Tyrosine-phosphorylated and Nonphosphorylated Sodium Channel β1 Subunits Are Differentially Localized in Cardiac Myocytes

Abstract

Voltage-gated sodium channel ␣ and ␤ subunits expressed in mammalian heart are differentially localized to t-tubules and intercalated disks. Sodium channel ␤ subunits are multifunctional molecules that participate in channel modulation and cell adhesion. Reversible, receptor-mediated changes in ␤1 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 ␤1 (pY␤1) and nonphosphorylated ␤1 subunits may be differentially localized in heart and thus interact with different cytoskeletal and signaling proteins. We developed an antibody that specifically recognizes pY␤1 and investigated the differential subcellular localization of ␤1 and pY␤1 in mouse ventricular myocytes. We found that pY␤1 colocalized with connexin-43, N-cadherin, and Na v 1.5 at intercalated disks but was not detected at the t-tubules. Anti-pY␤1 immunoprecipitates N-cadherin from heart membranes and from cells transfected with ␤1 and N-cadherin in the absence of other sodium channel subunits. pY␤1 does not associate with ankyrin B in heart membranes. N-cadherin and connexin-43 associate with Na v 1.5 in heart membranes as assessed by co-immunoprecipitation assays. We propose that sodium channel complexes at intercalated disks of ventricular myocytes are composed of Na v 1.5 and pY␤1 and that these complexes are in close association with both N-cadherin and connexin-43. ␤1 phosphorylation appears to regulate its localization to differential subcellular domains.

Sodium channel Scn1b null mice exhibit prolonged QT and RR intervals

“…7C), and connexin-43 (Fig. 7C,6I, 6J) were localized to the intercalated disks, consistent with previous studies [11,23,29]. We found similar results in studies on acutely dissociated ventricular myocytes performed in parallel (not shown).…”

“…P17 male mice were anesthetized and perfused and isolated hearts or individual myocytes were prepared for immunocytochemistry as described [11]. Anti-Na v 1.5, anti-β3, anti-β4, anti-Na v 1.6, anti-β1 ex , anti-connexin-43, and anti-ankyrin B were applied at 1:100 dilution; anti-Ncadherin at 1:50; anti-Na v 1.…”

“…β subunits are multi-functional molecules that regulate channel cell-surface expression levels and modulate channel function, affecting channel kinetics and voltage-dependence in vitro [8]. β subunits also function in vitro as homophilic and/or heterophilic cell adhesion molecules that recruit cytoskeletal ankyrin following homophilic cell adhesion [9][10][11].…”

Sodium Channel β1 Subunits Promote Neurite Outgrowth in Cerebellar Granule Neurons

“…Alternatively, sodium channel ␤ subunits may act indirectly through CAM homology domain-containing molecules. We have shown that ␤1 and N-cadherin interact in heart (41). Posttranslational modifications such as polysialylation have been shown to be important determinates in NCAM-mediated neurite outgrowth and neuronal fasciculation (4).…”

Voltage-Gated Na+Channel β1 Subunit-Mediated Neurite Outgrowth Requires Fyn Kinase and Contributes to Postnatal CNS DevelopmentIn Vivo

“…␤1 and ␤2 interact with tenascin-C and tenascin-R influencing cell migration, and participate in homophilic cell adhesion resulting in cellular aggregation and ankyrin recruitment (Srinivasan et al, 1998;Xiao et al, 1999;Malhotra et al, 2000Malhotra et al, , 2002. Furthermore, ␤1 interacts heterophilically with N-cadherin, contactin, neurofascin-155, neurofascin-186, NrCAM, and VGSC ␤2 (Kazarinova-Noyes et al, 2001;Malhotra et al, 2004;. Interactions between ␤1 and contactin, neurofascin-186, or ␤2 result in increased VGSC surface expression (Kazarinova-Noyes et al, 2001;.…”