J Biol Chem. 2005 Jun 3;280(22):21600-6. Epub 2005 Apr 17.
Molecular analysis of a store-operated and 2-acetyl-sn-glycerol-sensitive non-selective cation channel. Heteromeric assembly of TRPC1-TRPC3.
Liu, X., Bandyopadhyay, B. C., Singh, B. B., Groschner, K., Ambudkar, I. S.,
["Secretory Physiology Section, Gene Therapy and Therapeutics Branch, NIDCR, National Institutes of Health, DHHS, Bethesda, Maryland 20892, USA."]
["Secretory Physiology Section, Gene Therapy and Therapeutics Branch, NIDCR, National Institutes of Health, DHHS, Bethesda, Maryland 20892, USA."]
We have reported that internal Ca2+ store depletion in HSY cells stimulates a nonselective cation current which is distinct from I(CRAC) in RBL cells and TRPC1-dependent I(SOC) in HSG cells (Liu, X., Groschner, K., and Ambudkar, I. S. (2004) J. Membr. Biol. 200, 93-104). Here we have analyzed the molecular composition of this channel. Both thapsigargin (Tg) and 2-acetyl-sn-glycerol (OAG) stimulated similar non-selective cation currents and Ca2+ entry in HSY cells. The effects of Tg and OAG were not additive. HSY cells endogenously expressed TRPC1, TRPC3, and TRPC4 but not TRPC5 or TRPC6. Immunoprecipitation of TRPC1 pulled down TRPC3 but not TRPC4. Conversely, TRPC1 co-immunoprecipitated with TRPC3. Expression of antisense TRPC1 decreased (i) Tg- and OAG-stimulated currents and Ca2+ entry and (ii) the level of endogenous TRPC1 but not TRPC4. Antisense TRPC3 similarly reduced Ca2+ entry and endogenous TRPC3. Yeast two-hybrid analysis revealed an interaction between NTRPC1 and NTRPC3 (CTRPC1-CTRPC3, CTRPC3-CTRPC1, or CTRPC1-NTRPC3 did not interact), which was confirmed by glutathione S-transferase (GST) pull-down assays (GST-NTRPC3 pulled down TRPC1 and vice versa). Expression of NTRPC1 or NTRPC3 induced similar dominant suppression of Tg- and OAG-stimulated Ca2+ entry. NTRPC3 did not alter surface expression of TRPC1 or TRPC3 but disrupted TRPC1-TRPC3 association. In aggregate, our data demonstrate that TRPC1 and TRPC3 co-assemble, via N-terminal interactions, to form a heteromeric store-operated non-selective cation channel in HSY cells. Thus selective association between TRPCs generate distinct store-operated channels. Diversity of store-operated channels might be related to the physiology of the different cell types.
PMID: 15834157

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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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TRPC3 | Yeast two-hybrid | Not specified | 1-347 | Yeast | Not specified | 642-842 | Yeast | 15834157 |
TRPC1 |
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TRPC3 | Fusion protein-pull down assay | Not specified | 1-347 | E. coli | Not specified | Full-length | HEK293 lysates | 15834157 |
TRPC3 |
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TRPC1 | Yeast two-hybrid | Not specified | 642-842 | Yeast | Not specified | 642-842 | Yeast | 15834157 |
TRPC3 |
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TRPC1 | Fusion protein-pull down assay | Not specified | Full-length | HEK293 lysates | Not specified | 1-347 | E. coli | 15834157 |
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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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TRPC3 | Co-immunoprecipitation | HSY cell | 15834157 | |||||
TRPC3 |
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TRPC1 | Co-immunoprecipitation | HSY cell | 15834157 |
(
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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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TRPC3 | Yeast two-hybrid | Not specified | 1-73 | Not specified | 1-341 | 15834157 | ||
TRPC3 |
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TRPC1 | Yeast two-hybrid | Not specified | 1-341 | Not specified | 1-73 | 15834157 |
(
:
click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
