Mechanosensitive channel properties and membrane mechanics in mouse dystrophic myotubes

摘要

Muscular dystrophy is associated with increased activity of mechanosensitive channels (MSCs) and increased cell calcium levels. MSCs in patches from mdx mouse myotubes have higher levels of resting activity, compared to patches from wild-type mice, and a pronounced latency of activation and deactivation. Measurements of patch capacitance and geometry reveal that the differences are linked to cortical membrane mechanics rather than to differences in channel gating. We found unexpectedly that patches from mdx mice are strongly curved towards the pipette tip by actin pulling normal to the membrane. This force produces a substantial tension (∼5 mN m⁻¹) that can activate MSCs in the absence of overt stimulation. The inward curvature of patches from mdx mice is eliminated by actin inhibitors. Applying moderate suction to the pipette flattens the membrane, reducing tension, and making the response appear to be stretch inactivated. The pronounced latency to activation in patches from mdx mice is caused by the mechanical relaxation time required to reorganize the cortex from inward to outward curvature. The increased latency is equivalent to a three-fold increase in cortical viscosity. Disruption of the cytoskeleton by chemical or mechanical means eliminates the differences in kinetics and curvature between patches from wild-type and mdx mice. The stretch-induced increase in specific capacitance of the patch, ∼80 fF μm⁻², far exceeds the specific capacitance of bilayers, suggesting the presence of stress-sensitive access to large pools of membrane, possibly caveoli, T-tubules or portions of the gigaseal. In mdx mouse cells the intrinsic gating property of fast voltage-sensitive inactivation is lost. It is robust in wild-type mouse cells (observed in 50% of outside-out patches), but never observed in mdx cells. This link between dystrophin and inactivation may lead to increased background cation currents and Ca²⁺ influx. Spontaneous Ca²⁺ transients in mdx mouse cells are sensitive to depolarization and are inhibited by the specific MSC inhibitor GsMTx4, in both the d and l forms.