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 β_1a and α₂ -δ₁ subunits) and the generation of large Ca²⁺ currents, we found that without Stac3 the auxiliary γ₁ subunit also supported membrane expression of Ca_v 1.1/β_1a /α₂ -γ₁ , 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.