Identifying the STAC3/CaV1.1 interactions responsible for CaV1.1 expression in skeletal muscle.

Abstract

The adaptor protein STAC3 was shown to be crucial for membrane and functional expression of the voltage sensor of EC coupling CaV1.1. In fact, CaV1.1 membrane expression is reduced and calcium currents are abolished in STAC3 KO animals. Two distinct STAC3/CaV1.1 interactions have been identified: one between the II-III loop of CaV1.1 and the SH3-1 domain of STAC3 and a second between the proximal C-terminus of CaV1.1 channels and the linker region of STAC3. Here, we investigated whether a single interaction is responsible for the CaV1.1 membrane and functional expression or if both synergistically contribute to CaV1.1 expression. To this end, we generated two STAC3 fragments, STAC3-NT, consisting of the N-terminal part, including the C1 domain and the linker region, expected to interact with the proximal C-terminus of CaV1.1, and STAC3-CT, consisting of only the SH3 domains, expected to interact with the II-III loop of CaV1.1. Expression of either one of these fragments, in a novel C2C12 STAC3 KO cell line still promoted CaV1.1 membrane expression, but at a reduced level compared to full length STAC3, suggesting that both interactions are contributing to CaV1.1 membrane expression. However, only expression of STAC3-NT supported CaV1.1 functional expression in CaV1.1 reconstituted double CaV1.1/STAC3 KO cells, indicating that only the STAC3 interaction with the proximal C-terminus is essential for CaV1.1 function in muscle cells. In addition, we analysed the ability of each STAC3 fragment to colocalize with CaV1.1 in the triads of myotubes. While the STAC3-NT fragment is still able to incorporate in CaV1.1 clusters, albeit with a 50% reduction in the Pearson's coefficient compared to full length STAC3, the STAC3-CT fragment remained diffusely localized in the cytoplasm. Experiments are planned to establish the importance of each fragment for EC coupling.