Figure 7.

External Ca²⁺ reversibly block human TRPA1 channel in a state-dependent manner and distinctly block I751G/I752G mutant. (A) Representative current responses recorded from a HEK293T cell expressing wild-type TRPA1 (Aa and b; black traces) or I751G/I752G mutant (c and d; red traces), stimulated with 100 μM AITC in changing Ca²⁺ concentration at a constant potential of −70 mV. The application of AITC without (white horizontal bar) and with Ca²⁺ ions (1 mM, black horizontal bar) are indicated above the current traces. Similar effects were seen in 3–4 cells for each construct. (B) Time course of representative whole-cell currents of HEK293T expressing wild-type TRPA1 during voltage ramp protocol (from −80 to +80 mV, every 1 s) and application of 100 μM AITC in the control extracellular solution containing 1 mM Ca²⁺ (black horizontal bar) and after several removals of Ca²⁺ from the bath solution (white horizontal bar), measured at −70 mV and +70 mV. Below, the time course of reversal potential for the same cell and the same time period. (C) Neutralization of four negative charges within S1–S2 linker changed neither the voltage nor AITC response of TRPA1 channel. Average conductances of wild type (white circle, n = 121) and E754Q/D757N/E760Q/D763N quadruple mutant (violet diamond, n = 17) obtained from voltage step protocols as in Figure 4A. Lines are best fits to a Boltzmann function as described in Materials and Methods. (D) Time course of average whole-cell current responses induced by 100 μM AITC in the absence of Ca²⁺ (white horizontal bar) and subsequent addition of 1 mM Ca²⁺ to the bath solution (black horizontal bar), during continuous ramp protocol (from −80 to +80 mV, every 1 s), measured at −70 and +70 mV. Current responses through wild type are depicted as white circles and E754Q/D757N/E760Q/D763N mutant as violet diamonds. All data represent mean ± SEM (n indicated in brackets).