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

Transient receptor potential canonical 4 (TRPC4) forms non-selective cation channels implicated in the regulation of diverse physiological functions. Previously, TRPC4 was shown to be activated by the Gi/o subgroup of heterotrimeric G-proteins involving Gαi/o, rather than Gßγ, subunits. Because the lifetime and availability of Gα-GTP are regulated by regulators of G-protein signalling (RGS) and Gαi/o-Loco (GoLoco) domain-containing proteins via their GTPase-activating protein (GAP) and guanine-nucleotide-dissociation inhibitor (GDI) functions respectively, we tested how RGS and GoLoco domain proteins affect TRPC4 currents activated via Gi/o-coupled receptors. Using whole-cell patch-clamp recordings, we show that both RGS and GoLoco proteins [RGS4, RGS6, RGS12, RGS14, LGN or activator of G-protein signalling 3 (AGS3)] suppress receptor-mediated TRPC4 activation without causing detectable basal current or altering surface expression of the channel protein. The inhibitory effects are dependent on the GAP and GoLoco domains and facilitated by enhancing membrane targeting of the GoLoco protein AGS3. In addition, RGS, but not GoLoco, proteins accelerate desensitization of receptor-activation evoked TRPC4 currents. The inhibitory effects of RGS and GoLoco domains are additive and are most prominent with RGS12 and RGS14, which contain both RGS and GoLoco domains. Our data support the notion that the Gα, but not Gßγ, arm of the Gi/o signalling is involved in TRPC4 activation and unveil new roles for RGS and GoLoco domain proteins in fine-tuning TRPC4 activities. The versatile and diverse functions of RGS and GoLoco proteins in regulating G-protein signalling may underlie the complexity of receptor-operated TRPC4 activation in various cell types under different conditions.

Close spatio-association of the transient receptor potential canonical 4 (TRPC4) channel with Gαi in TRPC4 activation process.

TPRC channels are Ca(2+)-permeable, nonselective cation channels that are activated by a wide variety of stimuli, including G protein-coupled receptors (GPCRs). TRPC4 is commonly assumed to be activated by Gq/phospholipase C-coupled receptors. However, the other molecular mechanisms by which Gα proteins regulate TRPC4 remain unclear. Here, we found that Gαi2 regulates TRPC4 activation by direct binding. To investigate this mechanism, we used whole patch clamp and fluorescence resonance energy transfer (FRET). We tagged an isoform of mTRPC4 and G protein with CFP and YFP, respectively, and transiently transfected cells with the FRET pair. The FRET efficiency between TRPC4ß-CFP and the constitutively active mutant form of Gαi2 was nearly 15% and was greater than that observed with wild-type Gαi2 (nearly 5%). Gßγ and the TRPC4 channel showed a FRET efficiency lower than 6%. In HEK293 cells transfected with the M2 muscarinic receptor, the application of carbachol increased the FRET efficiency between TRPC4ß-CFP and Gαi2(WT)-YFP from 4.7 ± 0.4% (n = 7) to 12.6 ± 1.4% (n = 7). We also found that the TRPC4 channel directly interacts with Gαi2, but not with Gαq, when the channel is open. We analyzed the calcium levels in HEK293 cells expressing the channels and Gαi2 or Gαq using the calcium indicator YC6.1 (Yellow Cameleon 6.1). In response to the muscarinic agonist carbachol, M2-, Gαi2-, and TRPC4-expressing cells showed a prolonged Ca(2+) influx compared with cells expressing only M2. Together, these data suggest that Gαi2 activates the TRPC4 channel by direct binding, which then induces Ca(2+) entry.

Isoform- and receptor-specific channel property of canonical transient receptor potential (TRPC)1/4 channels.

Transient receptor potential canonical (TRPC) 1, the first mammalian homologue of Drosophila trp gene, is distributed widely in mammalian cells and is involved in many physiological functions. TRPC1 is reported to be functional following heteromeric formation with other TRPC channels such as TRPC4 or TRPC5. It is known that the composition of this widely distributed TRPC1 is far from simple; functionality of such channels has been highly controversial. Furthermore, TRPC1 gene is known to have two splicing variants; one encodes long (TRPC1α) and the other encodes short (TRPC1ß) TRPC1 isoforms, respectively. In this study, we examined the functionality of TRPC1/4 channels using various activation systems. Gq/11-coupled receptor (e.g., M1 or M3 receptors) stimulation significantly increased TRPC1α/4 currents but induced mild activation of TRPC1ß/4. In addition, when expressed with TRPC4, TRPC1α acted as a pore-constituting subunit and not a ß ancillary subunit. Multimerized with TRPC4, TRPC1α also generated strong pore field strength. We also found that Gi/o-coupled receptor (e.g., M2 receptor) stimulation was insufficient to activate TRPC1α/4 and TRPC1ß/4 channels but selectively activated TRPC4 homomeric channels. These findings demonstrate that TRPC1/4 channel shows dynamic gating property depending on TRPC1 isoform subtypes and receptor stimulation system. Therefore, careful discrimination of the specificity of TRPC1 isoforms and upstream activation system is important in thorough understanding of TRPC1 and TRPC1/4 channels.

Activation of TRPC4ß by Gαi subunit increases Ca2+ selectivity and controls neurite morphogenesis in cultured hippocampal neuron.

The ubiquitous transient receptor potential canonical (TRPC) channels function as non-selective, Ca(2+)-permeable channels. TRPC channels are activated by stimulation of Gαq-PLC-coupled receptors. Here, we report that TRPC4/TRPC5 can be activated by Gαi. We studied the essential role of Gαi subunits in TRPC4 activation and investigated changes in ion selectivity and pore dilation of the TRPC4 channel elicited by the Gαi2 subunit. Activation of TRPC4 by Gαi2 increased Ca2+ permeability and Ca2+ influx through TRPC4 channels. Co-expression of the muscarinic receptor (M2) and TRPC4 in HEK293 cells induced TRPC4-mediated Ca2+ influx. Moreover, both TRPC4ß and the TRPC4ß-Gαi2 signaling complex induced inhibition of neurite growth and arborization in cultured hippocampal neurons. Cells treated with KN-93, a CaMKII inhibitor, prevented TRPC4- and TRPC4-Gαi2(Q205L)-mediated inhibition of neurite branching and growth. These findings indicate an essential role of Gαi proteins in TRPC4 activation and extend our knowledge of the functional role of TRPC4 in hippocampal neurons.

Leptin promotes K(ATP) channel trafficking by AMPK signaling in pancreatic ß-cells.

Leptin is a pivotal regulator of energy and glucose homeostasis, and defects in leptin signaling result in obesity and diabetes. The ATP-sensitive potassium (K(ATP)) channels couple glucose metabolism to insulin secretion in pancreatic ß-cells. In this study, we provide evidence that leptin modulates pancreatic ß-cell functions by promoting K(ATP) channel translocation to the plasma membrane via AMP-activated protein kinase (AMPK) signaling. K(ATP) channels were localized mostly to intracellular compartments of pancreatic ß-cells in the fed state and translocated to the plasma membrane in the fasted state. This process was defective in leptin-deficient ob/ob mice, but restored by leptin treatment. We discovered that the molecular mechanism of leptin-induced AMPK activation involves canonical transient receptor potential 4 and calcium/calmodulin-dependent protein kinase kinase ß. AMPK activation was dependent on both leptin and glucose concentrations, so at optimal concentrations of leptin, AMPK was activated sufficiently to induce K(ATP) channel trafficking and hyperpolarization of pancreatic ß-cells in a physiological range of fasting glucose levels. There was a close correlation between phospho-AMPK levels and ß-cell membrane potentials, suggesting that AMPK-dependent K(ATP) channel trafficking is a key mechanism for regulating ß-cell membrane potentials. Our results present a signaling pathway whereby leptin regulates glucose homeostasis by modulating ß-cell excitability.

An essential role of PI(4,5)P2 for maintaining the activity of the transient receptor potential canonical (TRPC)4ß.

The transient receptor potential canonical 4 (TRPC4) channel is a Ca(2+)-permeable nonselective cation channel in mammalian cells and mediates a number of cellular functions. Many studies show that TRPC channels are activated by stimulation of Gαq-phospholipase C (PLC)-coupled receptors. However, our previous study showed that the TRPC4 current was inhibited by co-expression of a constitutively active form of Gαq (Gαq (Q209L)). A shortage of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] in Gαq (Q209L) may be responsible for reduced TRPC4 activity. Here, we tested this hypothesis by using a rapamycin-inducible system that regulates PI(4,5)P2 acutely and specifically. Our results showed that the TRPC4ß current was reduced by inducible Gαq (Q209L), but not by the mutants with impaired binding ability to PLCß. Depletion of PI(4,5)P2 by inducing the inositol polyphosphate 5-phosphatase to HEK293 cells that express TRPC4ß led to an irreversible inhibition of TRPC4ß currents. In contrast, inducing phosphatidylinositol 4-phosphate 5-kinase or intracellular PI(4,5)P2 application did not activate the TRPC4ß current. Finally, we revealed that PI(4,5)P2 is important in delaying the desensitization of TRPC4ß. Taken together, we suggest that PI(4,5)P2 is not the activator of TRPC4ß activation, but it is still necessary for regulating TRPC4ß activation.

The roles of G proteins in the activation of TRPC4 and TRPC5 transient receptor potential channels.

TRPC4 and TRPC5 channels are important regulators of electrical excitability in both gastrointestinal myocytes and neurons. Much is known regarding the assembly and function of these channels including TRPC1 as a homotetramer or a heteromultimer and the roles that their interacting proteins play in controlling these events. Further, they are one of the best-studied targets of G protein-coupled receptors and growth factors in general and Gαq protein coupled receptor or epidermal growth factor in particular. However, our understanding of the roles of Gαi/o proteins on TRPC4/5 channels is still rudimentary. We discuss potential roles for Gαi/o proteins in channel activation in addition to their known role in cellular signaling.

Gs cascade regulates canonical transient receptor potential 5 (TRPC5) through cAMP mediated intracellular Ca2+ release and ion channel trafficking.

Canonical transient receptor potential (TRPC) channels are Ca(2+)-permeable, non-selective cation channels those are widely expressed in mammalian cells. Various molecules have been found to regulate TRPC both in vivo and in vitro, but it is unclear how heterotrimeric G proteins transmit external stimuli to regulate the activity of TRPC5. Here, we demonstrated that TRPC5 was potentiated by the Gα(s) regulatory pathway. Whole-cell TRPC5 current was significantly increased by ß-adrenergic receptor agonist, isoproterenol (ISO, 246±36%, n=6), an activator of the adenylate cyclase, forskolin (FSK, 273±6%, n=5), or a membrane permeable cAMP analogue, 8-Br-cAMP (251±63%, n=7). In addition, robust Ca(2+) transient induced by isoproterenol was observed utilizing a Ca(2+) imaging technique. When intracellular [Ca(2+)](i) was buffered to 50nM, cAMP-induced potentiation was attenuated. We also found that the Ca(2+) release is mediated by IP(3) since intracellular IP(3) infusion attenuated the potentiation of TRPC5 by Gα(s) cascade. Finally, we identified that the membrane localization of TRPC5 was significantly increased by ISO (155±17%, n=3), FSK (172±39%, n=3) or 8-Br-cAMP (216±59%, n=3). In conclusion, these results suggest that the Gα(s)-cAMP pathway potentiates the activity of TRPC5 via facilitating intracellular Ca(2+) dynamics and increasing channel trafficking to the plasma membrane.

Selective Gαi subunits as novel direct activators of transient receptor potential canonical (TRPC)4 and TRPC5 channels.

The ubiquitous transient receptor potential canonical (TRPC) channels function as non-selective, Ca(2+)-permeable channels and mediate numerous cellular functions. It is commonly assumed that TRPC channels are activated by stimulation of Gα(q)-PLC-coupled receptors. However, whether the Gα(q)-PLC pathway is the main regulator of TRPC4/5 channels and how other Gα proteins may regulate these channels are poorly understood. We previously reported that TRPC4/TRPC5 can be activated by Gα(i). In the current work, we found that Gα(i) subunits, rather than Gα(q), are the primary and direct activators of TRPC4 and TRPC5. We report a novel molecular mechanism in which TRPC4 is activated by several Gα(i) subunits, most prominently by Gα(i2), and TRPC5 is activated primarily by Gα(i3). Activation of Gα(i) by the muscarinic M2 receptors or expression of the constitutively active Gα(i) mutants equally and fully activates the channels. Moreover, both TRPC4 and TRPC5 are activated by direct interaction of their conserved C-terminal SESTD (SEC14-like and spectrin-type domains) with the Gα(i) subunits. Two amino acids (lysine 715 and arginine 716) of the TRPC4 C terminus were identified by structural modeling as mediating the interaction with Gα(i2). These findings indicate an essential role of Gα(i) proteins as novel activators for TRPC4/5 and reveal the molecular mechanism by which G-proteins activate the channels.

Molecular determinants of PKA-dependent inhibition of TRPC5 channel.

Canonical transient receptor potential (TRPC) channels are Ca(2+)-permeable, nonselective cation channels that are widely expressed in numerous cell types. Here, we demonstrate a new mechanism of TPRC isofom 5 (TRPC5) regulation, via cAMP signaling via Gα(s). Monovalent cation currents in human embryonic kidney-293 cells transfected with TRPC5 were induced by G protein activation with intracellular perfusion of GTPγS or by muscarinic stimulation. This current could be inhibited by a membrane-permeable analog of cAMP, 8-bromo-cAMP, by isoproterenol, by a constitutively active form of Gα(s) [Gα(s) (Q227L)], and by forskolin. These inhibitory effects were blocked by the protein kinase A (PKA) inhibitors, KT-5720 and H-89, as well as by two point mutations at consensus PKA phosphorylation sites on TRPC5 (S794A and S796A). Surface expression of several mutated versions of TRPC5, quantified using surface biotinylation, were not affected by Gα(s) (Q227L), suggesting that trafficking of this channel does not underlie the regulation we report. This mechanism of inhibition was also found to be important for the closely related channel, TRPC4, in particular for TRPC4α, although TRPC4ß was also affected. However, this form of regulation was not found to be involved in TRPC6 and transient receptor potential vanilloid 6 function. In murine intestinal smooth muscle cells, muscarinic stimulation-induced cation currents were mediated by TRPC4 (>80%) and TRPC6. In murine intestinal smooth muscle cells, 8-bromo-cAMP, adrenaline, and isoproterenol decreased nonselective cation currents activated by muscarinic stimulation or GTPγS. Together, these results suggest that TRPC5 is directly phosphorylated by G(s)/cAMP/PKA at positions S794 and S796. This mechanism may be physiologically important in visceral tissues, where muscarinic receptor and ß(2)-adrenergic receptor are involved in the relaxation and contraction of smooth muscles.

Functional characteristics of TRPC4 channels expressed in HEK 293 cells.

The classical type of transient receptor potential (TRPC) channel is a molecular candidate for Ca(2+)-permeable cation channels in mammalian cells. Because TRPC4 and TRPC5 belong to the same subfamily of TRPC, they have been assumed to have the same physiological properties. However, we found that TRPC4 had its own functional characteristics different from those of TRPC5. TRPC4 channels had no constitutive activity and were activated by muscarinic stimulation only when a muscarinic receptor was co-expressed with TRPC4 in human embryonic kidney (HEK) cells. Endogenous muscarinic receptor appeared not to interact with TRPC4. TPRC4 activation by GTPgammaS was not desensitized. TPRC4 activation by GTPgammaS was not inhibited by either Rho kinase inhibitor or MLCK inhibitor. TRPC4 was sensitive to external pH with pK (a) of 7.3. Finally, TPRC4 activation by GTPgammaS was inhibited by the calmodulin inhibitor W-7. We conclude that TRPC4 and TRPC5 have different properties and their own physiological roles.

The specific activation of TRPC4 by Gi protein subtype.

The classical type of transient receptor potential channel (TRPC) is a molecular candidate for Ca(2+)-permeable cation channels in mammalian cells. Especially, TRPC4 has the similar properties to Ca(2+)-permeable nonselective cation channels (NSCCs) activated by muscarinic stimulation in visceral smooth muscles. In visceral smooth muscles, NSCCs activated by muscarinic stimulation were blocked by anti-Galphai/o antibodies. However, there is still no report which Galpha proteins are involved in the activation process of TRPC4. Among Galpha proteins, only Galphai protein can activate TRPC4 channel. The activation effect of Galphai was specific for TRPC4 because Galphai has no activation effect on TRPC5, TRPC6 and TRPV6. Coexpression with muscarinic receptor M2 induced TRPC4 current activation by muscarinic stimulation with carbachol, which was inhibited by pertussis toxin. These results suggest that Galphai is involved specifically in the activation of TRPC4.

Molecular determinant of sensing extracellular pH in classical transient receptor potential channel 5.

The classical transient receptor potential channel 5 (TRPC5) is a molecular candidate for nonselective cation channel (NSCC) activated by muscarinic receptor stimulation whereas extracellular pH inhibits or enhances NSCC activated by muscarinic receptor stimulation depending on extracellular cation compositions in native tissues. We investigated the effect of extracellular pH on TRPC5 and determined amino acid residues responsible for sensing extracellular pH. Extracellular acidosis inhibits TRPC5 with pKa of 6.24. Under 50 mM intracellular HEPES buffer condition, extracellular acidosis inhibits TRPC5 with pKa of 5.40. We changed titratable amino acids (C, D, E, H, K, R, Y) to nontitratable amino acids (A, N, Q, N, N, N, F) within pore region between transmembrane segments 5 and 6 in order to determine the residues sensing extracellular pH. Glutamate (at the position 543, 595, and 598), aspartate (at the position 548) and lysine (at the position 554) were responsible for sensing extracellular pH. The effect of extracellular pH in TRPC5 was also dependent on the composition of extracellular monovalent cations. In conclusion, TRPC5 is a molecular candidate for NSCC activated by muscarinic receptor stimulation, has glutamate amino acid residues responsible for sensing extracellular pH, and has a unique gating property depending on the composition of extracellular monovalent cations.