Localization of Cardiac Sodium Channels in Caveolin-Rich Membrane Domains
Regulation of Sodium Current Amplitude
This study demonstrates that caveolae, omega-shaped membrane invaginations, are involved in cardiac sodium channel regulation by a mechanism involving the α subunit of the stimulatory heterotrimeric G-protein, Gαs, via stimulation of the cell surface β-adrenergic receptor. Stimulation of β-adrenergic receptors with 10 μmol/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 β-adrenergic receptors, and thereby Gαs, promotes the presentation of cardiac sodium channels associated with caveolar membranes to the sarcolemma.
Regulation 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 β-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 β-adrenergic receptors (βARs) results in the activation of a signaling cascade involving the Gαs protein itself. This produces an increase in INa without changes in single-channel 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 Gαs to the cytoplasmic face of excised membrane patches mimics the effect of β-adrenergic receptor activation in increasing INa by increasing the number of functional channels at the plasma membrane.7 Thus, the direct effect of β-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 β-agonist washout and is reproducible by subsequent rounds of βAR 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 β-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 cascades 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), Gαs is of particular interest. Although Gαs can mimic the effect of βAR stimulation in increasing cardiac sodium current, Gβγ does not appear to be involved. Therefore, the action of the Gαs in this process must be dependent on the protein itself and other proteins with which it might interact. Specific regions of interaction between Gαi and caveolin-1, the caveolar scaffolding protein, have been identified, wherein Gαi binds to a region contained within the caveolin-1 scaffolding domain in a GTP/GDP-dependent fashion.23,25⇓ Although specific studies have not examined the interaction of Gαs with caveolin-3, the muscle-specific caveolin isoform, caveolin-3 is highly homologous to caveolin-1 in its scaffolding domain,26,27⇓ making an analogous interaction with Gαs 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 Gαs activation through βAR 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 Sprague-Dawley (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 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 Gαs 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 α1 and α2 isoforms of the Na+/K+ ATPase were a kind gift from Dr Kathleen J. Sweadner (Harvard University, Cambridge, Mass), and antibodies to both the α1 and α2 isoforms were purchased from Upstate Biotechnology. Anti-SP-19 (pan) sodium channel antibody and secondary HRP-conjugated 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) located in interdomain loop I-II of the cardiac voltage-gated sodium channel as similarly described by Cohen and Levitt.28
Samples were precipitated using the pyrogallol red-molybdate method29 and run on 7.5% to 12% polyacrylamide gel. Proteins were transferred to 0.45 μmol/L nitrocellulose membrane and blotted with appropriate antibodies.
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.
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.
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 μg/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.
Implication of Caveolin-3 in Adrenergically Mediated Increases in Sodium Current
Previous work2,7⇓ suggests that the direct G-protein effect of β-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 μmol/L 8-CPT-cAMP or 10 μmol/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 of 10 μmol/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 β-adrenergic increase in sodium current stimulated by 10 μmol/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 Gαs-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), 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 detergent-free 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 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 Gαs 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-Gαs 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 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 Gαs 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 Gαs actions, we also examined the presence of Gαs in the samples obtained by immunoprecipitation with anti-caveolin antibodies. Positive detection of Gαs in these samples (Figure 6) suggests that Gαs 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 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 gold-conjugated 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.
Because the direct Gαs 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 βAR. 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.
In the present study, we have been able to implicate the Gαs protein and caveolae in the direct β-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 Gαs 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 Gαs 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 Gαs 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 α-subunit and caveolin-1, wherein Gα proteins negatively regulate the function of caveolin-1. At the neuronal cell membrane, β-adrenergic stimulation and attendant Gα 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 β-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 CPT-cAMP or forskolin, an adenylyl cyclase activator, fails to elicit an increase in INa,2 suggesting that the second component involves Gαs directly. This direct effect elicited by β-adrenergic stimulation with ISO can be mimicked by the addition of short Gαs peptide sequences7 and is blocked by the addition of antibodies to Gαs7 or propranolol, but not by the addition of α-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 Gαs 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 β-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.
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.
Original received October 12, 2001; revision received January 7, 2002; accepted January 7, 2002.
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