Total flavone of flowers of Abelmoschus manihot (L.) Medic inhibits the expression of adhesion molecules in primary mesenteric arterial endothelial cells and ameliorates dextran sodium sulphate-induced ulcerative colitis in mice.

BACKGROUND: Flowers of Abelmoschus manihot (L.) medic (AM) is a traditional Chinese medicine used to treat chronic nephritis, nephrotic syndrome, diabetic nephropathy, and colonic inflammation. PURPOSE: This study aimed to explore the influence of the total flavone of AM flowers (TFA) on acute ulcerative colitis (UC) and the potential underlying mechanism. METHODS: Efficacy of TFA (30, 60, 120 mg/kg) on UC was evaluated in a dextran sodium sulphate (DSS)-induced colonic inflammatory mouse model by analyzing disease activity index (DAI), histopathological score, colon length, and cytokine expression. Expression levels of critical adhesion molecules and nuclear factor kappa B (NF-κB) were examined by qRT-PCR, Western blotting, or immunofluorescence labeling. Myeloperoxidase activity was examined using ELISA. In vitro THP-1 adhesion assay was used to evaluate monocyte adhesion. RESULTS: TFA significantly reduced DAI score, prevented colon shortening, and ameliorated histological injuries of colons in DSS-treated mice. TFA inhibited the expression of cytokines (IL-1ß and TNF-α) and adhesion molecules (ICAM-1, VCAM-1, and MAdCAM-1) in colon tissues of DSS mice. In vitro studies on mesenteric arterial endothelial cells (MAECs) showed that TFA attenuated TNF-α-induced upregulation of ICAM-1, VCAM-1, and MAdCAM-1, as well as THP-1 cell adhesion to MAECs. TFA also suppressed the phosphorylation and nuclear translocation of NF-κB in MAECs. CONCLUSION: TFA efficaciously ameliorates UC possibly by inhibiting monocyte adhesion through blocking TNF-α-induced NF-κB activation, which in turn suppresses the upregulation of adhesive molecules in colon endothelial cells. Inhibiting the expression of adhesion molecule in MAECs may represent a useful strategy for therapeutic development to treat UC, with TFA being a safe and efficacious therapeutic agent.

A gain-of-function TPC2 variant R210C increases affinity to PI(3,5)P2 and causes lysosome acidification and hypopigmentation.

Albinism is a group of inherited disorders mainly affecting skin, hair and eyes. Here we identify a de novo point mutation, p.R210C, in the TPCN2 gene which encodes Two Pore Channel 2 (TPC2) from a patient with albinism. TPC2 is an endolysosome and melanosome localized non-selective cation channel involved in regulating pigment production. Through inside-out recording of plasma membrane targeted TPC2 and direct recording of enlarged endolysosomal vacuoles, we reveal that the R210C mutant displays constitutive channel activation and markedly increased affinity to PI(3,5)P2. Mice harboring the homologous mutation, R194C, also exhibit hypopigmentation in the fur and skin, as well as less pigment and melanosomes in the retina in a dominant inheritance manner. Moreover, mouse embryonic fibroblasts carrying the R194C mutation show enlarged endolysosomes, enhanced lysosomal Ca2+ release and hyper-acidification. Our data suggest that R210C is a pathogenic gain-of-function TPC2 variant that underlies an unusual dominant type of albinism.

CAMK2/CaMKII activates MLKL in short-term starvation to facilitate autophagic flux.

MLKL (mixed lineage kinase domain like pseudokinase) is a well-known core component of necrosome that executes necroptotic cell death upon phosphorylation by RIPK3 (receptor interacting serine/threonine kinase 3). Recent studies also implicate a role of MLKL in endosomal trafficking, which is not always dependent on RIPK3. Using mouse Neuro-2a and L929 as well as human HEK293 and HT29 cells, we show here that MLKL is phosphorylated in response to serum and amino acid deprivation from the culture medium, in a manner that depends on CAMK2/CaMKII (calcium/calmodulin dependent protein kinase II) but not RIPK3. The starvation-induced increase in MLKL phosphorylation was accompanied by decreases in levels of lipidated MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta; LC3-II) and SQSTM1/p62 (sequestosome 1), markers of autophagosomes. These changes were prevented by disrupting either MLKL or CAMK2 by pharmacology and genetic manipulations. Moreover, disrupting MLKL or CAMK2 also inhibited the incorporation of LC3-II into autolysosomes, demonstrating a role of the CAMK2-MLKL pathway in facilitating autophagic flux during short-term starvation, in contrast to necroptosis which suppressed autophagic flux. Furthermore, unlike the necroptotic pathway, the starvation-evoked CAMK2-mediated MLKL phosphorylation protected cells from starvation-induced death. We propose that upon nutrient deprivation, MLKL is activated by CAMK2, which in turn facilitates membrane scission needed for autophagosome maturation, allowing the proper fusion of the autophagosome with lysosome and the subsequent substance degradation. This novel function is independent of RIPK3 and is not involved in necroptosis, implicating new roles for this pseudokinase in cell survival, signaling and metabolism.Abbreviations: CAMK2/CaMKII: calcium/calmodulin dependent protein kinase II; DIABLO/SMAC: direct inhibitor of apoptosis-binding protein with low pI/second mitochondria-derived activator of caspase; ECS: extracellular solution; ESCRT: endosomal sorting complexes required for transport; FBS: fetal bovine serum; GSK3B: glycogen synthase kinase 3 beta; HBSS: Hanks' balanced salt solution; KO: knockout; LC3-II: lipidated microtubule associated protein 1 light chain 3 beta; LDH: lactate dehydrogenase; MLKL: mixed lineage kinase domain like pseudokinase; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; N2a: Neuro-2a neuroblastoma; Nec-1: necrostatin-1; NSA: necrosulfonamide; PBS: phosphate-buffered saline; PI: propidium iodide; PK-hLC3: pHluorin-mKate2-human LC3; RIPK1: receptor interacting serine/threonine kinase 1; RIPK3: receptor interacting serine/threonine kinase 3; ROS: reactive oxygen species; RPS6KB1/S6K: ribosomal protein S6 kinase B1; shRNA: short hairpin RNA; siRNA: small interference RNA; SQSTM1/p62: sequestosome 1; TBS: Tris-buffered saline; TNF/TNF-α: tumor necrosis factor; TSZ, treatment with TNF + DIABLO mimetics + z-VAD-FMK.

Mechanisms of proton inhibition and sensitization of the cation channel TRPV3.

TRPV3 is a temperature-sensitive, nonselective cation channel expressed prominently in skin keratinocytes. TRPV3 plays important roles in hair morphogenesis and maintenance of epidermal barrier function. Gain-of-function mutations of TRPV3 have been found in both humans and rodents and are associated with hair loss, pruritus, and dermatitis. Here, we study the mechanisms of acid regulation of TRPV3 by using site-directed mutagenesis, fluorescent intracellular calcium measurement, and whole-cell patch-clamp recording techniques. We show that, whereas extracellular acid inhibits agonist-induced TRPV3 activation through an aspartate residue (D641) in the selectivity filter, intracellular protons sensitize the channel through cytoplasmic C-terminal glutamate and aspartate residues (E682, E689, and D727). Neutralization of the three C-terminal residues presensitizes the channel to agonist stimulation. Molecular dynamic simulations revealed that charge neutralization of the three C-terminal residues stabilized the sensitized channel conformation and enhanced the probability of α-helix formation in the linker between the S6 transmembrane segment and TRP domain. We conclude that acid inhibits TRPV3 function from the extracellular side but facilitates it from the intracellular side. These novel mechanisms of TRPV3 proton sensing can offer new insights into the role of TRPV3 in the regulation of epidermal barrier permeability and skin disorders under conditions of tissue acidosis.

TRPC4 as a coincident detector of Gi/o and Gq/11 signaling: mechanisms and pathophysiological implications.

TRPC channels are Ca2+-permeable nonselective cation channels activated downstream from phospholipase C (PLC). Although most TRPC channels can be activated by stimulating Gq/11-coupled receptors, TRPC4 requires simultaneous stimulation of Gi/o-coupled receptors, making it a perfect detector of coincident Gi/o and Gq/11 signaling. Evidence shows that activated Gαi/o proteins work together with PLCδ1 to induce robust TRPC4 activation and the process is accelerated by stimulation of other PLC isozymes, such as PLCß through Gq/11 proteins. Mechanistically, Gq/11-PLCß activation produces triggering proton and calcium signals to initiate self-propagating PLCδ1 activity, crucial for Gi/o-mediated TRPC4 function. Thus, TRPC4-containing channels are activated under conditions not only when coincident Gi/o and Gq/11 stimulation occurs, but also when Gi/o stimulation coincides with proton and Ca2+ signals. The resulting cytosolic Ca2+ rise and membrane depolarization switch the inhibitory Gi/o response to excitation. The conditions and implications of Gi/o-mediated TRPC4 activation in physiology and pathophysiology warrant further investigation.

Intracellular acidification facilitates receptor-operated TRPC4 activation through PLCδ1 in a Ca2+ -dependent manner.

KEY POINTS: Receptor-operated activation of TRPC4 cation channels requires Gi/o proteins and phospholipase-Cδ1 (PLCδ1) activation by intracellular Ca2+ . Concurrent stimulation of the Gq/11 pathway accelerates Gi/o activation of TRPC4, which is not mimicked by increasing cytosolic Ca2+ . The kinetic effect of Gq/11 was diminished by alkaline intracellular pH (pHi ) and increased pHi buffer capacity. Acidic pHi (6.75-6.25) together with the cytosolic Ca2+ rise accelerated Gi/o -mediated TRPC4 activation. Protons exert their facilitation effect through Ca2+ -dependent activation of PLCδ1. The data suggest that the Gq/11 -PLCß pathway facilitates Gi/o activation of TRPC4 through hydrolysing phosphatidylinositol 4,5-bisphosphate (PIP2 ) to produce the initial proton signal that triggers a self-propagating PLCδ1 activity supported by regenerative H+ and Ca2+ . The findings provide novel mechanistic insights into receptor-operated TRPC4 activation by coincident Gq/11 and Gi/o pathways and shed light on how aberrant activation of TRPC4 may occur under pathological conditions to cause cell damage. ABSTRACT: Transient Receptor Potential Canonical 4 (TRPC4) forms non-selective cation channels activated downstream from receptors that signal through G proteins. Our recent work suggests that TRPC4 channels are particularly coupled to pertussis toxin-sensitive Gi/o proteins, with a co-dependence on phospholipase-Cδ1 (PLCδ1). The Gi/o -mediated TRPC4 activation is dually dependent on and bimodally regulated by phosphatidylinositol 4,5-bisphosphate (PIP2 ), the substrate hydrolysed by PLC, and intracellular Ca2+ . As a byproduct of PLC-mediated PIP2 hydrolysis, protons have been shown to play an important role in the activation of Drosophila TRP channels. However, how intracellular pH affects mammalian TRPC channels remains obscure. Here, using patch-clamp recordings of HEK293 cells heterologously co-expressing mouse TRPC4ß and the Gi/o -coupled µ opioid receptor, we investigated the role of intracellular protons on Gi/o -mediated TRPC4 activation. We found that acidic cytosolic pH greatly accelerated the rate of TRPC4 activation without altering the maximal current density and this effect was dependent on intracellular Ca2+ elevation. However, protons did not accelerate channel activation by directly acting upon TRPC4. We additionally demonstrated that protons exert their effect through sensitization of PLCδ1 to Ca2+ , which in turn promotes PLCδ1 activity and further potentiates TRPC4 via a positive feedback mechanism. The mechanism elucidated here helps explain how Gi/o and Gq/11 co-stimulation induces a faster activation of TRPC4 than Gi/o activation alone and highlights again the critical role of PLCδ1 in TRPC4 gating.

Contribution of TRPC Channels in Neuronal Excitotoxicity Associated With Neurodegenerative Disease and Ischemic Stroke.

The seven canonical members of transient receptor potential (TRPC) proteins form cation channels that evoke membrane depolarization and intracellular calcium concentration ([Ca2+] i ) rise, which are not only important for regulating cell function but their deregulation can also lead to cell damage. Recent studies have implicated complex roles of TRPC channels in neurodegenerative diseases including ischemic stroke. Brain ischemia reduces oxygen and glucose supply to neurons, i.e., Oxygen and Glucose Deprivation (OGD), resulting in [Ca2+] i elevation, ion dyshomeostasis, and excitotoxicity, which are also common in many forms of neurodegenerative diseases. Although ionotropic glutamate receptors, e.g., N-methyl-D-aspartate receptors, are well established to play roles in excitotoxicity, the contribution of metabotropic glutamate receptors and their downstream effectors, i.e., TRPC channels, should not be neglected. Here, we summarize the current findings about contributions of TRPC channels in neurodegenerative diseases, with a focus on OGD-induced neuronal death and rodent models of cerebral ischemia/reperfusion. TRPC channels play both detrimental and protective roles to neurodegeneration depending on the TRPC subtype and specific pathological conditions involved. When illustrated the mechanisms by which TRPC channels are involved in neuronal survival or death seem differ greatly, implicating diverse and complex regulation. We provide our own data showing that TRPC1/C4/C5, especially TRPC4, may be generally detrimental in OGD and cerebral ischemia/reperfusion. We propose that although TRPC channels significantly contribute to ischemic neuronal death, detailed mechanisms and specific roles of TRPC subtypes in brain injury at different stages of ischemia/reperfusion and in different brain regions need to be carefully and systematically investigated.

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.

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

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 (PIP2), the mechanism by which the PLC pathway activates TRPC4/C5 remains unclear. We show here that TRPC4 activation requires coincident stimulation of Gi/o subgroup of G proteins and PLCδ, with a preference for PLCδ1 over PLCδ3, but not necessarily the PLCß pathway commonly thought to be involved in receptor-operated TRPC activation. In HEK293 cells coexpressing TRPC4 and Gi/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 PIP2. Reducing PIP2 through phosphatases abolished the biphasic kinetics and increased the probability of channel activation by weak Gi/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 Gi/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 Gi/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.

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.

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.