Localization of Cardiac Sodium Channels
in Caveolin-Rich Membrane Domains
Regulation of Sodium Current Amplitude
Tracy L. Yarbrough, Tong Lu, Hon-Chi Lee, Erwin F. Shibata
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Abstract—This study demonstrates that caveolae, omega-shaped membrane invaginations, are involved in cardiac sodium
channel regulation by a mechanism involving the ␣ subunit of the stimulatory heterotrimeric G-protein, G␣s, via
stimulation of the cell surface ␤-adrenergic receptor. Stimulation of ␤-adrenergic receptors with 10 ␮mol/L
isoproterenol in the presence of a protein kinase A inhibitor increased the whole-cell sodium current by a “direct”
cAMP-independent G-protein mechanism. The addition of antibodies against caveolin-3 to the cell’s cytoplasm via the
pipette solution abrogated this direct G protein–induced increase in sodium current, whereas antibodies to caveolin-1 or
caveolin-2 did not. Voltage-gated sodium channel proteins were found to associate with caveolin-rich membranes
obtained by detergent-free buoyant density separation. The purity of the caveolar membrane fraction was verified by
Western blot analyses, which indicated that endoplasmic/sarcoplasmic reticulum, endosomal compartments, Golgi
apparatus, clathrin-coated vesicles, and sarcolemmal membranes were excluded from the caveolin-rich membrane
fraction. Additionally, the sodium channel was found to colocalize with caveolar membranes by immunoprecipitation,
indirect immunofluorescence, and immunogold transmission electron microscopy. These results suggest that stimulation
of ␤-adrenergic receptors, and thereby G␣s, promotes the presentation of cardiac sodium channels associated with
caveolar membranes to the sarcolemma. (Circ Res. 2002;90:443-449.)
Key Words: caveolae 䡲 ion channel 䡲 signal transduction 䡲 cardiac 䡲 adrenergic
R
characteristics or shifts in voltage-dependent current activation as demonstrated by current-voltage relationships.2,7 In
the presence of inhibitors of PKA, the application of exogenous G␣s to the cytoplasmic face of excised membrane
patches mimics the effect of ␤-adrenergic receptor activation
in increasing INa by increasing the number of functional
channels at the plasma membrane.7 Thus, the direct effect of
␤-stimulation on INa appears to be the result of an increase in
the number of functional sodium channels at the sarcolemma
of cardiac myocytes. The source of the channels that mediate
this increase in INa is the focus of this study.
The time course of the direct effect (generally less than 10
minutes) makes synthesis of new channel proteins an unlikely
mechanism for the increased INa. In addition, the direct
increase in INa is reversible by ␤-agonist washout and is
reproducible by subsequent rounds of ␤AR stimulation.
These 2 factors are suggestive of a store of channel proteins
that can be functionally added to and removed from the
plasma membrane dependent on ␤-adrenergic signaling
events. We believe caveolae to be a prime candidate for such
a storage locale. Caveolae, subsarcolemmal membrane compartments, have been implicated in cellular trafficking cas-
egulation of voltage-gated ion channels at the plasma
membrane of excitable cells is essential in maintaining
cellular excitability and electrical impulse propagation. The
amplitude and slope of the action potential upstroke are
especially important in the control of cardiac conduction
velocity, and the maintenance of appropriate waves of excitation through the ventricles. In the nonpacemaker cells of the
heart, the voltage-gated sodium channel mediates the upstroke of the action potential. Neurohumoral regulation of this
sodium current, INa, via ␤-adrenergic stimulation has been
shown to increase the current by at least 2 known mechanisms: one “direct” and one “indirect”.1–7 The indirect, or
protein kinase A (PKA)– dependent, mechanism regulates INa
by phosphorylation of the channel protein at previously
identified sites,6 resulting in alterations of single-channel
voltage-dependent characteristics, including channel availability (inactivation).3,4,8 –12 The mechanism of the direct,
PKA-independent, effect is not well understood, though
evidence suggests that ligand binding to plasma membrane
␤-adrenergic receptors (␤ARs) results in the activation of a
signaling cascade involving the G␣s protein itself. This
produces an increase in INa without changes in single-channel
Original received October 12, 2001; revision received January 7, 2002; accepted January 7, 2002.
From the Departments of Physiology and Biophysics (T.L.Y., E.F.S.) and Internal Medicine (T.L., H.-C.L.), University of Iowa College of Medicine,
Iowa City, Iowa; and the Department of Veterans’ Affairs Medical Center (H.-C.L.), Iowa City, Iowa.
Correspondence to Erwin F. Shibata, PhD, Dept of Physiology and Biophysics, University of Iowa College of Medicine, 51 Newton Rd, 6-431 Bowen
Science Building, Iowa City, Iowa 52242-1109. E-mail [email protected]
© 2002 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
DOI: 10.1161/hh0402.105177
443
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cades involving adrenergic receptors13,14 and endothelial
nitric oxide synthase (eNOS),15 among other cell surface
proteins.16,17 Caveolae are capable of effectively removing
surface proteins from the plasma membrane through sequestration and endocytotic/transcytotic mechanisms on stimulation of specific receptors.18 –20 Importantly, caveolae may be
involved in mechanisms whereby the availability of water21
and volume-sensitive chloride channels17,22 at the plasma
membrane are mediated.
Among the many proteins localized to caveolar membrane
compartments by biochemical studies (reviewed in Okamoto
et al23 and Ostrom et al24), G␣s is of particular interest.
Although G␣s can mimic the effect of ␤AR stimulation in
increasing cardiac sodium current, G␤␥ does not appear to be
involved. Therefore, the action of the G␣s in this process must
be dependent on the protein itself and other proteins with
which it might interact. Specific regions of interaction between G␣i and caveolin-1, the caveolar scaffolding protein,
have been identified, wherein G␣i binds to a region contained
within the caveolin-1 scaffolding domain in a GTP/GDPdependent fashion.23,25 Although specific studies have not
examined the interaction of G␣s with caveolin-3, the musclespecific caveolin isoform, caveolin-3 is highly homologous to
caveolin-1 in its scaffolding domain,26,27 making an analogous interaction with G␣s likely. In this study, we investigate
the role of caveolae in the regulation of functional cardiac
sodium channel number at the sarcolemmal membrane. We
hypothesize that G␣s activation through ␤AR stimulation
results in the opening of caveolae, and thereby, the addition
of functional sodium channels to the sarcolemma.
Materials and Methods
Adult rat cardiac myocytes were obtained as outlined previously7
and as approved by the Animal Care and Use Committee at the
University of Iowa. Rats were procured from Harlan SpragueDawley (Indianapolis, Ind).
Purification of Caveolin-Rich Fraction
Isolation of caveolin-rich (CR) fraction was done using a previously
described detergent-free method,20 with some modifications. Myocyte lysates were separated on a 5% to 45% discontinuous sucrose
gradient, yielding a caveolin-rich membrane fraction (samples 5 and
6). For comparison, a heavy fraction (sample 11) was also collected.
Antibodies
Antibodies used were obtained as follows, and are referenced in the
online data supplement. Anti-cardiac ryanodine receptor (RyR) was
the kind gift of Dr Kevin P. Campbell (University of Iowa, Iowa
City). Antibodies to caveolin-3, caveolin-2, clathrin, Rab 5, Rab 11,
and secondary HRP-conjugated anti-mouse and anti-rabbit antibodies were obtained from Transduction Laboratories. Antibodies to
caveolin-1 and G␣s were obtained from Santa Cruz Biotechnology.
Antibodies to cytochrome oxidase subunit 2 (COX), plasma membrane calcium ATPase (PMCA), and mannosidase II were obtained
from Molecular Probes, Affinity Bioreagents, and Covance, respectively. Subtype-specific antibodies to the ␣1 and ␣2 isoforms of the
Na⫹/K⫹ ATPase were a kind gift from Dr Kathleen J. Sweadner
(Harvard University, Cambridge, Mass), and antibodies to both the
␣1 and ␣2 isoforms were purchased from Upstate Biotechnology.
Anti–SP-19 (pan) sodium channel antibody and secondary HRPconjugated anti-goat antibodies were obtained from Sigma. D492
polyclonal antibody was raised in goat to a 22-amino acid peptide
corresponding to a sequence (DRLPKSDSEDGPRALNQLSLC) lo-
cated in interdomain loop I-II of the cardiac voltage-gated sodium
channel as similarly described by Cohen and Levitt.28
Immunoblotting
Samples were precipitated using the pyrogallol red-molybdate method29 and run on 7.5% to 12% polyacrylamide gel. Proteins were
transferred to 0.45 ␮mol/L nitrocellulose membrane and blotted with
appropriate antibodies.
Immunoprecipitation
Caveolin-rich and heavy membrane fractions were pelleted by
centrifugation. Each was incubated with anti– caveolin-3 antibody
(1:500), followed by Protein G-agarose. The complexes were washed
and then boiled in 1X sample buffer. Supernatants were separated by
SDS-PAGE for immunoblotting.
Immunofluorescence
Immunolabeled frozen sections of adult rat ventricle were visualized
with a Zeiss confocal microscope. Primary antibodies were diluted as
follows: anti– caveolin-3 1:50; D492 1:200. Secondary immunofluorescent conjugates were diluted at 1:200.
Immunogold Labeling and Electron Microscopy
Ultrathin sections of CR fractions were fixed and labeled prior to
embedment, followed by silver-enhancement and counterstaining for
visualization with a H-7000 transmission electron microscope. Primary antibodies were diluted as follows: anti– caveolin-3 1:20; D492
1:100. Secondary gold-conjugates were diluted at 1:40.
Electrophysiological Protocol
Whole-cell voltage clamp data were generated by stepping membrane potential from ⫺100 mV to ⫺30 mV for 20 ms. In the
experimental groups, 100 ␮g/mL anti– caveolin-1, -2, or -3 antibody
was added to the pipette solution. In the control group, no antibody
was added. Raw data are plotted as INa relative to baseline INa (current
prior to bath addition of isoproterenol [ISO]). Histogram data are
presented as the mean⫾SEM. One-way ANOVA was used to
compare the percent change in INa among the control and experimental groups. Statistical significance is defined as P⬍0.05, with
n⫽number of experiments as indicated.
An expanded Materials and Methods section can be found in the
online data supplement available at http://www.circresaha.org.
Results
Implication of Caveolin-3 in Adrenergically
Mediated Increases in Sodium Current
Previous work2,7 suggests that the direct G-protein effect of
␤-adrenergic stimulation on cardiac sodium current is the
result of an increase in the number of functional channels
present at the sarcolemma. Increasing functional channel
number could be accomplished by modifications of nonfunctional channels in the membrane that make them conductive,
or by the recruitment of channels from a previously unrecognized intracellular storage location. We therefore sought to
determine the role of caveolae in the mechanism of channel
recruitment. Adult myocytes were clamped in the whole-cell
configuration with an intracellular pipette solution containing
0.022 mg/mL protein kinase A inhibitor (PKI), a concentration at which the effects of PKA on sodium current have been
shown to be completely inhibited;7 that is, the addition of 500
␮mol/L 8-CPT-cAMP or 10 ␮mol/L forskolin could not
induce an increase in INa in the presence of 0.022 mg/mL PKI.
Representative traces of the resulting currents obtained by
whole-cell patch clamp are shown in Figure 1. INa was
recorded before (baseline, BSLN) and after (ISO) the addition
Yarbrough et al
Sodium Channel Regulation by Cardiac Caveolae
445
Figure 2. Immunoblot for caveolin isoforms in isolated ventricular myocyte lysate. Caveolin-3 is detected in myocyte lysate,
caveolin-rich fraction, and heavy membrane fraction, whereas
neither caveolin-1 nor caveolin-2 is detected. Caveolins-1 and
-2 are detected in brain lysate only. BSA indicates bovine serum
albumin.
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Figure 1. Representative whole-cell patch clamp recordings of
INa in the presence of antibodies to 3 caveolin isoforms. All
experiments were performed in the presence of 22 ␮g/mL protein kinase A inhibitor. Currents are normalized to peak sodium
current before the addition of ISO. Baseline (BSLN) traces were
obtained prior to the addition of 10 ␮mol/L isoproterenol, and
ISO traces were obtained during superfusion with isoproterenol.
The addition of ISO induces an increase in the voltage-gated
sodium current in control cells wherein no antibody was added
to the pipette solution (A, control). Inclusion of 100 ␮g/mL anti–
caveolin-1 (B) or anti– caveolin-2 (C) antibody to the pipette
does not block ISO-mediated increase in INa, whereas the addition of 100 ␮g/mL anti– caveolin-3 antibody does (D). Panel D
contains 2 traces that superimpose. E, Relative percent change
of INa in response to 10 ␮mol/L ISO under conditions as outlined
in parts A through D. *P⬍0.05 vs the change in INa elicited by
superfusion of ISO in control cell population; n⫽8, 7, 5, and 5
for A through D, respectively.
of 10 ␮mol/L isoproterenol to the bath solution. Within 3
minutes of the onset of ISO superfusion (time course dependent on the speed of cell superfusion), voltage-dependent
sodium current was increased by up to 70.3⫾12.0%
(mean⫾SEM, n⫽8), as shown in Figure 1A. This increase
was both reversible on washout of ISO and reproducible by
repeated exposure to ISO. The addition of anti– caveolin-3
antibody to the pipette solution abolished any ISO-induced
increase (relative percent change in INa⫽3.6⫾5.5%, n⫽5,
P⬍0.05) in the voltage-dependent sodium current measured
under identical conditions (Figure 1D). Importantly, Figure 1
demonstrates that the addition of antibodies to either
caveolin-1 (Figure 1B) or caveolin-2 (Figure 1C) was unable
to block the ␤-adrenergic increase in sodium current stimulated
by 10 ␮mol/L ISO (relative percent change⫽71.9⫾16.3%, n⫽7,
P⬎0.05; and 76.7⫾25.2%, n⫽5, P⬎0.05, respectively), as
was anti– caveolin-3 antibody that had been preincubated
with its antigenic peptide (data not shown) (relative percent
change⫽149.7⫾76.7%, n⫽6, P⬎0.05). As shown in Figure
1E, the mean percent change in INa in the caveolin-1 and
caveolin-2 experimental groups is not significantly different
from control, whereas the mean of the caveolin-3 group is
(experimental groups versus control using a Dunnett comparisons test wherein significance is defined as P⬍0.05). Figure
2 demonstrates that neither caveolin-1 nor caveolin-2 is
detectable in the whole-cell lysate of isolated ventricular
cells, whereas caveolin-3 is detectable; rather, caveolins-1
and -2 can be detected in rat brain lysate. Furthermore,
caveolin-3 protein is detectable in caveolin-rich membrane
fractions obtained by sucrose gradient fraction, whereas
caveolins-1 and -2 are not. Taken together, the data presented
in Figures 1 and 2 suggest that caveolin-3 plays a specific
functional role in the direct G␣s-mediated increase in cardiac
sodium current. To further examine this hypothesis, it was
necessary to demonstrate that sodium channels localize to the
caveolar membrane compartment.
Isolation of Caveolin-Rich Membrane Fraction
From Adult Rat Cardiac Myocytes
The isolation of caveolin-rich membranes through a
detergent-free method that exploits the buoyant density of
caveolae by separation on sucrose density gradient has been
previously demonstrated.20,30,31 Myocyte lysates were separated using this discontinuous sucrose density gradient fractionation method, yielding 12 fractions (numbered 1 through
12, with sample 1 being the lightest [top] fraction), with
distinct protein compartmentalization. As shown in Figure 3,
a caveolin-rich fraction (samples 5 and 6, CR fraction),
occurred at the 25% sucrose cushion of the gradient.
Caveolin-3 could also be detected in heavier samples (samples 7 to 12), though enrichment for caveolin-3 occurred only
in samples 5 and 6 when samples were compared with the
whole-cell lysate. Electron microscopic examination of this
CR fraction demonstrated a relatively uniform population of
vesicles ranging in size from 75 to 150 nm (data not shown),
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Figure 3. Fractional distributions of caveolin-3 and clathrin proteins. Samples were obtained by sucrose density gradient fraction of myocyte lysate. Fractions numbered 1 through 12 from
top to bottom. A, Immunoblot with anti– caveolin-3 (cav-3) and
anti-clathrin antibodies demonstrates enrichment of caveolin-3
in fractions 5 and 6 with clathrin predominantly in fractions 9
through 12. B, Relative cav-3 (}) and clathrin (䢇) densities from
immunoblot as in A. Data are normalized to density of lysate
signal. Bottom bar indicates percent sucrose as layered on in
fractionation preparation.
consistent with previous estimations of caveolar size in adult
rat myocytes.21
During various states of cell growth and adaptation, caveolin and channel proteins are localized to intracellular compartments including endosomes and the Golgi apparatus.
Additionally, the process of lysate preparation by sonication
is likely to produce vesiculation of subcellular organelle
membranes. Work by other labs18,31,32 has suggested that
caveolin-rich fractions obtained exclusively by the detergentfree method may contain membrane derivatives (and, therefore, membrane components) not specific to caveolae. Therefore, it was essential to confirm that our detergent-free
preparation efficiently excluded potentially contaminating
membranes, and selectively enriched for caveolin-rich membranes. To examine the membrane purity of the CR fraction,
we performed Western blot analysis on the fractionation
samples using membrane markers for plasmalemmal and
subcellular organelle membranes.
Whereas caveolin-3 enriched membranes float in samples
5 and 6 (25% sucrose) on the gradient (see Figure 3),
clathrin-enriched membranes appear to settle at predominantly heavier levels of the gradient (mainly in fractions 10 to
12), suggesting the exclusion of cytoplasmic clathrin-coated
pits from the CR fraction and sequestration of these vesicles
(as well as those pits not yet fully dissociated from the
sarcolemma) at greater sucrose concentrations corresponding
to denser membrane fragments. Western analysis of our CR
fraction for the presence of several other organelle and
membrane compartments is shown in Figure 4. We probed
CR membrane fractions and were not able to detect the
following protein markers: Na⫹/K⫹ ATPase, plasma membrane calcium ATPase (PMCA), ryanodine receptor (RyR),
mannosidase II, cytochrome oxidase subunit IV (COX), Rab
Figure 4. Immunoblot for plasma membrane and subcellular
organelle marker proteins. Plasma membrane and organelle
marker proteins are detected in cardiac myocyte lysate and
heavy fraction (fraction 11), but not in caveolin-rich fraction
(fractions 5 and 6) obtained by sucrose gradient fractionation.
Caveolin-3 and sodium channel are detected in myocyte lysate,
caveolin-rich fraction, and heavy fraction.
5, and Rab 11. This suggests that, overall, we have been
successful in obtaining an enriched caveolar fraction, as
determined by Western analysis, that is not significantly
contaminated by sarcolemma or subcellular organelle membranes and vesicles, including SR (RyR), Golgi apparatus
(mannosidase II), mitochondria (COX), endosome (Rab 5 and
Rab 11), and clathrin-coated pits. In contrast, both caveolin-3
and sodium channels are detected in the caveolar membrane
fraction.
Localization of Sodium Channel to Caveolar
Membrane Domains
In support of our hypothesis, we sought to localize sodium
channel and G␣s proteins to gradient fractions rich in
caveolin-3. Samples obtained by sucrose density-gradient
fractionation were analyzed by Western blot and probed with
anti-caveolin, SP-19 (sodium channel), and anti-G␣s antibodies. As shown in Figure 5, sodium channel proteins are
detected in fractions 5 and 6, the caveolin-rich membrane
fractions, and in fractions at the bottom of the gradient where
Figure 5. Immunoblot analysis of samples obtained by sucrose
gradient fractionation. Sodium channel and G␣s proteins are
detected (using anti–SP-19 antibody) in caveolin-rich fraction
(fractions 5 and 6), with caveolin-3 immunoblot shown for
comparison.
Yarbrough et al
Sodium Channel Regulation by Cardiac Caveolae
447
Figure 6. Immunoblot analysis of samples immunoprecipitated
with anti-caveolin antibodies. Caveolin-rich and heavy fractions
were immunoprecipitated with specified primary antibodies (or
no primary antibody as indicated). Sodium channel (⬇240 kDa)
and G␣s (⬇45 kDa) proteins are detected in samples resulting
from immunoprecipitation with anti– caveolin-3 antibody, but not
in samples immunoprecipitated with antibodies against
caveolin-2.
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plasma membrane and T-tubule membranes rest (Figure 4).
Diffuse or double bands obtained on Western blot for sodium
channel proteins using SP-19 antibody reflects subtleties of
channel glycosylation.28,33–35 Additionally, Figure 5 demonstrates that G␣s proteins are distributed in the CR fraction and
in fractions at the bottom of the gradient. Caveolin-3 labeling
of the gradient is provided as a reference.
To confirm that the caveolin-3 and sodium channel proteins reside in the same membrane compartment, we subjected the CR fraction to immunoprecipitation with anti–
caveolin-3 antibodies. As shown in Figure 6, immunoblots
suggest that sodium channels localize to CR fraction membranes immunoprecipitated with anti– caveolin-3 antibody.
This was not true for samples immunoprecipitated with
caveolin-2 or no primary antibody. Additionally, sodium
channels were coimmunoprecipitated when “heavy” membrane fractions were immunoprecipitated with antibodies to
caveolin-3. This is expected in that some caveolin-3 rich
membrane remains in the denser regions of the gradient
following centrifugation (see Figure 5) and is presumably
made up of caveolar membranes that do not shear completely
from the sarcolemma during myocyte sonication. Because the
PKA-independent ISO-mediated increase in INa appears to be
a result of direct G␣s actions, we also examined the presence
of G␣s in the samples obtained by immunoprecipitation with
anti-caveolin antibodies. Positive detection of G␣s in these
samples (Figure 6) suggests that G␣s may have a physical role
in mediating the ISO-induced increase in INa in rat cardiac
myocytes.
As shown in Figure 7, we examined the localization of
sodium channel proteins by indirect immunofluorescence.
Labeling of frozen sections of rat ventricle with D492 Na⫹
channel antibody demonstrates the presence of sodium channel proteins throughout the cardiac sarcolemma (Figure 7A).
Colabeling with antibodies to caveolin-3 demonstrates punctate areas of colocalization of the 2 proteins along the plasma
membrane (Figure 7B), with the merged image shown in
panel C. Concurrent experiments performed in the absence of
primary antibody were negative (data not shown). With the
limitations on resolution inherent in fluorescent microscopy,
we further endeavored to colocalize sodium channel and
Figure 7. Indirect immunofluorescence of rat ventricle. Rat ventricle was double-labeled with D492 (Na⫹ channel) and anti–
caveolin-3 antibodies, followed by incubation with corresponding rabbit anti– goat-rhodamine conjugated and goat-antimouse-fluorescein conjugated antibodies. Punctate areas of
colocalization (represented in yellow) are apparent at the plasma
membrane of the ventricular cells. Region of zoom depicted in D is
represented by boxed area in C. Bar⫽20 ␮m for A through C and
bar⫽5 ␮m for D.
caveolin-3 proteins by immunogold labeling of our CR
fraction. As demonstrated in Figure 8, CR fraction membranes obtained by density gradient separation colabel with
sodium channel (large, 10 nm gold particle) and caveolin-3
(small, Ag⫹-enhanced ultra-small gold particle) antibodies,
with examples of clustering indicated by arrowheads and
boxes. Sections labeled with primary antibodies preincubated
with their antigenic peptide or with secondary goldconjugated antibodies alone were negative (data not shown).
This supports our hypothesis that sodium channels are localized to caveolae membrane compartments where they can be
presented to the plasma membrane on receptor stimulation.
Discussion
Because the direct G␣s effect appears to result in an increase
in the number of functional channels at the sarcolemma,7 we
hypothesized that functional channels were being recruited
from an intracellular pool, allowing for the rapid and controlled presentation of channels to the cell membrane on
stimulation of the ␤AR. Previous work by other labs36
supports the localization of channel proteins to distinct
membrane lipid raft compartments, including caveolae. In
addition, recent work by Zhou et al12 demonstrates that a
blocker of vesicular membrane trafficking can eliminate a
PKA-mediated increase in INa in Xenopus laevis oocytes
expressing the human heart sodium channel protein, hH1, by
blocking the stimulated increase in plasma membrane channel number.
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March 8, 2002
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Figure 8. Immunogold labeling of caveolin-rich fraction examined by transmission electron microscopy. Double labeling (primary antibodies: anti– caveolin-3 and D492; secondary antibodies: goat anti-mouse ultra-small gold conjugate; rabbit anti-goat
10-nm gold conjugate, respectively) of caveolar membranes
obtained by sucrose gradient fractionation demonstrates clustering of caveolin-3 (corresponding to small beads) and sodium
channel (corresponding to large beads), examples of which are
indicated by arrows and boxes. Boxes indicate enlarged regions
A, B, and C. Bar⫽0.1 ␮m.
In the present study, we have been able to implicate the
G␣s protein and caveolae in the direct ␤-adrenergic effect on
the cardiac sodium current. We demonstrate, with both
biochemical and functional evidence, that caveolae may be
involved in the receptor-mediated presentation of ion channels at the cell membrane. Use of the detergent-free buoyant
density method of caveolar membrane isolation routinely
yielded vesicles cross-reactive with antibodies to caveolin-3.
Western analysis of these density gradient fractions demonstrates that both the sodium channel and G␣s are associated
with caveolar membranes. In contrast, membrane markers for
several membrane compartments were not detectable in our
caveolin-rich fraction by Western analysis, suggesting that
caveolar membranes obtained in this way are relatively free
from contamination. Furthermore, purification of the
caveolin-rich fraction by immunoprecipitation with anti–
caveolin-3 antibodies yielded a fraction in which sodium
channel and G␣s proteins could be detected, suggesting a
physical association of both with the caveolar membrane.
These experiments suggest that voltage-gated sodium channels are physically associated with caveolin-3–rich membranes specifically. Additionally, immunocytochemistry
studies demonstrated colocalization of sodium channel and
caveolin proteins at the sarcolemma of cells at the tissue
level. Electrophysiological studies involving isolated cardiac
myocytes demonstrated that the direct G␣s effect on INa could
be specifically ablated by antibodies to caveolin-3. This
evidence substantiates the hypothesis that sodium channels
present within the caveolar membrane are functionally capable of mediating the PKA-independent isoproterenol-induced
increase in sodium current in the heart.
Evidence by Couet et al26 has suggested a physical association between the G-protein ␣-subunit and caveolin-1,
wherein G␣ proteins negatively regulate the function of
caveolin-1. At the neuronal cell membrane, ␤-adrenergic
stimulation and attendant G␣ activation results in a decrease
in the voltage-gated sodium current (reviewed in Catterall37).
In contrast, previous work by our laboratory and others has
shown that stimulation of ␤-adrenergic receptors with ISO
results in an increase in INa in cardiac myocytes. We have
demonstrated that this increase in INa in cardiac myocytes has
2 sources: a PKA-dependent component that results in
changes in both current amplitude and channel-gating (inactivation) kinetics, and a PKA-independent component that
increases current amplitude without changing single-channel
voltage-dependent parameters.2,7 When blocking concentrations of PKI are superfused across the cytoplasmic face of the
myocyte membrane, the addition of the cAMP analog CPTcAMP or forskolin, an adenylyl cyclase activator, fails to
elicit an increase in INa,2 suggesting that the second component involves G␣s directly. This direct effect elicited by
␤-adrenergic stimulation with ISO can be mimicked by the
addition of short G␣s peptide sequences7 and is blocked by
the addition of antibodies to G␣s7 or propranolol, but not by
the addition of ␣-adrenergic antagonists (data not shown).
The results of the present study demonstrate that the ISO
effect on INa is also blocked by the addition of anti– caveolin-3
antibodies to the intracellular milieu. Thus, our model of
caveolar function entails direct action by G␣s on caveolae,
resulting in the presentation of caveolar membrane components to the sarcolemma.
This work should be examined in light of its applicability
to other cell types and signaling pathways. It should be noted
that, although we focus on the mechanism whereby
␤-adrenergic stimulation regulates caveolar protein presentation at the sarcolemma, we expect that other signaling
cascades via different receptors may involve separate, although similar, mechanisms. Further experiments to determine which proteins are involved in caveolar kinesis and their
role, if any, in regulating receptor-mediated protein presentation at the cell membrane, are necessary. Some vesicle
transport molecules, including NSF and VAMP proteins,
have already been localized to caveolae derived from endothelium.38 The role of these proteins or homologous proteins
in muscle cells expressing caveolin-3 remains to be studied.
Finally, we employed cell isolation techniques that do not
account for in vivo regional cell origins (endocardium versus
myocardium versus epicardium39 or apex versus base). It is
likely that some variability in caveolar and sodium channel
density exists throughout the heart, and future studies in this
field are expected to further elucidate the subtleties of
caveolar regulation in different cell types and regions of the
heart. In spite of these limitations, this work does provide
interesting and important insight into the role of caveolae in
mediating rapid, reversible, and reproducible membrane
events that can affect overall cell excitability by regulating
plasma membrane protein presentation.
Yarbrough et al
Acknowledgments
This work was supported by an UNCF-Merck Graduate Research
Dissertation Fellowship, American Heart Association–Heartland Affiliate Grant 9951400Z, and National Institutes of Health Grant
1F31HL10011-01A1. We would like to express our thanks to Mark
A. Stamnes for consultation and critical manuscript review, Jianqiang Shao and Kenneth C. Moore of the Iowa Central Microscopy
Research Facility for expert technical assistance in immunomicroscopy techniques, and Prasad V.G. Katakam for technical assistance
in immunoblotting.
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Localization of Cardiac Sodium Channels in Caveolin-Rich Membrane Domains:
Regulation of Sodium Current Amplitude
Tracy L. Yarbrough, Tong Lu, Hon-Chi Lee and Erwin F. Shibata
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Circ Res. 2002;90:443-449; originally published online January 17, 2002;
doi: 10.1161/hh0402.105177
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3407/R2
online methods
Online Data Supplement
Erwin F. Shibata, PhD
“Localization of Cardiac Sodium Channels in Caveolin-Rich Membrane Domains:
Regulation of Sodium Current Amplitude”
ORIGINAL CONTRIBUTION
Expanded Methods
Myocyte Isolation. Adult rat cardiac myocytes were obtained as outlined previously1,
and as approved by the Animal Care and Use Committee at the University of Iowa.
Briefly, thoracotomy was performed on 50-70 day-old Sprague-Dawley male rats
anesthetized with methoxyflurane or isoflurane. The heart was excised and retrogradely
perfused for 4 minutes via the aorta on a modified Langendorff apparatus. The perfusing
solution was nominally calcium-free modified Tyrode’s solution that contained, in
mmol/L: 138 NaCl, 5.4 KCl, 0.5 MgCl2, 0.33 Na2HPO4, 5.5 HEPES, 5.5 dextrose, and
1mg/ml albumin; pH 7.38 with NaOH. Following this washout, the heart was then
perfused for 7-9 minutes at 37°C with nominally calcium-free Tyrode’s solution to which
0.25-0.3 mg/ml collagenase was added. Perfusion time and collagenase concentrations
were a function of the size and age of the animal. After perfusion, hearts were
mechanically dissociated by mincing the tissue with scissors, and cells were centrifuged
and washed in KB solution containing, in mmol/L: 70 KOH, 40 KCl, 1 K2HPO4, 0.5
MgCl2, 50 L-glutamic acid, 20 taurine, 0.5 EGTA, 10 HEPES, 5 creatine, 5 Na2ATP, 10
dextrose, and 5 pyruvic acid; pH 7.38 with KOH.
Isolation of Caveolin-rich Fraction. Isolation of caveolin-rich fraction was done using
a detergent-free method largely as described previously2, with some modifications. All
procedures were carried out at 4°C. After a final wash in KB solution, the rat cardiac
Page 1
myocyte pellet was resuspended in 2.0 ml of 500 mmol/L Na2CO3 plus 0.25 protease
inhibitor cocktail tablet (Boehringer Mannheim). Approximately 500 x 103 myocytes
were sonicated (3 times for 20 s using a Fisher Scientific 550 Sonic Dismembrator), and
the lysate was transferred to a centrifuge tube for discontinuous sucrose density gradient
separation using sucrose cushions of 5-45% (5%, 15%, 25%, 35%, and 45% w/v). The
gradient was centrifuged in a Beckman SW41 rotor at 39,000 rpm for 20 hours, and
twelve 1 ml-samples were collected, starting from the top of the gradient. We routinely
obtained a “caveolin-rich fraction” (samples 5 and 6, constituting the 25% sucrose
cushion in the density gradient). For comparison, a “heavy fraction” (sample 11) was
collected. To removed soluble proteins and pellet membranes, the caveolin-rich fraction
(and, similarly, the heavy fraction) was then washed in MBS (containing, in mmol/L: 25
Mes, 150 NaCl; pH 6.5) and centrifuged in the SW41 rotor at 40,000 rpm for 2 hours.
The pellet was then solubilized in Thorner buffer (4%SDS; 50mmol/L Tris; 8 mol/L urea;
pH 6.8 with NaOH) for use in protein assays and Western blotting. Protein
determinations were done using the copper/bicinchoninic acid assay (Pierce assay) from
Sigma.
Antibodies. Antibodies used were obtained as follows. Anti-cardiac ryanodine receptor
(RyR)3 was the kind gift of Dr. Kevin P. Campbell.
Monoclonal antibodies to caveolin-
34, caveolin-25, clathrin6, Rab 57, and Rab 118, and secondary HRP-conjugated antimouse and anti-rabbit antibodies were obtained from Transduction Laboratories.
Polyclonal antibodies to caveolin-19 and Gαs10 were obtained from Santa Cruz
Biotechnology. Antibodies to subunit 2 of cytochrome oxidase (COX)11, the plasma
Page 2
membrane calcium ATPase (PMCA)12, and mannosidase II13 were obtained from
Molecular Probes, Affinity Bioreagents, and Covance, respectively. Subtype-specific
antibodies14,15 to the α1 and α2 isoforms of the Na+/K+ ATPase were a kind gift from Dr.
Kathleen J. Sweadner, and antibodies to both the α1 and α2 isoforms were purchased
from Upstate Biotechnology. Anti-SP-19 (pan) sodium channel antibody16 used for
Western blot analysis and secondary HRP-conjugated anti-goat antibodies were obtained
from Sigma. D492 polyclonal antibody used for immunofluorescence and immunogold
electron microscopy was raised in goat to a 22-amino acid peptide corresponding to a
sequence (DRLPKSDSEDGPRALNQLSLC) located in interdomain loop I-II of the
cardiac voltage-gated sodium channel as similarly described by Cohen and Levitt
(1993)17.
Immunoblotting. Samples were precipitated using the pyrogallol red-molybdate method
as described previously for SDS-PAGE18. Precipitated samples were heated for six
minutes at 100°C in SDS-polyacrylamide electrophoresis buffer containing βmercaptoethanol. Samples were then run on 7.5-12% polyacrylamide gel at 200 V for 45
minutes, and proteins were transferred to 0.45 µM nitrocellulose membrane by
application of 0.3 mA current for 2 hours. Membranes were blocked for 25 minutes in
TBS-T containing, in mmol/L: 50 Tris, 150 NaCl, 3.8 HCl, 0.05% Tween-20, pH 7.6;
with 5% nonfat dry milk. The membranes were incubated with primary antibody at 4°C
overnight, washed twice in TBS-T, and incubated for 2 hours at room temperature in the
presence of HRP-conjugated secondary antibody. To remove excess antibody,
Page 3
membranes were then washed 5 times in TBS-T. Detection was performed using
enhanced chemiluminescence substrates from Amersham Pharmacia Biotech.
Immunoprecipitation. Enzymatically isolated adult rat ventricular cells were lysed by
sonication and membranes were separated on sucrose gradient cushion as described in
Isolation of caveolin-rich fraction above. Caveolin-rich fractions (gradient fractions 5
and 6, hereafter called “CR fraction”) and “heavy” fractions (gradient fraction 11)
obtained in this way were resuspended in buffer consisting of, in mmol/L: 150 NaCl, 5
EDTA, 1 phenylmethylsufonyl fluoride (PMSF), 25 Mes; pH 6.7 with NaOH. To this
was added anti-caveolin-3 monoclonal antibody (1:500) for incubation at 4°C overnight,
followed by incubation with Protein G-agarose beads overnight (16 hours) at 4°C. For
negative controls, fractions were incubated with anti-caveolin-2 (1:500) or no primary
antibody overnight. Samples were then treated similarly to those incubated with anticaveolin-3. Following incubation, the membrane-antibody-bead complexes were washed
five times with Buffer A containing, in mmol/L: 150 NaCl, 50 Tris-HCl, 1 EDTA, 0.5%
(w/v) Triton X-100. Following the final wash, the fraction was centrifuged and the
supernatant was collected. 1X Sample Buffer was added to the bead complexes, which
were then boiled, and the supernatant run on polyacrylamide gel for Western blotting.
Immunofluorescence. Adult rat ventricles were fixed in 4% paraformaldehyde solution,
infused with 30% sucrose solution for 2 hours, then frozen in liquid N2, and sectioned
using a cryostat. Sections collected on glass slides were incubated with primary
antibodies for one hour at room temperature, washed with PBS, and then incubated with
Page 4
fluorescent-conjugate secondary antibodies for one hour at room temperature. Sections
were then mounted and visualized on a Zeiss Laser Scanning Confocal Microscope.
Primary antibodies were diluted as follows: anti-caveolin-3 1:50; D492 1:200.
Immunofluorescent conjugates were diluted at 1:200.
Immunogold labeling and electron microscopy. CR fractions were fixed in 2%
paraformaldeyhyde/0.5% glutaraldehyde solution and labeled prior to embedment in
Eponate 12. Embedded capsules were sectioned with an ultramicrotome, with sections
collected on copper grids. Grids were incubated overnight at 4°C in primary antibodies
to the cardiac sodium channel (D492) and caveolin-3. After buffer washes, grids were
then incubated in secondary gold-conjugate for 2 hours at room temperature. Samples
were washed, fixed in 2.5% glutaraldehyde, and silver-enhanced. Finally,
counterstaining with uranyl acetate and lead citrate allowed for visualization with an H7000 transmission electron microscope. Primary antibodies were diluted as follows:
anti-caveolin-3 1:20; D492 1:100. Secondary gold-conjugates were diluted as follows:
goat anti-mouse ultra-small gold conjugate 1:40; rabbit anti-goat 10nm gold-conjugate
1:40.
Electrophysiological protocols and data analysis. Voltage pulse protocols were
generated and data was recorded using pCLAMP6.04 software and a personal computer
interfaced with an Axopatch 200 integrating patch-clamp amplifier (software and
amplifier from Axon Instruments). Solution-filled (for composition see below) pipette
resistances ranged from 0.5 to 1 MΩ and seal resistances were 20-50 GΩ. The signal
Page 5
was filtered at 5 kHz, and digitized at a sampling rate of 50 kHz. Whole-cell series
resistance was compensated to 80-85% of the uncompensated value. Due to spontaneous
shifts in the current inactivation-voltage relationship after establishing the whole-cell
configuration, INa data were recorded only after the currents became stable (usually
within 5 minutes). All experiments were performed at 21-23°C to help maintain voltageclamp control.
Whole-cell voltage clamp data was generated using test pulses wherein cell
membrane potential was held at –100 mV and then stepped to –40 mV for 20 ms, after
which it was returned to its resting level of –100 mV, with an interepisode interval of 1 s.
Pipette solutions contained, in mmol/L: 130 CsCl, 0.5 CaCl2, 2 MgCl2, 5 Na2ATP, 0.5
GTP, 5 EGTA, 10 HEPES, 0.022 mg/ml protein kinase A inhibitor (PKI); pH 7.25 with
KOH/HCl. Bath solution contained, in mmol/L: 130 choline chloride, 20 NaCl, 0.5 KCl,
1 CaCl2, 2 CoCl2, 10 HEPES, 5.5 dextrose; pH 7.38 with CsOH. In experiments with
anti-caveolin antibody, 100 µg/mL anti-caveolin –1, -2 or -3 antibody was added to the
pipette solution. In the corresponding control experiments, no antibody was added to the
pipette solution. Additional experiments were also performed in which 100 µg/ml anticaveolin-3 antibody + 100 µg/ml caveolin-3 antigenic peptide were added to the pipette
solution. Raw data are plotted as INa relative to baseline INa (sodium current prior to bath
addition of isoproterenol). Histogram data are presented as the mean ± S.E.M. One-way
analysis of variance (ANOVA) was used to compare the ISO-stimulated percent change
in INa among the control and experimental groups (populations in which antibodies
against caveolins -1, -2, or -3 were added to the pipette solution). Statistical significance
is defined as p<0.05, with n=number of experiments as indicated. In addition, Dunnett
Page 6
multiple comparisons test was performed to evaluate difference between each
experimental group versus control.
Page 7
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Page 10