Jacqueline Naylor

TRPC channel activation by extracellular thioredoxin

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 factor. Major research objectives are the identification of endogenous activators and the determination of cellular and tissue functions of these channels. Here we show the activation of TRPC5 (canonical TRP 5) homomultimeric and TRPC5–TRPC1 heteromultimeric channels by extracellular reduced thioredoxin, which acts 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 unknown. Particularly high extracellular concentrations of thioredoxin are apparent in rheumatoid arthritis, an inflammatory joint disease that disables millions of people worldwide. We show that TRPC5 and TRPC1 are expressed in secretory fibroblast-like synoviocytes from patients with rheumatoid arthritis, that endogenous TRPC5–TRPC1 channels of the cells are activated by reduced thioredoxin, and that blockade of the channels enhances secretory activity and prevents the suppression of secretion by thioredoxin. The data indicate the presence of a previously unrecognized ion-channel activation mechanism that couples extracellular thioredoxin to cell function.

Upregulated TRPC1 Channel in Vascular Injury In Vivo and Its Role in Human Neointimal Hyperplasia

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.

A Sphingosine-1–Phosphate-Activated Calcium Channel Controlling Vascular Smooth Muscle Cell Motility

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.

Sensing of Lysophospholipids by TRPC5 Calcium Channel

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.

Robotic multiwell planar patch-clamp for native and primary mammalian cells

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.

Pregnenolone Sulphate- and Cholesterol-Regulated TRPM3 Channels Coupled to Vascular Smooth Muscle Secretion and Contraction

Rationale: Transient receptor potential melastatin (TRPM)3 is a calcium-permeable ion channel activated by the neurosteroid pregnenolone sulfate and positively coupled to insulin secretion in &bgr; cells. Although vascular TRPM3 mRNA has been reported, there is no knowledge of TRPM3 protein or its regulation and function in the cardiovascular system. Objective: To determine the relevance and regulation of TRPM3 in vascular biology. Methods and Results: TRPM3 expression was detected at mRNA and protein levels in contractile and proliferating vascular smooth muscle cells. Calcium entry evoked by pregnenolone sulfate or sphingosine was suppressed by TRPM3 blocking antibody or knock-down of TRPM3 by RNA interference. Low-level constitutive TRPM3 activity was also detected. In proliferating cells, channel activity was coupled negatively to interleukin-6 secretion via a calcium-dependent mechanism. In freshly isolated aorta, TRPM3 positively modulated contractile responses independently of L-type calcium channels. Concentrations of pregnenolone sulfate required to evoke responses were higher than the known plasma concentrations of the steroids, leading to a screen for other stimulators. &bgr;-Cyclodextrin was one of few stimulators of TRPM3, revealing the channels to be partially suppressed by endogenous cholesterol, the precursor of pregnenolone. Elevation of cholesterol further suppressed channel activity and loading with cholesterol to generate foam cells precluded observation of TRPM3 activity. Conclusions: The data suggest functional relevance of TRPM3 in contractile and proliferating phenotypes of vascular smooth muscle cells, significance of constitutive channel activity, regulation by cholesterol, and potential value of pregnenolone sulfate in therapeutic vascular modulation.

Production of a specific extracellular inhibitor of TRPM3 channels.

Isoform‐specific ion channel blockers are useful for target validation in drug discovery and can provide the basis for new therapeutic agents and aid in determination of physiological functions of ion channels. The aim of this study was to generate a specific blocker of human TRPM3 channels as a tool to help investigations of this member of the TRP cationic channel family.

Short-Term Stimulation of Calcium-Permeable Transient Receptor Potential Canonical 5–Containing Channels by Oxidized Phospholipids

Objective—To determine whether calcium-permeable channels are targets for the oxidized phospholipids: 1-palmitoyl-2-glutaroyl-phosphatidylcholine (PGPC) and 1-palmitoyl-2-oxovaleroyl-phosphatidylcholine (POVPC). Methods and Results—Oxidized phospholipids are key factors in inflammation and associated diseases, including atherosclerosis; however, the initial reception mechanisms for cellular responses to the factors are poorly understood. Low micromolar concentrations of PGPC and POVPC evoked increases in intracellular calcium in human embryonic kidney 293 cells that overexpressed human transient receptor potential canonical 5 (TRPC5) but not human TRP melastatin (TRPM) 2 or 3. The results of electrophysiological experiments confirmed stimulation of TRPC5. To investigate relevance to endogenous channels, we studied proliferating vascular smooth muscle cells from patients undergoing coronary artery bypass surgery. PGPC and POVPC elicited calcium entry that was inhibited by anti-TRPC5 or anti-TRPC1 antibodies or dominant-negative mutant TRPC5. Calcium release did not occur. The effect was functionally relevant because it enhanced cell migration. The actions of PGPC and POVPC depended on Gi/o proteins but not on previously identified G protein-coupled receptors for oxidized phospholipids. Conclusion—Stimulation of calcium-permeable TRPC5-containing channels may be an early event in cellular responses to oxidized phospholipids that couples to cell migration and requires an unidentified G protein-coupled receptor.

Pregnenolone sulphate-independent inhibition of TRPM3 channels by progesterone

Transient Receptor Potential Melastatin 3 (TRPM3) is a widely expressed calcium-permeable non-selective cation channel that is stimulated by high concentrations of nifedipine or by physiological steroids that include pregnenolone sulphate. Here we sought to identify steroids that inhibit TRPM3. Channel activity was studied using calcium-measurement and patch-clamp techniques. Progesterone (0.01–10 μM) suppressed TRPM3 activity evoked by pregnenolone sulphate. Progesterone metabolites and 17β-oestradiol were also inhibitory but the effects were relatively small. Dihydrotestosterone was an inhibitor at concentrations higher than 1 μM. Corticosteroids lacked effect. Overlay assays indicated that pregnenolone sulphate, progesterone and dihydrotestosterone bound to TRPM3. In contrast to dihydrotestosterone, progesterone inhibited nifedipine-evoked TRPM3 activity or activity in the absence of an exogenous activator, suggesting a pregnenolone sulphate-independent mechanism of action. Dihydrotestosterone, like a non-steroid look-alike compound, acted as a competitive antagonist at the pregnenolone sulphate binding site. Progesterone inhibited endogenous TRPM3 in vascular smooth muscle cells. Relevance of TRPM3 or the progesterone effect to ovarian cells, which have been suggested to express TRPM3, was not identified. The data further define a chemical framework for competition with pregnenolone sulphate at TRPM3 and expand knowledge of steroid interactions with TRPM3, suggesting direct steroid binding and pregnenolone sulphate-independent inhibition by progesterone.

Stereo-selective inhibition of transient receptor potential TRPC5 cation channels by neuroactive steroids

BACKGROUND AND PURPOSE Transient receptor potential canonical 5 (TRPC5) channels are widely expressed, including in the CNS, where they potentiate fear responses. They also contribute to other non‐selective cation channels that are stimulated by G‐protein‐coupled receptor agonists and lipid and redox factors. Steroids are known to modulate fear and anxiety states, and we therefore investigated whether TRPC5 exhibited sensitivity to steroids.