Signaling Mechanism for Receptor-activated Canonical Transient Receptor Potential 3 (TRPC3) Channels

Abstract

Canonical transient receptor potential 3 (TRPC3) is a receptor-activated, calcium permeant, non-selective cation channel. TRPC3 has been shown to interact physically with the N-terminal domain of the inositol 1,4,5-trisphosphate receptor, consistent with a "conformational coupling" mechanism for its activation. Here we show that low concentrations of agonists that fail to produce levels of inositol 1,4,5-trisphosphate sufficient to induce Ca 2؉ release from intracellular stores substantially activate TRPC3. By several experimental approaches, we demonstrate that neither inositol 1,4,5-trisphosphate nor G proteins are required for TRPC3 activation. However, diacylglycerols were sufficient to activate TRPC3 in a protein kinase C-independent manner. Surface receptor agonists and exogenously applied diacylglycerols were not additive in activating TRPC3. In addition, inhibition of metabolism of diacylglycerol slowed the reversal of receptor-dependent TRPC3 activation. We conclude that receptor-mediated activation of phospholipase C in intact cells activates TRPC3 via diacylglycerol production, independently of G proteins, protein kinase C, or inositol 1,4,5-trisphosphate.In non-excitable cells, depletion of internal Ca 2ϩ stores activates store-operated channels (SOCs) 1 mediating calcium entry across the plasma membrane, a process known as capacitative calcium entry (CCE) (1-3). Despite considerable attention, the molecular identity of SOCs and their gating mechanism(s) remain unknown. Two major hypotheses for the mechanism of activation of CCE have been proposed. The first involves the release of a soluble factor from the endoplasmic reticulum (4, 5). The second, the "conformational coupling" model, proposes a direct interaction of inositol 1,4,5-trisphosphate (IP 3 ) receptors in the endoplasmic reticulum with SOCs in the plasma membrane (2, 6). The latter hypothesis has gained widespread support in the last few years.TRPC3 is a member of the canonical transient receptor potential (TRPC) family of Ca 2ϩ -permeant, non-selective cation channels (7) whose members have been hypothesized to form, or contribute to the formation of the elusive SOC channel (7-9). TRPC3 and its close structural homologs, TRPC6 and TRPC7, in many expression systems, including HEK293 cells, behave as receptor-operated cation channels (10 -12) that can be activated by exogenous application of diacylglycerols (DAG) independently of store-depletion (13, 14). However, Kiselyov et al. (15,16) reported that TRPC3 channels in excised patches from TRPC3-expressing HEK293 cells could be stimulated with IP 3 and IP 3 receptors. These authors suggested that TRPC3 activation involves interaction with IP 3 receptors in their ligandbound state, consistent with the conformational coupling hypothesis. Ma et al. (17) also proposed a requirement of IP 3 receptors for native SOC and TRPC3 channel activation based on experiments with the membrane-permeant IP 3 receptor antagonist, 2-aminoethoxydiphenyl borane (2-APB). Finally, Boulay et al. (18) provided bi...

Obligatory Role of Src Kinase in the Signaling Mechanism for TRPC3 Cation Channels

“…This they interpreted as being indicative of a coupling, lipase-independent role of PLC␥ in agonist-dependent cation entry regulation. However, our experiments showing the inability of BCR stimulation to activate TRPC3 when expressed in the PLC␥2-deficient DT40 cell line indicate a requirement for PLC-dependent DAG generation for channel activity (8,14,40). This is supported by the finding that stimulation of the Gq/PLC␤-coupled muscarinic receptor pathway or challenging the cells with OAG, efficiently activated TRPC3-mediated cation entry in PLC␥-KO DT40 cells, i.e.…”

“…Despite the existence of multiple potential phosphorylation sites in the TRPC3 primary sequence, there are very few studies specifically addressing the role of kinases in TRPC3 activity. Recent studies provided evidence that PKC (14,41) and protein kinase G negatively regulate TRPC3 (42). By using HEK293 cells stably expressing TRPC3 we found that the non-receptor tyrosine kinase Src is absolutely required for signaling to TRPC3, whether through receptor activation, or through application of OAG.…”

Thapsigargin activates Ca2+ entry both by store-dependent, STIM1/Orai1-mediated, and store-independent, TRPC3/PLC/PKC-mediated pathways in human endothelial cells

“…When exogenously expressed some TRPC channels displayed SOCE activity [12][13][14]. However, numerous studies also demonstrated that TRPC channels did not operate as SOCE channels, but rather are gated by second messengers like Ca 2+ or diacylglycerol (DAG; [15][16][17]). Hence, the involvement of TRPC channels in the SOCE mechanism is a controversial issue, and appears to dependent on the cell types investigated, and importantly on the expression level of the TRPC [14,18,19].…”

Homer 1 Mediates Store- and Inositol 1,4,5-Trisphosphate Receptor-dependent Translocation and Retrieval of TRPC3 to the Plasma Membrane

“…That at least one of the PM proteins interacting with IP 3 Rs is TRPC3 is shown in Fig. 4C, which shows that IP of TRPC3 co-immunoprecipitated IP 3 R3, the co-IP was reduced by cell stimulation, and the IP 3 Rs-TRPC3 complex was reformed by inhibition of carbachol-stimulated cells with atropine.Although several studies (17,19,28,45,46), including ours (18, 30), suggested that TRPC3 can function as SOCs, others concluded that this is not the case (15,47,48). However, the behavior of TRPC3 in native cells was examined before only in one study using pontine neurons from P3-P8 rats.…”