Role of Muscle Specific Caveolin-3 in Mechanosensitive Channel Regulation

Abstract

Caveolin-3 (Cav-3) is a small muscle specific scaffolding protein that acts to complex signaling proteins within cholesterol rich raft domains through a small cytoplasmic binding domain. Cav-3 has the unique function of inserting only into the inner membrane leaflet causing expansion of the inner surface that induces curvature and membrane involutions called caveolae. In muscle the dystroglycan complex is localized to the caveolae and is the cortical cytoskeleton attachment point for dystrophin and other proteins which regulate sarcolemma tension. These attributes make caveolae ideal mechanosensing structures and likely locations for mechanosensitive channels (MSCs). To understand the role of Cav-3 in MSC regualtion we depleted membrane cholesterol with methyl β-cyclodextran which caused disruption and internalization of the caveolae protein and increased MSC activity in patch pipette recordings. Overexpression of a Cav-3-GFP fusion protein in well differentiated myotubes also leads to increased MSC activity. Reduction of Cav-3 with miRNA produced no change in MSC activity, though baseline MSC activity is already low in differentiated myotubes. To rapidly and specifically inhibit Cav-3 interactions, we fused the 20 amino acid Cav-3 scaffolding domain the 16 amino acid antennapedia membrane translocation signaling domain (Anten-CSD). Treatment of myotubes with 20 μM Anten-CSD caused internal Ca2+ to increase, large blebs to formed on the sarcolemma and Cav-3-GFP aggregation on the surface. There was an increase in MSC activity within 20 minutes after Anten-CBD treatment. We are currently investigating a link between Cav-3 and TRP channels. These studies suggest that Cav-3 may be involved in both localization of MSCs to the sarcolemma and regulating channel activity in myotubes through its ability to complex MSCs with the cortical cytoskeleton.