—Mammalian counterparts of the Drosophila trp gene have been suggested to encode store-operated Ca ²⁺ channels. These specialized channels are widely distributed and may have a general function to reload Ca ²⁺ 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 Ca ²⁺ 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 Ca ²⁺ 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 Ca ²⁺ channel subunit in arterial smooth muscle cells.

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 (EC(50) 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 Ca(2+) 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.

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. 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. 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. In whole‐cell and excised membrane patch recordings, 2‐APB acts from the extracellular but not intracellular face of the membrane. Block of TRPC5 by 2‐APB is less at positive voltages, suggesting that it enters the electric field or acts by modulating channel gating. 2‐APB also blocks TRPC6 and TRPM3 expressed in HEK‐293 cells, but not TRPM2. 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. 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. British Journal of Pharmacology (2005) 145, 405–414. doi:10.1038/sj.bjp.0706197

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 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-β-cyclodextrin (mβcd, 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 ET _A receptor) was reduced by mβcd and increased by cholesterol. Neither ligand binding nor colocalization of ET _A and caveolin-1 was affected by mβcd. Ca ²⁺ inflow via store-operated channels after depletion of intracellular Ca ²⁺ stores was reduced in mβcd-treated caudal arteries, as shown by Mn ²⁺ quench rate and intracellular [Ca ²⁺ ] 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 mβcd, as seen by immunofluorescence. Part of the contractile response to ET-1 was inhibited by Ni ²⁺ (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 mβcd. Increased store-operated channel activity in basilar arteries after organ culture correlated with increased sensitivity of ET-1 contraction to mβcd. These results suggest that cholesterol influences vascular reactivity to ET-1 by affecting the caveolar localization of TRPC1.