FIGURE 2.
rTRPV1 Tyr511 substitutions exhibit impaired capsaicin sensitivity. A, current traces of whole cell recordings from HEK293T cells expressing the wt rTRPV1 (top panel), and the mutants rTRPV1 containing the Y511W (middle panel), and Y511M (bottom panel) substitutions at a holding potential of −40 mV. Cells were first exposed to pH 5.5 (empty bars; as a reference for channel expression) followed by applications of 1 and 30 μm capsaicin (Cap; gray and black bars, respectively). Bars above the trace indicate the time course of each activator application. B, mean/scatter-dot plot representing the normalized amplitude of whole cell currents in cells expressing rTRPV1 with indicated Tyr511 substitutions, evoked by 1 (gray triangles) or 30 μm (black circles) capsaicin, normalized to the current amplitude of the pH 5.5 response. Statistical significance between responses to different capsaicin concentrations for each construct was determined with paired Student's t test, where *** represents p ≤ 0.001 and ns represents a non-significant difference (n = 5–9 cells). Note a reduction in the capsaicin-evoked response in all tested mutants. Importantly, the recordings from Y511M and Y511I constructs could only be obtained when overexpressed. C, normalized concentration-response relationships to protons of wt and the indicated mutant receptors. Points represent the mean ± S.E. response of 6–7 HEK293T cells and solid lines are fit to the Hill equation: rTRPV1 (full circles, black line; nH = 1.1 ± 0.2; EC50 = pH 5.7 ± 0.3), rTRPV1 (Y511W) (empty triangle, dark gray line; nH = 1.1 ± 0.1; EC50 = pH 5.9 ± 0.1), and rTRPV1 (Y511F) (empty circle, light gray line; nH = 0.9 ± 0.1; EC50 = pH 5.8 ± 0.3). Holding potential of −40 mV.