Molecular mechanisms of the STAC3-CaV1.1 interaction in skeletal muscle EC coupling

Skeletal muscle excitation-contraction (EC) coupling is a fundamental process in muscle physiology, in which an electrical signal, the action potential, is transduced 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 membrane depolarization and physically activating the opening of calcium release channels (ryanodine receptors; RyR1) in the sarcoplasmic reticulum. The resulting cytoplasmic calcium signal regulates the force of muscle contraction. Loss of any one of the participating channel proteins, e.g CaV1.1, the auxiliary subunit CaVb1a or the RyR1, causes paralysis and consequently death by asphyxia. Additionally, dysfunction of the same proteins results in muscle diseases. Recently, an essential component 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. STAC3 participates in the EC coupling protein complex and its genetic deletion in mouse or fish disrupted EC coupling. One report recently suggested that STAC3 is necessary for the membrane targeting of CaV1.1 in non-muscle cells. Additionally, a point mutation in STAC3 causes Native American Myopathy (NAM), a rare muscle disease characterized by multiple clinical features, including muscle weakness, susceptibility to malignant hyperthermia and dysmorphic facial features, and causing the death of one third of the affected individuals by the age of 18. However, the mechanism of STAC3 function in skeletal muscle EC coupling cells remains incompletely understood. In this research proposal, we will take advantage of unique skeletal muscle cell culture models to characterize the properties and identify the molecular determinants of the STAC3/CaV1.1 interaction and to identify the specific function of STAC3 in skeletal muscle EC coupling. We hypothesize that if STAC3 is a fundamental element of the EC coupling apparatus, STAC3 is stably localized in the membrane of skeletal muscle and forms specific interactions with one or several components of the EC coupling apparatus (e.g. CaV1.1 and CaVb1a). In addition, if STAC3 is indispensable for CaV1.1 membrane expression in native muscle cells, STAC3 deletion in myotubes will result in reduced CaV1.1 membrane expression. The proposed experiments, including molecular genetics, advanced microscopy techniques, and electrophysiology, will allow us to discriminate between these possibilities. The expected results will reveal the molecular mechanism of STAC3 action in normal 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 its possible involvement in a rare muscle disease.

Skeletal muscle excitation-contraction (EC) coupling is a fundamental process in muscle physiology, in which an electrical signal, the action potential, is transduced 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 membrane depolarization and physically activating the opening of calcium release channels (ryanodine receptors; RyR1) in the sarcoplasmic reticulum. The resulting cytoplasmic calcium signal regulates the force of muscle contraction. Loss of any one of the participating channel proteins, e.g CaV1.1, the auxiliary subunit CaV1a or the RyR1, causes paralysis and consequently death by asphyxia. Additionally, dysfunction of the same proteins results in muscle diseases. Recently, an essential component 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. STAC3 participates in the EC coupling protein complex and its genetic deletion in mouse or fish disrupted EC coupling. Additionally, a point mutation in STAC3 causes Native American Myopathy (NAM), a rare muscle disease characterized by multiple clinical features, including muscle weakness, susceptibility to malignant hyperthermia and dysmorphic facial features, and causing the death of one third of the affected individuals by the age of 18. However, the mechanism of STAC3 function in skeletal muscle EC coupling cells remained incompletely understood. In this research proposal, we took advantage of unique skeletal muscle cell culture models to characterize the properties and identify the molecular determinants of the STAC3/CaV1.1 interaction. We discovered that STAC3 establishes two distinct interactions with CaV1.1, which involve distinct domains of the two proteins. The first interaction, which is disrupted in NAM, is essential for the functional coupling of CaV1.1 and the RyR1, while the second interaction is crucial for the stable incorporation of STAC3 in the CaV1.1 channel complex. Additionally, the second interaction was found to be conserved among other calcium channels and STAC proteins, which are expressed in the brain and other tissues, and to have important physiological consequences: association of STAC proteins to these calcium channels result in inhibition of an important negative feedback mechanism, calcium-dependent inactivation, and therefore in increased calcium influx, leading to different cellular responses. Finally, we reported that STAC3 establishes an interaction with an additional protein, apart from the CaV1.1 channel. Although the identity of this protein remains to be elucidated, this finding suggests that STAC3 may indeed act as a linker between CaV1.1 and the RyR1.