title: TRPC3 controls agonist-stimulated intracellular Ca2+ release by mediating the interaction between inositol 1,4,5-trisphosphate receptor and RACK1.
abstract: 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.
authors.affiliation: Secretory Physiology Section, Molecular Physiology and Therapeutics Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Bidhan C
authors.initials: BC
authors.lastname: Bandyopadhyay
authors.forename: Hwei L
authors.initials: HL
authors.lastname: Ong
authors.forename: Timothy P
authors.initials: TP
authors.lastname: Lockwich
authors.forename: Xibao
authors.initials: X
authors.lastname: Liu
authors.forename: Biman C
authors.initials: BC
authors.lastname: Paria
authors.forename: Brij B
authors.initials: BB
authors.lastname: Singh
authors.forename: Indu S
authors.initials: IS
authors.lastname: Ambudkar