Molecular determinants mediating gating of Transient Receptor Potential Canonical (TRPC) channels by stromal interaction molecule 1 (STIM1)

Abstract

Transient receptor potential canonical (TRPC) channels mediate a critical part of the receptor-evoked Ca(2+) influx. TRPCs are gated open by the endoplasmic reticulum Ca(2+) sensor STIM1. Here we asked which stromal interaction molecule 1 (STIM1) and TRPC domains mediate the interaction between them and how this interaction is used to open the channels. We report that the STIM1 Orai1-activating region domain of STIM1 interacts with the TRPC channel coiled coil domains (CCDs) and that this interaction is essential for opening the channels by STIM1. Thus, disruption of the N-terminal (NT) CCDs by triple mutations eliminated TRPC surface localization and reduced binding of STIM1 to TRPC1 and TRPC5 while increasing binding to TRPC3 and TRPC6. Single mutations in TRPC1 NT or C-terminal (CT) CCDs reduced interaction and activation of TRPC1 by STIM1. Remarkably, single mutations in the TRPC3 NT CCD enhanced interaction and regulation by STIM1. Disruption in the TRPC3 CT CCD eliminated regulation by STIM1 and the enhanced interaction caused by NT CCD mutations. The NT CCD mutations converted TRPC3 from a TRPC1-dependent to a TRPC1-independent, STIM1-regulated channel. TRPC1 reduced the FRET between BFP-TRPC3 and TRPC3-YFP and between CFP-TRPC3-YFP upon stimulation. Accordingly, knockdown of TRPC1 made TRPC3 STIM1-independent. STIM1 dependence of TRPC3 was reconstituted by the TRPC1 CT CCD alone. Knockout of Trpc1 and Trpc3 similarly inhibited Ca(2+) influx, and inhibition of Trpc3 had no further effect on Ca(2+) influx in Trpc1(-/-) cells. Cell stimulation enhanced the formation of Trpc1-Stim1-Trpc3 complexes. These findings support a model in which the TRPC3 NT and CT CCDs interact to shield the CT CCD from interaction with STIM1. The TRPC1 CT CCD dissociates this interaction to allow the STIM1 Orai1-activating region within STIM1 access to the TRPC3 CT CCD and regulation of TRPC3 by STIM1. These studies provide evidence that the TRPC channel CCDs participate in channel gating.

Keywords: Calcium; Calcium Signaling; Gating; Gating Mechanism; STIM1; Signaling; TRP Channels.

FIGURE 1.

Interaction of TRPC channels with SOAR and effect of N-terminal triple point mutations on the interaction and on TRPC channel surface expression. a, SOAR interacts with TRPC1, TRPC4, and TRPC5 but not with TRPC3, TRPC6, and TRPC7. IP, immunoprecipitation. b and c, mutations of three residues in the NT coiled coil domains of the indicated TRPC channels eliminate their surface expression and reduce the interaction of TRPC1, TRPC4, and TRPC5 with SOAR but enhance the interaction of TRPC3 and TRPC6 with SOAR. The mutants used were as follows: TRPC1, Y224S/L227S/L238S; TRPC3, L241S/L245S/L248S; TRPC5, Y241S/L244S/L255S; and TRPC6, L300S/L304S/L307S. The blots in a–c are one of three similar experiments. d, the predicted N-terminal and C-terminal CCDs of TRPC1 and TRPC3, the N-terminal CCDs of TRPC5 and TRPC6, and the tested mutations in the hydrophobic region that make up the coiled coil-mediated interactions.

FIGURE 2.

Role of N- and C-terminal single point mutations in TRPC1 interaction with and regulation by STIM1. HEK cells were transfected with STIM1 and the TRPC1 wild type or mutants (a–d) and the M3 receptor (c and d). a and b show the effect of mutations in the N- or C-terminal coiled coil domains of TRPC1, respectively, on its interaction with STIM1. Anti-HA-HRP (TRPC1) and anti-GFP (STIM1) were used for detection of TRPC1 and STIM1. The blots are representative of three experiments. IP, immunoprecipitation. c and d, example of activated TRPC1 current trace and current/voltage relationship. Here and in all experiments, the channels were activated by 100 μm M3 receptor agonist carbachol. The current/voltage relationship was taken at the time marked by ■ and after subtraction of the leak current measured in the presence of NMDG. pA, picoampere; pF, picofarad. d, TRPC1-independent current was taken as the current measured with the channel-dead mutant TRPC1(F562A) (see Ref. 29). *, p < 0.05 or better with respect to TRPC1 current; #, p < 0.01 with respect to the current measured in the absence of STIM1. The results are the mean ± S.E. of the number of cells analyzed, which are listed in the columns.

FIGURE 3.

N- and C-terminal single point mutants increase TRPC3 interaction with SOAR and STIM1. HEK cells were transfected with the indicated TRPC3 mutants and SOAR (a) or STIM1 (b and c). Interaction was evaluated by coimmunoprecipitation (IP) (a–c) and surface localization by biotinylation. The blot in a is one of three experiments, and the blot in b is one of two experiments. c, effect of the C-terminal CCD TRPC3(I807S) mutation on the interaction of the wild type, TRPC3(L241S), and TRPC3(L245S) with STIM1. The blots are representative of four experiments. TRPC3 was detected with antibodies recognizing FLAG and STIM1 and recognizing GFP. The minor high molecular weight band in a may represent TRPC3 multimers, and the lower band in b is due to some STIM1 degradation.

FIGURE 4.

Effect of scavenging STIM1 by various amounts of Orai1(R91W) on current density of TRPC3, TRPC3(L245S), TRPC3(I807S), and TRPC3(I807S/L245S). a, HEK cells were treated with different concentrations of STIM1 siRNA for 72 h prior to evaluation of STIM1 abundance by Western blot analyses using anti-STIM1 antibodies in two separate experiments (IB). HEK cells were treated with scrambled (SCR) or the indicated concentrations of STIM1 siRNA. After 48 h, the cells were transfected with TRPC3 or TRPC3(L245S) and the M3 receptor, and, 24 h later, they were used to measure TRPC3 current. Each point is the mean ± S.E. of six to eight experiments. b–d, HEK cells were transfected with wild-type TRPC3 or TRPC3(L245S) and different amounts of Orai1(R91W). b, example current/voltage relationship in medium containing 2 mm Ca²⁺. The columns in c and d show the effect of Orai1(R91W) and Orai1(R91W)+STIM1 on current density. pA, picoampere; pF, picofarad. e, HEK cells were transfected with TRPC3(I807S) or TRPC3(L245S/I807S), the M3 receptor, and the indicated concentrations of Orai1(R91W). The results in c–e are the mean ± S.E. of the number of cells analyzed, which are listed in the columns. *, p < 0.05; #, p < 0.01 compared with the respective controls.

FIGURE 5.

Effect of TRPC1 C-terminal coiled coil domain on regulation of TRPC3 by STIM1. a and b, example current/voltage relationship in Ca²⁺-free medium and summary (b) examining the effect of scavenging STIM1 by 0.5 μg Orai1(R91W) on TRPC3(L245S) function in cells treated with scrambled (Scr) and TRPC1 siRNA. The results in b are the mean ± S.E. of nine cells. pA, picoampere; pF, picofarad; Con, control. c, the BFP-TRPC3 channel tagged at the N terminus with BFP and TRPC3-YFP tagged at the C terminus with YFP were transfected alone or together with AH-tagged TRPC1 in HeLa cells. After 24 h, the FRET between BFP and YFP was measured before and after stimulation with 100 μm ATP. d, the CFP-TRPC3-YFP channel tagged at both the N terminus with CFP and at the C terminus with YFP was transfected alone or together with AH-tagged TRPC1 in HeLa cells. After 24 h, the FRET between CFP and YFP was measured before and after stimulation with 100 μm ATP. The traces in c and d are the mean ± S.E. of the indicated number of analyzed cells. e, effect of TRPC1 C and N terminus CCDs on the interaction of TRPC3 and mutants with STIM1 and their interaction with STIM1 as analyzed by coimmunoprecipitation (IP). The blots are representative of three similar experiments. f, effect of the isolated TRPC1 CT CCD and NT CCD on the regulation of TRPC3 by STIM1 in cells treated with TRPC1 siRNA. The average carbachol-activated current densities are given as mean ± S.E. of the number of cells analyzed as listed in the columns.

FIGURE 6.

Role and interaction of endogenous Trpc1 and Trpc3 in salivary gland ducts. a, Ca²⁺ was measured in Fura-2-loaded salivary gland ducts isolated from wild-type, Trpc1^−/−, and Trpc3^−/− mice. The ducts were perfused with solutions containing 1.5 mm Ca²⁺ or 0.2 mm EGTA (Ca²⁺ free conditions) as indicated by the bars. b and c, Ca²⁺ was measured in salivary gland ducts from wild-type (b) and Trpc1^−/− mice (c) treated with 3 μm Trpc3 inhibitor pyrazole 10 (Pyr 10). The traces are the mean ± S.E. of at least five duct fragments analyzed in each experiment obtained in three (a) or two (b and c) experiments in ducts isolated from three (a) or two (b and c) mice of each phenotype. d, mutual coimmunoprecipitation (IP) of Trpc1/Stim1/Trpc3 in resting and stimulated salivary gland cells. Coimmunoprecipitation was tested in resting cells (R) and cells stimulated with the M3 receptor ligand carbachol (100 μm) and treated with the SERCA pump inhibitor CPA 25 μm (S). In the inputs, the heavy bands are not specific, and the bands representing the probed proteins are marked by arrows and identified by expression of the proteins in HEK cells as controls. The antibodies recognizing Trpc1 did not recognize the protein without enrichment to show the Trpc1 input. Combinations of monoclonal and polyclonal antibodies were used to immunoprecipitate Stim1 that coimmunoprecipitated Trpc1 and Trpc3 and to immunoprecipitate Trpc1 that coimmunoprecipitated Trpc3 and Stim1. The experiments are separate duplicates of resting and stimulated cells and are one of three similar experiments. IB, immunoblot.

FIGURE 7.

A model depicting gating of TRPC3 by STIM1. In the inactive state, the TRPC1 and TRPC3 N- and C-terminal CCDs interact to shield the STIM1 C-terminal CCD in both channels. TRPC1 can be directly activated by STIM1 when STIM1 interacts with a domain formed by the interacting TRPC1 NT and CT CCDs (right). Another conformation of TRPC1 is formed by receptor stimulation when TRPC1 is in complex with TRPC3 and serves to gate TRPC3. When in complex with TRPC3, receptor stimulation dissociates the TRPC1 CCDs, allowing the TRPC1 CT CCD to access the TRPC3 CCDs to dissociate them and expose the TRPC3 CT CCD so that it can interact with the STIM1 SOAR domain and present the polybasic domain to the conserved positive charges, which then open the channel.