Critical roles of G _i/o proteins and phospholipase C-δ1 in the activation of receptor-operated TRPC4 channels

Abstract

Transient Receptor Potential Canonical (TRPC) proteins form nonselective cation channels commonly known to be activated downstream from receptors that signal through phospholipase C (PLC). Although TRPC3/C6/C7 can be directly activated by diacylglycerols produced by PLC breakdown of phosphatidylinositol 4,5-bisphosphate (PIP 2 ), the mechanism by which the PLC pathway activates TRPC4/C5 remains unclear. We show here that TRPC4 activation requires coincident stimulation of G i/o subgroup of G proteins and PLCδ, with a preference for PLCδ1 over PLCδ3, but not necessarily the PLCβ pathway commonly thought to be involved in receptor-operated TRPC activation. In HEK293 cells coexpressing TRPC4 and G i/o -coupled μ opioid receptor, μ agonist elicited currents biphasically, with an initial slow phase preceding a rapidly developing phase. The currents were dependent on intracellular Ca 2+ and PIP 2 . Reducing PIP 2 through phosphatases abolished the biphasic kinetics and increased the probability of channel activation by weak G i/o stimulation. In both HEK293 cells heterologously expressing TRPC4 and renal carcinoma-derived A-498 cells endogenously expressing TRPC4, channel activation was inhibited by knocking down PLCδ1 levels and almost completely eliminated by a dominant-negative PLCδ1 mutant and a constitutively active RhoA mutant. Conversely, the slow phase of G i/o -mediated TRPC4 activation was diminished by inhibiting RhoA or enhancing PLCδ function. Our data reveal an integrative mechanism of TRPC4 on detection of coincident G i/o , Ca 2+ , and PLC signaling, which is further modulated by the small GTPase RhoA. This mechanism is not shared with the closely related TRPC5, implicating unique roles of TRPC4 in signal integration in brain and other systems.Canonical TRPs (TRPC1-7) are the most homologous to the prototypical Drosophila TRP and are believed to be activated downstream of phospholipase C (PLC) (1). In both heterologous and native systems, stimulating PLCβ via the G q/11 subgroup of G proteins is commonly used to activate TRPC channels. Recent studies, however, also suggest a role for G i/o subgroup of G proteins in the activation of TRPC4/C5 (2-4).TRPC4 is implicated in the regulation of microvascular permeability (5), renal cancer proliferation (6, 7), neurotransmitter release (8), intestinal contraction and motility (9), neurite extension (10), epileptiform burst firing, and seizure-induced neurodegeneration (11). The channel mediates Na + and Ca 2+ influx, causing membrane depolarization and intracellular Ca 2+ concentration ([Ca 2+ ] i ) elevation, which in turn alter cell function (12). Although advances have been made in demonstrating TRPC4 channel activation under G i/o and/or PLC stimulation, as well as its dependence on [Ca 2+ ] i , a precise description of signaling events underlying the mechanism of TRPC4 activation remains elusive.Here, we distinguished the contributions of G q/11 and G i/o pathways to TRPC4 activation and uncovered a strict codependence on G i/o and PLC pathways. We focuse...

Dynamic NHERF interaction with TRPC4/5 proteins is required for channel gating by diacylglycerol

“…Interestingly, TRPC4/5 channels are not only activated via the G q/11 -protein-PLC pathway, but probably also subsequent to G i/o -coupled receptor activation (45,46). However, the mechanisms of G i/o -mediated TRPC4/5 activation are still not completely understood and might involve either direct G i/o protein interaction (45) or activation of PLCδ1 (46).…”

“…Interestingly, TRPC4/5 channels are not only activated via the G q/11 -protein-PLC pathway, but probably also subsequent to G i/o -coupled receptor activation (45,46). However, the mechanisms of G i/o -mediated TRPC4/5 activation are still not completely understood and might involve either direct G i/o protein interaction (45) or activation of PLCδ1 (46). It was speculated that PLC-mediated PIP 2 depletion might be involved in TRPC4 and -5 channel activation (11,12), even though there was initial evidence that TRPC5 channels might be DAG sensitive as well (10,14).…”

Regulator of G-protein signalling and GoLoco proteins suppress TRPC4 channel function via acting at Gαi/o

“…TRPC4 currents in response to activation of M 2 R using a muscarinic receptor agonist, CCh, were recorded by the whole-cell voltage clamp technique. In these cells, an endogenous G q/11 -coupled muscarinic receptor type, probably M 3 R, is also present and has been shown to facilitate G i/o -mediated TRPC4 activation [12]. To further help the development of TRPC4 currents, a Cs + -based internal solution was used throughout and the bath was replaced with a Cs + -rich external solution soon after the establishment of whole-cell configuration in the Na + -based normal Tyrode's solution before agonist application.…”

“…These activities are believed to be related to Ca 2+ and Na + influx mediated by TRPC4 channels, which triggers Ca 2+ signalling and membrane depolarization. To achieve strong control of the cellular function, the TRPC4 channels are tightly regulated through multiple levels of cross-talk among signalling networks [12]. …”

“…In particular, the activation of TRPC4 depends on activated Gα i/o subunits [18], which are usually produced through stimulation of a subset of G-protein-coupled receptors (GPCRs), known as G i/o -coupled receptors. To fully activate TRPC4, the stimulation by G i/o proteins also needs to coincide with Ca 2+ -dependent activation of PLCδ1, which forms a positive-feedback loop, allowing sustained TRPC4 activity [12]. …”