Application of eVSP to TRPC6. (A) Schematic view of the protocol used to test the PI(4,5)P₂ sensitivity of TRPC6 using VSP in the whole-cell patch configuration. The bath solution was always perfused, and OAG, a TRPC6 activator, was added just before measurement. While TRPC6 channel is activated by OAG (see Materials and methods for details), we repeatedly tested TRPC6 current with VSP by depolarization pulse as shown in the bottom panel. A single 500- or 1,000-ms depolarizing pulse was stepped to 100 mV. The holding potential was −50 mV. (B) Representative TRPC6 current traces recorded from HEK293T cells coexpressing CiDr-VSP^mChe WT (upper), L223F (middle, eVSP), or Dr-VSP C302S (lower). Dashed lines indicate 0 level. (C) Enlarged and superimposed view of current traces during the depolarizing test pulse shown in B. The gray and black lines show CiDr-VSP^mChe WT and CiDr-VSP^mChe L223F (eVSP), respectively. The scale bar refers commonly to the TRPC6 current waveform with CiDr-VSP^mChe WT (gray line) and the TRPC6 current waveform with CiDr-VSP^mChe L223F (black line). Arrows indicate the time lag between the onset of the depolarization (+100 mV) and the onset of the current suppression (cross points of two dotted lines). Traces are superimposed from B by y axis offset without a scale change. (D) Plots of the time lag of three constructs. Data are the mean ± SD from two to six cells, and each symbol shows the data from each cell. Values in parentheses indicate the numbers of cells. In the case of Dr-VSP C302S, the time lag was not calculated because of no current decay. Upper bars show P values from a two-tailed Student’s t test.