This study demonstrates that caveolae, omega-shaped membrane invaginations, are involved in cardiac sodium channel regulation by a mechanism involving the alpha subunit of the stimulatory heterotrimeric G-protein, Galpha(s), via stimulation of the cell surface beta-adrenergic receptor. Stimulation of beta-adrenergic receptors with 10 micromol/L isoproterenol in the presence of a protein kinase A inhibitor increased the whole-cell sodium current by a "direct" cAMP-independent G-protein mechanism. The addition of antibodies against caveolin-3 to the cell's cytoplasm via the pipette solution abrogated this direct G protein-induced increase in sodium current, whereas antibodies to caveolin-1 or caveolin-2 did not. Voltage-gated sodium channel proteins were found to associate with caveolin-rich membranes obtained by detergent-free buoyant density separation. The purity of the caveolar membrane fraction was verified by Western blot analyses, which indicated that endoplasmic/sarcoplasmic reticulum, endosomal compartments, Golgi apparatus, clathrin-coated vesicles, and sarcolemmal membranes were excluded from the caveolin-rich membrane fraction. Additionally, the sodium channel was found to colocalize with caveolar membranes by immunoprecipitation, indirect immunofluorescence, and immunogold transmission electron microscopy. These results suggest that stimulation of beta-adrenergic receptors, and thereby Galpha(s), promotes the presentation of cardiac sodium channels associated with caveolar membranes to the sarcolemma.