Mammalian homologues of Drosophila melanogaster transient receptor potential (TRP) are a large family of multimeric cation channels that act, or putatively act, as sensors of one or more chemical factor1,2. Major research objectives are the identification of endogenous activators and the determination of cellular and tissue functions of these novel channels. Here we show activation of TRPC5 homomultimeric and TRPC5-TRPC1 heteromultimeric channels3-5 by extracellular reduced thioredoxin acting by breaking a disulphide bridge in the predicted extracellular loop adjacent to the ion-selectivity filter of TRPC5. Thioredoxin is an endogenous redox protein with established intracellular functions, but it is also secreted and its extracellular targets are largely unknown6-9. Particularly high extracellular concentrations of thioredoxin are apparent in rheumatoid arthritis8,10-12, an inflammatory joint disease disabling millions of people worldwide13. We show that TRPC5 and TRPC1 are expressed in secretory fibroblast-like synoviocytes from patients with rheumatoid arthritis, endogenous TRPC5-TRPC1 channels of the cells are activated by reduced thioredoxin, and blockade of the channels enhances secretory activity and prevents suppression of secretion by thioredoxin. The data suggest a novel ion channel activation mechanism that couples extracellular thioredoxin to cell function.Striking activators of TRPC5 are extracellular lanthanide ions4,14,15. Effects of these ions depend on a glutamic acid residue at position 54314 in the predicted extracellular loop adjacent to the ion pore (Supplementary Fig. 1-2). This structural feature may, therefore, have functional importance in enabling extracellular factors to activate the channels. Because lanthanides are unlikely physiological activators we were interested in alternatives and developed a hypothesis based on amino acid sequence alignment which showed two cysteine residues near glutamic acid 543 that are conserved in TRPC5, TRPC4 and TRPC1 ( Supplementary Fig. 2), a subset of the seven TRPC channels1-5. TRPC5 and TRPC4 have similar functional properties4 and both form heteromultimers with TRPC13-5, a subunit that has weak targeting to the plasma membrane when expressed in isolation3,16. Pairs of cysteine residues may be covalently linked by a disulphide bridge that can be cleaved by reduction. We therefore applied the chemical reducing agent dithiothreitol (DTT) to HEK 293 cells expressing TRPC515,16. There was channel activation with the characteristic current-voltage relationship (I-V) of TRPC5 and block by 2-APB, an inhibitor of TRPC55 (Fig. 1a, b, d). Current recovered on wash-out of DTT (data not shown). Similarly, the membrane-impermeable disulphide reducing agent TCEP (Fig. 1c, d) activated TRPC5, whereas the thiol reagent MTSET had no effect (Fig. 1d). TRPC5 was inhibited by cadmium ions only after pre-treatment with DTT ( Fig. 1e, f), consistent with the metal ion acting by re-engaging cysteines17. Other TRP channels lacking the cysteine pair in a similar po...

Abstract-In a screen of potential lipid regulators of transient receptor potential (TRP) channels, we identified sphingosine-1-phosphate (S1P) as an activator of TRPC5. We explored the relevance to vascular biology because S1P is a key cardiovascular signaling molecule. TRPC5 is expressed in smooth muscle cells of human vein along with TRPC1, which forms a complex with TRPC5. Importantly, S1P also activates the TRPC5-TRPC1 heteromultimeric channel. Because TRPC channels are linked to neuronal growth cone extension, we considered a related concept for smooth muscle. We find S1P stimulates smooth muscle cell motility, and that this is inhibited by E3-targeted anti-TRPC5 antibody. Ion permeation involving TRPC5 is crucial because S1P-evoked motility is also suppressed by the channel blocker 2-aminoethoxydiphenyl borate or a TRPC5 ion-pore mutant. S1P acts on TRPC5 via two mechanisms, one extracellular and one intracellular, consistent with its bipolar signaling functions. The extracellular effect appears to have a primary role in S1P-evoked cell motility. The data suggest S1P sensing by TRPC5 calcium channel is a mechanism contributing to vascular smooth muscle adaptation. Key Words: vascular smooth muscle Ⅲ vein Ⅲ sphingosine-1-phosphate Ⅲ transient receptor potential Ⅲ calcium channel S phingosine-1-phosphate (S1P) has emerged as a major endogenous signaling phospholipid with diverse roles in yeast, plants, and mammals. 1 Proposed functions include the regulation of cell proliferation, migration, programmed death, and pathological processes including cancer, asthma, inflammation, and trauma. There has been particular interest in the role of S1P in the cardiovascular system, where it accumulates in atherosclerotic lesions and plays a role in ischemic preconditioning of the heart. 2-5 S1P is derived from the phosphorylation of sphingosine catalyzed by sphingosine kinase, sphingosine being from ceramide and ceramide from sphingomyelin, a constituent lipid of signaling microdomains of plasma membrane lipid rafts and caveolae. 3 S1P is detected in serum at almost 1 mol/L, although protein binding impacts on the available concentration and local concentrations may vary substantially. 6 S1P is quite unusual among signaling molecules in having separate intracellular and extracellular effects. 1,4,7,8 It affects vascular smooth muscle cell migration, 9,10 evokes contraction of rat mesenteric artery, 11 and slows pacemaker activity of the sino-atrial node of the heart. 12 The underlying mechanisms are only partially worked out, but vascular smooth muscle cells respond to S1P with transient followed by sustained elevation of the cytosolic Ca 2ϩ concentration, 10,11,13,14 whereas cardiac myocytes show activation of potassium current and S1P-evoked "Ca 2ϩ deregulation," depending on extracellular Ca 2ϩ . 12,15 Despite positive effects on Ca 2ϩ signaling, the molecular basis of a Ca 2ϩ channel stimulated by S1P is unknown. L-type voltage-gated Ca 2ϩ channels are inhibited by S1P. 12 The Drosophila transient receptor potential ...

Occlusive vascular disease is a widespread abnormality leading to lethal or debilitating outcomes such as myocardial infarction and stroke. It is part of atherosclerosis and is evoked by clinical procedures including angioplasty and grafting of saphenous vein in bypass surgery. A causative factor is the switch in smooth muscle cells to an invasive and proliferative mode, leading to neointimal hyperplasia. Here we reveal the importance to this process of TRPC1, a homolog of Drosophila transient receptor potential. Using 2 different in vivo models of vascular injury in rodents we show hyperplasic smooth muscle cells have upregulated TRPC1 associated with enhanced calcium entry and cell cycle activity. Neointimal smooth muscle cells after balloon angioplasty of pig coronary artery also express TRPC1. Furthermore, human vein samples obtained during coronary artery bypass graft surgery commonly exhibit an intimal structure containing smooth muscle cells that expressed more TRPC1 than the medial layer cells. Veins were organ cultured to allow growth of neointimal smooth muscle cells over a 2-week period. To explore the functional relevance of TRPC1, we used a specific E3-targeted antibody to TRPC1 and chemical blocker 2-aminoethoxydiphenyl borate. Both agents significantly reduced neointimal growth in human vein, as well as calcium entry and proliferation of smooth muscle cells in culture. The data suggest upregulated TRPC1 is a general feature of smooth muscle cells in occlusive vascular disease and that TRPC1 inhibitors have potential as protective agents against human vascular failure.

The channels are thought to have structural similarity to ␣-subunits of voltage-gated K ϩ channels, with intracellular amino and carboxy termini and four proteins required for coordination of a single ion pore. As with K ϩ channels, heteromultimerization confers greater diversity. However, unlike voltage-gated K ϩ channels, membrane depolarization is not the primary trigger for channel activity. Instead, chemical factors are considered to be primary stimuli. Details of the chemical sensing properties are becoming apparent and hold promise for revealing further complexity and novelty. In addition, important roles of TRP channels have emerged, including in sensation and cell survival, but we are far from a full appreciation of the purposes of these channels and, in some cases, there is relatively little understanding of TRP family members -one example being TRPM3.

TRPC calcium channels are emerging as a ubiquitous feature of vertebrate cells, but understanding of them is hampered by limited knowledge of the mechanisms of activation and identity of endogenous regulators. We have revealed that one of the TRPC channels, TRPC5, is strongly activated by common endogenous lysophospholipids including lysophosphatidylcholine (LPC) but, by contrast, not arachidonic acid. Although TRPC5 was stimulated by agonists at G-protein-coupled receptors, TRPC5 activation by LPC occurred downstream and independently of G-protein signaling. The effect was not due to the generation of reactive oxygen species or because of a detergent effect of LPC. LPC activated TRPC5 when applied to excised membrane patches and thus has a relatively direct action on the channel structure, either because of a phospholipid binding site on the channel or because of sensitivity of the channel to perturbation of the bilayer by certain lipids. Activation showed dependence on side-chain length and the chemical head-group. The data revealed a previously unrecognized lysophospholipid-sensing capability of TRPC5 that confers the property of a lipid ionotropic receptor.

Non-alcoholic fatty liver disease and steatohepatitis are highly associated with obesity and type 2 diabetes mellitus. Cotadutide, a GLP-1R/GcgR agonist, was shown to reduce blood glycemia, body weight and hepatic steatosis in patients with T2DM. Here, we demonstrate that the effects of Cotadutide to reduce body weight, food intake and improve glucose control are predominantly mediated through the GLP-1 signaling, while, its action on the liver to reduce lipid content, drive glycogen flux and improve mitochondrial turnover and function are directly mediated through Gcg signaling. This was confirmed by the identification of phosphorylation sites on key lipogenic and glucose metabolism enzymes in liver of mice treated with Cotadutide. Complementary metabolomic and transcriptomic analyses implicated lipogenic, fibrotic and inflammatory pathways, which are consistent with a unique therapeutic contribution of GcgR agonism by Cotadutide in vivo . Significantly, Cotadutide also alleviated fibrosis to a greater extent than Liraglutide or Obeticholic acid (OCA), despite adjusting dose to achieve similar weight loss in 2 preclinical mouse models of NASH. Thus Cotadutide, via direct hepatic (GcgR) and extra-hepatic (GLP-1R) effects, exerts multi-factorial improvement in liver function and is a promising therapeutic option for the treatment of steatohepatitis.

Nutrient excess, a major driver of obesity, diminishes hypothalamic responses to exogenously administered leptin, a critical hormone of energy balance. Here, we aimed to identify a physiological signal that arises from excess caloric intake and negatively controls hypothalamic leptin action. We found that deficiency of the gastric inhibitory polypeptide receptor ( Gipr ) for the gut-derived incretin hormone GIP protected against diet-induced neural leptin resistance. Furthermore, a centrally administered antibody that neutralizes GIPR had remarkable antiobesity effects in diet-induced obese mice, including reduced body weight and adiposity, and a decreased hypothalamic level of SOCS3, an inhibitor of leptin actions. In contrast, centrally administered GIP diminished hypothalamic sensitivity to leptin and increased hypothalamic levels of Socs3 . Finally, we show that GIP increased the active form of the small GTPase Rap1 in the brain and that its activation was required for the central actions of GIP. Altogether, our results identify GIPR/Rap1 signaling in the brain as a molecular pathway linking overnutrition to the control of neural leptin actions.

Robotic multiwell planar patch-clamp has become common in drug development and safety programs because it enables efficient and systematic testing of compounds against ion channels during voltage-clamp. It has not, however, been adopted significantly in other important areas of ion channel research, where conventional patch-clamp remains the favored method. Here, we show the wider potential of the multiwell approach with the ability for efficient intracellular solution exchange, describing protocols and success rates for recording from a range of native and primary mammalian cells derived from blood vessels, arthritic joints and the immune and central nervous systems. The protocol involves preparing a suspension of single cells to be dispensed robotically into 4-8 microfluidic chambers each containing a glass chip with a small aperture. Under automated control, giga-seals and whole-cell access are achieved followed by preprogrammed routines of voltage paradigms and fast extracellular or intracellular solution exchange. Recording from 48 chambers usually takes 1-6 h depending on the experimental design and yields 16-33 cell recordings.