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.
Department of Pharmacology, University of Michigan, Ann Arbor, Michigan 48109-0632, USA.
Jyoti D
JD
Malhotra
Veena
V
Thyagarajan
Chunling
C
Chen
Lori L
LL
Isom
D000818:Animals; D017487:Ankyrins / Q000378:metabolism; D015820:Cadherins / Q000737:chemistry / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D018031:Connexin 43 / Q000378:metabolism; D006224:Cricetinae; D003599:Cytoskeleton / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D007150:Immunohistochemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D009124:Muscle Proteins / Q000737:chemistry; D009206:Myocardium / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000378:metabolism; D062554:NAV1.5 Voltage-Gated Sodium Channel; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000737:chemistry; D011233:Precipitin Tests; D011485:Protein Binding; D015222:Sodium Channels / Q000737:chemistry*; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry* / Q000378:metabolism
C498995:Ank2 protein, mouse; D017487:Ankyrins; D015820:Cadherins; D018031:Connexin 43; D018076:DNA, Complementary; C502403:FAT1 protein, human; C502405:Fat1 protein, rat; D009124:Muscle Proteins; D062554:NAV1.5 Voltage-Gated Sodium Channel; C568320:SCN5A protein, human; C568321:Scn5a protein, mouse; C568322:Scn5a protein, rat; D015222:Sodium Channels; C502406:fat1 protein, mouse; D019000:Phosphotyrosine; D014443:Tyrosine