TRPC3 controls agonist-stimulated intracellular Ca²⁺ release by mediating the interaction between inositol 1,4,5-trisphosphate receptor and RACK

Activation of TRPC3 channels is concurrent with inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R)-mediated intracellular Ca²⁺ 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 IP3R with RACK1 and IP₃-dependent intracellular Ca²⁺ 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-IP₃R complex and increased surface expression of TRPC3 and Ca²⁺ entry. Mutation of the putative RACK1 binding sequence in TRPC3 disrupted plasma membrane localization of the channel. CCh-stimulated recruitment of TRPC3-RACK1-IP₃R complex as well as increased surface expression of TRPC3 and receptor-operated Ca²⁺ entry were also attenuated. Importantly, CCh-induced intracellular Ca²⁺ release was significantly reduced as was RACK1-IP₃R association without any change in thapsigargin-stimulated Ca²⁺ release and entry. Knockdown of endogenous TRPC3 also decreased RACK1-IP₃R association and decreased CCh-stimulated Ca²⁺ entry. Furthermore, an oscillatory pattern of CCh-stimulated intracellular Ca²⁺ release was seen in these cells compared with the more sustained pattern seen in control cells. Similar oscillatory pattern of Ca²⁺ release was seen after CCh stimulation of cells expressing the TRPC3 mutant. Together these data demonstrate a novel role for TRPC3 in regulation of IP₃R function. We suggest TRPC3 controls agonist-stimulated intracellular Ca²⁺ release by mediating interaction between IP₃R and RACK1.