Duplex signaling by CaM and Stac3 enhances CaV1.1 function and provides insights into congenital myopathy.

CaV1.1 is essential for skeletal muscle excitation-contraction coupling. Its functional expression is tuned by numerous regulatory proteins, yet underlying modulatory mechanisms remain ambiguous as CaV1.1 fails to function in heterologous systems. In this study, by dissecting channel trafficking versus gating, we evaluated the requirements for functional CaV1.1 in heterologous systems. Although coexpression of the auxiliary β subunit is sufficient for surface-membrane localization, this baseline trafficking is weak, and channels elicit a diminished open probability. The regulatory proteins calmodulin and stac3 independently enhance channel trafficking and gating via their interaction with the CaV1.1 carboxy terminus. Myopathic stac3 mutations weaken channel binding and diminish trafficking. Our findings demonstrate that multiple regulatory proteins orchestrate CaV1.1 function via duplex mechanisms. Our work also furnishes insights into the pathophysiology of stac3-associated congenital myopathy and reveals novel avenues for pharmacological intervention.