaking at 265 RPM at 37°C for 2 h,
followed by a 10 minute spin at 10,000 g and 4°C. To remove excess DAz, supernatant was
transferred to Amicon Microcon YM-3 spin columns and spun at room temperature, 10,000 g for
90 min. Retentate was resuspended in fresh lysis buffer and spun for another 60 min. For
Staudinger ligation, lysate (at a concentration of 2 mg/mL) was incubated in 100µM p-biotin and
5 mM DTT for 2 hours, nutating at 37°C. Samples were then resuspended in SDS sample
buffer and frozen at -20°C prior to streptavidin western blotting. For labeling sulfenic acidmodified Kv1.5 in cell lysates, LTK or HL-1 cells were harvested in non-denaturing lysis buffer
containing 150 mM NaCl, 50 mM Tris-Cl, 5 mM EDTA, 1% Triton X-100, and 5 mM DAz, pH 7.5
with protease inhibitors (Roche), followed by gentle rocking at 4°C for 20 min. Lysates were
then dounce homogenized, followed by centrifugation at 16,000 g for 4 min at 4°C to remove
cell debris. Supernatant was then incubated at 37°C for 2-2.5h with gentle rocking to allow
labeling of sulfenic acid modified proteins with DAz2. Protein (250 µg) was immunoprecipitated
overnight at 4°C with 1.5 µL of anti-V5 antibody conjugated to 60 µL of Protein G-agarose
beads. The following day, the beads were washed twice with wash buffer containing protease
inhibitors: 150 mM NaCl, 50 mM Tris-Cl, 5 mM EDTA, and .1% Triton X-100. For the ligation
reaction, beads were resuspended in wash buffer with 250 µM p-biotin and incubated 2h at
37°C with gentle rocking, prior to electrophoresis and streptavidin-HRP Western blotting.
Perfusion of Isolated Rat Heart and Immunoprecipitation of Kv1.5
Rat hearts were excised and Langendorff perfused with Krebs-Henseleit buffer (KHB) with or
without 200µM diamide for 55 min., followed by isolation of membranes as previously
described3. Briefly, 200-300 mg tissue was minced with a razor blade and homogenized in
buffer containing 50 mM tris, 10 mM EGTA, 5 mM DAz, and protease inhibitors (pH 7.4).
Homogenate was then centrifuged at 4°C for 10 min. at 2000 RPM. Supernatant was collected
and membranes were isolated via ultracentrifugation at 45000g for 90 min. at 4°C. The
supernatant was then discarded and the pellet resuspended in homogenization buffer (see
above) + 1% NP-40, followed by incubation at 37°C for 2 hours to allow DAz to bind sulfenic
acid-modified proteins. Kv1.5 immunoprecipitation was then performed overnight using 1.5 µL
anti-Kv1.5 antibody (Alomone Labs APC-150), 75 µL protein A-sepharose beads, and 1 mg total
protein. Staudinger ligation and electrophoresis were performed the next day as outlined
above.
N-Glycosidase Treatment of Kv1.5
Vehicle-treated cells were lysed in the presence of DAz, and Kv1.5 was immunoprecipitated and
labeled with p-biotin as outlined in the method. After washing, sample on the beads was
incubated in 1X glycoprotein denaturing buffer for 10 min at 100°C, followed by incubation
overnight in 1 µl of 10x G7 reaction buffer, 1 µl of 10% NP-40, and 1 µl of N-glycosidase F at
37°C. Supernatant from beads was analyzed via western blot.
Immunocytochemistry and Confocal Imaging
For surface, internalization and recycling assays, immunocytochemistry was performed as
described previously4, with minor modifications.
Surface labeling of Kv1.5 in HL-1 cells: 48h post transfection, HL-1 cells transiently expressing
Kv1.5-GFP were administered treatments, followed by live cell staining on ice to stop further
internalization/recycling of Kv1.5. Cells were washed twice with ice-cold PBS, incubated with a
2 polyclonal anti-GFP antibody (1:500) in 2% goat serum for 30 minutes, washed three times with
PBS, incubated with goat anti-rabbit AlexaFluor 594 secondary antibody (1:500) in 2% goat
serum for 30 minutes, washed three times with PBS, fixed with 4% paraformaldehyde and
mounted with ProLong Gold anti-fade reagent (Invitrogen).
Internalization of Kv1.5: HL-1 cells transiently expressing Kv1.5-GFP were live-cell stained with
anti-GFP antibody (1:500) for 30 min on ice. Following this incubation, the cells were
administered treatments at 37°C. Cells were then placed back on ice and any remaining surface
labeled channels were saturated with goat anti-rabbit AlexaFluor 405 secondary antibody
(1:200) for 30 minutes. Cells were then fixed with 4% paraformaldehyde, permeabilized with
0.1% Triton X-100 in 2% goat serum/PBS. Internalized, labeled channel was then detected by
incubating with biotin-conjugated goat anti-rabbit secondary antibody (1:500) 30 minutes,
followed by incubation with Cy5-conjugated streptavidin antibody (1:500) 30 minutes.
Recycling of Kv1.5: HL-1 cells transiently expressing Kv1.5-GFP were live cell stained with an
anti-GFP antibody (1:500) for 30 min on ice. Following this incubation, treatments were
administered for 30 minutes at 37°C, with the remaining treatment duration performed on ice.
Cells were then stained with goat anti-rabbit AlexaFluor 405 secondary antibody (1:200) for 30
min on ice to saturate remaining surface labeled Kv1.5, then returned to 37°C for 60 minutes to
allow recycling of Kv1.5. After this, recycled Kv1.5 was labeled with biotin- conjugated goat
anti-rabbit secondary antibody (1:500) for 30 min on ice, followed by incubation with Cy5conjugated streptavidin antibody (1:500) for 30 min on ice. Cells were then fixed, washed, and
mounted with ProLong Gold.
Staining for colocalization of Kv1.5 with HSP70 or EEA1: For colocalization with HSP70, HL-1
cells transiently expressing Kv1.5-GFP were live-cell stained with anti-GFP antibody (1:500) for
30 min on ice. Following this incubation, the cells were administered treatments at 37°C. Cells
were then placed back on ice and any remaining surface labeled channels were saturated with
goat anti-rabbit AlexaFluor 405 secondary antibody (1:200) for 30 minutes. Cells were then fixed
with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in 2% goat serum/PBS.
Internalized, labeled channel was then detected by incubating with biotin-conjugated goat antirabbit secondary antibody (1:500) 30 minutes, followed by incubation with Cy5-conjugated
streptavidin antibody (1:500) 30 min. Cells were then stained with anti-HSP70 antibody (1:500)
overnight at 4°C, followed by goat anti-mouse AlexaFluor 594 secondary antibody (1:500) for 60
min at room temperature. Cells were then washed and mounted with ProLong Gold. For
colocalization with EEA1, HL-1 cells transiently expressing Kv1.5-GFP were administered
treatments at 37°C. After treatment, cells were fixed with 4% paraformaldehyde, permeabilized
with 0.1% Triton X-100 in 2% donkey serum/PBS, followed by incubation with goat anti-EEA1
antibody (1:100) in 2% donkey serum/PBS. Cells were then incubated with donkey anti-goat
AlexaFluor 594 secondary antibody (1:500) for 30 min at room temperature and mounted with
ProLong Gold. Staining for internalized Kv1.5 was omitted because of cross-reactivity between
donkey anti-goat secondary antibody and the goat antibodies used for the internalization assay.
Imaging: Transfected cells displaying fluorescent signals were acquired on an Olympus
Fluoview 500 confocal microscope using a 60X oil objective. Images were obtained by taking a
series of stacks every 0.5 µm through the cell and combining the images into a composite stack.
To measure oxidant-induced changes in Kv1.5 surface expression, internalization and recycling,
Z-stacks were compressed, and total fluorescence was calculated for total Kv1.5 (eGFP),
surface Kv1.5 (Texas Red or DAPI), and internalized/recycled Kv1.5 (Cy5) using NIH ImageJ
software. Background fluorescence was determined by measuring the background signal for all
channels tested. To determine specific fluorescence, the background signal was subtracted
from the total fluorescent signal. For quantification, Kv1.5 fluorescent signal (surface,
internalized and recycled) was normalized to total Kv1.5-GFP fluorescence in each cell.
3 Electrophysiology
Ionic currents were recorded at room temperature using the whole cell configuration of the patch
clamp technique, as described previously5, with minor modifications. HL-1 cells transiently
expressing WT or cysteine mutant Kv1.5-GFP 48h post-transfection were administered
treatments at 37°C, trypsinized and allowed to settle in the recording chamber. The bath
solution contained (in mmol/L): NaCl 110, KCl 4, MgCl2 1, CaCl2 1.8, HEPES 10, and glucose
10. The pH was adjusted to 7.35 with NaOH. Borosilicate glass pipettes with resistance of 2-4
Mohm were filled with a solution containing (in mmol/L): KCl 20, NaCl 8, HEPES 10, K2BAPTA
10, K2ATP 4, potassium aspartate 110, CaCl2 1, MgCl2 1. The pH was adjusted to 7.2 with
KOH. Transfected cells were identified by GFP fluorescence, and a gigaohm seal was obtained
by gentle suction. To measure Kv channel activation, the membrane voltage was stepped from
-80 to +60 mV in 10-mV increments. Eighty percent of series resistance was compensated.
Steady state currents at the end of each depolarizing pulse were measured and normalized to
cell capacitance to obtain current density. For IKur experiments, rat cardiac myocytes were
isolated and recordings were conducted following treatment with dimedone, diamide or vehicle.
Ca+ current was inhibited by the addition of 200uM CdCl to our bath solution mM (110 NaCl, 4
KCl, 1 MgCl2•H2O, 1.8 CaCl2, 10 HEPES, 1.8 glucose, pH 7.35). Pipette solution consisted of
mM (20 KCl, 8 NaCl2, 1 MgCl2•H2O, 1 CaCl2, 110 L-Aspartic acid potassium salt, 10 HEPES, 10
K4BAPTA, 4 K2ATP, pH to 7.2). IKslow (IKur + Iss) was obtained using a 100ms pre-pulse to -40mV
from a holding potential of -80mV, to inhibit Na+ current and current mediated by Kv4 channels
(Ito), followed by a 500ms pulse to +30mV. Iss, current mediated by Kv2.1 and hERG, was
obtained using the same protocol as above with the addition of 50uM 4-Aminopyridine (4-AP), to
inhibit IKur. Current specific to Kv1.5 (IKur) was then obtained by determining the difference
between IKslow and Iss.
Proteasome Activity Assay
An optimized method was used for determining heart tissue chymotrypsin-like activity6.
Reported values are without ATP and were averaged from 3 independent experiments. Values
for dimedone-treated cell lysates were expressed as a percentage of values for untreated cells
for each experiment.
Statistics
Statistics were performed using Prism software Version 5 from Graphpad Prism Software (San
Diego, CA). All data are expressed as mean +/- SEM. Data were analyzed using one-way
ANOVA followed by Tukey’s post hoc test, or unpaired, two-tailed t-test. A p value of <0.05 was
considered significant.
4 SUPPLEMENTAL FIGURES
Online Figure I: Labeling sulfenic acid-modified proteins with DAz. A, Strategy for
detecting sulfenic acid-modified Kv1.5 using the novel chemical probe, DAz. Cells are lysed in
the presence of DAz, which covalently binds to sulfenic acid-modified proteins. DAz labeled
proteins are then conjugated to a secondary, biotinylated agent, enabling detection of the
sulfenic acid modification via streptavidin western blot. B, HRP-streptavidin western blot
depicting DAz-labeled sulfenic acid in whole tissue lysate from mouse heart treated with tBOOH
or vehicle.
5 Online Figure II: Human atrial fibrillation is accompanied by a global increase in sulfenic
acid-modified proteins. Representative HRP-streptavidin western blot depicting global
sulfenic acid-modified proteins in a human patient with chronic atrial fibrillation, compared to an
unmatched, healthy donor.
6 Online Figure III: DAz recognizes sulfenic acid-modified Kv1.5. A, LTK cells stably
expressing Kv1.5-WT were labeled with DAz. Samples were then treated overnight at 37°C
with reaction buffer (control, lane 1) or PNGase (lane 2) to deglycosylate Kv1.5, which abolished
the doublet, leaving a single, non-glycosylated band. The blot was then stripped and re-probed
with anti-V5 antibody to verify efficient immunoprecipitation. B, LTK cells stably expressing
Kv1.5-WT were labeled with DAz, alone or concurrently with dimedone, the parent compound of
DAz. Dimedone blocks DAz labeling of sulfenic acid-modified Kv1.5.
7 Online Figure IV: Sulfenic acid modification of Kv1.5 is redox-sensitive in cardiac
myocytes. A, HL-1 cells stably expressing Kv1.5-WT were treated for 60 min. with tBOOH,
followed by labeling with DAz. Results from three separate experiments were quantified via
densitometry analyzed via unpaired t-test. *indicates p<.05 relative to control. B, HL-1 cells
stably expressing Kv1.5-WT were treated for 60 min. with diamide, followed by labeling with
DAz. Results were quantified via densitometry and analyzed via unpaired t-test. *indicates
p<.05 relative to control.
8 Online Figure V: Diamide treatment produces a global increase in sulfenic acid-modified
proteins in freshly isolated rat heart. A, Western blot depicting lysates from HEK cells
transfected with Kv1.5-WT or Kv1.5-GFP and probed for Kv1.5 using a commercially available
antibody (Alomone Labs). B, Representative HRP-streptavidin western blot depicting sulfenic
acid-modified proteins in isolated rat hearts perfused with normal KHB solution, or KHB solution
containing diamide.
9 Online Figure VI: IKur, the Kv1.5-encoded current, is redox-sensitive. Rat cardiac
myocytes were isolated sterilely, cultured, and used within 24 hrs. IKur currents were recorded
following 10 min. treatment with M199+ medium alone or with diamide (n=4 cells per treatment).
Data were analyzed using unpaired t-test * denotes p<0.05, ** denotes p<0.01.
10 Online Figure VII: Oxidant treatment causes a significant, time-dependent reduction in
surface levels of Kv1.5 in HL-1 atrial myocytes.
A, Cartoon illustrating the Kv1.5-GFP construct. B, HL-1 cells stably expressing wild type
Kv1.5-GFP were treated with tBOOH or vehicle 60 min., followed by immunoprecipitation and
labeling with DAz and HRP-streptavidin western blot. C, Cells expressing Kv1.5-GFP were
treated with tBOOH or vehicle for 30 or 60 min. Surface channel (red) was quantified using NIH
ImageJ software, normalized to total GFP (i.e. total Kv1.5) fluorescence, and analyzed via oneway ANOVA. n=60+ cells per condition, three experiments. *** indicates p<.0001 relative to
vehicle. D, Levels of total GFP fluorescence (total Kv1.5 expression) were quantified and
analyzed as in (C) Levels are not significantly different (p>.05).
11 Online Figure VIII: The reduction in Kv1.5 surface expression in HL-1 atrial myocytes is
not oxidant-specific. A-B, HL-1 cells transiently expressing Kv1.5-GFP were treated with
H2O2 or vehicle (A) for 60 min. or diamide or vehicle (B) for 30 min., followed by labeling of
surface Kv1.5 (red). Surface channel was quantified using NIH ImageJ software, normalized to
total GFP (i.e. total Kv1.5) fluorescence, and analyzed via unpaired t-test. n=60+ cells per
condition, three experiments. ** indicates p<.01, *** indicates p<.001 relative tovehicle. GFP
levels are not significantly different (p>.05) data not shown. Scale bars: 30 microns.
12 Online Figure IX: Short-term exposure to oxidative stress does not significantly affect
Kv1.5 protein expression. A, Representative western blot image of HL-1 cells stably
expressing V5-tagged Kv1.5-GFP. Cells were treated with vehicle or tBOOH for 60 min. An
additional sample received dimedone alone for 90 min. A final sample was pre-treated with
dimedone for 30 min., followed by 60 min. tBOOH treatment in the continued presence of
dimedone. B, Data from three separate experiments in (A) were quantified via densitometry.
V5 band (Kv1.5-GFP) area density was normalized to alpha tubulin, converted to ratios (relative
to vehicle) and analyzed via 1-way ANOVA, followed by Tukey’s post-hoc test. p>.05, nonsignificant. C-G, GFP fluorescence, used as an internal control for Kv1.5-GFP expression, was
quantified using NIH ImageJ software. Results were analyzed via Kruskal-Wallis test followed
by Dunn's post test, or unpaired t-test (G), p>.05 for all samples.
13 Online Figure X: Internalization and recycling assays. Diagram of live-cell internalization
(A) or recycling (B) assays in HL-1 atrial myocytes. C, HL-1 cells transiently expressing Kv1.5GFP were live cell stained and treated with dimedone or vehicle as outlined in Detailed
Methods. Zero minutes recycling is the time point after dimedone or vehicle treatment, but prior
to returning cells to 37°C to allow recycling of internalized Kv1.5. At this point, as expected,
there is no recycled Kv1.5 (middle panel).
14 Online Figure XI: Oxidant and dimedone treatments do not interfere with function of the
proteasome. A, HL-1 cells were treated with vehicle, tBOOH, dimedone, MG132, or MG132 +
dimedone. After 15 hrs, cell lysates were generated and analyzed via western blotting with
monoclonal anti-ubiquitin antibody. Blot was stripped and re-probed with alpha-tubulin antibody
as a loading control. tBOOH and dimedone treatment do not inhibit the proteasomal
degradation of ubiquitinated proteins. B, HL-1 cells were treated with dimedone or vehicle.
After 15 hrs, cell lysates were generated and an optimized proteasomal activity method was
used for determining heart tissue chymotrypsin-like activity. Reported values are without ATP
and were averaged from three independent experiments. Values for dimedone-treated cell
lysates were expressed as a percentage of values for untreated cells for each experiment and
analyzed via unpaired t-test, p>.05
15 SUPPLEMENTAL TABLE
Online Table I: Oxidant treatment does not significantly alter the biophysical properties
of Kv1.5. Currents were recorded from HL-1atrial myocytes following vehicle or tBOOH
treatment, using the patch clamp technique as described in the detailed methods.
Current Density
V50 Activation
V50 Inactivation
(+60mV)
(+60mV)
(+60mV)
Kv1.5-GFP
428.52 +/- 187.5
- 9.49 +/ - 1.2
-11.71 +/ - 1.6
Kv1.5-GFP + 200uM
171.87 +/-
-10.23 +/ - 0.8
-13.38 +/ - 2.3
47.1
tBOOH
16 1.
2.
3.
4.
5.
6.
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17