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.
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...
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,Ntetraacetic 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.The non-voltage-gated TRP Ca 2ϩ channel encoded by the transient receptor potential (trp) 1 gene has a major role in the phospholipase C-dependent light response of the Drosophila photoreceptor (1). Since this discovery, many trp-related Ca 2ϩ channels have been discovered in mammals, beginning with TRPC1 (e.g. Ref.2), which is a subunit of some store-operated Ca 2ϩ channels (e.g. Ref.3). There are now known to be at least 20 trp-related mammalian genes, all apparently encoding cationic channels, many of which are Ca 2ϩ permeable. They would appear to be the molecular basis of the many non-voltage-gated cationic channels with diverse functions and expression profiles in mammalian systems. On the basis of amino acid sequence the mammalian TRPs are divided into three subgroups, TRPC (C, canonical), TRPV (V, vanilloid receptor), and TRPM (M, melastatin receptor) (4). Increasingly it is becoming apparent that the regulation of these proteins is complex, with gating factors as diverse as temperature, menthol, diacylglycerol, arachidonic acid, and osmotic stress (5, 6).TRPM2 (also called TRPC7 or LTRPC2) is a recently characterized member of the TRPM family (7-11). It forms a cationic channel activated by intracellular ADP-ribose, -NAD ϩ , or arachidonic acid. The sensitivity of the channel to -NAD ϩ is thought to couple TRPM2 to the redox state of the cell (10). T...
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--cyclodextrin (mcd, 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 mcd and increased by cholesterol. Neither ligand binding nor colocalization of ET A and caveolin-1 was affected by mcd. Ca 2ϩ inflow via store-operated channels after depletion of intracellular Ca 2ϩ stores was reduced in mcd-treated caudal arteries, as shown by Mn 2ϩ quench rate and intracellular [Ca 2ϩ ] 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 mcd, as seen by immunofluorescence. Part of the contractile response to ET-1 was inhibited by Ni 2ϩ (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 mcd. Increased store-operated channel activity in basilar arteries after organ culture correlated with increased sensitivity of ET-1 contraction to mcd. These results suggest that cholesterol influences vascular reactivity to ET-1 by affecting the caveolar localization of TRPC1. Key Words: arterial smooth muscle Ⅲ methyl--cyclodextrin Ⅲ caveolae Ⅲ endothelin Ⅲ store-operated Ca 2ϩ channels H ypercholesterolemia increases reactivity to endothelin-1 (ET-1) in experimental animals and humans. [1][2][3][4] This has been pointed out as one possible factor in the progression of atherosclerosis. [5][6][7] The mechanism of action has not been elucidated, although both endothelial dysfunction and altered smooth muscle reactivity have been proposed. 5 Lipoprotein particles may directly influence endothelial membrane-associated endothelial NO synthase activity by interfering with cholesterol-rich domains referred to as caveolae. 8 Although these effects modulate the endothelial influence on vascular tone, less is known regarding direct effects of cholesterol on vascular smooth muscle functions.Caveolae are 50-to 100-nm membrane invaginations that integrate many cellular receptor functions. 9 For instance, ET A receptors expressed in COS cells colocalize with the caveolae-associated protein caveolin. 10,11 The caveolar structure is disrupted after depletion of cholesterol with cyclodextrins, 12 and this correlates with a decreased contractility to ET-1, but not to depolarization or ␣ 1 -receptor stimulation, in endothelium-denuded rat caudal arteries. 13 Cholesterol might thus modulate the strength of caveolae-associated signaling, providing a basis for altered contractility in response to ET-1.Activation of the ET A receptor stimulates Ca 2ϩ inflow ov...
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