Abstract 1503: Protein Kinase A-Independent and Gsα-Mediated Regulation of Caveolar Cardiac Sodium Channels by β-Adrenergic Stimulation.
Beta-adrenergic regulation of switching caveolae from the closed configuration to the open state by Gsα-mediated pathway plays an important role in rapidly increasing the surface membrane channel density in cardiac ventricular myocytes. We show that a subpopulation of NaV1.5 channels that is localized in caveolae of rat ventricle myocytes is part of a signaling complex directly regulated by Gsα. This regulatory signaling pathway is PKA-independent and depends upon the interaction of the Gsα protein with caveolin-3 scaffolding protein. In whole-cell electrophysiological experiments, 1mM QX-314, a permanently charged, membrane impermeable triethyl derivative of lidocaine, was applied to the extracellular side of the membrane to completely and irreversibly inhibit surface Na+ channel conductance. Subsequent to washout of QX-314, β-adrenergic receptor stimulation with 10 μM isoproterenol (ISO) in the presence of PKA-inhibitor (22 μg/ml), revealed only TTX resistant Na+ current. The ISO-enhanced Na+ current is inhibited in the presence of intracellular application of anti-caveolin-3 mAb suggesting that this current originates from Na+ channels within the caveolae membrane. Western blot analysis of caveolae-rich fractions and immunolabeling of plasma membrane sheets from ventricle myocytes also demonstrated that NaV1.5 channels are specifically colocalized to caveolin-3 membranes. The direct effect elicited by β-adrenergic stimulation with ISO can be mimicked by the addition of a 16 a.a. peptide (27–42 a.a. of the rat and human Gsα protein). The effect depended upon the presence of a histidine at residue 41 of Gsα. Substitutions of H41A abolished the activation of caveolae-localized Na+ channels. These findings demonstrate that subcellular localization of NaV1.5 channels to caveolar macromolecular signaling plays a specific functional role in the direct Gsα-mediated increase in rat cardiac ventricular sodium current.