Figure 3.

Changes in heat sensitivity produced by alanine mutations in the putative inner-pore region of TRPV1. A, Bar graphs show effects of alanine substitution on the temperature threshold (top graph) and temperature coefficient (bottom graph) measured from the rising phase of whole-cell membrane currents induced by a 25–48°C heat ramp (10°C/s). The dotted line in the bottom graph indicates the value at which the temperature dependence of the thermally induced currents is considered to be nonspecific [i.e., close to that of the aqueous diffusion limit (∼2)]. B, Arrhenius plot of whole-cell currents obtained from representative cells transfected with wild-type (open circles) or I679A-TRPV1 (open squares) normalized at 45°C (ordinate, log scale) and plotted against the reciprocal of the absolute temperature (abscissa). The temperature coefficients Q₁₀ were determined for each cell over the temperature range at which the Arrhenius plot was linear (dashed lines). C, Activation of channels by depolarizing voltage steps recorded at 25°C in extracellular control solution (holding potential, −70 mV; voltage steps from −140 to +140 mV; increment, +20 mV). Averaged voltage–current relationships were constructed from responses obtained from three to five independent recordings, such as shown in the inset. They normalized in L669A (open circles), G683A (filled squares), wild-type channel (small filled circles, dotted line). D, Effects of alanine substitution on voltage-dependent gating properties of TRPV1. Voltage-dependent gating parameters, the half-activation voltage V_1/2, and equivalent charge z were estimated from steady-state current–voltage (I--V) relationships obtained at the end of 60 ms voltage steps from −140 mV to +140 mV by fitting them to the following Boltzmann equation: I = g × (V − E_rev)/(1 + exp(−zF(V − V_1/2)/RT)), where g is the whole-cell conductance, E_rev is the reversal potential, and F, R, and T have their usual thermodynamic meaning.