Effect of cholesterol depletion for 90 min treatment with 2.5 mM M βCD:cholesterol (10:1) but not for 15 min treatment with 10 mM M β CD on proton-activated TRPV1. (A) Representative traces of TRPV1 whole-cell response of cells treated with MβCD:cholesterol, activated with pH 5.5, clamped at −60mV (extracellular buffer 4, intracellular buffer 5), containing two currents, I1 and I2. (B) The initial current density upon activation I1was unaffected by treatment with MβCD:cholesterol whereas the following current density I2 was 60% lower for cells treated with MβCD:cholesterol compared to control cells (n = 6, p < 0.05). (C) YO-PRO uptake was observed during stimulation with pH 5.5 (extracellular buffer 4) in cells treated with MβCD. Fluorescence uptake rate increased during activation with pH 5.5, but no significant difference in rate could be determined from the treatment (n = 34–46). (D) YO-PRO uptake was observed during stimulation with pH 5.5 (extracellular buffer 4) in cells treated for 90 min with 2.5 mM MβCD:cholesterol (10:1). Fluorescence uptake rate increased during activation with pH 5.5, the MβCD:cholesterol-treatment resulted in a reduction by ∼70% of the uptake rate in treated cells compared to control cells (n = 15–30, p < 0.001) after ∼30 s of stimulation.