Mutations that lead to muscular dystrophy often create deficiencies in 
cytoskeletal support of the muscle sarcolemma causing hyperactive 
mechanosensitive cation channel (MSC) activity and elevated intracellular 
Ca(2+). Caveolae are cholesterol-rich microdomains that form mechanically 
deformable invaginations of the sarcolemma. Mutations to caveolin-3, the main 
scaffolding protein of caveolae in muscle, cause Limbe-Girdle muscular 
dystrophy. Using genetic and acute chemical perturbations of developing myotubes 
we investigated whether caveolae are functionally linked to MSCs. MSC 
sensitivity was assayed using suction application to patches and probe-induced 
indentation during whole-cell recordings. Membrane mechanical stress in patches 
was monitored using patch capacitance/impedance. Cholesterol depletion disrupted 
caveolae and caused a large increase in MSC current. It also decreased the 
membrane mechanical relaxation time, likely reflecting cytoskeleton dissociation 
from the bilayer. Reduction of Cav3 expression with miRNA also increased MSC 
current and decreased patch relaxation time. In contrast Cav3 overexpression 
produced a small decrease in MSC currents. To acutely and specifically inhibit 
Cav3 interactions, we made a chimeric peptide containing the antennapedia 
membrane translocation domain and the Cav3 scaffolding domain (A-CSD3). A-CSD3 
action was time dependent initially producing a mild Ca(2+) leak and increased 
MSC current, while longer exposures decreased MSC currents coinciding with 
increased patch stiffening. Images of GFP labeled Cav3 in patches showed that 
Cav3 doesn't enter the pipette, showing patch composition differed from the cell 
surface. However, disruption via cholesterol depletion caused Cav3 to become 
uniformly distributed over the sarcolemma and Cav3 appearance in the patch dome. 
The whole-cell indentation currents elicited under the different caveolae 
modifying conditions mirror the patch response supporting the role of caveolae 
in MSC function. These studies show that normal expression levels of Cav3 are 
mechanoprotective to the sarcolemma through multiple mechanisms, and Cav3 
upregulation observed in some dystrophies may compensate for other mechanical 
deficiencies.