Multiple Sites of Interaction May Be Involved in the Regulation of CaV1.1 by Stac3

Stac3, which is highly expressed only in skeletal muscle, can be subdivided into an N-terminal region, a PKC C1 domain, a linker, and tandem SH3 domains. Stac3 (i) facilitates the trafficking of CaV1.1 to the plasma membrane, (ii) modulates the function of CaV1.1 as a Ca2+ channel, and (iii) is required for the ability of CaV1.1 to activate Ca2+ release via RyR1. Previous work from our lab has shown that function (iii) likely depends on binding of the tandem SH3 domains to residues 745-765 of the CaV1.1 II-III loop, an interaction which is supported (Wong King Yuen et al., 2017) by crystallographic and calorimetric analysis of isolated segments of the two proteins. Here, by using electrophysiology, and by testing for colocalization of fluorescently tagged fragments, we systematically tested the ability of discrete Stac3 domains to interact with intact CaV1.1 and its cytoplasmic domains in tsA201 cells. We found that a mutation (P758L), recently reported to cause malignant hyperthermia susceptibility, abolishes the interaction of Stac3 with the CaV1.1 II-III loop. We also found that the tandem SH3 domains (residues 243-360) co-localize with the wild-type CaV1.1 II-III loop and promote insertion of full-length CaV1.1 into the plasma membrane, where it produces gating charge movements but not ionic currents. However, expression of Stac3 residues 1-242 together with residues 243-360 restored ionic currents almost identical to those restored by full-length Stac3, indicative of interaction site(s) between CaV1.1 and Stac3 other than the II-III loop and the SH3 tandem. Consistent with this possibility, expression of only Stac3 residues 1-242, together with CaV1.1, was sufficient to restore Ca2+ currents almost similar in magnitude to those for full-length Stac3.