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.
Highlights
Activation of TRPC3 channels is concurrent with inositol 1,4,5-trisphosphate (IP3) receptor (IP3R)-mediated intracellular Ca2؉ release and associated with phosphatidylinositol 4,5bisphosphate hydrolysis and recruitment to the plasma membrane
Our data demonstrate that agonist stimulation of cells results in recruitment of a TRPC3-receptor for activated C-kinase-1 (RACK1)-IP3R ternary complex that is critical for both internal Ca2ϩ release via IP3R and Ca2ϩ entry via TRPC3
The data presented above suggest that interactions between RACK1, TRPC3, and IP3R generate a dynamically regulated ternary complex that determines Ca2ϩ entry via TRPC3 and internal Ca2ϩ release via IP3R
Summary
Cell Culture, Transfection, and Crude Membrane Preparation—Human embryonic kidney cell 293 (HEK293) cells culture, transfection, and lysis were performed as previously described (8 –10). We used the strategy employed previously to identify RACK1 binding domains in protein kinase C2, RGS11, and PDE4D5 [18, 19, 23, 24] to examine possible RACK1 binding sequences in N3-TRPC3 This approach revealed a plasmids isolated from the positive clones did not interact with consensus acidic repeat motif (Glu-232, Asp-233, Glu-240, and CTRPC3 (data not shown). In rat hippocampal neurons endogenous TRPC3 (Fig. 1C, the was lower (Fig. 2D, data with the deletion mutant was not green signal detected using anti-TRPC3 antibody) was localized shown). All the studies discussed below have been done with in the plasma membrane of the cell body and in the neurites mut-C3 either stably or transiently expressed in HEK293 cells where it co-localized with endogenous RACK1 (Fig. 1C, yellow (both conditions yielded similar data).
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