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. 2009 Jun 1;587(Pt 11):2429-42.
doi: 10.1113/jphysiol.2009.172254. Epub 2009 Mar 30.

TRPC1 and STIM1 mediate capacitative Ca2+ entry in mouse pulmonary arterial smooth muscle cells

Lih Chyuan Ng  1 , Mary D McCormack, Judith A Airey, Cherie A Singer, Phillip S Keller, Xiao-Ming Shen, Joseph R Hume
Affiliations

TRPC1 and STIM1 mediate capacitative Ca2+ entry in mouse pulmonary arterial smooth muscle cells

Lih Chyuan Ng et al. J Physiol. .

Abstract

Previous studies in pulmonary arterial smooth muscle cells (PASMCs) showed that the TRPC1 channel mediates capacitative Ca(2+) entry (CCE), but the molecular signal(s) that activate TRPC1 in PASMCs remains unknown. The aim of the present study was to determine if TRPC1 mediates CCE through activation of STIM1 protein in mouse PASMCs. In primary cultured mouse PASMCs loaded with fura-2, cyclopiazonic acid (CPA) caused a transient followed by a sustained rise in intracellular Ca(2+) concentration ([Ca(2+)](i)). The transient but not the sustained rise in [Ca(2+)](i) was partially inhibited by nifedipine. In addition, CPA increased the rate of Mn(2+) quench of fura-2 fluorescence that was inhibited by SKF 96365, Ni(2+), La(3+) and Gd(3+), exhibiting pharmacological properties characteristic of CCE. The nifedipine-insensitive sustained rise in [Ca(2+)](i) and the increase in Mn(2+) quench of fura-2 fluorescence caused by CPA were both inhibited in cells pretreated with antibody raised against an extracellular epitope of TRPC1. Moreover, STIM1 siRNA reduced the rise in [Ca(2+)](i) and Mn(2+) quench of fura-2 fluorescence caused by CPA, whereas overexpression of STIM1 resulted in a marked increase in these responses. RT-PCR revealed TRPC1 and STIM1 mRNAs, and Western blot analysis identified TRPC1 and STIM1 proteins in mouse PASMCs. Furthermore, TRPC1 was found to co-immunoprecipitate with STIM1, and the precipitation level of TRPC1 was increased in cells subjected to store depletion. Taken together, store depletion causes activation of voltage-operated Ca(2+) entry and CCE. These data provide direct evidence that CCE is mediated by TRPC1 channel through activation of STIM1 in mouse PASMCs.

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Figures

Figure 1
Figure 1. Store depletion increases [Ca2+]i in mouse PASMCs
Store depletion increases [Ca2+]i in cultured mouse PASMCs. A, in control experiments, removal of external Ca2+ caused a decrease in fluorescence ratio below basal level. Subsequent addition of 2 mm Ca2+ caused a very small transient increase in fluorescence ratio, which slowly returned to basal levels (dotted line). B, when applied in Ca2+-free solution, depletion of intracellular stores with 10 μm CPA transiently elevated fura-2 fluorescence ratio, indicating Ca2+ release from the intracellular stores (arrow). Re-addition of 2 mm Ca2+ in the continued presence of CPA caused a transient followed by a sustained increase in fluorescence ratio. The transient but not the sustained component was reduced by 10 μm nifedipine (Nif). C, mean changes in [Ca2+]i compared to the resting [Ca2+]i in control (n= 22) and store depletion (n= 117) experiments. Filled bars indicate mean decrease in [Ca2+]i. Shaded and open bars indicate mean transient and sustained rise in [Ca2+]i, respectively. **P < 0.01 and ++P < 0.01, compared to the resting [Ca2+]i (ANOVA). D, bar graph showing the mean changes in transient and sustained rise in [Ca2+]i caused by store depletion after re-addition of 2 mm Ca2+, in the absence (n= 117) and presence (n= 296) of 10 μm nifedipine. *P < 0.05 (unpaired t test).
Figure 2
Figure 2. Store depletion increases the rate of Mn2+ quench of fura-2 fluorescence in mouse PASMCs
A, depletion of intracellular Ca2+ stores with 10 μm CPA increased the rate of Mn2+ quench of fura-2 fluorescence in the presence of 10 μm nifedipine. B–E, the increase in Mn2+-quench of fura-2 activated by store depletion was inhibited by 50 μm SKF 96365 (B), 500 μm Ni2+ (C), 100 μm La3+ (D) and 100 μm Gd3+ (E). F, bar graph showing percentage change in fura-2 quench rate after store depletion, in the absence (n= 212) and presence of SKF96365 (n= 92), Ni2+ (n= 51), La3+ (n= 67) and Gd3+ (n= 47). **P < 0.01 (ANOVA).
Figure 3
Figure 3. TRPC1 and STIM1 expression in mouse PASMCs
A, upper panel, RT-PCR products from cultured mouse PASMCs amplified using primers for mouse TRPC1 (516 bp) and β-actin (498 bp). Three separate RT-PCR reactions were performed in the presence (+) and absence (–) of reverse transcriptase (RT). Lower panel, TRPC1 protein and GAPDH were detected in cultured mouse PASMCs using Western blot analysis. A negative control was performed by pre-incubating TRPC1 antibody with the antigen peptide. Experiments were performed in 5 separate Western blot analyses. B, upper panel, RT-PCR products from cultured mouse PASMCs amplified using primers for mouse STIM1 (473 bp) and β-actin (498 bp). Data shown for β-actin and STIM1 expressions are from two separate part of a same gel. Three separate RT-PCR reactions were performed in the presence (+) and absence (–) of reverse transcriptase (RT). Lower panel, STIM1 protein and GAPDH were detected in cultured mouse PASMCs using Western blot analysis. Experiments were performed in 6 separate Western blot analyses.
Figure 4
Figure 4. TRPC1 mediates CCE in mouse PASMCs
A, TRPC1 antibody (1 : 100) inhibited the CPA-induced sustained but not transient increase in fura-2 fluorescence ratio in the presence of 10 μm nifedipine. B, bar graph showing mean changes in transient and sustained increase in [Ca2+]i caused by 10 μm CPA after re-addition of 2 mm Ca2+ in the presence of 10 μm nifedipine, in control cells (filled bars, TRPC1 Ab+peptide, n= 156) and in cells treated with TRPC1 antibody (open bars, n= 139). **P < 0.01 (unpaired t test). C, TRPC1 antibody (1 : 100) inhibited the increase in Mn2+ quench of fura-2 fluorescence caused by 10 μm CPA in the presence of 10 μm nifedipine. D, bar graph showing percentage change in fura-2 quench rate after store depletion in the presence of 10 μm nifedipine, in control cells (filled bar, TRPC1 Ab+peptide, n= 117) and in cells treated with TRPC1 antibody (open bar, n= 48). **P < 0.01 (unpaired t test).
Figure 5
Figure 5. siRNA knockdown of STIM1 reduces CCE in mouse PASMCs
A, STIM1 protein and GAPDH were detected in non-transfected mouse PASMCs and in PASMCs transfected with 200 nm scrambled siRNA (negative control). The expression of STIM1 but not GAPDH reduced significantly in cells transfected with 200 nm STIM1 siRNA. Experiments were performed in 3 separate Western blot analyses. B, siRNA knockdown of STIM1 reduced the CPA-induced transient and sustained increase in fura-2 fluorescence ratio in the presence of 10 μm nifedipine. C, bar graph showing mean changes in transient and sustained increase in [Ca2+]i caused by 10 μm CPA after re-addition of 2 mm Ca2+ in the presence of 10 μm nifedipine, in negative control cells (filled bars, n= 51) and in STIM1 siRNA-transfected cells (open bars, n= 84). **P < 0.01 (unpaired t test). D, siRNA knockdown of STIM1 reduced the increase in Mn2+ quench of fura-2 fluorescence caused by 10 μm CPA in the presence of 10 μm nifedipine. E, bar graph showing percentage change in fura-2 quench rate after store depletion in the presence of 10 μm nifedipine, in negative control cells (filled bar, n= 119) and in STIM1 siRNA-transfected cells (open bar, n= 107). **P < 0.01 (unpaired t test).
Figure 7
Figure 7. Overexpression of STIM1 enhances CCE in mouse PASMCs
A, overexpression of STIM1 enhanced the increase in CPA-induced transient and sustained rise in fura-2 fluorescence ratio in the presence of 10 μm nifedipine. B, bar graph showing mean changes in transient and sustained increase in [Ca2+]i caused by 10 μm CPA after re-addition of 2 mm Ca2+ in the presence of 10 μm nifedipine, in Ad-GFP cells (filled bars, n= 56) and in Ad-GFP-STIM1 cells (open bars, n= 75). **P < 0.01, *P < 0.05 (unpaired t test). C, overexpression of STIM1 enhanced the increase in Mn2+ quench of fura-2 fluorescence caused by 10 μm CPA in the presence of 10 μm nifedipine. D, bar graph showing percentage change in fura-2 quench rate after store depletion in the presence of 10 μm nifedipine, in Ad-GFP cells (n= 88) and in Ad-GFP-STIM1 cells (n= 103). **P < 0.01 (unpaired t test).
Figure 6
Figure 6. Overexpression of STIM1 in mouse PASMCs
A, STIM1 protein and GAPDH were detected in non-infected mouse PASMCs and in PASMCs infected with adenovirus containing GFP (Ad-GFP). The expression of STIM1 but not GAPDH increased markedly in cells infected with STIM1-GFP-adenovirus (Ad-GFP-STIM1). Experiments were performed in 3 separate Western blot analyses. B, bar graph showing mean changes in transient and sustained increase in [Ca2+]i caused by 10 μm CPA after re-addition of 2 mm Ca2+ in the presence of 10 μm nifedipine, in non-infected cells (filled bars, n= 61) and Ad-GFP cells (open bars, n= 62).
Figure 8
Figure 8. STIM1 associates with TRPC1 to mediate CCE in mouse PASMCs
A, in cultured mouse PASMCs overexpressing STIM1, TRPC1 antibody (1 : 100) reduced the CPA-induced transient and sustained increase in fura-2 fluorescence ratio in the presence of 10 μm nifedipine. B, bar graph showing mean changes in transient and sustained increase in [Ca2+]i caused by 10 μm CPA after re-addition of 2 mm Ca2+ in the presence of 10 μm nifedipine, in Ad-GFP-STIM1 cells under control condition (filled bars, TRPC1 Ab+peptide, n= 48) and in cells treated with TRPC1 antibody (open bars, n= 63). **P < 0.01 (unpaired t test). C, in cultured mouse PASMCs overexpressing STIM1, TRPC1 antibody (1 : 100) inhibited the increase in Mn2+ quench of fura-2 fluorescence caused by 10 μm CPA in the presence of 10 μm nifedipine. D, bar graph showing percentage change in fura-2 quench rate after store depletion in the presence of 10 μm nifedipine, in Ad-GFP-STIM1 cells under control condition (filled bar, TRPC1 Ab+peptide, n= 44) and in cells treated with TRPC1 antibody (open bar, n= 31). **P < 0.01 (unpaired t test).
Figure 9
Figure 9. TRPC1 interacts with STIM1 to form SOCs in mouse PASMCs
A, left panel, TRPC1 was detected in cultured mouse PASMCs in the absence and presence of store depletion. Right panel, bar graph showing expression levels of TRPC1 measured relative to GAPDH in control cells (denoted as 1, filled bar), and in cells subjected to store depletion (open bar). Data are means ±s.e.m. of 3 separate Western blot analyses. B, left panel, STIM1 was detected in cultured mouse PASMCs in the absence and presence of store depletion. Right panel, bar graph showing expression levels of STIM1 measured relative to GAPDH in control cells (denoted as 1, filled bar), and in cells subjected to store depletion (open bar). Data are means ±s.e.m. of 3 separate Western blot analyses. C, STIM1 co-immunoprecipitated TRPC1 in cultured mouse PASMCs in the absence and presence of store depletion. STIM1 was first immunoprecipitated (IP) with EXBIO STIM1 antibody (10 μg) and the blot was subsequently probed with BD Biosciences STIM1 antibody (WB, 1 : 100). The blot was then probed for co-IP of TRPC1 expression using TRPC1 antibody (WB, 1 : 100, Alomone). Experiments were performed in 3 separate co-IP procedures and Western blot analyses.
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