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. 2010 Dec 3;285(49):38666-73.
doi: 10.1074/jbc.M110.155036. Epub 2010 Oct 6.

STIM1-dependent and STIM1-independent function of transient receptor potential canonical (TRPC) channels tunes their store-operated mode

Kyu Pil Lee  1 , Joseph P Yuan, Insuk So, Paul F Worley, Shmuel Muallem
Affiliations

STIM1-dependent and STIM1-independent function of transient receptor potential canonical (TRPC) channels tunes their store-operated mode

Kyu Pil Lee et al. J Biol Chem. .

Abstract

Ca(2+) influx by store-operated Ca(2+) channels is a key component of the receptor-evoked Ca(2+) signal. In all cells examined, transient receptor potential canonical (TRPC) channels mediate a significant portion of the receptor-stimulated Ca(2+) influx. Recent studies have revealed how STIM1 activates TRPC1 in response to store depletion; however, the role of STIM1 in TRPC channel activation by receptor stimulation is not fully understood. Here, we established mutants of TRPC channels that could not be activated by STIM1 but were activated by the "charge-swap" mutant STIM1(K684E,K685E). Significantly, WT but not mutant TRPC channels were inhibited by scavenging STIM1 with Orai1(R91W), indicating the STIM1 dependence and independence of WT and mutant TRPC channels, respectively. Importantly, mutant TRPC channels were robustly activated by receptor stimulation. Moreover, STIM1 and STIM1(K684E,K685E) reciprocally affected receptor-activated WT and mutant TRPC channels. Together, these findings indicate that TRPC channels can function as STIM1-dependent and STIM1-independent channels, which increases the versatility of TRPC channel function and their role in receptor-stimulated Ca(2+) influx.

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Figures

FIGURE 1.
FIGURE 1.
Interaction of STIM1 with wild-type and mutant TRPC channels. A, HEK cells were transfected with eYFP-STIM1 and HA-TRPC1 (C1), HA-TRPC1(D639K,D640K) (C1 DDKK), HA-TRPC4 (C4), and HA-TRPC4(E645K,E649K) (C1 EEKK) and were used to immunoprecipitate (IP) eYFP (STIM1) and blot for HA (TRPC1 and TRPC4). B, HEK cells were transfected with eYFP-STIM1 and FLAG-TRPC3 (C3), FLAG-TRPC3(D697K,D698K) (C3 DDKK), FLAG-TRPC6 (C6), and FLAG-TRPC6(E755K,E756K) (C6 EEKK) and were used to immunoprecipitate eYFP (STIM1) and blot for FLAG (TRPC3 and TRPC6). C, HEK cells were transfected with eYFP-STIM1 and FLAG-TRPC5 (C5) or FLAG-TRPC5(D651K,E652K) (C5 DEKK) and used to immunoprecipitate eYFP (STIM1) and blot for FLAG (TRPC5). Note that the DD/E-to-KK mutations had no effect on the interaction of STIM1 with the TRPC channels.
FIGURE 2.
FIGURE 2.
Inhibition of native SOC by the charge-swap TRPC mutants and rescue of SOC by STIM1(K684E,K685E). A, alignment of a C-terminal region of TRPC channels encompassing the conserved negative charges that are highlighted in boldface colored letters. The two negative charges were mutated to the positively charged lysines. B and C, HEK cells were transfected with GFP (black traces) and the following TRPC channel mutants: TRPC3(D697K,D698K) (TRPC3(DD/KK); blue traces and bars), TRPC4(E648K,E649K) (TRPC4(EE/KK); green traces and bars), TRPC5(D651K,E652K) (TRPC5(DE/KK); red traces and bars), and TRPC6(E755K,E756K) (TRPC6(EE/KK); magenta traces and bars). The cells were also transfected with empty vector (B) or STIM1(K684E,K685E) (S1(KK/EE); C). The Fura-2-loaded cells were incubated in Ca2+-free medium, treated with 25 μm cyclopiazonic acid (CPA) for 7.5 min to deplete the stores, and then exposed to medium containing 2 mm Ca2+ to assay Ca2+ influx. D, mean ± S.E. of at least 15 cells from two separate experiments.
FIGURE 3.
FIGURE 3.
STIM1-dependent and STIM1-independent function of receptor-activated TRPC channels. Black traces are for HEK cells transfected with GFP and M3 receptors only and show the native receptor-stimulated Ca2+ influx. HEK cells were also transfected with M3 receptors, the indicated TRPC channel mutants, and empty vector (green traces) with wild-type STIM1 (blue traces) or with STIM1(K684E,K685E) (S1(KK/EE); red traces). The cells in Ca2+-free medium were stimulated with 0.1 mm carbachol (Carb) and then incubated with medium containing carbachol and 2 mm Ca2+ to assay Ca2+ influx. A, TRPC3(D697K,D698K) (TRPC3(DD/KK), C3(DD/KK)); B, TRPC4(E648K,E649K) (TRPC4(EE/KK), C4(EE/KK)); C, TRPC5(D651K,E652K) (TRPC5(DE/KK), C5(DE/KK)); D, TRPC6(E755K,E756K) (TRPC6(EE/KK), C6(EE/KK)). The traces are the mean ± S.E. of at least five cells and are representative of three more experiments with similar results.
FIGURE 4.
FIGURE 4.
Scavenging STIM1 with Orai1(R91W) inhibits the TRPC3 but not TRPC3(D697K,D698K) current. HEK cells were transfected with M3 receptors; wild-type TRPC3 (A and B) or TRPC3(D697K,D698K) (TRPC3(DD/KK), C3(DD/KK); C and D); and empty vector (thin black traces), Orai1(R91W) (thick black traces), or Orai1(R91W) + STIM1 (gray traces). A and C, examples of the time dependence of the current; B and D, I/V at peak current; E, mean ± S.E. of current density at −80 mV in pA/pF from the indicated number of experiments. *, p < 0.01 of TRPC3 + Orai1(R91W) (O1(R91W)) relative to wild-type TRPC3. All others were not different from wild-type TRPC3.
FIGURE 5.
FIGURE 5.
Effect of STIM1 and STIM1(K684E,K685E) on TRPC3 and TRPC3(D697K,D698K) currents. HEK cells were transfected with M3 receptors; TRPC3 (A–C) or TRPC3(D697K,D698K) (TRPC3(DD/KK); D–F); and empty vector (thin black traces), wild-type STIM1 (gray traces), or STIM1(K684E,K685E) (STIM1(KK/EE); thick black traces). The Na+ current was measured as described under “Materials and Methods.” A and D, examples of the time dependence of the current; B and E, I/V at peak current; C and F, mean ± S.E. of current density in pA/pF from seven experiments. *, p < 0.05 or better relative to the respective controls.
FIGURE 6.
FIGURE 6.
Effect of Orai1(R91W), STIM1, and STIM1(K684E,K685E) on TRPC5 and TRPC5(D651K,E652K) currents. A–C, HEK cells were transfected with M3 receptors; TRPC5 (C5); and empty vector (thin black traces), Orai1(R91W) (thick black traces), or Orai1(R91W) + STIM1 (O1(R91W)+S1; gray traces). D–F, HEK cells were transfected with M3 receptors; TRPC5(D651K,E652K) (TRPC5(DE/KK), C5(DE/KK)); and empty vector (thin black traces), Orai1(R91W) (thin gray traces), STIM1 (thick gray traces), or STIM1(K684E,K685E) (S1(KK/EE); thick black traces). The Na+ current was measured as described under “Materials and Methods.” A and D, examples of the time dependence of the current; B and E, I/V at peak current; C and F, mean ± S.E. of current density in pA/pF from the indicated number of experiments. *, p < 0.01 relative to the respective controls.
FIGURE 7.
FIGURE 7.
Effect of wild-type STIM1 and STIM1(K684E,K685E) on the TRPC6(E755K,E756K) current. HEK cells were transfected with M3 receptors; TRPC6(E755K,E756K) (TRPC6(EE/KK)); and empty vector (thin black traces), STIM1 (thick black traces), or STIM1(K684E,K685E) (STIM1(KK/EE); gray traces). The Na+ current was measured as described under “Materials and Methods.” A, examples of the time dependence of the current; B, I/V at peak current; C, mean ± S.E. of current density in pA/pF from 15–19 experiments. *, p < 0.01.
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