Published online before print March 8, 2004, doi: 10.1161/01.CIR.0000121421.61896.24
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(Circulation. 2004;109:1421-1427.)
© 2004 American Heart Association, Inc.
Basic Science Reports |
Distinct Subcellular Localization of Different Sodium Channel 
and ß Subunits in Single Ventricular Myocytes From Mouse Heart
From the Department of Pharmacology, University of Washington, Seattle (S.K.G.M. R.E.W., K.A.M., T.S., W.A.C.); Medizinische Universitätsklinik Würzburg, Würzburg, Germany (S.K.G.M.); and Elixir Pharmaceuticals, Cambridge, Mass (R.C.).
Correspondence to William A. Catterall, PhD, Department of Pharmacology, Box 357280, University of Washington, Seattle, WA 98195-7280. E-mail wcatt{at}u.washington.edu
Received November 21, 2002; de novo received September 19, 2003; revision received December 5, 2003; accepted December 9, 2003.
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Abstract |
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Background— Voltage-gated sodium channels composed of pore-forming
and auxiliary ß subunits are responsible for the rising phase of the action potential in cardiac muscle, but their localizations have not yet been clearly defined.
Methods and Results— Immunocytochemical studies show that the principal cardiac subunit isoform Nav1.5 and the ß2 subunit are preferentially localized in intercalated disks, identified by immunostaining of connexin 43, the major protein of cardiac gap junctions. The brain subunit isoforms Nav1.1, Nav1.3, and Nav1.6 are preferentially localized with ß1 and ß3 subunits in the transverse tubules, identified by immunostaining of -actinin, a cardiac z-line protein. The ß1 subunit is also present in a small fraction of intercalated disks. The recently cloned ß4 subunit, which closely resembles ß2 in amino acid sequence, is also expressed in ventricular myocytes and is localized in intercalated disks as are ß2 and Nav1.5.
Conclusions— Our results suggest that the primary sodium channels present in ventricular myocytes are composed of Nav1.5 plus ß2 and/or ß4 subunits in intercalated disks and Nav1.1, Nav1.3, and Nav1.6 plus ß1 and/or ß3 subunits in the transverse tubules.
Key Words: ion channels • sodium • myocytes • ventricles • immunohistochemistry
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Introduction |
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Voltage-gated sodium channels are responsible for the initiation of action potentials in most excitable cells. They are composed of a pore-forming
subunit and 1 or 2 auxiliary ß subunits.1 Ten genes encoding subunits have been identified, and 9 have been functionally expressed.2 The different subunit isoforms have distinct patterns of development and localization in the nervous system and skeletal and cardiac muscle, and they have different physiological and pharmacological properties. Isoforms preferentially expressed in the central nervous system (Nav1.1, Nav1.2, Nav1.3, Nav1.6) are inhibited by nanomolar concentrations of tetrodotoxin, as is the isoform present in adult skeletal muscle (Nav1.4). In contrast, the primary cardiac isoform (Nav1.5) requires micromolar concentrations of tetrodotoxin for inhibition because of the presence of a cysteine instead of an aromatic residue in the pore region of domain I.3 Recent studies show that the brain-type isoforms Nav1.1, Nav1.3, and Nav1.6 are expressed in ventricular myocytes and have distinct subcellular localization and function.4
The 4 known ß subunits of sodium channels divide into 2 groups: ß1 and ß3 are most similar in sequence and are noncovalently associated with subunits,5,6 while the ß2 and ß4 subunits are also closely related in amino acid sequence and are disulfide-linked to subunits.7,8 The ß subunits are multifunctional because they modulate channel gating, regulate the level of expression at the plasma membrane, and function as cell adhesion molecules through interaction with the cytoskeleton, extracellular matrix, and other cell adhesion molecules that regulate cell migration and aggregation.9 mRNA for the ß1 subunit has been reported in rat5 and human heart,10 but its presence in mouse heart is controversial.11,12 Protein and mRNA for the ß2 and ß3 subunits are expressed primarily in neuronal tissue6,7 but are also detected in the heart.6,12,13 Protein and mRNA of the recently discovered ß4 subunit are expressed in many tissues, including brain, heart, and skeletal muscle.8
Alterations in sodium channel expression and function are known to have severe effects on excitability. In the nervous system, mutations in sodium channel and ß subunits cause epilepsy and febrile seizures.14,15 In the heart, mutations in the gene encoding Nav1.5 cause inherited hyperexcitability syndromes, including long QT syndrome type III and Brugada syndrome, which can lead to sudden cardiac death.16 Differential expression and localization of sodium channel subunits are likely to be important determinants of electric excitability of cardiac myocytes. This study defines the localization of sodium channel ß subunits in single ventricular myocytes and determines the composition of cardiac sodium channel complexes by correlation with the differential localization of subunits. Our results indicate that the cardiac sodium channels are composed of Nav1.1, Nav1.3, and Nav1.6 plus ß1 or ß3 subunits in the transverse (t-) tubular system and Nav1.5 plus ß2 and/or ß4 subunits in the intercalated disks.
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Methods |
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All experimental procedures used in this study were approved by the institutional animal care and use committee of the University of Washington. Biotinylated goat anti-rabbit IgG, avidin D fluorescein, avidin, biotin, Vectashield, and normal goat serum were purchased from Vector; mouse laminin was obtained from BD Biosciences. All other chemicals were purchased from Sigma.
Ventricular myocytes were isolated from adult male (aged 8 to 10 weeks) wild-type B6129F1 mice (n=10) or knockout mice with targeted deletions of the ß1 (n=4) or ß2 (n=4) subunits,17,18 which are viable but have arrhythmias. Tissue sections from littermates were cut for parallel analysis, and the distributions of sodium channel subunits and marker proteins were analyzed by immunocytochemistry and confocal microscopy as described previously.4 We viewed 80 to 100 myocytes per animal for each antibody used in this study, and the results were consistent for all animals examined. The anti-SP19 antibody against a conserved region of intracellular loop between domains III and IV and anti-SP20 against the intracellular loop between domains II and III of the subunit of sodium channels have been shown previously to recognize sodium channel subunits specifically.19,20 The anti-Nav1.1,21 anti-Nav1.3, and anti–connexin 43 antibodies were purchased from Chemicon International. Anti-Scn8a against Nav1.6 was obtained from Alomone Labs, and anti–-actinin was obtained from Sigma. The anti-SH1 antibody was generated against the peptide SH1 (KTEPQAPGCGETPEDS), corresponding to residues 1122 to 1137 of the subunit of Nav1.5.22 The anti-ß1,23 anti-ß2,23 anti-ß3, and anti-ß48 antibodies were generated against peptides corresponding to residues 164 to 191, 59 to 73, 45 to 66, and 81 to 97, respectively. Antibody specificity for the anti-SH1,4 anti-ß1, anti-ß2,23 and anti-ß48 antibodies was established as described previously, and specificity for ß subunits was confirmed in this study by immunoblotting ß subunits expressed in tsA-201 cells.23 Control sections included preincubation of the primary antibodies with their respective peptides or incubation with no primary antibody. In all cases, specific staining was abolished, and no staining was observed above background.
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Results |
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Immunolocalization of Sodium Channel ß Subunits in Ventricular Cardiomyocytes
To examine whether the different ß subunits are present in adult mouse heart and to determine their subcellular localization, we used subunit-specific antibodies and visualized the bound antibodies by immunocytochemistry. In control experiments, immunoblot staining of expressed ß subunits by these antibodies was specific, with no cross-reaction between subunits (Figure 1). The anti-ß1 subunit antibody recognized the endogenous ß1A splice variant in tsA-201 cells in every gel lane as well as the smaller ß1 subunit in the left lane containing a cell sample in which ß1 was exogenously expressed.
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In immunocytochemical experiments, we observed strikingly different localizations of the ß subunits within the ventricular cell. ß1 was localized in a striated pattern (Figure 2A), very similar to the staining of Nav1.1, Nav1.3, and Nav1.6 described previously.4 In addition, the ß1 subunit was also observed in clusters at the margin of the cell in a small fraction of the intercalated disks (Figure 2A, asterisks). In contrast, the ß2 subunit was concentrated in clusters at the cell margins, resembling the previously reported distribution of Nav1.5 in the intercalated disks (Figure 2C).4 Myocytes from mice lacking ß1 or ß2 subunits17,18 were not immunostained with our antibodies, confirming their specificity (Figure 2B, 2D). The same localization of ß2 subunits in intercalated disks that we observed in dissociated myocytes can also be seen in intact ventricular tissue from adult mice (Figure 2E), and we also found the same localization of other subunits in tissue sections as shown here for dissociated myocytes (not shown). The ß3 subunit was observed in a striated pattern, but staining was distinctly less intense than for ß1 (Figure 2G). Similar to the distribution of the ß2 subunits, the ß4 subunits were localized in punctate clusters in the intercalated disks (Figure 2H). Myocytes incubated without primary antibody (Figure 2F) or with antibodies preabsorbed with their specific peptide antigens (data not shown) were not stained.
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Localization of Sodium Channel ß Subunits by Double Immunolabeling
To further specify the expression patterns of the different ß subunits, we compared their localizations with the specific marker proteins -actinin and connexin 43. -Actinin is an actin-binding protein associated with the z-lines in which transverse tubules are located,24,25 as shown by immunostaining with a monoclonal anti–-actinin antibody in Figure 3A. Gap junctions in intercalated disks were identified by labeling with a specific antibody directed against the major cardiac gap junction protein, connexin 4326 (Figure 3B).27,28 We found localization of the ß1 (Figure 3C) and ß3 subunits (Figure 3E) at punctate spots along the z-lines identified by anti–-actinin labeling, supporting localization of these ß subunits in t-tubules. Examination of a z-series of images showed that these ß subunits are located in t-tubules through the depth of the cell.
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In contrast to ß1 and ß3, the ß2 (Figure 3D) and ß4 subunits (Figure 3F) are localized in punctate clusters in some of the intercalated disks labeled with connexin 43 at the margin of the myocytes, most often at the ends of the cylindrical cells. Neither the ß2 nor the ß4 subunit localization completely overlaps the staining pattern of connexin 43, indicating that the sodium channels cluster at specific sites within the intercalated disks.
Localization of Sodium Channel Subunits by Double Immunolabeling
Using 2 different antibodies that recognize a broad range of sodium channels (anti-SP19 and anti-SP20), we observed sodium channel clusters at the margins of the myocytes in the intercalated disks (yellow) as well as in a striated pattern (green; Figure 4A, 4B). In contrast, subunit-specific immunostaining for Nav1.5 demonstrates its localization at the intercalated disks but not in transverse tubules (Figure 4F). Two new splice variants of Nav1.5 have been reported in the mouse heart.29 We obtained the same staining pattern using an additional antibody designed to recognize all known Nav1.5 splice variants, confirming localization of all known isoforms of Nav1.5 at the intercalated disks. Not all connexin 43–positive areas are also positive for Nav1.5, implying that sodium channel complexes are not present in the entire area of the intercalated disk but are clustered at unique sites within the disks. These results are similar to our findings with ß2 and ß4 subunits (Figure 3D, 3F).
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Double labeling with the broad-specificity antibodies showed localization of sodium channels at the z-line in a striated pattern (green; Figure 4A, 4B). Brain-type sodium channel subunits are expressed in a similar striated pattern4 within the myocyte,12 consistent with localization in the transverse tubules. Double labeling of brain-type Nav1.6 (Figure 4C), Nav1.1, or Nav1.3 channels (not shown) and connexin 43 reveals a striated pattern of sodium channel staining (green) and no overlapping regions with connexin 43 (yellow), demonstrating that these sodium channel isoforms are present in transverse tubules but are not detectable at the intercalated disks. In contrast, double labeling of Nav1.6 (Figure 4D), Nav1.1 (Figure 4E), and Nav1.3 (not shown) with -actinin shows punctate colocalization at the z-lines, confirming t-tubular localization of these brain sodium channel subtypes.
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Discussion |
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Our results indicate that sodium channels in intercalated disks of ventricular myocytes contain Nav1.5 plus ß2 and/or ß4 subunits. The ß1 subunit is also present in smaller fractions of the intercalated disks. In addition, we found that Nav1.1, Nav1.3, and Nav1.6 plus ß1 and/or ß3 subunits are present in the t tubules. The ß subunits have different effects on voltage-dependent gating of skeletal muscle and neuronal sodium channels,5–8,12,13,30–32 and therefore differential interaction of the ß subunits with these brain-type
subunits in cardiac myocytes is expected to differentially regulate their function. The distinct localization of the principal cardiac sodium channel in intercalated disks and the brain-type sodium channels in t tubules suggests different physiological functions.
Sodium Channels in Intercalated Disks of Ventricular Myocytes
Voltage-gated sodium channels are concentrated in the intercalated disks of ventricular myocytes.4,33 Our results show that this represents preferential, and possibly exclusive, localization of the Nav1.5 subunits at these specialized cell-cell junctions.4 Our experiments presented here using double labeling of connexin 43 and Nav1.5 channels confirm the specific localization of this subtype of sodium channels to the intercalated disk. Moreover, our results show that only a fraction of the intercalated disk membrane containing connexin 43 also contains Nav1.5 channels. Evidently, Nav1.5 channels are clustered at unique sites within the intercalated disks and serve to initiate and propagate action potentials over the cell surface of the ventricular myocyte from these specific locations. Regions of the intercalated disks having connexin 43 but not sodium channels would depolarize the disk membrane of the downstream myocyte electronically and thereby contribute to activation of sodium channels nearby.
Our experiments show that sodium channel ß2 and ß4 subunits are also preferentially localized in intercalated disks in both dissociated ventricular myocytes and slices of intact ventricular tissue. These results imply that the major sodium channel complex in the intercalated disks is composed of Nav1.5 subunits plus ß2 and/or ß4 subunits. This conclusion is also supported by coimmunoprecipitation experiments in which Nav1.5 subunits and ß2 subunits were found to be associated.12 ß1 and ß3 subunits have been shown to modulate gating kinetics of Nav1.5 in mammalian cell expression systems,12,13 whereas ß2 and ß4, which are similar in structure to each other, seem to have no major modulatory effect on Nav1.5 function.8,12 However, ß2 and ß4 subunits contain extracellular immunoglobulin-like domains that are implicated in cell-cell interactions.7,9 Therefore, the principal role of these auxiliary subunits may be to support insertion and immobilization of Nav1.5 channels in intercalated disks.
Our experiments also detect ß1 subunits in a fraction of intercalated disks. In contrast to ß2 and ß4 subunits, coexpression of ß1 subunits with Nav1.5 subunits has a substantial functional effect.3,34 Therefore, ß1 subunits may modify the functional properties of Nav1.5 channels in the intercalated disks. Our results do not determine whether ß1 subunits are associated with Nav1.5 subunits alone or with complexes of Nav1.5 subunits and ß2 or ß4 subunits. However, previous studies indicate that brain sodium channels can contain both ß1 and ß2 subunits.1 Thus, interaction of ß1 subunits with Nav1.5 may modulate the function of a fraction of sodium channels at intercalated disks.
Sodium Channels in Transverse Tubules
In contrast to intercalated disks, Nav1.5 channels are not localized in transverse tubules.4 Unexpectedly, our previous work suggests that the brain sodium channels Nav1.1, Nav1.3, and Nav1.6 are localized there.4 In confirmation of this conclusion, we observed punctate clusters of these brain-type sodium channels along the z-line identified by -actinin labeling. The localization of these brain-type sodium channels in transverse tubules is therefore clearly distinct from the cardiac Nav1.5 channels.
Our immunocytochemical experiments show that the ß1 and ß3 subunits are preferentially localized in transverse tubules with the brain-type Nav1.1, Nav1.3, and Nav1.6 subunits. These ß subunits differentially modulate the functional properties of brain-type Nav1.2 channels expressed in mammalian cells32 and therefore may differentially affect the function of these brain-type sodium channels expressed in transverse tubules. The presence of multiple subtypes of brain sodium channels in transverse tubules along with 2 distinct ß subunits may allow the excitability of the t-tubule membrane to be tuned to the requirements for efficient coupling of cell surface depolarization to contraction.
These results on immunolocalization of sodium channels in the transverse tubules of ventricular myocytes have both similarities and important differences compared with previous reports. The present results agree with those of Malhotra et al,12 who found Nav1.1 and ß1 subunits in t- tubules in sections of mouse heart. In contrast, Cohen33 presented evidence for Nav1.5 in the t-tubular system, and Malhotra et al12 presented evidence for ß2 subunits in transverse tubules and for coimmunoprecipitation of ß2 subunits with Nav1.1 subunits from cultured neonatal myocytes, in contrast to the expectations from our work.4 To verify our results, we determined the localization of Nav1.5 and ß2 subunits in both single ventricular myocytes and slices of ventricular tissue to exclude artifacts caused by cell isolation. We found that both of these subunits are specifically localized to the intercalated disks in intact slices of ventricular tissue as well as in ventricular myocytes4 (Figure 2D). The staining of t-tubules by antibodies against Nav1.5 in the experiments of Cohen33 likely represents cross-reaction of the antibodies used with Nav1.3, as we reported previously.4 We do not have an explanation for the apparent difference in localization of ß2 subunits between this study and that of Malhotra et al.12 However, we note that most of the studies of Malhotra et al12 were performed on cultures of neonatal myocytes in which t-tubules are poorly developed, and therefore expression and assembly of sodium channel complexes may differ from the adult myocytes used in our work.
Functional Significance of Differential Sodium Channel Localization in Ventricular Myocytes
The results of this report and our previous study4 support the unexpected conclusion that brain-type sodium channels are preferentially localized in transverse tubules, while the Nav1.5 channel that is primarily expressed in cardiac tissue is preferentially localized in the intercalated disks in ventricular myocytes. What are the physiological roles of these differentially localized sodium channels? We have previously proposed that the Nav1.5 channels in intercalated disks are involved primarily in initiation and propagation of the cardiac action potential from cell to cell.4 They may function in a manner similar to sodium channels at nodes of Ranvier in myelinated nerves by mediating rapid saltatory conduction of the action potential from cell to cell across the myocardium. In contrast, sodium channels in transverse tubules may function in coordinating and synchronizing the conduction of the action potential from the cell surface of the myocyte into the interior via the transverse tubules. The more negative voltage dependence of gating and more rapid activation and inactivation of brain-type sodium channels3 may be required for this function. The specialized localization and function of these 2 sets of sodium channels in ventricular myocytes potentially allow differential regulation of action potential propagation across the cell surface and into the transverse tubules by cellular signaling processes and by specific pharmacological agents.
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Acknowledgments |
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This work was supported by a fellowship of the Deutsche Forschungsgemeinschaft to Dr Maier (Ma 2252/1-1) and by research grant PO1 HL44948 from the National Institutes of Health to Dr Catterall. We thank Dr Charlotte F. Ratcliffe for characterization of the anti-ß3 antibody and Thuy Vien, Bridget Coila, and Elizabeth Catterall for excellent technical assistance. We thank Dr Lori Isom (University of Michigan) for providing the founder mice for the ß1 and ß2 knockout mice.
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 D. Johnson and E. S. Bennett Isoform-specific Effects of the beta2 Subunit on Voltage-gated Sodium Channel Gating J. Biol. Chem., September 8, 2006; 281(36): 25875 - 25881. [Abstract] [Full Text] [PDF]
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F. Brette and C. H. Orchard No Apparent Requirement for Neuronal Sodium Channels in Excitation-Contraction Coupling in Rat Ventricular Myocytes Circ. Res., March 17, 2006; 98(5): 667 - 674. [Abstract] [Full Text] [PDF]
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 P. J. Stocker and E. S. Bennett Differential Sialylation Modulates Voltage-gated Na+ Channel Gating throughout the Developing Myocardium J. Gen. Physiol., February 27, 2006; 127(3): 253 - 265. [Abstract] [Full Text] [PDF]
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G. K. Wang, T. Edrich, and S.-Y. Wang Time-Dependent Block and Resurgent Tail Currents Induced by Mouse {beta}4154-167 Peptide in Cardiac Na+ Channels J. Gen. Physiol., February 27, 2006; 127(3): 277 - 289. [Abstract] [Full Text] [PDF]
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L.S. Meadows and L.L. Isom Sodium channels as macromolecular complexes: Implications for inherited arrhythmia syndromes Cardiovasc Res, August 15, 2005; 67(3): 448 - 458. [Abstract] [Full Text] [PDF]
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L. Lagostena, D. Avitabile, E. De Falco, A. Orlandi, F. Grassi, M. G. Iachininoto, G. Ragone, S. Fucile, G. Pompilio, F. Eusebi, et al. Electrophysiological properties of mouse bone marrow c-kit+ cells co-cultured onto neonatal cardiac myocytes Cardiovasc Res, June 1, 2005; 66(3): 482 - 492. [Abstract] [Full Text] [PDF]
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 V. Haufe, J. A. Camacho, R. Dumaine, B. Gunther, C. Bollensdorff, G. S. von Banchet, K. Benndorf, and T. Zimmer Expression pattern of neuronal and skeletal muscle voltage-gated Na+ channels in the developing mouse heart J. Physiol., May 1, 2005; 564(3): 683 - 696. [Abstract] [Full Text] [PDF]
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J. N. Dominguez, F. Navarro, D. Franco, R. P. Thompson, and A. E. Aranega Temporal and spatial expression pattern of {beta}1 sodium channel subunit during heart development Cardiovasc Res, March 1, 2005; 65(4): 842 - 850. [Abstract] [Full Text] [PDF]
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V. Haufe, J.M. Cordeiro, T. Zimmer, Y.S. Wu, S. Schiccitano, K. Benndorf, and R. Dumaine Contribution of neuronal sodium channels to the cardiac fast sodium current INa is greater in dog heart Purkinje fibers than in ventricles Cardiovasc Res, January 1, 2005; 65(1): 117 - 127. [Abstract] [Full Text] [PDF]
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