Fanning Zeng

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.

Block of TRPC5 channels by 2-aminoethoxydiphenyl borate : a differential, extracellular and voltage-dependent effect

1 2‐Aminoethoxydiphenyl borate (2‐APB) has been widely used to examine the roles of inositol 1,4,5‐trisphosphate receptors (IP3Rs) and store‐operated Ca2+ entry and is an emerging modulator of cationic channels encoded by transient receptor potential (TRP) genes. 2 Using Ca2+‐indicator dye and patch‐clamp recording we first examined the blocking effect of 2‐APB on human TRPC5 channels expressed in HEK‐293 cells. 3 The concentration–response curve has an IC50 of 20 μM and slope close to 1.0, suggesting one 2‐APB molecule binds per channel. The blocking effect is not shared by other Ca2+ channel blockers including methoxyverapamil, nifedipine, N‐propargylnitrendipine, or berberine. 4 In whole‐cell and excised membrane patch recordings, 2‐APB acts from the extracellular but not intracellular face of the membrane. 5 Block of TRPC5 by 2‐APB is less at positive voltages, suggesting that it enters the electric field or acts by modulating channel gating. 6 2‐APB also blocks TRPC6 and TRPM3 expressed in HEK‐293 cells, but not TRPM2. 7 Block of TRP channels by 2‐APB may be relevant to cell proliferation because 2‐APB has a greater inhibitory effect on proliferation in cells overexpressing TRPC5. 8 Our data indicate a specific and functionally important binding site on TRPC5 that enables block by 2‐APB. The site is only available via an extracellular route and the block shows mild voltage‐dependence.

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.

Human TRPC5 channel activated by a multiplicity of signals in a single cell

Here we explore the activation mechanisms of human TRPC5, a putative cationic channel that was cloned from a region of the X chromosome associated with mental retardation. No basal activity was evident but activity was induced by carbachol stimulation of muscarinic receptors independently of Ca2+ release. This is ‘receptor activation’, as described for mouse TRPC5. In addition, and in the absence of receptor stimulation, extracellular gadolinium (0.1 mm) activated TRPC5, an effect that was mimicked by 5–20 mm extracellular Ca2+ with intracellular Ca2+ buffered. We refer to this as ‘external ionic activation’. TRPC5 was also activated by modest elevation of [Ca2+]i in the absence of GTP –‘calcium activation’. A putative fourth activation mechanism is a signal from depleted intracellular Ca2+ stores. Consistent with this idea, human TRPC5 was activated by a standard store‐depletion/Ca2+ re‐entry protocol, an effect that was difficult to explain by calcium activation. Multiplicity of TRPC5 activation was demonstrated in single cells and thus not dependent on heterogeneity of expression levels or cellular context. Therefore, human TRPC5 is activated by a range of stimuli, avoiding dependence on a single critical activator as in many other ion channels. One of these stimuli would seem to be a change in Ca2+ handling by the endoplasmic reticulum.

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.

Generation of functional ion-channel tools by E3 targeting.

Here we describe a strategy for generating ion-channel inhibitors. It takes advantage of antibody specificity combined with a pattern recognition approach that targets the third extracellular region (E3) of a channel. To test the concept, we first focused on TRPC5, a member of the transient receptor potential (TRP) calcium channel family, the study of which has been hindered by poor pharmacological tools. Extracellular application of E3-targeted anti-TRPC5 antibody led to a specific TRPC5 inhibitor, enabling TRPC5 to be distinguished from its closest family members, and TRPC5 function to be explored in a relatively intractable physiological system. E3 targeting was further applied to voltage-gated sodium channels, leading to discovery of a subtype-specific inhibitor of NaV1.5. These examples illustrate the potential power of E3 targeting as a systematic method for producing gene-type specific ion-channel inhibitors for use in routine assays on cells or tissues from a range of species and having therapeutic potential.

Calcium-sensing mechanism in TRPC5 channels contributing to retardation of neurite outgrowth

The calcium‐ and sodium‐permeable transient receptor potential channel TRPC5 has an inhibitory role in neuronal outgrowth but the mechanisms governing its activity are poorly understood. Here we propose a mechanism involving the neuronal calcium sensor‐1 (NCS‐1) protein. Inhibitory mutants of TRPC5 and NCS‐1 enhance neurite outgrowth similarly. Mutant NCS‐1 does not inhibit surface‐expression of TRPC5 but generally suppresses channel activity, irrespective of whether it is evoked by carbachol, store depletion, lanthanides or elevated intracellular calcium. NCS‐1 and TRPC5 are in the same protein complex in rat brain and NCS‐1 directly binds to the TRPC5 C‐terminus. The data suggest protein–protein interaction between NCS‐1 and TRPC5, and involvement of this protein complex in retardation of neurite outgrowth.

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.