Tyrosine-phosphorylated and nonphosphorylated sodium channel beta1 subunits are differentially localized in cardiac myocytes.

Voltage-gated sodium channel alpha and beta subunits expressed in mammalian heart are differentially localized to t-tubules and intercalated disks. Sodium channel beta subunits are multifunctional molecules that participate in channel modulation and cell adhesion. Reversible, receptor-mediated changes in beta1 tyrosine phosphorylation modulate its ability to recruit and associate with ankyrin. The purpose of the present study was to test our hypothesis that tyrosine-phosphorylated beta1 (pYbeta1) and nonphosphorylated beta1 subunits may be differentially localized in heart and thus interact with different cytoskeletal and signaling proteins. We developed an antibody that specifically recognizes pYbeta1 and investigated the differential subcellular localization of beta1 and pYbeta1 in mouse ventricular myocytes. We found that pYbeta1 colocalized with connexin-43, N-cadherin, and Nav1.5 at intercalated disks but was not detected at the t-tubules. Anti-pYbeta1 immunoprecipitates N-cadherin from heart membranes and from cells transfected with beta1 and N-cadherin in the absence of other sodium channel subunits. pYbeta1 does not associate with ankyrinB in heart membranes. N-cadherin and connexin-43 associate with Nav1.5 in heart membranes as assessed by co-immunoprecipitation assays. We propose that sodium channel complexes at intercalated disks of ventricular myocytes are composed of Nav1.5 and pYbeta1 and that these complexes are in close association with both N-cadherin and connexin-43. beta1 phosphorylation appears to regulate its localization to differential subcellular domains.

tricular myocytes (3,9). The tetrodotoxin-sensitive (TTX-S) channels, Na v 1.1, Na v 1.3, and Na v 1.6, colocalize with ␣-sarcomeric actin and are thus proposed to be expressed in the ttubular system (2,3). Furthermore, Na v 1.1 and Na v 1.3 channels are found in the SA node, whereas Na v 1.5 is not (4,8). We have shown immunolocalization of ␤1 and ␤2 subunits at ttubules of ventricular myocytes (2). We also showed that ␤1 and ␤2 subunits associate with both Na v 1.1 and Na v 1.5 channels in heart membranes and isolated cardiac myocytes (2). ␤1, ␤2, ␤3, and ␤4 subunits are colocalized with Na v 1.1 and Na v 1.3 in SA nodal cells (4). This arrangement may have functional implications; Na v 1.5 expressed in intercalated disks may be primarily responsible for action potential conduction between cells, whereas the TTX-S channels in the t-tubule system are proposed to play important roles in coupling depolarization of the cell surface membrane to contraction (3,4,7). Finally, the TTX-S channels located in the SA node may contribute to automaticity (4).
Sodium channel ␤ subunits are multifunctional molecules (10). ␤1, ␤2, ␤3, and ␤4 modulate the cell surface expression levels and kinetic behaviors of the TTX-S sodium channel ␣ subunits (11)(12)(13). Although ␤1 and ␤2 associate with Na v 1.5 in heart (2), their functional effects on Na v 1.5 channels are less clear, and much of the literature remains in disagreement over the importance of these interactions (14). In addition to their roles as channel modulators, ␤1 and ␤2 are cell adhesion molecules (CAMs) of the immunoglobulin superfamily (15). ␤1 and ␤2 interact homophilically in vitro, resulting in ankyrin recruitment to points of cell-cell contact in transfected cells (16), and interact with the extracellular matrix molecules tenascin-C and tenascin-R (17,18), resulting in cellular repulsion in vitro (17). ␤1 interacts heterophilically with the CAMs contactin and neurofascin-186, and ␤1-contactin interactions result in increased sodium channel density at the cell surface (19 -21).
Reversible receptor-mediated changes in ␤1 tyrosine phosphorylation may modulate its ability to recruit and associate with ankyrin (22). The association of ankyrin with ␤1 is mediated through a 16-amino acid segment of the ␤1 intracellular domain containing a tyrosine residue (Tyr 181 ). This event occurs independently of ␤1 association with the ion-conducting pore. Phosphorylation of residue Tyr 181 (or expression of a ␤1 mutant mimicking phosphorylation of Tyr 181 , ␤1Y181E) had no effect on ␤1-mediated cell adhesion but resulted in the inhibition of ␤1mediated ankyrin recruitment in transfected Drosophila S2 cells as well as the inhibition of ␤1-ankyrin association as assessed by co-immunoprecipitation from transfected fibroblasts. Similar results were obtained in cells expressing wild type ␤1 following treatment with fibroblast growth factor (FGF) and orthovanadate to phosphorylate ␤1 on tyrosine residues.
FIGQY phosphorylation of the intracellular domain of L1 family CAMs, such as neurofascin, abolishes their ability to interact with ankyrin, establishing specialized ankyrin-dependent and ankyrin-independent microdomains in neurons and epithelial cells (23)(24)(25)(26). Nonphosphorylated neurofascin interacts with ankyrin G at nodes of Ranvier, whereas phosphorylated L1 CAMs are found at other specialized sites of cell-cell contact such as paranodes of sciatic nerve, neuromuscular junctions, adherens junctions, and regions of neuronal migration and axon extension (23,27). The FIGQY/H mutation in human L1 results in clinical disease, demonstrating that this tyrosine residue is critical for normal development of the nervous system (28 -32). These results suggest the presence of a novel signaling pathway that is important for the regulation of L1 family CAMs during developmental processes.
We propose that tyrosine-phosphorylated ␤1 (pY␤1, containing phospho-Tyr 181 ) and nonphosphorylated ␤1 may be differentially localized in excitable cells and thus interact with different cytoskeletal and signaling proteins. To test our hypothesis, we developed an antibody that specifically recognizes pY␤1. We then investigated the differential subcellular localization of ␤1 and pY␤1 in mouse ventricular myocytes. We found that although an antibody that recognized the extracellular region of ␤1, anti-␤1 ex (which detects both ␤1 and pY␤1), showed staining at the t-tubules as well as the intercalated disks, anti-pY␤1 stained intercalated disks and not t-tubules. Anti-␤1 ex staining colocalized with that of phalloidin and ankyrin B at z-lines/t-tubules as well as with connexin-43 at intercalated disks. Anti-pY␤1 colocalized with connexin-43, Ncadherin, and Na v 1.5 at intercalated disks but not with z-line/ t-tubule markers. ␤1Y181E, a ␤1 mutant that mimics tyrosine phosphorylation of residue Tyr 181 , does not associate with ankyrin in transfected cells and does not modulate sodium currents (21,22). In the present study we show that anti-pY␤1 immunoprecipitates N-cadherin from heart membranes and from cells transfected with ␤1 and N-cadherin in the absence of sodium channel ␣ subunits, indicating that these two proteins associate. Na v 1.5 channels and connexin-43 also associate with N-cadherin in heart as assessed by co-immunoprecipitation assays. In contrast, pY␤1 does not associate with ankyrin B in heart membranes. Thus, we propose that sodium channel complexes at intercalated disks of ventricular myocytes are composed of tetrodotoxin-resistant Na v 1.5 and pY␤1 and that these complexes are in close association with both N-cadherin and connexin-43. Sodium channels at t-tubules of cardiac myocytes are composed of TTX-S channels, such as Na v 1.1 and Na v 1.6, and nonphosphorylated ␤1 subunits. These complexes are proposed to be in close association with ankyrin B .
Cell Transfection-1610 Chinese hamster lung (CHL) cells (17) were transfected with cDNAs, as indicated in the legends of Fig. 1 or Fig. 5 using FuGENE (Roche Applied Science) according to the manufacturer's instructions. Protein expression was confirmed by Western blot. For Western blot analysis, cells were solubilized in 5% SDS and boiled in SDS-PAGE sample buffer containing 5% ␤-mercaptoethanol. Rat brain or heart membranes, prepared as described previously (33), were also solubilized and used as positive controls. Samples were separated on a 10% polyacrylamide gel and transferred to nitrocellulose. Western blots were probed with antibodies as indicated in the figure legends. Immunoreactive bands were visualized with WestDura chemiluminescent substrate (Pierce).
Isolation of Cardiac Myocytes-All animal procedures were performed in accordance with University of Michigan guidelines for animal use and care. P17-18 ␤1 (ϩ/ϩ) mice (34) congenic on the C57BL/6 background were anesthetized by intraperitoneal injection of Beuthanasia-D (Schering-Plough Animal Health Corp., Kenilworth, NJ). Individual ventricular myocytes were isolated as described previously (35). Briefly, intact hearts were dissected into warmed Basic Tyrode's solution (137 mM NaCl, 5.4 mM KCl, 0.5 mM MgCl 2 , 0.16 mM NaH 2 PO 4 , 3 mM NaHCO 3 , 5 mM HEPES, pH 7.4) containing 0.1 mg/ml heparin. They were then mounted on a Langendorff apparatus and retrogradely perfused with oxygenated modified Tyrode's solution (MT; 137 mM NaCl, 5.4 mM KCl, 4.5 mM MgCl 2 , 0.16 mM NaH 2 PO 4 , 3 mM NaHCO 3 , 20 mM HEPES, 10 mM glucose, and 1.3 mg/ml taurine, pH 7.4) supplemented with 50 M EGTA for 8 -10 min. The perfusion solution was then switched to MT containing 0.6 mg/ml collagenase type II (Worthington Biochemical Corp., Lakewood, NJ), 0.28 mg/ml hyaluronidase, and 0.12 mg/ml protease IX (Sigma), 10 M CaCl 2 for a further 10 -15 min. Following collagenase treatment, the hearts were removed from the perfusion apparatus, placed in MT with 25 mg/ml albumin and 0.15 M CaCl 2 , and cut lengthwise into ϳ2 ϫ 4-mm strips using a razor blade. Individual myocytes were released by dissociation using a series of fire-polished Pasteur pipettes of diminishing bore diameter. All solutions used for myocyte extraction were maintained at 35°C.
Co-immunoprecipitation Analyses-Immunoprecipitations were performed from mouse heart membranes prepared as previously described (2), isolated mouse cardiac myocytes, or transfected cells as indicated in the figure legends. Proteins were solubilized in 1.25% Triton X-100, and the soluble fractions were incubated overnight at 4°C with primary antibody (as indicated in the figure legends). Protein A-Sepharose (50 l of a 1:1 suspension) was then added, and the incubation continued for 2 h at 4°C. The protein A-Sepharose beads were precipitated and washed with 50 mM Tris-HCl, pH 7.5, containing 0.1% Triton X-100. Immunoprecipitated proteins were eluted from the protein A-Sepharose with SDS-PAGE sample buffer and separated on polyacrylamide gels. Proteins were transferred to nitrocellulose and probed with antibodies (as indicated in the figure legends). Chemiluminescent detection was accomplished with WestDura reagent (Pierce).
Immunocytochemical Analysis of Heart Sections and Isolated Myocytes-Mice were anesthetized by intraperitoneal injection of Beuthanasia-D (Schering-Plough Animal Health Corp., Kenilworth, NJ). The heart was exposed and washed by injection of 50 ml of prewashing buffer (8 g/liter NaCl, 4 g/liter dextrose, 8 g/liter sucrose, 0.23 g/liter CaCl 2 , 0.34 g/liter sodium cacodylate) and then perfused by injection of 50 ml of perfusion solution (40 g/liter sucrose, 40 g/liter paraformaldehyde, 14.34 g/liter sodium cacodylate). The heart was removed and incubated in perfusion solution at 4°C overnight with constant rotation. The next day the solution was changed to 30% sucrose, and the incubation was continued overnight at 4°C with constant rotation. Cryostat sections (0.4 m thick) were cut, postfixed in 2% paraformaldehyde, treated for 10 min with 0.5% Triton X-100 in TBS buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl), and then blocked at room temperature in the same solution containing 5% newborn calf serum. Primary antibodies (as indicated in the figure legends) were then added in the above solution that also contained 0.1% Tween 20 and incubated for 1.5 h at room temperature. The sections were washed with TBS containing 0.1% Tween 20. Secondary antibodies (also as indicated in the figure legends) were then added, and the incubation continued for 1.5 h at room temperature. Sections were washed, mounted with Gelmount (Biomeda Corp., Foster City, CA), and visualized with an Olympus FluoView 500 confocal laser-scanning microscope or a Zeiss LSM 510 confocal microscope mounted on a Zeiss Axiovert 100 M inverted microscope in the Microscopy and Image Analysis Laboratory Core Facility at the University of Michigan.

RESULTS
Specificity of the Antiphosphotyrosine ␤1 Subunit Antibody, Anti-pY␤1-The goal of this study was to determine the subcellular localization of tyrosine-phosphorylated sodium channel ␤1 subunits (pY␤1) in cardiac myocytes. To accomplish our goal we generated a polyclonal antibody against the peptide Ac-QENAS-E(pY)LAITC-amide. To demonstrate the specificity of anti-pY␤1 for pY␤1 subunits over nonphosphorylated ␤1, we used a previously described ␤1 mutant construct, ␤1Y181A (22), and a plasmid encoding wild type ␤1 (33). Substitution of alanine for tyrosine at position 181 prevents tyrosine phosphorylation of ␤1 subunit protein (22). CHL cells were transfected with wild type ␤1 or ␤1Y181A cDNAs and then treated with FGF and orthovanadate as described previously to obtain tyrosine phosphorylation of ␤1 subunits (22). Western blot analysis was performed on cell extracts prepared in the presence of FGF, orthovanadate, and protease inhibitors as described previously (Fig. 1) (22). The blot was first probed with anti-pY␤1 to detect tyrosine-phosphorylated ␤1 subunits (left) and then stripped and reprobed with anti-␤1 ex to detect both phosphorylated and nonphosphorylated forms (right). Fig. 1 shows that only wild type ␤1 subunits and not ␤1Y181A subunits are recognized by anti-pY␤1 under phosphorylating conditions, whereas both wild type ␤1 and ␤1Y181A subunits are recognized by anti-␤1 ex . The presence of an immunoreactive signal in the ␤1Y181A lane with anti-␤1 ex shows that ␤1 protein was indeed expressed but not recognized by anti-pY␤1. Thus, anti-pY␤1 appears to be specific for tyrosine-phosphorylated ␤1 subunits.
Immunocytochemical Analysis of Ventricular Sections-To investigate the subcellular localization of pY␤1 relative to Na v 1.5 and connexin-43 in ventricular myocytes, we performed immunohistochemistry and analyzed our results using confocal microscopy ( Fig. 2). We reported previously that ␤1, ␤2, and Na v 1.1 colocalize with the z-line marker ␣-sarcomeric actin in ventricular myocytes (2), suggesting that these sodium channel subunits may be expressed in t-tubules. However, we did not analyze intercalated disks in that study. Using the same Na v 1.5 antibody as Maier et al. (3), our present results confirm that Na v 1.5 is indeed expressed at intercalated disks, as shown by its colocalization with connexin-43 ( Fig. 2A). Anti-␤1 ex staining also coincides with connexin-43, as shown in Fig. 2B. Anti-pY␤1 stains intercalated disks (Fig. 2C) but not t-tubules/zlines (data not shown), suggesting that tyrosine phosphorylation of ␤1 subunits may result in differential subcellular targeting and thus interaction with signaling molecules present in that microdomain. To ensure that the observed anti-pY␤1 signal was not contaminated by the anti-connexin-43 signal, we performed a single label experiment (Fig.  2E) in which pY␤1 remained localized at intercalated disks. In agreement with previous results, we observed N-cadherin staining at intercalated disks ( Fig. 2D) (36) and ␤1 ex (2) and ankyrin B (37) staining in the regions of z-lines/t-tubules using phalloidin to indicate actin localization (Fig. 2, F and G).
pY␤1 Associates with a Sodium Channel Complex in Heart-Our immunohistochemical results suggest that sodium channel complexes located at intercalated disks of cardiac myocytes are composed of Na v 1.5 and pY␤1 (and possibly nonphosphorylated ␤1) and that these complexes may also interact with N-cadherin and/or connexin-43, critical structural molecules for for-mation of cardiac mechanical and electrical junctions, respectively (38). Our results also suggest that nonphosphorylated ␤1 subunits associate with TTX-S sodium channels and ankyrin B at the t-tubules. We next investigated the potential interaction of Na v 1.5 and/or ␤1 subunits with N-cadherin, connexin-43, and ankyrin B using biochemical methods. Both anti-␤1 ex and anti-pY␤1 immunoprecipitate N-cadherin from mouse heart membranes (Fig. 3A), suggesting that ␤1 and N-cadherin are associated in a complex in heart. ␤1 also associates with ankyrin B in heart (Fig. 3B, left). However, only anti-␤1 ex and not anti-pY␤1 is able to co-immunoprecipitate ankyrin B (Fig.  3B, right), strengthening our hypothesis that tyrosine phosphorylation of ␤1 may negatively regulate its ability to interact with ankyrin and instead target ␤1 to alternative subcellular domains. Anti-Na v 1.5 immunoprecipitates N-cadherin (Fig.  3C, left) and connexin-43 (Fig. 3C, right) from heart membranes. Thus, our results suggest that a sodium channel complex consisting of Na v 1.5 and pY␤1 is present at intercalated disks of cardiac myocytes and that this complex is closely FIG. 1. Specificity of anti-pY␤1. Confluent 100-mm dishes of CHL cells transfected with ␤1Y181A or wild type ␤1 were stimulated with 50 ng/ml FGF ϩ 1 mM orthovanadate for 30 min at 37°C, solubilized in SDS-PAGE sample buffer, separated on 10% polyacrylamide gels, transferred to nitrocellulose, and probed with anti-pY␤1 antibody (1: 500, left). The same blot was then stripped and reprobed with anti-␤1 ex antibody (1:500 dilution, right). In both panels, the primary antibody was followed with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:100,000 dilution). Chemiluminescent detection of immunoreactive bands was accomplished with WestDura reagent. Molecular weight markers are indicated in kDa. The arrow indicates the position of ␤1 immunoreactive bands.
FIG. 2. Immunocytochemical localization of ␤1 and pY␤1 in cardiac myocytes. Mouse heart sections or acutely isolated myocytes were prepared for immunohistochemistry as described under "Experimental Procedures," incubated in 10 mM Tris-HCl, pH 7.5, 150 mM NaCl (TBS buffer) containing 0.5% Triton X-100 (TBS-T), and then blocked at room temperature in the same solution containing 5% newborn calf serum. Sections were incubated in primary antibodies for 1.5 h at room temperature and washed in TBS-T. Secondary antibodies were then added, and the incubation continued for 1.5 h at room temperature. Sections were washed, and then a second round of primary-secondary antibody staining was performed to achieve double labeling. Sections were washed, mounted with Gelmount, and visualized with an Olympus FluoView 500 confocal laser-scanning microscope or a Zeiss LSM 510 confocal microscope mounted on a Zeiss Axiovert 100 M inverted microscope in the Microscopy and Image Analysis Laboratory Core associated with N-cadherin and connexin-43. Nonphosphorylated ␤1 subunits may also be present in this complex, but we are unable to determine that using our available antibodies. Because anti-␤1 ex staining coincides with t-tubules/z-line markers and anti-pY␤1 does not, we propose that nonphosphorylated ␤1 is targeted to the t-tubules where it is available to associate with TTX-S sodium channels and ankyrin B .
We next performed co-immunoprecipitation experiments with anti-pY␤1 or anti-␤1 ex to investigate whether phosphorylated and nonphosphorylated ␤1 subunits differentially associ-ate with sodium channel ␣ subunits in heart. For these experiments we used isolated mouse cardiac myocytes to avoid contamination of the preparation with neuronal sodium channels. As shown in Fig. 4, Na v 1.1, Na v 1.5, and Na v 1.6 can be immunoprecipitated from isolated solubilized cardiac myocytes with anti-␤1 ex or anti-pY␤1. Anti-␤1 ex and anti-pY␤1 immunoprecipitate similar levels of Na v 1.5 protein (Fig. 4, center). Because the immunoreactive sodium channel band in the anti-␤1 ex lane should represent the total level of Na v 1.5 associated with ␤1 subunits (phosphorylated and nonphosphorylated) in the membrane preparation, these data suggest that a high percentage of Na v 1.5 is associated with pY␤1. In contrast, higher levels of Na v 1.1 and Na v 1.6 are immunoprecipitated with anti-␤1 ex than with anti-pY␤1 (Fig. 4, left and right), suggesting that a higher percentage of these TTX-S channels associate with nonphosphorylated ␤1 compared with pY␤1 subunits. These data are consistent with our immunohistochemical results showing that Na v 1.5 and pY␤1 colocalize at intercalated discs, whereas nonphosphorylated ␤1 is localized to t-tubules/z-lines, as shown previously for Na v 1.1 and Na v 1.6 (2, 3).
To investigate the association of N-cadherin with ␤1 more closely, we performed co-immunoprecipitation experiments from transfected CHL␤1 (17), CHL␤1 STOP (16), CHL␤1Y181E (22), or CHL␤1Y181A (22) cells using anti-␤1 ex and anti-Ncadherin antibodies. CHL cells express moderate levels of endogenous N-cadherin, as shown by the Western blot in Fig. 5A (CHL lane), but do not express endogenous sodium channel ␣ or ␤ subunits (33). ␤1 and N-cadherin associate in the absence of Na v 1.5 in CHL␤1 cells (Fig. 5A, ␤1 ex lane). The ␤1 mutant constructs ␤1Y181E (mimicking phosphorylation of Tyr 181 ) and ␤1Y181A (abolishing phosphorylation of Tyr 181 ) (22) both interact with N-cadherin in transfected cells (Fig. 5B), suggesting that the phosphorylation state of ␤1 does not affect its ability to interact with N-cadherin and instead may only affect targeting of the ␤1 subunit to N-cadherin-containing subcellular domains in polarized cells in vivo. In support of this hypothesis, we observed that ␤1 STOP , a truncation mutant lacking the ␤1 intracellular domain (16), also interacts with N-cadherin in FIG. 3. Association of ␤1 and Na v 1.5 with N-cadherin, connexin-43, or ankyrin B in heart. Mouse heart membranes were solubilized in 1.25% Triton X-100, and the soluble fraction was incubated for 4 h at 4°C with anti-␤1 ex , anti-pY␤1, anti-Na v 1.5, anti-N-cadherin, or non-immune IgG as indicated. Protein A-Sepharose was added, and the incubation continued for 2 h at 4°C. The protein A-Sepharose beads were precipitated in a microcentrifuge and washed in 50 mM Tris-HCl, pH 7.5, 0.1% Triton X-100. Immunoprecipitates were eluted from the protein A-Sepharose with SDS-PAGE sample buffer and separated on 10% polyacrylamide gels. Proteins were then transferred to nitrocellulose and probed with anti-N-cadherin (1:500 dilution), anti-ankyrin B (1:500), or anti-connexin-43 (1:500) antibody followed by horseradish peroxidase-conjugated goat anti-rabbit IgG (1:100,000 dilution). Chemiluminescent detection of immunoreactive bands was accomplished with WestDura reagent. IgG, non-immune serum. Molecular weight markers are indicated in kDa. A, ␤1 and pY␤1 associate with N-cadherin in rat heart. Anti-N-cadherin, anti-␤1 ex , anti-pY␤1, or IgG was used as the immunoprecipitating (IP) antibody as indicated. Anti-N-cadherin (N-Cad) was used to probe the Western blot. The arrow indicates the position of immunoreactive N-cadherin bands. Lower bands in all lanes are contributed by IgG. B, left, ␤1 associates with ankyrin B in rat heart. Anti-␤1 ex or IgG was used as the immunoprecipitating (IP) antibody as indicated. Heart membranes (HM) were run directly on the gel without immunoprecipitation as a positive control for ankyrin B staining. Antiankyrin B (ank B ) was used to probe the Western blot. The arrow indicates the position of immunoreactive ankyrin B bands. Right, ␤1 but not pY␤1 associates with ankyrin B in rat heart. Anti-␤1 ex , anti-pY␤1, or IgG was used as the immunoprecipitating (IP) antibody as indicated. Antiankyrin B was used to probe the Western blot. C, left, Na v 1.5 associates with N-cadherin in rat heart. Anti-Na v 1.5 or IgG was used as the immunoprecipitating (IP) antibody as indicated. Anti-N-cadherin was used to probe the Western blot. Right, Na v 1.5 associates with connexin-43 in rat heart. Anti-Na v 1.5 or IgG was used as the immunoprecipitating (IP) antibody as indicated. Anti-connexin-43 (Cx-43) was used to probe the Western blot. Bands at ϳ50 kDa are contributed by IgG. Arrows in both panels of C indicate positions of immunoreactive Ncadherin or connexin-43 bands, respectively.
FIG. 4. Association of sodium channel ␣ subunits with ␤1 versus pY␤1 in isolated cardiac myocytes. Isolated cardiac myocytes (100 g/aliquot) were solubilized in 1.25% Triton X-100, and equal aliquots of the soluble fraction were incubated overnight at 4°C with anti-␤1 ex or anti-pY␤1 as indicated. Protein A-Sepharose was added, and the incubation continued for 2 h at 4°C. The protein A-Sepharose beads were precipitated in a microcentrifuge and washed in 50 mM Tris-HCl, pH 7.5, 0.1% Triton X-100. Immunoprecipitates were eluted from the protein A-Sepharose with SDS-PAGE sample buffer, and equal aliquots were separated on 4 -15% gradient polyacrylamide gels. Proteins were then transferred to nitrocellulose and probed with specific sodium channel ␣ subunit antibodies (Na v 1.1, Na v 1.5, or Na v 1.6) as indicated, followed by horseradish peroxidase-conjugated goat antirabbit IgG (1:100,000 dilution). Chemiluminescent detection of immunoreactive bands was accomplished with WestDura reagent. The molecular weight marker is indicated in kDa. The arrow indicates immunoreactive ␣ subunit bands.
CHL cells (Fig. 5B), suggesting that the intracellular domain of ␤1 is not required for ␤1-N-cadherin association and that these two CAMs interact through their transmembrane or extracellular Ig loop domains or that a secondary protein, endogenous to CHL cells, is an intermediary between ␤1 and N-cadherin. DISCUSSION Sodium channel ␤1 subunits are multifunctional molecules that regulate channel electrophysiological properties and cell surface expression in vitro and in vivo (10,34). ␤1 is a member of the Ig superfamily of CAMs that participates in homophilic and heterophilic cell adhesion, interacts with extracellular matrix molecules, and recruits ankyrin to points of cell-cell contact as a result of homophilic cell adhesive interactions (15). ␤1mediated ankyrin recruitment in heterologous expression systems is dependent on phosphorylation of tyrosine residue 181, located in the ␤1 intracellular domain. Treatment of ␤1-transfected cells with FGF and orthovanadate or expression of ␤1Y181E does not affect ␤1-mediated cell adhesion but results in the inhibition of ankyrin recruitment as well as the abolishment of ␤1-ankyrin association as assessed by co-immunoprecipitation (22). Interestingly, substitution of ␤1Y181E for wild type ␤1 in a cell line expressing Na v 1.2 channels abolishes ␤1-mediated changes in electrophysiological properties but preserves ␣-␤1 association (21), suggesting that this residue plays a critical role in ankyrin recruitment and channel modulation. The goal of the present study was to determine the differential subcellular localization of ␤1 versus pY␤1 in cardiac myocytes as well as their differential association with ankyrin B versus N-cadherin to test the hypothesis that ␤1 tyrosine phosphorylation regulates its localization within the myocyte as well as its ability to interact with signaling or cytoskeletal molecules.
To accomplish our goals, we developed a polyclonal pY␤1specific antiserum and used it to localize pY␤1 in cardiac ventricular myocytes in relationship to anti-␤1 ex (which recognizes nonphosphorylated and phosphorylated ␤1 subunits) and known markers of t-tubules, z-lines, and intercalated disks. We observed that although anti-␤1 ex staining overlaps with phalloidin and ankyrin B at z-lines and t-tubules, respectively, and with connexin-43 and N-cadherin at intercalated disks, anti-pY␤1 staining colocalizes with connexin-43, N-cadherin, and Na v 1.5 but not with phalloidin and ankyrin B . Biochemical analyses showed that anti-␤1 ex co-immunoprecipitates ankyrin B , whereas anti-pY␤1 does not. In contrast, pY␤1 associates with N-cadherin, consistent with its localization at intercalated disks (7,39). ␤1 interacts with N-cadherin via its extracellular and/or transmembrane domains in transfected cells in the absence of Na v 1.5. The phosphorylation state of ␤1 does not appear to influence its ability to interact with Ncadherin. Phosphorylation may instead affect ␤1 targeting to alternative subcellular domains such as intercalated disks, where it is able to come into contact with specific signaling proteins. Anti-␤1 ex and anti-pY␤1 immunoprecipitate comparable levels of Na v 1.5 channels. Higher levels of the TTX-S channels Na v 1.1 and Na v 1.6 appear to associate with anti-␤1 ex compared with anti-pY␤1. Thus, we propose that the sodium channels present at intercalated disks of cardiac myocytes are composed of Na v 1.5 and pY␤1 and that this complex is closely associated with N-cadherin and connexin-43, proteins that are critical for cardiac mechanical and electrical junctions, respectively (39). Sodium channels located at the t-tubules include Na v 1.1 and Na v 1.6 in association with nonphosphorylated ␤1 subunits and ankyrin B .
A physiological function of ␤1 tyrosine phosphorylation may be to regulate ␤1-ankyrin interactions, resulting in differential ␤1 localization to subcellular microdomains, as proposed previously for FIGQY-phosphorylated L1 CAMs (23). The sequence 181 YLAI in ␤1 fits the consensus for a YXXA-type intracellular sorting signal that is recognized by clathrinassociated adaptor molecules and is involved in subcellular targeting of proteins in polarized cells (13,40,41). Phosphorylation of this critical tyrosine residue in other CAMs abolishes binding to clathrin adaptor proteins and creates binding sites for other signaling molecules such as tyrosine kinases (for review, see Ref. 41), suggesting that tyrosine-based sorting motifs are sites of dynamic regulation. L1 CAMs contain a similar sorting signal and associate with the clathrin adaptor molecule AP2 in brain via this sorting motif, resulting in endocytosis (42). L1 CAMs that are phosphorylated at this critical tyrosine residue no longer interact with ankyrin and instead interact with doublecortin, a protein that may participate in directing neuronal migration (43). Thus, FIGQY phosphorylation of L1 CAMs may function as a positive signal in addition to inhibiting ankyrin binding (43). The presence of this tyrosinebased sorting motif is required for axonal growth cone target- FIG. 5. Association of ␤1 and N-cadherin in transfected cells in the absence of sodium channel ␣ subunits. Confluent 100-mm dishes of CHL cells were transfected with ␤1, ␤1Y181E, ␤1Y181A, or ␤1 STOP cDNA constructs as indicated. Cells were solubilized in 1.25% Triton X-100, and the soluble fraction was incubated overnight at 4°C with anti-␤1 ex or non-immune IgG as indicated. Protein A-Sepharose was added, and the incubation continued for 2 h at 4°C. The protein A-Sepharose beads were precipitated in a microcentrifuge and washed in 50 mM Tris-HCl, pH 7.5, 0.1% Triton X-100. Immunoprecipitates were eluted from the protein A-Sepharose with SDS-PAGE sample buffer and separated on 7.5% polyacrylamide gels. Proteins were then transferred to nitrocellulose and probed with anti-N-cadherin (1:500 dilution) antibody followed by horseradish peroxidase-conjugated goat anti-rabbit IgG (1:100,000 dilution). Chemiluminescent detection of immunoreactive bands was accomplished with WestDura reagent. IgG, non-immune serum. CHL, solubilized untransfected 1610 cells run directly on the gel without immunoprecipitation to show endogenous N-cadherin expression. RB, rat brain membranes run directly on the gel without immunoprecipitation as a positive control for N-cadherin expression. Molecular weight markers are indicated in kDa. Arrows indicate immunoreactive N-cadherin bands. A, untransfected CHL cells express N-cadherin endogenously (CHL lane). ␤1 and N-cadherin associate in CHL-␤1 cells in the absence of sodium channel ␣ subunits (␤1 ex lane). B, the intracellular domain of ␤1 is not required for N-cadherin association. N-cadherin is immunoprecipitated with anti-␤1 ex antibody from CHL cells transfected with the ␤1 mutants ␤1Y181E, ␤1Y181A, and ␤1 STOP or wild type ␤1.
ing of L1 CAM in dorsal root ganglion neurons (44). In addition and similar to the results of the present study, FIGQY-phosphorylated L1-type CAMs are concentrated at adherens junctions of epithelial cells (23,27).
Phosphorylation of ␤1 at Tyr 181 , similar to L1 CAMs, may result in differential targeting of ␤1 in polarized cells, establishing spatial segregation between ␤1 and ankyrin and allowing ␤1 to participate in signaling pathways at specialized sites of cell-cell contact. When phosphorylated, we propose that ␤1 is no longer able to associate with ankyrin and is instead targeted to alternative subcellular domains where it interacts with a different set of cell adhesive, cytoskeletal, and signaling molecules. In cardiac myocytes, we propose that nonphosphorylated ␤1 associates with ankyrin-containing domains at the t-tubules, whereas pY␤1 is targeted to intercalated disks where it is able to interact with N-cadherin and its associated set of phosphotyrosine adaptor proteins (45). At intercalated disks, pY␤1 may contribute to the structural integrity of these mechanically stressed sites as a CAM. Na v 1.5 channels may be targeted or anchored to these sites through interactions with pY␤1. We have shown recently that ␤1Y181E, a mutant construct that mimics ␤1 tyrosine phosphorylation, does not modulate Na v 1.2 sodium currents expressed in transfected cells (21). Although we have not yet tested this construct in combination with Na v 1.5, we propose that pY␤1 may not affect cardiac Na v 1.5 sodium currents and may play a strictly cell adhesive/structural role. We have not yet identified the kinase(s) responsible for ␤1 phosphorylation; however, preliminary results indicate that ␤1 associates with fyn kinase in heart. 2 ␤1 has been shown to associate with the receptor phosphotyrosine phosphatase ␤ (RPTP␤) in neurons and transfected cells (46). The receptor phosphotyrosine phosphatases interact directly with and modulate the phosphorylation of cadherin-catenin complexes (for review, see Ref. 47) and may thus play a role in regulating the phosphorylation of ␤1 at intercalated disks of cardiac myocytes.
In adult heart, cardiomyocytes are linked together at intercalated disks, where adherens junctions, gap junctions, and desmosomes make up the cardiac intercellular junctions that mediate mechanical and electrical coupling throughout the heart (38). A close association of adherens junctions with mechanical junctions at the intercalated disks is thought to be necessary for conduction, and expression of N-cadherin at adherens junctions is a prerequisite for subsequent gap junction formation (36,39). The present results demonstrate that pY␤1 interacts with Na v 1.5 and N-cadherin, an adherens junctional protein, at intercalated disks and that the Na v 1.5 signaling complex also associates with connexin-43, a gap junctional protein. The intracellular domain of cadherin interacts with the catenins, proteins that ultimately communicate with the actin cytoskeleton and play roles in the regulation of cell adhesion and intracellular signaling (45). Disruption of the Factin-based and microtubular cytoskeleton in myocytes modulates the coupling between availability and activation of cardiac sodium channels, suggesting that cytoskeletal integrity may be a requirement for normal propagation of cardiac action potentials and thus regulation of excitability (48,49). In addition to N-cadherin, pY␤1 subunits may interact with cytoskeletal anchoring proteins located at intercalated disks such as the PDZ domain containing molecule syntrophin ␥2, which also interacts with Na v 1.5 (50), or ZO-1, a PDZ domain-containing molecule that associates with connexin-43 (51,52).
The present study, combined with previous results, suggests that nonphosphorylated ␤1 interacts with TTX-S sodium chan-nels and ankyrin B in the t-tubules of cardiac myocytes and that this arrangement mediates coupling of electrical excitation to contraction in cardiac muscle (2,3,22). A loss-of-function mutation in ankyrin B results in dominantly inherited type 4 long-QT cardiac arrhythmia in humans, and ankyrin B (Ϯ) mice display a similar phenotype that includes the disruption of targeting of ankyrin B -binding proteins to the t-tubules of cardiac myocytes (9,37). Surprisingly, the subcellular localizations of sodium channel Na v 1.5 and Na v 1.6 ␣ subunits are normal in cardiac myocytes isolated from these mice (9). It will be interesting to investigate the subcellular organization of nonphosphorylated versus phosphorylated ␤1 subunits in ankyrin B (Ϯ) myocytes, as this arrangement might be disrupted and may contribute to the arrhythmic phenotype, possibly through altered sodium channel density or functional modulation. Na v 1.5 (Ϯ) mice have reduced Na v 1.5 channel density, leading to slowed conduction with a diverse set of clinical phenotypes (53). Because ␤1 subunits have been shown to modulate the current densities and/or function of Na v 1.5 and the TTX-S sodium channels in heterologous systems, one might predict that mutations in the ␤1 gene might also lead to arrhythmias and conduction defects.