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. 2017 Jan 31;10:16. doi: 10.3389/fnmol.2017.00016

Figure 4.

Figure 4

Mutations of isoleucines 751 and 752 alter voltage and AITC sensitivity of TRPA1. (A) Representative currents from wild type (WT, black), I751A/I752A (green) and I751G/I752G (red) mutant TRPA1 in response to indicated voltage step protocol (holding potential −70 mV; 100 ms voltage steps from −80 mV to +200 mV, increment 20 mV) recorded in control extracellular solution ~1 min after whole-cell formation. (B) Average conductances of wild type (WT, white circles, n = 121), I751A/I752A (green triangles, n = 22) and I751G/I752G (red squares, n = 31) mutants obtained from the voltage step protocols as in (A). Depolarizing voltage activated human TRPA1 to a maximal amplitude of 4.8 ± 0.2 nA (measured at +200 mV). Voltage-dependent gating parameters were estimated by fitting the conductance G = I/(V-Vrev) as a function of test potential V to the Boltzmann equation: G = [(Gmax-Gmin)/(1+exp (−zF(V-V1/2)/RT))] + Gmin, where z is the apparent number of gating charges, V1/2 is the half-activation voltage, Gmin and Gmax are the minimum and maximum whole cell conductance, Vrev is the reversal potential, and F, R, and T have their usual thermodynamic meanings. Compared to the wild type, the double mutant channels exhibited lower voltage sensitivity, a drop in maximal conductances and a shift in V1/2 values toward higher depolarizing potentials (V1/2 = 154.6 ± 4.9 mV, z = 0.98 ± 0.11 e0, n = 13 for I751A/I752A and V1/2 = 152.1 ± 4.9 mV, z = 0.87 ± 0.05 e0, n = 20 for I751G/I752G). Data represent mean ± SEM. (C) Time course of representative current responses of wild type, I751A/I752A (D) and I751G/I752G (E) mutant channels evoked by 100 μM AITC in control extracellular solution during a continuous ramp protocol (from −80 to +80 mV, every 1 s). The current responses were assessed at −70 and +70 mV. The horizontal bar above the records indicates the period of AITC application. In cells expressing wild-type TRPA1 channels, AITC produced robust (7.7 ± 0.9 nA; n = 18; at +70 mV) and rapid current responses at both positive and negative membrane potentials (time of peak 8.4 ± 1.0 s at +70 mV) followed by immediate desensitization. In contrast, both the I751A/I752A and I751G/I752G mutants exhibited much slower kinetics of AITC-induced responses (time of peak 23.2 ± 3.8 s, n = 5 and 29.8 ± 3.0 s, n = 4; at +70 mV). Notably, both the double mutants exhibited a significant reduction in AITC-induced amplitudes selectively at negative membrane potentials, as is clearly visible in the bottom current-voltage (I-V) relationships obtained from the cells shown above at the peak of AITC responses.