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. 2008 Nov 7;32(3):439-48.
doi: 10.1016/j.molcel.2008.09.020.

STIM1 gates TRPC channels, but not Orai1, by electrostatic interaction

Weizhong Zeng  1 , Joseph P Yuan, Min Seuk Kim, Young Jin Choi, Guo N Huang, Paul F Worley, Shmuel Muallem
Affiliations

STIM1 gates TRPC channels, but not Orai1, by electrostatic interaction

Weizhong Zeng et al. Mol Cell. .

Abstract

The receptor-evoked Ca(2+) signal includes activation of the store-operated channels (SOCs) TRPCs and the Orais. Although both are gated by STIM1, it is not known how STIM1 gates the channels and whether STIM1 gates the TRPCs and Orais by the same mechanism. Here, we report the molecular mechanism by which STIM1 gates TRPC1, which involves interaction between two conserved, negatively charged aspartates in TRPC1((639)DD(640)) with the positively charged STIM1((684)KK(685)) in STIM1 polybasic domain. Charge swapping and functional analysis revealed that exact orientation of the charges on TRPC1 and STIM1 are required, but all positive-negative charge combinations on TRPC1 and STIM1, except STIM1((684)EE(685))+TRPC1((639)RR(640)), are functional as long as they are reciprocal, indicating that STIM1 gates TRPC1 by intermolecular electrostatic interaction. Similar gating was observed with TRPC3((697)DD(698)). STIM1 gates Orai1 by a different mechanism since the polybasic and S/P domains of STIM1 are not required for activation of Orai1 by STIM1.

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Figures

Fig. 1
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).
Fig. 2
Fig. 2. Orientation of both STIM1(K684) and STIM1(K685) with TRPC1(D639) and TRPC1(D640) is required for TRPC1 channel activity
TRPC1 current was measured in HEK cells transfected with wild-type TRPC1 (○), TRPC1+STIM1(K684E) (Δ) or TRPC1+STIM1(K685E) (□) (a), TRPC1(D639K) alone (◊;), TRPC1(D639K)+STIM1(K684E) (Δ) or TRPC1(D639K)+STIM1(K685E) (□) (b), TRPC1(D640K) alone (◊), TRPC1(D640K)+STIM1(K684E) (Δ) or TRPC1(D640K)+STIM1(K685E) (□) (c). Panel (d) shows the mean±s.e.m. of 7 experiments with TRPC1 and 5 experiments with all other conditions. Current was also measured in HEK cells transfected with TRPC1 (○), TRPC1+STIM1(672EE673) (Δ), TRPC1(639KK640)+STIM1(672EE673) (□) or TRPC1 with STIM1X681 (▽) (e), TRPC1+STIM1 (○), TRPC1+STIM1(Ins596G) (Δ) or TRPC1+S1(Δ596L) (□) (f). The columns in (g) are the mean±sem of the indicated number of experiments.
Fig. 3
Fig. 3. Electrostatic interaction between TRPC1(639DD640) and STIM1(684KK685) is independent of receptor stimulation
a-b. HEK cells treated with TRPC1 siRNA (filled diamonds) or HEK cells treated with STIM1 siRNA (siSTIM1) (○) were transfected with 0.25 µg/ml cDNA of TRPC1 (C1(DD), a), TRPC1(639KK640) (C1(KK), b), STIM1CT (S1CT(KK), a) and STIM1CT(639EE640) (S1CT(EE), b). (c) shows the mean±s.e.m of 3 experiments at each condition; doted columns depict the spontaneous current and striped columns depict the current stimulated by 100 µM carbachol. (d) HEK cells were transfected with the TRPC1 mutants alone (first column) or with the indicated STIM1CT mutants. The protocol of panel (a) was used to measure the maximal TRPC1 current after carbachol stimulation. The results are the mean±s.e.m. of at least 4 experiments for each condition. Highlighted in black bold are combinations that were expected to have current but showed no current. Highlighted in bold and underlined are near maximal or maximal rescues. Panel (e) shows that carbachol enhances the interaction between TRPC1 and STIM1CT.
Fig. 4
Fig. 4. Electrostatic gating is independent of TRPC1-STIM1 expression levels, observed with TRPC3 and activation of TRPC1 by store depletion
In (a–c) HEK cells were treated with TRPC1 siRNA (siC1) and transfected with empty vector (black symbols), or 50 ng of siRNA protected (sm) TRPC1(639KK640) (blue symbols), smTRPC1+STIM1 (green symbols) or smTRPC1(639KK640)+ STIM1(684EE685) (red symbols). Note the low current level. (d–f) Current was measured in HEK cells transfected with the mutants TRPC3(697KK698) (black symbols) or TRPC3(697KK698) and STIM1(684EE685) (red symbols). (g–i) HEK cells transfected with TRPC1 (black, blue, red symbols) or TRPC1(639KK640)+STIM1(684EE685) (green symbols) were dialyzed with pipette solutions containing 70 nM Ca2+ buffered with 5 mM EGTA (black symbols) or 10 mM BAPTA with no added Ca2+ (blue, red and green symbols) and bathed in solutions containing 2 mM EGTA (black, green and red symbols) or 10 mM Ca2+ (blue symbols). (b, e, h) are the corresponding I/Vs, and the columns in (c, f, i) show the mean±s.s.m. of 4–5 experiments.
Fig. 5
Fig. 5. STIM1 lysine (K) and serine/proline (S/P) domains are not required for activation of Orai1
(a–d) HEK cells were transfected with Orai1 (O1) and STIM1(ΔK) (S1ΔK, green symbols), STIM1(684EE685) (S1(K/E), red symbols), STIM1(ΔS/P) (S1ΔS/P, yellow symbols) or STIM1(ΔS/PΔK) (S1ΔS/PΔK, blue symbols), and Orai1 current was measured in the presence of 10 mM Ca2+ or divalent-free media (DFM). (c) shows typical Icrac current I/V curves for Orai1 and all STIM1 constructs and (d) shows the mean±s.e.m currents from the indicated number of experiments. (e–g) HEK cells were transfected with 0.25 µg/ml TRPC1 and 0.25 μg/ml STIM1 (red symbols), S1ΔK (green symbols), S1ΔS/P (yellow symbols) or S1ΔS/PΔK (blue symbols), and the TRPC1 current was measured. (g) shows the mean±s.e.m currents in the indicated number of experiments. h) shows that the STIM1 constructs do not affect total or surface expression of TRPC1 or STIM1. TRPC1 was detected with anti-HA, and STIM1 was detected with anti-myc.
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References

    1. Ambudkar IS, Ong HL, Liu X, Bandyopadhyay B, Cheng KT. TRPC1: the link between functionally distinct store-operated calcium channels. Cell Calcium. 2007;42:213–223. - PubMed
    1. Berridge MJ, Bootman MD, Roderick HL. Calcium signalling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol. 2003;4:517–529. - PubMed
    1. Chang WC, Di Capite J, Singaravelu K, Nelson C, Halse V, Parekh AB. Local Ca2+ influx through Ca2+ release-activated Ca2+ (CRAC) channels stimulates production of an intracellular messenger and an intercellular pro-inflammatory signal. The Journal of biological chemistry. 2008;283:4622–4631. - PubMed
    1. Chang WC, Nelson C, Parekh AB. Ca2+ influx through CRAC channels activates cytosolic phospholipase A2, leukotriene C4 secretion, and expression of c-fos through ERK-dependent and -independent pathways in mast cells. Faseb J. 2006;20:2381–2383. - PubMed
    1. Chang WC, Parekh AB. Close functional coupling between Ca2+ release-activated Ca2+ channels, arachidonic acid release, and leukotriene C4 secretion. The Journal of biological chemistry. 2004;279:29994–29999. - PubMed

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