Cavβ2 Subunit Associates with Caveolin-3 and Regulates Trafficking and β2adrenergic Receptor Regulation of the Caveolar L-Type Ca2+ Channels

The auxiliary CaVβ subunits (CaVβ1-CaVβ4) influence the trafficking and functional properties of pore forming α subunits of L-type Ca2+ channels. Recently we have demonstrated a subpopulation of CaV1.2 channels in caveolae microdomains in ventricular myocytes that are specifically regulated by the β2-adrenergic receptor stimulation. We hypothesize that a specific Cavβ subunit isoform is essential for the localization and regulation of caveolar CaV1.2 channels. Immunogold labeling and electron microscopy demonstrated that Cavβ2c but not Cavβ3co-localized with Cav-3 in ventricular myocytes. GST-Cav-3 pull-down experiments using various Cav-3 domain fusion proteins confirmed that Cav-3 directly associates with CaVβ2 subunit but not with CaVβ1, CaVβ3, or CaVβ4. Immunoprecipitation experiments from transfected HEK293 cells demonstrated that Cav-3 co-immunoprecipitate with Cav1.2 subunit when coexpressed with Cavβ2c subunit. However, Cav1.2 did not co-IP with Cav-3 when Cav1.2 was coexpressed with either CaVβ1b, CaVβ3, CaVβ4 or Cav1.2 subunit alone in HEK293 cells, suggesting Cavβ2c is required for caveolar targeting of Cav1.2 channels. The functional role of Cavβ2c subunit on caveolar Ca2+ channels was analysed by patch-clamp technique in neonatal mouse cardiomyocytes transfected with either a control siRNA or siRNA specific to Cavβ2c. In the control siRNA transfected myocytes both β1AR (norepinephrine, 10uM, prazosin, 10uM) and β2AR specific (salbutamol, 1uM, atenelol 1uM) stimulation significantly increased ICa,L by 100% and 60% respectively. However, when the Cavβ2C subunit expression was knocked down by specific Cavβ2C siRNA transfection into the myocytes, the β2AR specific stimulation of ICa,L was abolished, where as β1AR stimulation of ICa,L was intact. siRNA mediated knockdown of Cavβ2C subunit was confirmed by immunostaining and confocal microscopy. We conclude that Cavβ2C subunit is specifically responsible for the targeting and functional regulation of the caveolar Cav1.2 channels in ventricular myocytes.