Cardiac sodium channels (Nav1.5) reside in both the plasmalemma and membrane invaginations called caveolae. Opening of the caveolar neck permits resident channels to become functional. In cardiac myocytes, caveolar opening can be stimulated by applying -receptor agonists, which initiates an interaction between the G-protein subunit Gs and Caveolin-3. This study shows that, in adult rat ventricular myocytes, a functional Gs-Caveolin-3 interaction occurs even in the absence of the caveolin-binding sequence motif of Gs. Consistent with previous data, whole-cell experiments conducted in the presence of intracellular PKA-inhibitor (PKI), stimulation with -receptor agonists increased the sodium current (INa) by 35.9±8.6% (P<0.05), and this increase was mimicked by application of Gs protein. Inclusion of anti-Caveolin3 antibody abolished this effect. These findings suggest that Gs and Caveolin-3 are components of a PKA-independent pathway that leads to the enhancement of INa. In this study, alanine scanning mutagenesis of Gs (40THR42) in conjunction with voltage-clamp studies demonstrated that the histidine residue at position 41 of Gs (H41) is a critical residue for the functional increase of INa. Protein interaction assays suggest that GsFL (full-length) binds to Caveolin-3, but the enhancement of INa is observed only in the presence of Gs H41. We conclude that Gs H41 is a critical residue in the regulation of the increase in sodium current in ventricular myocytes.