I317C and H319C in the central vestibule are accessible to MTSET but not to MTS-TPAE. Superimposed scaled current traces on the left show that extracellular application of MTS-TPAE (blue bar) has little effect on currents evoked by ATP (black bar). Extracellular application of MTSET, even after the application of MTS-TPAE, irreversibly modified currents evoked by ATP (right traces). (A) Deactivation of I317C mutant became faster after MTSET application. (B) ATP-induced current of H319C was potentiated by MTSET application. ATP was applied every 3 min. The control traces (black traces) in A and B are the same in each of the three panels. (C) Quantitative comparisons of current modifications by MTS reagents. Averaged current modifications by MTSET (left) or MTS-TPAE (right) at each Cys mutant (n = 3–9). Error bars are SEM. Orange bar highlights H319C, a position where MTSET causes pronounced current potentiation. Although I317C showed little change in current amplitude after MTSET application, the MTS reagent accelerated current deactivation after ATP removal; therefore, the residue is highlighted with an asterisk to indicate MTS reactivity.