Critical importance of the interaction between IS6 and IIS6 to induction. (A) A view of the XtTPC3 structural model in the PI(3,4)P₂-bound open state, depicting the regions related to PI(3,4)P₂ binding and coupling with voltage dependence. Domain I and domain II in one monomer are depicted in green and aqua, respectively. Those in the other monomer are shown in white and gray, and the VSDs are omitted for clarity. VSDs and pore domains are shown as ribbon and cylinder models, respectively. The region indicated by the dotted circle shows the interaction that bridges the DI-S6 and DII-S6 (Tyr293, Arg297, and Glu665). (B) Structural view of the proximal region between the cytosolic parts of IS6 and IIS6 in the superimposed structures of XtTPC3 and MmTPC1. The carbon atoms of side chains are shown in green for XtTPC3 and in white for MmTPC1. Dashed lines indicate putative hydrogen bonds with distances of 2.6–3.3 Å in the model structures. (C) Representative current traces of a series of channels with mutations at Tyr293, Arg297, and Glu665 before (black) and after (red) induction. Currents were evoked by a 100-mV pulse from −60 mV. (D) G–V relationships of channels with mutations at Tyr293, Arg297, or Glu665. (E) Plots of V_1/2 in a series of mutants of Tyr293, Arg297, and Glu665 before (black) and after (red) induction. #, the V_1/2 values before induction in E665Q and Y293H could not be obtained because the currents were too small. Error bars represent the SD of five to seven independent experiments. **, P < 0.01.