J Biol Chem. 2007 Mar 23;282(12):9105-16. Epub 2007 Jan 15.
Dynamic assembly of TRPC1-STIM1-Orai1 ternary complex is involved in store-operated calcium influx. Evidence for similarities in store-operated and calcium release-activated calcium channel components.
Ong, H. L., Cheng, K. T., Liu, X., Bandyopadhyay, B. C., Paria, B. C., Soboloff, J., Pani, B., Gwack, Y., Srikanth, S., Singh, B. B., Gill, D. L., Ambudkar, I. S.,
["Secretory Physiology Section, Gene Therapy and Therapeutics Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892, USA."]
["Secretory Physiology Section, Gene Therapy and Therapeutics Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892, USA."]
Store-operated calcium entry (SOCE) is a ubiquitous mechanism that is mediated by distinct SOC channels, ranging from the highly selective calcium release-activated Ca2+ (CRAC) channel in rat basophilic leukemia and other hematopoietic cells to relatively Ca2+-selective or non-selective SOC channels in other cells. Although the exact composition of these channels is not yet established, TRPC1 contributes to SOC channels and regulation of physiological function of a variety of cell types. Recently, Orai1 and STIM1 have been suggested to be sufficient for generating CRAC channels. Here we show that Orai1 and STIM1 are also required for TRPC1-SOC channels. Knockdown of TRPC1, Orai1, or STIM1 attenuated, whereas overexpression of TRPC1, but not Orai1 or STIM1, induced an increase in SOC entry and I(SOC) in human salivary gland cells. All three proteins were co-localized in the plasma membrane region of cells, and thapsigargin increased co-immunoprecipitation of TRPC1 with STIM1, and Orai1 in human salivary gland cells as well as dispersed mouse submandibular gland cells. In aggregate, the data presented here reveal that all three proteins are essential for generation of I(SOC) in these cells and that dynamic assembly of TRPC1-STIM1-Orai1 ternary complex is involved in activation of SOC channel in response to internal Ca2+ store depletion. Thus, these data suggest a common molecular basis for SOC and CRAC channels.
PMID: 17224452

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Validation: In vitro validation
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Assay with recombinant proteins | Reference | |||||||||
TRP channel construct | Interactor construct | |||||||||
TRP channel | Interactor | Method | Species | Region | Expression system | Species | Region | Expression system | ||
TRPC1 |
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STIM1 | Fusion protein-pull down assay | Not specified | 1-347 | E. coli | Not used | HSG cell lysates | 17224452 |
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)

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Validation: In vivo validation
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Assay with endogenous proteins | Assay with overexpressed proteins | Reference | ||||||||
Cell or tissue | Cell or tissue | TRP channel construct | Interactor construct | |||||||
TRP channel | Interactor | Method | Species | Region | Species | Region | ||||
TRPC1 |
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STIM1 | Co-immunofluorescence staining | HSG cell | Human | Full-length | Not specified | Full-length | 17224452 | |
TRPC1 |
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STIM1 | Co-immunoprecipitation | HEK293 | Not used | Not specified | Full-length | 17224452 | ||
TRPC1 |
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STIM1 | Co-immunoprecipitation | A7r5 | Not used | Not specified | Full-length | 17224452 |
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)

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Characterization
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Binding region mapping | Stoichiometry | Affinity (Kd) | Reference | |||||||
TRP channel | Interactor | |||||||||
TRP channel | Interactor | Method | Species | Region | Species | Region | ||||
TRPC1 |
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STIM1 | Fusion protein-pull down assay | Human | 1-347 | Human | Not determined | 17224452 |
(
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
