Bernhard Flucher

The skeletal muscle Ca V 1.1 channel functions as the voltage‐sensor of excitation−contraction (EC) coupling. Recently, the adaptor protein STAC3 was found to be essential for both Ca V 1.1 functional expression and EC coupling. Interestingly, STAC proteins were also reported to inhibit calcium‐dependent inactivation (CDI) of L‐type calcium channels (LTCC), an important negative feedback mechanism in calcium signaling. The same could not be demonstrated for Ca V 1.1, as STAC3 is required for its functional expression. However, upon strong membrane depolarization, Ca V 1.1 conducts calcium currents characterized by very slow kinetics of activation and inactivation. Therefore, we hypothesized that the negligible inactivation observed in Ca V 1.1 currents reflects the inhibitory effect of STAC3. Here, we inserted a triple mutation in the linker region of STAC3 (ETLAAA), as the analogous mutation abolished the inhibitory effect of STAC2 on CDI of Ca V 1.3 currents. When coexpressed in Ca V 1.1/STAC3 double knockout myotubes, the mutant STAC3‐ETLAAA failed to colocalize with Ca V 1.1 in the sarcoplasmic reticulum/membrane junctions. However, combined patch‐clamp and calcium recording experiments revealed that STAC3‐ETLAAA supports Ca V 1.1 functional expression and EC coupling, although at a reduced extent compared to wild‐type STAC3. Importantly, STAC3‐ETLAAA coexpression dramatically accelerated the kinetics of activation and inactivation of Ca V 1.1 currents, suggesting that STAC3 determines the slow Ca V 1.1 currents kinetics. To examine if STAC3 specifically inhibits the CDI of Ca V 1.1 currents, we performed patch‐clamp recordings using calcium and barium as charge carriers in HEK cells. While Ca V 1.1 displayed negligible CDI with STAC3, this did not increase in the presence of STAC3‐ETLAAA. On the contrary, our data demonstrate that STAC3 specifically inhibits the voltage‐dependent inactivation (VDI) of Ca V 1.1 currents. Altogether, these results designate STAC3 as a crucial determinant for the slow activation kinetics of Ca V 1.1 currents and implicate STAC proteins as modulators of both components of inactivation of LTCC.