Fig. 1. Gating of TRPC1 by STIM1 is mediated by electrostatic interaction of TRPC1(⁶³⁹DD⁶⁴⁰) with STIM1(⁶⁸⁴KK⁶⁸⁵).

a. Alignment of the indicated C terminal sequences of TRPC channels. Highlighted in blue are mutations that have no effect of TRPC1 activity or its regulation by STIM1. Highlighted in red are the conserved negative charges (DD, EE or DE) in TRPCs. The TRP box is underlined. Also shown is the STIM1 K-rich domain. In this Fig., in Table 1 and in Fig. 2, as indicated by the bars, the cells were incubated in Na⁺-containing media, then stimulated with 100 µM carbachol and finally incubated in Na⁺-free, NMDG⁺-containing media to determine the zero current. b-e: cells were transfected with HA-TRPC1 and myc-STIM1 constructs. (b) shows the current of control cell transfected with YFP (black symbols) and cells transfected with wild type TRPC1(⁶³⁹DD⁶⁴⁰) and STIM1(⁶⁸⁴KK⁶⁸⁵) (red symbols), inhibition of TRPC1(⁶³⁹DD⁶⁴⁰) by STIM1(⁶⁸⁴AA⁶⁸⁵) (blue symbols) and a lack of current by TRPC1(⁶³⁹AA⁶⁴⁰) (green symbols). (c) shows the lack of current of TRPC1(⁶³⁹KK⁶⁴⁰) (red symbols) and its rescue by STIM1(⁶⁸⁴EE⁶⁸⁵) (blue symbols) and STIM1(⁶⁸⁴DD⁶⁸⁵) (green symbols). (d) shows the lack of current of TRPC1(⁶³⁹EE⁶⁴⁰) (red symbols), inhibition of wild-type TRPC1(⁶³⁹DD⁶⁴⁰) by STIM1(⁶⁸⁴RR⁶⁸⁵) (blue symbols) and rescue of TRPC1(⁶³⁹EE⁶⁴⁰) by STIM1(⁶⁸⁴RR⁶⁸⁵) (green symbols). (e) shows representative I/Vs of the indicated TRPC1+STIM1 combinations. In (f), HEK cells were co-transfected with wild-type HA-TRPC1 and the indicated myc-STIM1 mutants, biotinylated and used to determine effect of the mutants on co-IP of STIM1 and TRPC1 and total and surface expression of TRPC1. TRPC1 was detected with anti-HA, and STIM1 was detected with anti-myc. (g) the same experiment as in (e) except that effect of the STIM1 mutants was measured on expression of TRPC1(⁶³⁹KK⁶⁴⁰).