Characterization of the STAC3-CaV1.1 Interaction in Skeletal Muscle Excitation-Contraction Coupling

Abstract

Skeletal muscle EC coupling critically depends on the physical interaction between the voltage-gated calcium channel CaV1.1 (DHPR), acting as voltage sensor, and the SR calcium release channel (RyR1). The skeletal muscle-specific DHPR components essential for EC coupling are the CaV1.1 II-III loop and the auxiliary β1a subunit. Recently, STAC3 (src homology 3 and cysteine rich domain 3) has been identified as an additional essential EC coupling protein. Further investigations indicated that STAC3 functions in membrane trafficking of CaV1.1 and linked STAC3 to a rare muscle disease Native American Myopathy (NAM). In contrast STAC3 is not expressed in the heart and thus appears not to participate in cardiac muscle EC coupling. These findings raise the question as to whether STAC3 binds exclusively to the skeletal muscle DHPR, and whether its interaction with the EC coupling complex is stable or dynamic. Here, we applied fluorescence recovery after photobleaching (FRAP) in dysgenic (CaV1.1-null) myotubes reconstituted with CaV1.1 and GFP-tagged STAC3 to demonstrate that STAC3 forms stable complexes with the skeletal muscle DHPR. Furthermore, we examined the subcellular localization of STAC3 and of the other two members of the STAC family of adaptor proteins, STAC1 and STAC2, in dysgenic myotubes reconstituted with either CaV1.1 or CaV1.2. Together these experiments demonstrate that the three members of the STAC family display differential interactions with the skeletal muscle and cardiac DHPRs. These functional differences allow the use of chimeric proteins to identify the molecular domains involved in the association of STAC3 with the skeletal muscle EC coupling apparatus. This work is supported by Austrian Research Fund FWF (P23479, P27031).