Estimation of the effect of surface charge on the kinetics of block by TPA and single-channel conductance. (A) The TPA (20 µM) block kinetics with 4 µM capsaicin (top) or 5 µM LPA (bottom). The superimposed black and red lines represent the exponential fits. (B) Voltage dependence of block for currents elicited by capsaicin (circles, solid fit, n = 4) or LPA (squares, dashed fit, n = 8). Data are plotted as mean ± SEM. From the exponential fit we obtained the following parameters: k_b(0) = 1.10⁶ ± 4.6 10⁵ s⁻¹ M⁻¹ for capsaicin, and k_b(0) = 1.32 × 10⁶ ± 3.5 10⁵ s⁻¹ M⁻¹ for LPA (P 0.01), while Z_app = 0.43 ± 0.08 for capsaicin, and Z_app = 0.34 ± 0.05 for LPA (P 0.01). The dashed curve is the increase in the blocking rate expected if LPA contributes −14 mV of surface potential, as calculated in the text. This shows that the blocker entrance to the pore is electrostatically shielded from the membrane surface potential. (C) Representative traces from single-channel recordings of TRPV1 channels in the inside-out configuration at +60 mV, in response to 4 µM capsaicin (black) or 50 nM capsaicin + 5 µM LPA 18:0 (orange). (D) Comparison of the single-current amplitude elicited by capsaicin alone (black) or in combination with LPA 18:0 (orange) for experiment shown in (C). The mean single-current amplitude elicited for four independent experiments was 7.33 ± 0.21 pA for capsaicin 4 µM and 7.27 ± 0.17 pA for capsaicin in combination with LPA 18:0 (P 0.01). Macroscopic current recordings were performed in the inside-out configuration of the patch clamp, the holding potential was 0 mV, and 100-ms-long square pulses from 40 to 140 mV (in 20-mV increments) were applied.