David J. Beech

TrpC1 Is a Membrane-Spanning Subunit of Store-Operated Ca2+ Channels in Native Vascular Smooth Muscle Cells

Abstract— Mammalian counterparts of the Drosophila trp gene have been suggested to encode store-operated Ca2+ channels. These specialized channels are widely distributed and may have a general function to reload Ca2+ into sarcoplasmic reticulum as well as specific functions, including the control of cell proliferation and muscle contraction. Heterologous expression of mammalian trp genes enhances or generates Ca2+ channel activity, but the crucial question of whether any of the genes encode native subunits of store-operated channels remains unanswered. We have investigated if TrpC1 protein (encoded by trp1 gene) is a store-operated channel in freshly isolated smooth muscle cells of resistance arterioles, arteries, and veins from human, mouse, or rabbit. Messenger RNA encoding TrpC1 was broadly expressed. TrpC1-specific antibody targeted to peptide predicted to contribute to the outer vestibule of TrpC1 channels revealed that TrpC1 is localized to the plasma membrane and has an extracellular domain. Peptide-specific binding of the antibody had a functional effect, selectively blocking store-operated Ca2+ channel activity. The antibody is a powerful new tool for the study of mammalian trp1 gene product. The study shows that TrpC1 is a novel physiological Ca2+ channel subunit in arterial smooth muscle cells.

Non-selective cationic channels of smooth muscle and the mammalian homologues of Drosophila TRP.

Throughout the body there are smooth muscle cells controlling a myriad of tubes and reservoirs. The cells show enormous diversity and complexity compounded by a plasticity that is critical in physiology and disease. Over the past quarter of a century we have seen that smooth muscle cells contain – as part of a gamut of ion‐handling mechanisms – a family of cationic channels with significant permeability to calcium, potassium and sodium. Several of these channels are sensors of calcium store depletion, G‐protein‐coupled receptor activation, membrane stretch, intracellular Ca2+, pH, phospholipid signals and other factors. Progress in understanding the channels has, however, been hampered by a paucity of specific pharmacological agents and difficulty in identifying the underlying genes. In this review we summarize current knowledge of these smooth muscle cationic channels and evaluate the hypothesis that the underlying genes are homologues of Drosophila TRP (transient receptor potential). Direct evidence exists for roles of TRPC1, TRPC4/5, TRPC6, TRPV2, TRPP1 and TRPP2, and more are likely to be added soon. Some of these TRP proteins respond to a multiplicity of activation signals – promiscuity of gating that could enable a variety of context‐dependent functions. We would seem to be witnessing the first phase of the molecular delineation of these cationic channels, something that should prove a leap forward for strategies aimed at developing new selective pharmacological agents and understanding the activation mechanisms and functions of these channels in physiological systems.

Critical Intracellular Ca2+ Dependence of Transient Receptor Potential Melastatin 2 (TRPM2) Cation Channel Activation

TRPM2 is a member of the melastatin-related TRP (transient receptor potential) subfamily. It is expressed in brain and lymphocytes and forms a cation channel that is activated by intracellular ADP-ribose and associated with cell death. In this study we investigated the calcium dependence of human TRPM2 expressed under a tetracycline-dependent promoter in HEK-293 cells. TRPM2 expression was associated with enhanced hydrogen peroxide-evoked intracellular calcium signals. In whole-cell patch clamp recordings, switching from barium- to calcium-containing extracellular solution markedly activated TRPM2 as long as ADP-ribose was in the patch pipette and exogenous intracellular calcium buffering was minimal. We suggest this effect reveals a critical dependence of TRPM2 channel activity on intracellular calcium. In the absence of extracellular calcium we observed concentration-dependent activation of TRPM2 channels by calcium delivered from the patch pipette (EC50 340 nm, slope 4.9); the maximum effect was at least as large as that evoked by extracellular calcium. Intracellular dialysis of cells with high concentrations of EGTA or 1,2-bis(o-Aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) strongly reduced the amplitude of the extracellular calcium response, and the residual response was abolished by a mixture of high and low affinity calcium buffers. TRPM2 channel currents in inside-out patches showed a strong requirement for Ca2+ at the intracellular face of the membrane. We suggest that calcium entering via TRPM2 proteins acts at an intracellular calcium sensor closely associated with the channel, providing essential positive feedback for channel activation.

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.

Cholesterol Depletion Impairs Vascular Reactivity to Endothelin-1 by Reducing Store-Operated Ca2+ Entry Dependent on TRPC1

Abstract— The reactivity of the vascular wall to endothelin-1 (ET-1) is influenced by cholesterol, which is of possible importance for the progression of atherosclerosis. To elucidate signaling steps affected, the cholesterol acceptor methyl-&bgr;-cyclodextrin (m&bgr;cd, 10 mmol/L) was used to manipulate membrane cholesterol and disrupt caveolae in intact rat arteries. In endothelium-denuded caudal artery, contractile responsiveness to 10 nmol/L ET-1 (mediated by the ETA receptor) was reduced by m&bgr;cd and increased by cholesterol. Neither ligand binding nor colocalization of ETA and caveolin-1 was affected by m&bgr;cd. Ca2+ inflow via store-operated channels after depletion of intracellular Ca2+ stores was reduced in m&bgr;cd-treated caudal arteries, as shown by Mn2+ quench rate and intracellular [Ca2+] response. Expression of TRPC1, 3, and 6 was detected by reverse transcriptase–polymerase chain reaction, and colocalization of TRPC1 with caveolin-1 was reduced by m&bgr;cd, as seen by immunofluorescence. Part of the contractile response to ET-1 was inhibited by Ni2+ (0.5 mmol/L) and by a TRPC1 blocking antibody. In the basilar artery, exhibiting less store-operated channel activity than the caudal artery, ET-1–induced contractions were insensitive to the TRPC1 blocking antibody and to m&bgr;cd. Increased store-operated channel activity in basilar arteries after organ culture correlated with increased sensitivity of ET-1 contraction to m&bgr;cd. These results suggest that cholesterol influences vascular reactivity to ET-1 by affecting the caveolar localization of TRPC1.

Piezo1 integration of vascular architecture with physiological force

The mechanisms by which physical forces regulate endothelial cells to determine the complexities of vascular structure and function are enigmatic. Studies of sensory neurons have suggested Piezo proteins as subunits of Ca2+-permeable non-selective cationic channels for detection of noxious mechanical impact. Here we show Piezo1 (Fam38a) channels as sensors of frictional force (shear stress) and determinants of vascular structure in both development and adult physiology. Global or endothelial-specific disruption of mouse Piezo1 profoundly disturbed the developing vasculature and was embryonic lethal within days of the heart beating. Haploinsufficiency was not lethal but endothelial abnormality was detected in mature vessels. The importance of Piezo1 channels as sensors of blood flow was shown by Piezo1 dependence of shear-stress-evoked ionic current and calcium influx in endothelial cells and the ability of exogenous Piezo1 to confer sensitivity to shear stress on otherwise resistant cells. Downstream of this calcium influx there was protease activation and spatial reorganization of endothelial cells to the polarity of the applied force. The data suggest that Piezo1 channels function as pivotal integrators in vascular biology.

Attenuation of store-operated Ca2+ current impairs salivary gland fluid secretion in TRPC1(-/-) mice.

Agonist-induced Ca2+ entry via store-operated Ca2+ (SOC) channels is suggested to regulate a wide variety of cellular functions, including salivary gland fluid secretion. However, the molecular components of these channels and their physiological function(s) are largely unknown. Here we report that attenuation of SOC current underlies salivary gland dysfunction in mice lacking transient receptor potential 1 (TRPC1). Neurotransmitter-regulated salivary gland fluid secretion in TRPC1-deficient TRPC1(−/−) mice was severely decreased (by 70%). Further, agonist- and thapsigargin-stimulated SOC channel activity was significantly reduced in salivary gland acinar cells isolated from TRPC1(−/−) mice. Deletion of TRPC1 also eliminated sustained Ca2+-dependent potassium channel activity, which depends on Ca2+ entry and is required for fluid secretion. Expression of key proteins involved in fluid secretion and Ca2+ signaling, including STIM1 and other TRPC channels, was not altered. Together, these data demonstrate that reduced SOC entry accounts for the severe loss of salivary gland fluid secretion in TRPC1(−/−) mice. Thus, TRPC1 is a critical component of the SOC channel in salivary gland acinar cells and is essential for neurotransmitter-regulation of fluid secretion.

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.

TRPC1: store-operated channel and more.

Transient receptor potential canonical 1 (TRPC1) is a transmembrane protein expressed in a range of vertebrate cells including smooth muscle, endothelium, neurones and salivary gland cells. It functions as an element of a mixed cationic Ca2+-permeable channel, probably commonly as part of a heterotetrameric assembly involving other related proteins such as TRPC5. Wide-ranging biological roles of TRPC1 are suggested, including regulation of smooth muscle and stem cell proliferation, endothelin-evoked arterial contraction, salivary gland secretion, endothelial permeability, glutamatergic neurotransmission, growth cone turning, neuroprotection, neuronal differentiation, lipid raft integrity and the nuclear factor of activated T-cell transcription factor. The mechanisms by which TRPC1 serves these functions are starting to emerge. At one level, it is apparent that TRPC1 is subcellularly compartmentalised, at least in part in cholesterol-rich caveolae closely associated with sub-plasmalemmal endoplasmic reticulum. At another level, TRPC1 is embedded in a protein complex that can include inositol trisphosphate receptor, homer, calmodulin, caveolin-1, FKBP25, I-mfa, MxA, GluR1α, bFGFR-1, Gq/11 protein, phospholipase C-β/γ, protein kinase C-α and RhoA. It is also apparent that TRPC1 responds to general stimuli—not only depletion of intracellular Ca2+ stores, but also receptor activation, and membrane stretch. We are at the early stages of understanding of how these various signals and components integrate to form a functional channel, and this article provides a brief overview of current progress.