Stac3 has a direct role in skeletal muscle-type excitation-contraction coupling that is disrupted by a myopathy-causing mutation

In skeletal muscle, conformational coupling between Ca(v)1.1 in the plasma membrane and type 1 ryanodine receptor (RyR1) in the sarcoplasmic reticulum (SR) is thought to underlie both excitation-contraction (EC) coupling Ca2+ release from the SR and retrograde coupling by which RyR1 increases the magnitude of the Ca2+ current via Ca(v)1.1. Recent work has shown that EC coupling fails in muscle from mice and fish null for the protein Stac3 (SH3 and cysteine-rich domain 3) but did not establish the functional role of Stac3 in the Ca(v)1.1-RyR1 interaction. We investigated this using both tsA201 cells and Stac3 KO myotubes. While confirming in tsA201 cells that Stac3 could support surface expression of Ca(v)1.1 (coexpressed with its auxiliary beta(1a) and alpha(2)-delta(1) subunits) and the generation of large Ca2+ currents, we found that without Stac3 the auxiliary gamma(1) subunit also supported membrane expression of Ca(v)1.1/beta(1a)/alpha(2)-delta(1), but that this combination generated only tiny Ca2+ currents. In Stac3 KO myotubes, there was reduced, but still substantial Ca(v)1.1 in the plasma membrane. However, the Ca(v)1.1 remaining in Stac3 KO myotubes did not generate appreciable Ca2+ currents or EC coupling Ca2+ release. Expression of WT Stac3 in Stac3 KO myotubes fully restored Ca2+ currents and EC coupling Ca2+ release, whereas expression of Stac3(W280S) (containing the Native American myopathy mutation) partially restored Ca2+ currents but only marginally restored EC coupling. We conclude that membrane trafficking of Ca(v)1.1 is facilitated by, but does not require, Stac3, and that Stac3 is directly involved in conformational coupling between Ca(v)1.1 and RyR1.