TRPC3 controls agonist-stimulated intracellular Ca2+ release by mediating the interaction between inositol 1,4,5-trisphosphate receptor and RACK1.

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.

Analysis of TRPC3-interacting proteins by tandem mass spectrometry.

Mammalian transient receptor potential canonical (TRPC) channels are a family of nonspecific cation channels that are activated in response to stimulation of phospholipase C (PLC)-dependent hydrolysis of the membrane lipid phosphatidylinositol 4,5-bisphosphate. Despite extensive studies, the mechanism(s) involved in regulation of mammalian TRPC channels remains unknown. Presence of various protein-interacting domains in TRPC channels have led to the suggestion that they associate with proteins that are involved in their function and regulation. This study was directed toward identifying the proteins associated with native TRPC3 using a shotgun proteomic approach. Anti-TRPC3 antibody was used to immunoprecipitate TRPC3 from solubilized rat brain crude membranes under conditions that allow retention of TRPC3 function. Proteins in the TRPC3 (using anti-TRPC3 antibody) and control (using rabbit IgG) immunoprecipitates were separated by SDS-PAGE, the gel was sectioned, and the resolved proteins were digested by trypsin in situ. After extraction of the peptides, the peptides were separated by HPLC and sequences derived by MS/MS. Analysis of the data revealed 64 specific TRPC3-associated proteins which can be grouped in terms of their cellular location and involvement in specific cellular function. Many of the proteins identified have been previously reported as TRPC3-regulatory proteins, such as IP3Rs and vesicle trafficking proteins. In addition, we report novel putative TRPC3-interacting proteins, including those involved in protein endocytosis and neuronal growth. To our knowledge, this is the first comprehensive proteomic analysis of a native TRPC channel. These data reveal potential TRPC3 regulatory proteins and provide novel insights of the mechanism(s) regulating TRPC3 channels as well as the possible cellular functions where the channel might be involved.

Functional organization of TRPC-Ca2+ channels and regulation of calcium microdomains.

TRP family of proteins are components of unique cation channels that are activated in response to diverse stimuli ranging from growth factor and neurotransmitter stimulation of plasma membrane receptors to a variety of chemical and sensory signals. This review will focus on members of the TRPC sub-family (TRPC1-TRPC7) which currently appear to be the strongest candidates for the enigmatic Ca(2+) influx channels that are activated in response to stimulation of plasma membrane receptors which result in phosphatidyl inositol-(4,5)-bisphosphate (PIP(2)) hydrolysis, generation of IP(3) and DAG, and IP(3)-induced Ca(2+) release from the intracellular Ca(2+) store via inositol trisphosphate receptor (IP(3)R). Homomeric or selective heteromeric interactions between TRPC monomers generate distinct channels that contribute to store-operated as well as store-independent Ca(2+) entry mechanisms. The former is regulated by the emptying/refilling of internal Ca(2+) store(s) while the latter depends on PIP(2) hydrolysis (due to changes in PIP(2) per se or an increase in diacylglycerol, DAG). Although the exact physiological function of TRPC channels and how they are regulated has not yet been conclusively established, it is clear that a variety of cellular functions are controlled by Ca(2+) entry via these channels. Thus, it is critical to understand how cells coordinate the regulation of diverse TRPC channels to elicit specific physiological functions. It is now well established that segregation of TRPC channels mediated by interactions with signaling and scaffolding proteins, determines their localization and regulation in functionally distinct cellular domains. Furthermore, both protein and lipid components of intracellular and plasma membranes contribute to the organization of these microdomains. Such organization serves as a platform for the generation of spatially and temporally dictated [Ca(2+)](i) signals which are critical for precise control of downstream cellular functions.

VAMP2-dependent exocytosis regulates plasma membrane insertion of TRPC3 channels and contributes to agonist-stimulated Ca2+ influx.

The mechanism(s) involved in agonist-stimulation of TRPC3 channels is not yet known. Here we demonstrate that TRPC3-N terminus interacts with VAMP2 and alphaSNAP. Further, endogenous and exogenously expressed TRPC3 colocalized and coimmunoprecipitated with SNARE proteins in neuronal and epithelial cells. Imaging of GFP-TRPC3 revealed its localization in the plasma membrane region and in mobile intracellular vesicles. Recovery of TRPC3-GFP fluorescence after photobleaching of the plasma membrane region was decreased by brefeldin-A or BAPTA-AM. Cleavage of VAMP2 with tetanus toxin (TeNT) did not prevent delivery of TRPC3 to the plasma membrane region but reduced its surface expression. TeNT also decreased carbachol and OAG, but not thapsigargin, stimulated Ca2+ influx. Importantly, carbachol, not thapsigargin, increased surface expression of TRPC3 that was attenuated by TeNT and not by BAPTA. In aggregate, these data suggest that VAMP2-dependent exocytosis regulates plasma membrane insertion of TRPC3 channels and contributes to carbachol-stimulation of Ca2+ influx.

Plasma membrane localization of TRPC channels: role of caveolar lipid rafts.

GPCR-mediated activation of the Ca2+ signalling cascade leads to stimulation of Ca2+ influx into non-excitable cells. Both store-dependent and independent channels likely contribute towards this Ca2+ influx. However, the identity of the channels and exact mechanism by which they are activated remains elusive. The TRPC family of proteins has been proposed as molecular components of these channels. Studies from our laboratory and others have shown that mammalian TRPC proteins are assembled in a multiprotein complex that includes various key Ca2+ signalling proteins. However, relatively little is known regarding the mechanisms involved in the assembly of the TRPC channel complex in the plasma membrane. We have reported that TRPC1 and TRPC3 signalling complexes are associated with caveolar lipid raft domains (LRDs) in the plasma membrane. Recently we have examined the role of caveolin-1 in the regulation of TRPC channels and store-operated Ca2+ entry (SOCE). Based on our studies, we suggest that (1) caveolin 1 has a potentially critical role in the localization of TRPC channels plasma membrane caveolar LRDs, and (2) the molecular architecture of caveolae can facilitate intramolecular interactions between TRPC channels and associated proteins that are involved in activation and/or inactivation of SOCE.