Interaction of TRPC6 with TRPC4 mediates regulation of TRPC6 by STIM1. (a) Native STIM1 specifically coimmunoprecipitates TRPC4 and TRPC6. HEK cells were transfected with Flag–TRPC6 alone (C6), or C6 with HA–TRPC1 (C6+C1), HA–TRPC4 (C6+C4) or HA–TRPC5 (C6+C5) and the extracts were used to immunoprecipitate native STIM1 and probed for coimmunoprecipitation of TRPC6. (b) STIM1 is required for TRPC4–TRPC6 coimmunopreciption. HEK cells treated with scrambled (first and second lanes) or STIM1 siRNA (third lane) were transfected with Flag–TRPC6 (C6) or C6+HA–TRPC4 (C6+C4; last two lanes) and the extracts were used to immunoprecipitate TRPC4 and probe for coimmunoprecipitation of TRPC6. (c, d) TRPC4, but not TRPC1, is required for regulation of TRPC6 by STIM1. Current density was measured in cells transfected with TRPC6 alone or TRPC6 and ΔERM–STIM1D76A. TRPC6 and TRPC6+ΔERM–STIM1D76A were expressed in cells treated with scrambled siRNA (control; c, d), TRPC4 siRNA (c) or TRPC1 siRNA (d). The spontaneous, stimulated and total current densities are shown. The error bars indicate the mean ± s.e.m. of five experiments with TRPC4 siRNA and four experiments with TRPC1 siRNA.