STAC3 determines the slow activation kinetics of Ca_V 1.1 currents and inhibits its voltage-dependent inactivation

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.