J Biol Chem. 2008 Nov 21;283(47):32821-30. doi: 10.1074/jbc.M805382200. Epub 2008 Aug 28.
TRPC3 controls agonist-stimulated intracellular Ca2+ release by mediating the interaction between inositol 1,4,5-trisphosphate receptor and RACK1.
Bandyopadhyay, B. C., Ong, H. L., Lockwich, T. P., Liu, X., Paria, B. C., Singh, B. B., Ambudkar, I. S.,
["Secretory Physiology Section, Molecular Physiology and Therapeutics Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892, USA."]
["Secretory Physiology Section, Molecular Physiology and Therapeutics Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892, USA."]
Activation of TRPC3 channels is concurrent with inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R)-mediated intracellular Ca(2+) release and associated with phosphatidylinositol 4,5-bisphosphate hydrolysis and recruitment to the plasma membrane. Here we report that interaction of TRPC3 with receptor for activated C-kinase-1 (RACK1) not only determines plasma membrane localization of the channel but also the interaction of IP(3)R with RACK1 and IP(3)-dependent intracellular Ca(2+) release. We show that TRPC3 interacts with RACK1 via N-terminal residues Glu-232, Asp-233, Glu-240, and Glu-244. Carbachol (CCh) stimulation of HEK293 cells expressing wild type TRPC3 induced recruitment of a ternary TRPC3-RACK1-IP(3)R complex and increased surface expression of TRPC3 and Ca(2+) entry. Mutation of the putative RACK1 binding sequence in TRPC3 disrupted plasma membrane localization of the channel. CCh-stimulated recruitment of TRPC3-RACK1-IP(3)R complex as well as increased surface expression of TRPC3 and receptor-operated Ca(2+) entry were also attenuated. Importantly, CCh-induced intracellular Ca(2+) release was significantly reduced as was RACK1-IP(3)R association without any change in thapsigargin-stimulated Ca(2+) release and entry. Knockdown of endogenous TRPC3 also decreased RACK1-IP(3)R association and decreased CCh-stimulated Ca(2+) entry. Furthermore, an oscillatory pattern of CCh-stimulated intracellular Ca(2+) release was seen in these cells compared with the more sustained pattern seen in control cells. Similar oscillatory pattern of Ca(2+) release was seen after CCh stimulation of cells expressing the TRPC3 mutant. Together these data demonstrate a novel role for TRPC3 in regulation of IP(3)R function. We suggest TRPC3 controls agonist-stimulated intracellular Ca(2+) release by mediating interaction between IP(3)R and RACK1.
PMID: 18755685

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Screening
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Experimental screening | Non-experimental screening | Reference | ||||||||
TRP channel construct | Interactor source | |||||||||
TRP channel | Interactor | Method | Species | Region | Species | Organ/tissue | Sample type | |||
TRPC3 |
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RACK1 | Yeast two-hybrid | Not specified | 1-341 | Human | Embroynic kidney | cDNA library | 18755685 |
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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 | ||||
TRPC3 |
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RACK1 | Co-immunoprecipitation | HEK293 | Not specified | Full-length | Not used | 18755685 | ||
TRPC3 |
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RACK1 | Co-immunoprecipitation | Rat brain lysates | 18755685 | |||||
TRPC3 |
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RACK1 | Co-immunofluorescence staining | HEK293 | Not specified | Full-length | Not used | 18755685 |
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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 | ||||
TRPC3 |
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RACK1 | Yeast two-hybrid | Not specified | 221-342 | Human | Not determined | 18755685 |
(
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
