Dissecting the functions of multiple interactions of STAC3

Skeletal muscle excitation-contraction (EC) coupling is a fundamental process in muscle physiology, in which an electrical signal, the action potential in the motor neuron, is converted into a mechanical response, muscle contraction. Protein complexes called voltage-gated calcium channels, consisting of the pore-forming CaV1.1 and additional CaV auxiliary subunits, mediate this signal transduction process by sensing the electrical signal and physically activating the opening of calcium release channels (ryanodine receptors; RyR1) in an intracellular compartment, the sarcoplasmic reticulum. Recently, an essential protein of the skeletal muscle EC coupling apparatus, STAC3, has been identified. STAC3 belongs to a family of adaptor proteins, which facilitate protein-protein interactions and the generation of bigger signaling complexes. In addition, mutations in STAC3 have been linked to a severe muscle disease, Native American myopathy. STAC3 was found to have two different functions in EC coupling: (1) it is essential for the stability and function of CaV1.1 and (2) it is crucial for the communication between CaV1.1 and RyR1. In a previous FWF project (T855), we could demonstrate that two different regions of STAC3 establish two distinct interactions with CaV1.1. In this research proposal, we will take advantage of a unique skeletal muscle cell culture model, in which STAC3 has been genetically deleted, to identify the specific functions of the two distinct STAC3/CaV1.1 interactions in skeletal muscle EC coupling. The proposed experiments, including molecular genetics, advanced microscopy techniques, and electrophysiology, will allow us to determine whether each STAC3/CaV1.1 interaction is responsible for each STAC3 function in EC coupling or whether both STAC3/CaV1.1. interactions are synergistically contributing to both functions. In addition, the generation of the STAC3 null model will allow us to test the physiological consequences of novel STAC3 mutations that have been linked to Native American myopathy. The expected results will reveal the molecular functions of the interactions established by STAC3 in EC coupling as well as the pathophysiological mechanisms involved in Native American Myopathy. Thus, the proposed research project will advance our understanding of a fundamental process in muscle physiology and STAC3 involvement in a rare muscle disease.

Excitation-contraction (EC) coupling in skeletal muscle is a tightly regulated process that converts membrane depolarization into calcium release from the sarcoplasmic reticulum (SR), ultimately leading to muscle contraction. At the core of this mechanism lies the L-type calcium channel CaV1.1, located in the transverse (T)-tubule membrane, which acts as the voltage sensor and interacts directly with the ryanodine receptor type 1 (RyR1) on the SR membrane. The adaptor protein STAC3 (SH3 and cysteine-rich domain 3) has emerged as a critical regulator of this process. Genetic studies have shown that loss-of-function mutations in Stac3 lead to a congenital myopathy-referred to as STAC3 disorder-characterized by severe muscle weakness and impaired EC coupling. While STAC3 is established as essential for the functional expression of CaV1.1 and its conformational coupling with RyR1, the molecular mechanisms underlying these roles remained incompletely understood. Notably, STAC3 interacts with two key cytoplasmic regions of CaV1.1: the intracellular II-III loop and the proximal C-terminus. Previous work has emphasized the importance of the II-III loop in EC coupling; however, the respective contributions of each interaction domain have not been clearly delineated. Moreover, the role of STAC3 in modulating the biophysical properties of CaV1.1 currents-particularly their unusually slow gating kinetics-has remained largely unexplored. Emerging evidence from related STAC family proteins (e.g., STAC1 and STAC2) suggests that these proteins can modulate the inactivation kinetics of L-type calcium channels, such as CaV1.2 and CaV1.3, by suppressing calcium-dependent inactivation (CDI). However, the extent to which STAC3 contributes to similar regulatory mechanisms in skeletal muscle CaV1.1, especially in terms of voltage-dependent inactivation (VDI) and gating kinetics, has not been directly addressed. In this research grant, we sought to dissect the dual role of STAC3 in (1) regulating CaV1.1 surface expression and EC coupling via its domain-specific interactions, and (2) modulating the kinetic and inactivation properties of CaV1.1 currents. Using structure-function analysis, electrophysiology, and patient-derived mutations, we demonstrated that STAC3 binding to the CaV1.1 C-terminus is essential for channel expression and minimal EC coupling, while its interaction with the II-III loop enhances coupling efficiency. Furthermore, we showed that STAC3 shapes the biophysical profile of CaV1.1 by suppressing voltage-dependent inactivation and slowing current kinetics, properties that are abolished in a triple-mutant STAC3 (ETLAAA) variant. These findings establish STAC3 as a multifunctional regulator of skeletal muscle calcium signaling and provide mechanistic insights relevant to both normal physiology and STAC3-related myopathies.