AJP - Heart Journal of Applied Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 294: H1693-H1699, 2008. First published February 15, 2008; doi:10.1152/ajpheart.01337.2007
0363-6135/08 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Material
Right arrow All Versions of this Article:
294/4/H1693    most recent
01337.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Palygin, O. A.
Right arrow Articles by Shibata, E. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Palygin, O. A.
Right arrow Articles by Shibata, E. F.

Regulation of caveolar cardiac sodium current by a single Gs{alpha} histidine residue

Oleg A. Palygin, Janette M. Pettus, and Erwin F. Shibata

Department of Molecular Physiology and Biophysics, Carver College of Medicine, The University of Iowa, Iowa City, Iowa

Submitted 14 November 2007 ; accepted in final form 12 February 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cardiac sodium channels (voltage-gated Na+ channel subunit 1.5) reside in both the plasmalemma and membrane invaginations called caveolae. Opening of the caveolar neck permits resident channels to become functional. In cardiac myocytes, caveolar opening can be stimulated by applying β-receptor agonists, which initiates an interaction between the stimulatory G protein subunit-{alpha} (Gs{alpha}) and caveolin-3. This study shows that, in adult rat ventricular myocytes, a functional Gs{alpha}-caveolin-3 interaction occurs, even in the absence of the caveolin-binding sequence motif of Gs{alpha}. Consistent with previous data, whole cell experiments conducted in the presence of intracellular PKA inhibitor stimulation with β-receptor agonists increased the sodium current (INa) by 35.9 ± 8.6% (P < 0.05), and this increase was mimicked by application of Gs{alpha} protein. Inclusion of anti-caveolin-3 antibody abolished this effect. These findings suggest that Gs{alpha} and caveolin-3 are components of a PKA-independent pathway that leads to the enhancement of INa. In this study, alanine scanning mutagenesis of Gs{alpha} (40THR42), in conjunction with voltage-clamp studies, demonstrated that the histidine residue at position 41 of Gs{alpha} (H41) is a critical residue for the functional increase of INa. Protein interaction assays suggest that Gs{alpha}FL (full length) binds to caveolin-3, but the enhancement of INa is observed only in the presence of Gs{alpha} H41. We conclude that Gs{alpha} H41 is a critical residue in the regulation of the increase in INa in ventricular myocytes.

voltage-gated sodium channel subunit 1.5; caveolae; caveolin; G protein; ventricle

β-ADRENERGIC RECEPTOR (β-AR) stimulation is of fundamental importance in the heart. The classical paradigm is for β-adrenergic-activated stimulatory G protein {alpha}-subunit (Gs{alpha}) to stimulate adenylyl cyclase. cAMP then activates protein kinase A (PKA) and phosphorylates proteins that contain the PKA-Ser/Thr motif. PKA-mediated phosphorylation of voltage-gated Na+ channel subunit 1.5 (Nav1.5) channels typically results in increases in both current amplitude and the rate of current decay (10, 15, 17, 21, 34). In addition to this classic paradigm, another pathway has been identified through studies in which pipette dialysis was used to apply the PKA inhibitor (PKI) peptide to adult ventricular myocytes (15). Under these conditions, Gs{alpha} increases Nav1.5 current amplitude through a parallel PKA-independent pathway without altering voltage dependence or kinetics of the sodium current (INa). Overall, these studies suggest that the increase in current amplitude is mediated by activating additional channels from a cellular reservoir of Nav1.5 channels (15). Furthermore, the addition of a short NH2-terminal Gs{alpha}-derived peptide (residues 28–42) to the pipette solution used in whole cell patch-clamp recordings can mimic the effect of β-AR stimulation and is prevented by the addition of anti-Gs{alpha} antibodies (15, 33).

Recent studies have identified both cardiac sodium channels (Nav1.5) (27, 28, 33) and L-type calcium channels [voltage-gated calcium channel subunit 1.2a (Cav1.2a)] (2, 16, 28) in cardiomyocyte cellular reservoirs-caveolae. Caveolae are cholesterol- and sphingolipid-enriched plasma membrane invaginations containing a signature scaffolding integral membrane protein called caveolin (24). Caveolae are immobile vesicles (31) whose neck opening is regulated in a dynamic fashion, and this is known to modulate the continuity between the intracaveolar and extracellular spaces (28). However, the mechanism(s) responsible for this regulation has not been clearly defined. This study begins to identify the proteins involved in this pathway.

Three caveolin-encoding genes (cav1, cav2, and cav3) are translated to express six known subtypes of the protein [caveolin-1{alpha} and -1β, caveolin-2{alpha}, -2β, and -2{gamma}, and caveolin-3 (Cav-3)] (22, 25). Cav-3 is primarily expressed in skeletal muscle, smooth muscle, and cardiac muscle. Adult cardiac myocytes express predominantly Cav-3 (13).

From in vitro Gs{alpha} binding studies, Gs{alpha} has been shown to interact with caveolin-1 (Cav-1) at the caveolin scaffolding domain (CSD) (amino acid 82–101 of Cav-1) (25). The CSD of Cav-1 was shown to be a potent inhibitor of heterotrimeric G proteins in GTP hydrolysis assays (14). Although subsequent studies have shown the CSD of Cav-1 to function as a negative regulator of signaling molecules, others have found the interaction with the scaffolding domain to function as activators, as well as having no function at all (9). The Cav-3 CSD is highly homologous to the Cav-1 CSD (7), suggesting that an analogous Gs{alpha} interaction may take place. Gs{alpha} contains a caveolin-binding sequence (CBS) motif ({Phi}X{Phi}XXXX{Phi}XX{Phi}, where {Phi} is an aromatic amino acid, i.e., Trp, Phe, or Tyr; FTFKDLHFKMF in Gs{alpha}) (7, 20) (amino acid 212–222; Fig. 1A). Since this motif exists within most caveolae-associated proteins, it is thought to mediate the interaction between caveolin binding proteins and the CSD of caveolin. However, functional data of the Gs{alpha}-Cav-3 interaction are limited and require further detailed in vivo studies.


Figure 1
View larger version (52K):
[in this window]
[in a new window]

  		Fig. 1. Schematic diagram of stimulatory G protein {alpha}-subunit (Gs{alpha}). A: linear schematic representation of Gs{alpha} showing the locations of the Gs{alpha} peptide (amino acid 28–42) and the caveolin-binding sequence (CBS) motif (amino acid 212–222) in the full-length Gs{alpha} protein (Gs{alpha}FL). B: ribbon diagram depiction of Gs{alpha}-MgGTP{gamma}S. Residues of the CBS motif and the Gs{alpha} peptide histidine 41 (His41) with the imidazole side chain in stick representation are located at the surface of the molecule (shown in black). The sphere represents the Mg2+ ion, and GTP{gamma}S is shown in stick representation, which is located in the nucleotide binding pocket. The structure was generated using the PDB entry 1AZT (30) and PyMOL 0.99 (8).

 
A 16-amino acid peptide from the Gs{alpha} NH2-terminus (amino acid 28–42; see Fig. 1A) has been previously found to mimic the β-AR enhancement of INa (15), although it is not part of the Gs{alpha} CBS motif. This does not necessarily rule out an involvement of the Gs{alpha} CBS motif in the functional interaction between Gs{alpha} and caveolin proteins. For example, the Gs{alpha} CBS motif could potentially facilitate an interaction between the Gs{alpha} NH2-terminus and an unknown substrate that is required to open the caveolar neck, and such an interaction would regulate the continuity between the extracellular space and the intracaveolar environment.

To test the hypothesis that specific residues at the Gs{alpha} NH2-terminus are critical in enhancing the INa, we undertook a series of experiments to determine which residue(s) is critical to the functional interaction between Gs{alpha} and caveolin proteins. Our results show that there are at least two interacting sites of Gs{alpha} with Cav-3: the Gs{alpha}FL (full length) H41A mutant binds Cav-3 without increasing the cardiac INa and is thus nonfunctional, and a second binding site at the Gs{alpha} NH2-terminus (H41), which appears necessary to enhance the INa in the PKA-independent pathway.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Adult male Sasco Sprague-Dawley rats were obtained from Charles River Laboratories and housed in the Animal Care Facility at the University of Iowa. Rat cardiac myocytes were obtained as approved by the Animal Care and Use Committee at the University of Iowa. An expanded MATERIALS AND METHODS section is available online. (The online version of this article contains supplemental data.)

Isolation of rat cardiac ventricular myocytes. Single ventricular myocytes from adult rat hearts were isolated using established enzymatic digestion techniques, as previously described (33). After enzymatic digestion of the heart, pieces of left ventricular tissue were gently triturated, washed, and gently centrifuged in Kraftbrühe solution (3x) and stored on ice.

Enrichment of caveolin-rich fraction. Caveolin-rich fractions (CRF) were obtained by sucrose gradient fractionation using a detergent-free method, with some modification from protocols that had been described previously (26, 29). The resulting solubilized CRF sample was used in protein interaction assays and Western blotting.

The protein-protein interaction assay. The cardiac ventricular myocyte lysate preparation, sucrose gradient fractionation, CRF preparation, pHis6Gs{alpha} wild-type (WT) and mutant protein expression, protein purification, and column immobilization were carried out using standard protocols. The protocols are detailed in the online data supplements.

The CRF sample was loaded onto the Ni-His columns containing the immobilized Gs{alpha} protein and allowed to enter the resin by gravity flow. The Ni-His-Gs{alpha}-CRF protein complexes were washed 3x with 5 ml of 1x binding and wash buffer. The bound protein complexes were eluted from the resin using elution buffer (in mmol/l): 20 Tris·HCl, pH 8.0, 500 NaCl, and 500 imidazole. Four fractions were collected: first a 250-µl fraction, followed by three 600-µl fractions. Aliquots of the eluates were reserved for SDS-PAGE.

Site-directed mutagenesis of the phis6-Gs{alpha}. Two mutants of Gs{alpha}, the single mutant H41A and the double mutant T40A/R42A, were generated using the Stratagene QuikChange II Site-directed Mutagenesis Kit by PCR amplification on the pHis6-Gs{alpha} (poly-histidine tagged Gs{alpha}) template, using the following mutagenic sense and antisense primers: for Gs{alpha}FL H41A, 5'-CAAGCAGGTCTACCGGGCCACGGCTCGTCTGCTGCTGC-3' and 5'-GCAGCAGCAGACGAGCCGTGGCCCGGTAGACCTGCTTG-3'; and for Gs{alpha}FL T40A/R42A, 5'-CAAGCAGGTCTACCGGGCCGCTCACGCTCTGCTGCTGC-3' and 5'-GCAGCAGCAGAGCGTGAGCGGCCCGGTAGACCTGCTTG-3'.

The resulting PCR products were used to transform XL-Blue cells. Randomly selected colonies for each mutant were cultured and the DNA purified using the QIAprep Spin Miniprep Kit. The presence of the desired mutations was confirmed by sequencing the entire Gs{alpha} sequence in each selected clone. Transformations, protein expression, and purification of WT and mutant pHis6-Gs{alpha} were carried out as detailed in the online data supplements.

Electrophysiological protocol. Voltage-dependent INa were measured using standard patch-clamp techniques. Whole-cell INa were recorded with an Axopatch 200B amplifier. The analog signal was filtered using an eight-pole Bessel filter with a bandwidth of 5 kHz and digitized at a sampling rate of 50 kHz. Borosilicate glass capillaries (VWR) were used to fabricate patch pipettes. Electrode resistances ranged from 0.8 to 1.2 M{Omega}, and seal resistances were 1–5 G{Omega}. Whole cell voltage-clamp data were elicited from a holding potential of –120 mV to membrane potentials ranging from –110 to +30 mV. Pipette seal resistances were compensated to >95% of the uncompensated value. The whole cell bath solution contained the following (in mmol/l): 10 NaCl, 130 choline chloride, 4.5 KCl, 1.0 CaCl2, 2.0 MgCl2, 2.0 CoCl2, 10.0 HEPES, and 5.5 glucose (pH 7.35, titrated with KOH). The pipette solution contained the following (in mmol/l): 130 CsCl, 0.5 CaCl2, 2 MgCl2, 5 Na2ATP, 0.5 GTP, 5 EGTA, 10 HEPES (pH 7.25, titrated with CsOH). The intracellular concentration of free sodium is estimated to be ~5 mM (18, 32), and the sodium reversal potential is calculated to be +17.6 mV (22°C). In all experiments, the pipette solution contained 22 µg/ml PKI, a concentration at which the effects of PKA on INa have been shown to be completely inhibited (17). Anti-Cav-3 monoclonal antibody (MAb) (BD Transduction Laboratories) was added to the pipette solution at a concentration of 0.34 µmol/l. Control antibody experiments were performed using anti-Cav-1 and anti-Cav-3 MAbs (BD Transduction Laboratories), showing no inhibition of the INa increase with isoproterenol (ISO) when anti-Cav-1 MAb was in the pipette solution. Specificity of the antibodies was also shown with anti-Cav-1, -2, and -3 MAb (33). In this study, anti-Gs{alpha} polyclonal antibody (PAb) was added to the pipette solution at a concentration of 0.44 µmol/l (Santa Cruz Biotechnology). The antibody solutions were sodium azide free. The 16-amino acid Gs{alpha} peptides (Chemicon International), WT Gs{alpha}FL protein, and Gs{alpha}FL mutant proteins were added to the pipette solution at a concentration of 1 µmol/l. Gs{alpha}FL protein was checked for degradation products using matrix-assisted laser desorption ionization-time of flight mass spectrometry. Efficacy was tested during storage in pipette solution without any preservatives. The proteins showed full effectiveness for 9 days without significant decrease in enhancing INa.

Statistical analysis. Electrophysiological data were collected and analyzed using pCLAMP 9.0 software (Axon Instruments), and OriginPro 7.5 (OriginLab) ANOVA was used to compare the nominal change in INa among the control and experimental means. All data are shown as means ± SE. Statistical significance is defined as P < 0.05, with N number of experiments as indicated.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
To investigate how INa is modulated by a PKA-independent pathway in acutely disassociated adult rat ventricular myocytes, whole cell patch-clamp recordings were performed with PKI in the pipette solution. As previously shown (15, 28) and confirmed in this study, 10 µmol/l ISO (a β-AR agonist) increased the peak INa density by 36% (from 61.2 ± 7.1 to 83.2 ± 5.3 pA/pF, n = 15, P < 0.05). The involvement of Gs{alpha} and Cav-3 is illustrated by the family of current traces in Fig. 2A. From a holding potential of –120 mV, a series of test pulses from –110 to +30 mV was elicited in 10-mV steps. These current traces reveal that the presence of Gs{alpha}FL protein in the pipette solution can mimic the enhancing effect of ISO (control 61.2 ± 7.1 pA/pF, n = 15 vs. Gs{alpha}FL 85.3 ± 4.3 pA/pF, n = 10, P < 0.05). The involvement of the caveolae signature scaffolding protein, Cav-3, in the regulatory mechanism of INa density enhancement was demonstrated using a MAb to an NH2-terminal epitope of Cav-3. Application of ISO in the presence of 0.34 µmol/l anti-Cav3 MAb in the pipette solution abrogated the increase of β-adrenergic stimulation (33), suggesting that Cav-3 acts in the signaling pathway that regulates the current-enhancing mechanism. In addition, Fig. 2A shows that anti-Cav-3 MAb can prevent the Gs{alpha}-mediated effect (Gs{alpha}FL + anti-Cav-3 MAb 54.6 ± 4.8 pA/pF, n = 8). Figure 2B (top) shows that the Gs{alpha}-mediated increase of INa density was similar to the ISO increase in the presence of PKI in all investigated cells (n = 10). Additional studies shown in Fig. 2B (bottom) indicate that, in the presence of anti-Gs{alpha} PAb (0.44 µmol/l) and PKI, ISO failed to significantly increase the peak INa density (Gs{alpha} PAb 51.9 ± 2.4 pA/pF, n = 10 vs. Gs{alpha} PAb + ISO 58.8 ± 2.2 pA/pF, n = 10, P < 0.05). Moreover, the bath addition of ISO with Gs{alpha}FL (85.2 ± 4.0 pA/pF, n = 10) or Gs{alpha}FL + anti-Cav-3 MAb (54.6 ± 4.8, n = 8) in the pipette solution did not further increase the amplitude of INa density (Fig. 2, A and C). These data suggest that other signaling pathways, either alternative or parallel, do not significantly affect the enhancement of INa. Summary data are shown in Fig. 2C.


Figure 2
View larger version (25K):
[in this window]
[in a new window]

  		Fig. 2. Sodium current (INa) modulation through the caveolin (Cav)-3 and Gs{alpha}-dependent pathway in rat cardiomyocytes. A: superimposed families of current traces, from a holding potential of –120 mV to potentials elicited by voltages ranging from –110 mV to +30 mV in 10-mV increments. The superimposed raw current traces include a control (105 pF, cell capacitance), Gs{alpha}FL protein (95 pF), Gs{alpha} polyclonal antibody (PAb) + superfusion of isoproterenol (ISO) (102 pF), and Gs{alpha}FL plus the anti-Cav-3 monoclonal antibody (MAb) (80 pF). Gs{alpha}FL protein and antibodies were applied via the pipette, and all traces are normalized to cell capacitance. B, top: averaged current-voltage (I-V) relationship of control (n = 15) and intracellular application of purified Gs{alpha}FL protein (n = 10). This application mimics the enhancement by ISO on INa. B, bottom: in the presence of the anti-Gs{alpha} PAb in the pipette, and perfusion of ISO (n = 10), INa density did not differ from that produced by the anti-Gs{alpha} PAb, suggesting the specificity of a Gs{alpha} effect and not a Gβ{gamma} effect. C: summary plots of experiments, with application of either Gs{alpha}FL plus anti-Gs{alpha} PAb or anti-Cav-3 MAb. In control, the application of ISO increased INa density. In the presence of Gs{alpha}FL alone, application of ISO did not increase INa density, suggesting that the increase in current is due to Gs{alpha} function. When ISO was applied along with anti-Gs{alpha} PAb, ISO did not further increase the INa density. Anti-Cav-3 MAb abolished the effect of Gs{alpha}FL on INa, and the addition of ISO did not change the INa density. PKI, PKA inhibitor. *Statistical difference was tested by analysis of variance with P < 0.05.

 
These findings reveal that 1) Gs{alpha}FL can mimic the increase of INa density seen in response to ISO; 2) the Gs{alpha} enhancement of INa requires Cav-3; 3) the ISO-mediated increase of INa density depends on Gs{alpha}; and 4) ISO cannot further increase the INa density in the presence of anti-Gs{alpha} or anti-Cav-3 antibody. These results suggest that Gs{alpha} and Cav-3 are components of the signaling pathway that leads to an increased INa density in the PKA-independent pathway.

Previous studies had shown that a 16-amino acid peptide near the NH2-terminus of Gs{alpha} (28–42) could fully mimic the properties of the PKA-independent INa increase induced by native Gs{alpha} protein in rat cardiac ventricular myocytes (15). In studies using overlapping 10-mer peptides of the 16-amino acid peptide, only the 10-mer, which included the 40THR42 of Gs{alpha} residues, was active in mimicking the ISO-activated increase of INa (15). Detailed mutation studies were, therefore, conducted focusing on the THR residues. Further investigation into the role of H41 was studied using single- and double-point mutations of the Gs{alpha}FL protein: a Gs{alpha}FL H41A single mutant and a Gs{alpha}FL T40A/R42A double mutant. Although application of the WT Gs{alpha}FL protein or the Gs{alpha}FL T40A/R42A enhanced the INa in rat cardiomyocytes [85.3 ± 4.3 pA/pF, n = 10, and 86.9 ± 9 pA/pF, n = 7 vs. 62.1 ± 6.9 pA/pF (control) P < 0.05] (Fig. 3A), the histidine to alanine mutant Gs{alpha}FL H41A did not augment the current significantly (54.8 ± 3 pA/pF, n = 9) (Fig. 3B). Figure 3B also shows that subsequent addition of ISO with Gs{alpha}FL H41A led to a significant increase in current density (76.1 ± 6.5 pA/pF, n = 9) by activating endogenous Gs{alpha}. The findings from the functional experiments involving the WT Gs{alpha}FL protein and the Gs{alpha}FL mutants are summarized in Fig. 3C. The results showing that both the Gs{alpha}FL protein and the Gs{alpha}FL T40A/R42A led to a similar increase in INa density substantiate our hypothesis that H41 of Gs{alpha} is the key to the PKA-independent, Cav-3-dependent increase of INa.


Figure 3
View larger version (27K):
[in this window]
[in a new window]

  		Fig. 3. Involvement of His41 in the regulation of INa density by Gs{alpha}. A: the wild-type (WT) Gs{alpha}FL (n = 10) and the Gs{alpha}FL T40A/R42A (n = 9) had the ability to enhance the INa. B: the Gs{alpha}FL H41A fails to augment the current. Addition of ISO under these conditions generated a significant increase in INa density (n = 7). C: summary of the experiments with Gs{alpha}FL and mutants. Controls represent INa in myocytes in the absence of Gs{alpha}FL protein, without ISO (solid bars) or with ISO (open bars). In the presence of Gs{alpha}FL, as well as the Gs{alpha}FL T40A/R42A, a similar increase in INa density was generated. The addition of ISO did not further enhance the INa. The Gs{alpha}FL H41A, however, did not produce changes in INa density compared with controls. *Statistical difference was tested by analysis of variance with P < 0.05. D: WT Gs{alpha}FL and mutant Gs{alpha}FL proteins all captured Cav-3 from a solubilized caveolin-rich fractions (CRF) prepared from adult cardiac ventricular myocytes in a protein-protein assay. Ni-ATA resin immobilized pHis6-Gs{alpha}FL proteins, and interacting proteins were eluted from the resin, and the resulting protein complexes were analyzed by SDS-PAGE and immunoblotting with PAbs to Gs{alpha} and Cav-3. A Cav-3 protein band was detected in the elutate from columns 4–6, containing Gs{alpha}FL protein. The interaction assay included two control columns: lane 2 (without Gs{alpha}FL and with CRF) and lane 3 (with Gs{alpha}FL and without CRF). Lanes 1 and 7 are the immunoblot protein migration control lanes. Expanded protocols are detailed in the online data supplement.

 
We next investigated if the H41 of Gs{alpha} is also critical to the binding interaction of Gs{alpha} and Cav-3 in an in vitro protein interaction assay. We utilized the WT and mutant Gs{alpha}FL proteins that were used in the electrophysiological experiments. In this assay, Ni-ATA resin immobilized poly-histidine-tagged Gs{alpha}FL proteins; both WT and mutant were used as the bait proteins. A dialyzed, solubilized Cav-3-enriched sucrose gradient fraction (CRF) from adult cardiac ventricular myocytes was used as the prey protein-containing lysate. Interestingly, we found that the WT Gs{alpha}FL protein and the Gs{alpha}FL mutants were all able to capture and hold significant amounts of Cav-3 protein from the CRF (Fig. 3D). It is important to note that the WT Gs{alpha}FL and mutant Gs{alpha}FL proteins all contain an intact CBS motif, having been mutagenized at residues 40–42 only (see Fig. 1A).

The molecular actions of Gs{alpha} were next studied in greater detail. We used the 16-amino acid peptide near the NH2-terminus of Gs{alpha} (28–42) as a starting point to identify the Gs{alpha} residues most relevant to the functional interaction. The application of this peptide to inside-out patches reproducibly increased INa by ~30%, without any change in the time constant of current decay (15). In this whole cell recording study, we introduced 1.0 µmol/l of the Gs{alpha} peptide THR (C-28KQKQKDKQVYRATHR42), in the presence of PKI, and measured changes in peak INa density. The current-voltage relationship in Fig. 4A shows that the INa increase was similar in magnitude to that observed when Gs{alpha}FL protein was applied (Gs{alpha}FL 85.3 ± 4.3 pA/pF, n = 10 vs. Gs{alpha} peptide THR 85.6 ± 5.3 pA/pF, n = 4, P < 0.05). Moreover, as shown in the superimposed current traces, intracellular application of the Gs{alpha} peptide THR did not alter the activation kinetics, the time to peak current, or the rate of current decay (Fig. 4A, inset). We hypothesized that, if alternative pathways regulate this change in INa density in ventricular myocytes, then application of ISO to the Gs{alpha} peptide THR-stimulated current would result in a further enhancement of the current. However, Fig. 4A shows that bath application of ISO to the cell after internal dialysis of the Gs{alpha} peptide THR did not result in any significant change to the INa density (Gs{alpha} peptide THR 85.6 ± 5.3 pA/pF, n = 4 vs. Gs{alpha} peptide THR with ISO 85.0 ± 5.2 pA/pF, n = 4, P < 0.05).


Figure 4
View larger version (28K):
[in this window]
[in a new window]

  		Fig. 4. Effects on INa density in the presence of Gs{alpha} mutant peptides in the pipette solution. A: Gs{alpha} peptide THR induced an increase in INa density. Addition of ISO in the presence of Gs{alpha} peptide THR did not further increase the current (n = 4). Two superimposed current traces of Gs{alpha} peptide THR, with and without ISO, are shown in the inset [holding potential (HP) = –120 mV, test potential = –30 mV, 20 pA/pF, 5-ms calibration]. B: the Gs{alpha} peptide AAA (T40A, H41A, R42A, triple mutation) abolished the augmentation of current density. ISO enhanced the INa in the presence of the Gs{alpha} peptide AAA (n = 4). Examples of current traces are shown as an inset (HP = –120 mV, test potential = –30 mV, 20 pA/pF, 5-ms calibration), along with the superimposed normalized traces. C: the Gs{alpha} peptide AHA (T40A/R42A, double mutant) had a similar effect to Gs{alpha} peptide THR, with or without ISO (n = 4). D: application of Gs{alpha} peptide TAR (H41A, single mutant) resulted in a phenotype similar to that produced by the triple mutant Gs{alpha} peptide AAA. ISO enhanced the INa density when applied together with the single mutant peptide (n = 4).

 
We next used alanine scanning mutational analysis to identify which amino acid or combination of amino acids (40THR42) is required for the enhancement of the Gs{alpha} and Cav-3-dependent INa. Figure 4, B–D, shows triple, double, and single mutations of the 40THR42 residues, respectively. In each series of experiments, Gs{alpha} peptide mutant was added to the pipette solution. Whole cell patch-clamp data shown in Fig. 4B indicate that, although a mutation of 40THR42 to 40AAA42 completely abolished the increase in INa density, the subsequent application of ISO, nevertheless, increased the current by 36%. Figure 4C shows the response to internal perfusion of a double mutant with the histidine intact, Gs{alpha} peptide AHA (T40A/R42A). This peptide increased the peak current to the same level as the nonmutant Gs{alpha} peptide THR (40THR42). Subsequent application of ISO did not further increase the peak INa density. Finally, Fig. 4D shows data for the Gs{alpha} peptide TAR (H41A), in which only the histidine is replaced with an alanine. This mutant did not alter the current-voltage relationship compared with that of the control; however, the subsequent application of ISO enhanced the INa to values similar to those for the control Gs{alpha} peptide THR. The full summary of the mutant peptides that we tested (Fig. 5) shows that H41 modulates the activity of Gs{alpha} peptide THR, and that this residue is critical for the ISO-induced increase of INa density.


Figure 5
View larger version (41K):
[in this window]
[in a new window]

  		Fig. 5. Top: summary bar graph of the effects of WT and Gs{alpha} peptide mutants on INa density in the absence and presence of ISO. Only the peptides that contained a histidine at position 41 mimicked the effects of ISO. The voltage-clamp parameters to generate the bar graphs were HP = –120 mV, test potential = –30 mV. Full I–V relationships of important peptide mutants are shown in Fig. 4. Bottom: table indicates peptide sequences and corresponding abbreviations. *Statistical difference was tested by analysis of variance with P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study provides support for our model that a Gs{alpha}-Cav-3 interaction leads to an augmentation of the cardiac INa, due to an increase in the number of active sodium channels. The results of our study begin to dissect the PKA-independent contribution to a pathway whereby β-AR stimulation increases the magnitude of INa in cardiac myocytes.

Because it is well-recognized that Gs{alpha} has a multiplicity of effects in vivo (9, 11, 12, 19, 22), we tested whether Gs{alpha}-Cav-3-independent mechanisms existed. Previous work on G protein and caveolin interactions has focused primarily on Cav-1; however, Gs{alpha} localization to Cav-3 has also been reported (11, 12). Although Cav-1 and Cav-3 exhibit extensive homology in the CSD domain (80%), certain differences appear to have important physiological consequences (3–6, 23). There is emerging evidence that the signaling regulation of caveolin may be different in different cell types and that negative regulation of caveolin is not a universal effect (9). The Gs{alpha} effect of increasing INa may also be due to a direct effect of Gs{alpha} with the sodium channel. However, recent studies showed that interaction of Gs{alpha} and noncaveolar sodium channels that reside in the plasmalemma do not contribute to the PKA-independent increase of INa (28). It was previously shown that INa in the presence of PKI exhibited no change in the voltage dependence and kinetics of INa and that the single-channel amplitude, open time as well as the closed time, was unaltered (15). Furthermore, it was shown that a permanently charged, membrane-impermeant quaternary derivative of the local anesthetic lidocaine (QX-314) can inhibit cardiac sodium channels at extracellular as well as intracellular binding sites and is irreversible for >1 h (1). Subsequent to complete inhibition of INa and washout of QX-314, ISO was consistently able to activate INa by ~20–30% of control (28). It was concluded that, since QX-314 could not enter the closed caveolae, only the surface plasmalemmal sodium channels would be inhibited. The subsequent ISO enhancement of INa was due to the opening of the caveolae neck and making caveolar sodium channels functional by establishing electrical continuity of the intracaveolar space with the extracellular space. This enhancement was inhibited with intracellular application of anti-Gs{alpha} antibody and in a different cell with application of anti-Cav-3 antibody. These studies suggest that, in adult ventricular myocytes, Gs{alpha} does not directly interact with the sodium channel protein. In the present study, protein interaction assay data suggest that Gs{alpha}FL and the Gs{alpha}FL H41A mutant interacted with Cav-3, as previously shown for Cav-1 (14). However, the electrophysiological data show that the Gs{alpha}FL H41A was not capable of enhancing the INa. These functional experiments with Gs{alpha}FL show that, in cardiac ventricular myocytes, the molecular interaction of Gs{alpha} with Cav-3 does not negatively regulate Gs{alpha}. This is in contrast to the in vitro binding assay studies, where Cav-1 functions to inactivate or negatively regulate the activation state of Gs{alpha} (14, 25). Thus the CBS motif of Gs{alpha} that interacts with caveolin does not appear to have any regulatory activity of the Gs{alpha}-Cav-3 functional interaction. Only in the presence of histidine at residue 41 does Gs{alpha} have the ability to act as the critical switch to increase INa. The experiments using the Gs{alpha}FL protein were corroborated by testing the effects of short Gs{alpha} peptides, which lack the CBS motif. Increases in current amplitude could only be induced by application of ISO when the peptide did not contain a histidine at residue 41. However, application of ISO in the presence of the Gs{alpha}FL H41A or Gs{alpha} peptide H41A mutant was able to increase INa by stimulating endogenous Gs{alpha}. As shown in Figs. 3 and 5, mutations of the 40THR42 residues confirmed that H41 of Gs{alpha} mediates the functional effect of the Gs{alpha}-Cav3 interaction.

The three-dimensional ribbon structure representation of Gs{alpha} (30) (Fig. 1B) shows the surface location of the CBS motif and His41 of Gs{alpha}, supporting that these two distinct and exposed regions are good candidates for interaction(s) with Cav-3. Together, our data suggest that Gs{alpha} binds to Cav-3 to stabilize the interaction of the two proteins, and the single histidine at position 41 of Gs{alpha} is the key to unlocking the caveolae and thereby making the caveolae-resident sodium channels functional. Further studies will be required to determine the details of the interaction between Gs{alpha} H41 and Cav-3. While direct interaction between this residue and Cav-3 is certainly possible, future studies may identify additional binding partners that link Gs{alpha}(H41) and Cav-3 in the regulation of β-adrenergic-mediated cardiac sodium channel activity in cardiac myocytes.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the National Heart, Lung, and Blood Institute Grant HL075541 (to E. F. Shibata).


   ACKNOWLEDGMENTS
 
We acknowledge the excellent technical assistance of Carol Butters and the assistance with the 3D modeling of Gs{alpha} by Brandy Barron.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. F. Shibata, Dept. of Molecular Physiology and Biophysics, Carver College of Medicine, The Univ. of Iowa, 6-450 Bowen Science Bldg., Iowa City, IA 52242-1109 (e-mail: erwin-shibata{at}uiowa.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Alpert L, Fozzard H, Hanck D, Makielski J. Is there a second external lidocaine binding site on mammalian cardiac cells? Am J Physiol Heart Circ Physiol 257: H79–H84, 1989.[Abstract/Free Full Text]
  2. Balijepalli R, Foell J, Hall D, Hell J, Kamp T. Localization of cardiac L-type Ca2+ channels to a caveolar macromolecular signaling complex is required for β2-adrenergic regulation. Proc Natl Acad Sci USA 103: 7500–7505, 2006.[Abstract/Free Full Text]
  3. Barouch L, Harrison R, Skaf M, Rosas G, Cappola T, Kobeissi Z, Hobal I, Lemmon C, Burnett A, O'Rourke B, Rodriguez E, Huang P, Lima J, Berkowitz D, Hare J. Nitric oxide regulates the heart by spatial confinement of nitric oxide synthase isoforms. Nature 416: 337–340, 2002.[Medline]
  4. Bucci M, Gratton JP, Rudic R, Acevedo L, Roviezzo F, Cirino G, Sessa W. In vivo delivery of the caveolin-1 scaffolding domain inhibits nitric oxide synthesis and reduces inflammation. Nat Med 6: 1362–1367, 2000.[CrossRef][Web of Science][Medline]
  5. Carozzi A, Roy S, Morrow I, Pol A, Wyse B, Clyde-Smith J, Prior I, Nixon S, Hancock J, Parton R. Inhibition of lipid raft-dependent signaling by a dystrophy-associated mutant of caveolin-3. J Biol Chem 277: 17944–17949, 2002.[Abstract/Free Full Text]
  6. Cohen A, Hnasko R, Schubert W, Lisanti M. Role of caveolae and caveolins in health and disease. Physiol Rev 84: 1341–1379, 2004.[Abstract/Free Full Text]
  7. Couet J, Li S, Okamoto T, Ikezu T, Lisanti MP. Identification of peptide and protein ligands for the caveolin-scaffolding domain. J Biol Chem 272: 6525–6533, 1997.[Abstract/Free Full Text]
  8. DeLano W. The PyMol Molecular Graphics System. Palo Alto, CA: Delano Scientific, 2002.
  9. Folco E, Liu GX, Koren G. Caveolin-3 and SAP97 form a scaffolding protein complex that regulates the voltage-gated potassium channel Kv1.5. Am J Physiol Heart Circ Physiol 287: H681–H690, 2004.[Abstract/Free Full Text]
  10. Frohnwieser B, Chen LQ, Schreibmayer W, Kallen R. Modulation of the human cardiac sodium channel {alpha}-subunit by cAMP-dependent protein kinase and the responsible sequence domain. J Physiol 498: 309–318, 1997.[Abstract/Free Full Text]
  11. Head B, Patel H, Roth D, Lai N, Niesman I, Farquhar M, Insel P. G-protein-coupled receptor signaling components localize in both sarcolemmal and intracellular caveolin-3 microdomains in adult cardiac myocytes. J Biol Chem 280: 31036–31044, 2005.[Abstract/Free Full Text]
  12. Insel P, Head B, Ostrom R, Patel H, Swaney J, Tang CM, Roth D. Caveolae and lipid rafts: G protein-coupled receptor signaling microdomains in cardiac myocytes. Ann NY Acad Sci 1047: 166–172, 2005.[CrossRef][Web of Science][Medline]
  13. Kawabe JI, Grant BS, Yamamoto M, Schwencke C, Okumura S, Ishikawa Y. Changes in caveolin subtype protein expression in aging rat organs. Mol Cell Endocrinol 176: 91–95, 2001.[CrossRef][Web of Science][Medline]
  14. Li S, Okamoto T, Chun M, Sargiacomo M, Casanova J, Hansen S, Nishimoto I, Lisanti M. Evidence for a regulated interaction between heterotrimeric G proteins and caveolin. J Biol Chem 270: 15693–15701, 1995.[Abstract/Free Full Text]
  15. Lu T, Lee HC, Kabat J, Shibata E. Modulation of rat cardiac sodium channel by the stimulatory G-protein {alpha}-subunit. J Physiol 518: 371–384, 1999.[Abstract/Free Full Text]
  16. Maguy A, Herbert T, Nattel S. Involvement of lipid rafts and caveolae in cardiac channel function. Cardiovasc Res 69: 798–807, 2006.[Abstract/Free Full Text]
  17. Matsuda J, Lee HC, Shibata E. Enhancement of rabbit cardiac sodium channels by β-adrenergic stimulation. Circ Res 70: 199–207, 1992.[Abstract/Free Full Text]
  18. Melchior N. Sodium and potassium complexes of adenosine-triphosphate: equilibrium studies. J Biol Chem 208: 615–627, 1954.[Free Full Text]
  19. Oh P, Schnitzer J. Segregation of heterotrimeric G proteins in cell surface microdomains: Gq binds caveolin to concentrate in caveolae, whereas Gi and Gs target lipid rafts by default. Mol Biol Cell 12: 685–698, 2001.[Abstract/Free Full Text]
  20. Okamoto T, Schlegel A, Scherer P, Lisanti M. Caveolins, a family of scaffolding proteins for organizing "preassembled signaling complexes" at the plasma membrane. J Biol Chem 273: 5419–5422, 1996.[CrossRef]
  21. Ono K, Fozzard H, Hanck D. Mechanism of cAMP-dependent modulation of cardiac sodium channel current kinetics. Circ Res 72: 807–815, 1993.[Abstract/Free Full Text]
  22. Parton R, Simon K. The multiple faces of caveolae. Nat Rev Mol Cell Biol 8: 185–194, 2007.[CrossRef][Web of Science][Medline]
  23. Patel H, Murray F, Insel P. Caveolae as organizers of pharmacologically relevant signal transduction molecules. Annu Rev Pharmacol Toxicol 48: 14.11–14.33, 2007.
  24. Razani B, Lisanti M. Caveolins and caveolae: molecular and functional relationships. Exp Cell Res 271: 36–44, 2001.[CrossRef][Web of Science][Medline]
  25. Razani B, Woodman S, Lisanti M. Caveolae: from cell biology to animal physiology. Pharmacol Rev 54: 431–446, 2002.[Abstract/Free Full Text]
  26. Rybin V, Xu X, Steinberg S. Activated protein kinase C isoform targets to cardiomyocyte caveolae: stimulation of local protein phosphorylation. Circ Res 84: 980–988, 1999.[Abstract/Free Full Text]
  27. Scriven D, Klimek A, Asghari P, Bellve K, Moore E. Caveolin-3 is adjacent to a group of extradyadic ryanodine receptors. Biophys J 89: 1893–1901, 2005.[CrossRef][Web of Science][Medline]
  28. Shibata E, Brown T, Washburn Z, Bai J, Revak T, Butters C. Autonomic regulation of voltage-gated cardiac ion channels. J Cardiovasc Electrophysiol 17: S34–S42, 2006.[CrossRef][Medline]
  29. Smart E, Ying Y, Mineo C, Anderson R. A detergent-free method for purifying caveolae membrane from tissue culture cells. Proc Natl Acad Sci USA 92: 10104–10108, 1995.[Abstract/Free Full Text]
  30. Sunahara R, Tesmer J, Gilman A, Sprang S. Crystal structure of the adenylyl cyclase activator Gs{alpha}. Science 278: 1943–1947, 1997.[Abstract/Free Full Text]
  31. Van Deurs B, Roepstorff K, Hommelgaard A, Sandvig K. Caveolae: anchored, multifunctional platforms in the lipid ocean. Trends Cell Biol 13: 92–100, 2003.[CrossRef][Web of Science][Medline]
  32. Wilson J, Chin A. Chelation of divalent cations by ATP, studied by titration calorimetry. Anal Biochem 193: 16–19, 1991.[CrossRef][Web of Science][Medline]
  33. Yarbrough T, Lu T, Lee HC, Shibata E. Localization of cardiac sodium channels in caveolin-rich membrane domains: regulation of sodium current amplitude. Circ Res 90: 443–449, 2002.[Abstract/Free Full Text]
  34. Zhou H, Yi J, Hu N, George A Jr, Murray K. Activation of protein kinase A modulates trafficking of the human cardiac sodium channel in Xenopus oocytes. Circ Res 87: 33–38, 2000.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Material
Right arrow All Versions of this Article:
294/4/H1693    most recent
01337.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Palygin, O. A.
Right arrow Articles by Shibata, E. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Palygin, O. A.
Right arrow Articles by Shibata, E. F.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2008 by the American Physiological Society.