MSC patch currents increase when caveolae are disrupted or reduced.

<p>ΔCp kinetics indicates cytoskeletal interactions with the membrane. Reduced interactions are normally associated with a more rapid patch mechanical relaxation rate. Ensemble averaged MSC currents at −110 mV (A) and patch stress represented by ΔCp (B) are shown for control (n = 27) Cav3 overexpression (n = 16), Cav3 reduction by inhibitory RNA (n = 16, Cav3-miRNA) and cholesterol depletion (n = 9, 5 mM MβCD). The exponential fits to the rising phase relaxation curves of the ensemble capacitance records are shown as dashed lines through the data and extended out to 1.2 seconds. Ensemble averaged ΔCp (C), channel current at −120 mV (D) and patch conductance at 0 mV (E) over the range of pressures studied are shown (SEM error bars). Patches from MβCD treated cells were fragile so data was collected at −70 mmHg first, before patch rupture which often occurred before other pressures could be sampled. Cav3 overexpression had no effect on the MSC activity or patch mechanics suggesting saturation of caveolae components. Patches from cells expressing Cav3-miRNA showed a significant increase in MSC current, patch conductance and increased ΔCp where the faster relaxation suggested a more compliant patch (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072894#pone.0072894.s004" target="_blank">Fig. S4B</a> for statistical analysis). Patches from cells treated with 5 mM MβCD showed the largest increase in all parameters measured. The parameters from the ΔCp curve fits are shown in (F). Either first or second (MβCD) order exponentials were used and the fitting data shows that the relaxation rates decreased as patch current increased, but the predicted steady state ΔCp stayed the same. This shows that factors that disrupt caveolin do not change patch size but do affect the relaxation rate.