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. Author manuscript; available in PMC: 2009 Jun 19.
Published in final edited form as: Nat Cell Biol. 2007 May 7;9(6):636–645. doi: 10.1038/ncb1590

Figure 3.

Figure 3

Regulation of TRPC3 by STIM1 requires TRPC1. (a) Rescue of TRPC3 activity by cytosolic STIM1. HEK cells treated with STIM1 siRNA were transfected with HA–TRPC1 (C1) alone, C1 and YFP–STIM1 (C1+Y–S), Flag–TRPC3 alone (C3) or C3 and YFP–STIM1 (C3+Y–S) and receptor-stimulated current density was measured. The error bars indicate the mean ± s.e.m. of four experiments. (b) TRPC1 is required for regulation of TRPC3 by STIM1. The insert shows coimmunoprecipitation of TRPC3 with native TRPC1 from cells treated with scrambled or STIM1 siRNA and transfected with Flag–TRPC3. The native TRPC1 was immunoprecipitated with the Sigma anti-TRPC1 and probed for coimmunoprecipitation of TRPC3 expressed at low levels. Current density was measured in cells transfected with TRPC3 alone or TRPC3+ΔERM–STIM1D76A. Expression was in cells treated with scrambled siRNA (control) and TRPC1 siRNA or TRPC4 siRNA. The yellow columns represent the percent residual current recorded for TRPC3+ΔERM–STIM1D76A in scrambled siRNA cells versus cells treated with TRPC1 siRNA. The error bars indicate the mean ± s.e.m. of five experiments with TRPC1 siRNA and four experiments with TRPC4 siRNA. (c) STIM1 is not obligatory for TRPC3 activity. Control and STIM1 siRNA-treated cells were transfected with low and high TRPC3. The blot shows the level of native and expressed TRPC3. For native and expressed TRPC3 the blot was probed with anti-TRPC3 and developed for 15 s and 5 s, respectively. Cells were stimulated with carbachol to record the TRPC3 current. Note that overexpression of TRPC3 overcomes the dependence of TRPC3 activation on STIM1. The error bars indicate the mean ± s.e.m. of five experiments. (d) Faster activation of heteromultimeric TRPC3. HEK cells transfected with TRPC1 or TRPC3 were repeatedly stimulated with carbachol. Example traces and the average half time for activation of TRPC1 and TRPC3 by the fist and second stimulation are shown. The TRPC3 current is approximately twice as large as the TRPC1 current, but it was rescaled for illustration purposes. The error bars indicate the mean ± s.e.m. of four experiments. Uncropped images of the blots are shown in the Supplementary Information, Fig. S1.