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...

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 Ca 2+ , 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.

No abstract

. E3-targeted anti-TRPC5 antibody inhibits storeoperated calcium entry in freshly isolated pial arterioles. Am J Physiol Heart Circ Physiol 291: H2653-H2659, 2006. First published July 21, 2006 doi:10.1152/ajpheart.00495.2006.-Smooth muscle cells in arterioles have pivotal roles in the determination of blood pressure and distribution of local blood flow. The cells exhibit calcium entry in response to passive store depletion, but the mechanisms and relevance of this phenomenon are poorly understood. Previously, a role for canonical transient receptor potential 1 (TRPC1) was indicated, but heterologous expression studies showed TRPC1 to have poor function in isolation, suggesting a requirement for additional proteins. Here we test the hypothesis that TRPC5 is such an additional protein, because TRPC5 forms heteromultimeric channels with TRPC1, and RNA encoding TRPC5 is present in arterioles. Recordings were from arteriolar fragments freshly isolated from rabbit pial membrane. Ionic current in response to store depletion has properties like that of the TRPC1/TRPC5 heteromultimer, and so the effect of the E3-targeted, externally acting, anti-TRPC5 blocking antibody (T5E3) was explored. T5E3 suppressed calcium entry in store-depleted arterioles but had no effect in the absence of store depletion. T5E3 preadsorbed to its antigenic peptide did not inhibit calcium entry. TRPC6 is commonly detected in smooth muscle and is present in the arterioles, but T5E3 had no effect on TRPC6. The data suggest that calcium entry occurring in response to passive store depletion in smooth muscle cells of arterioles involves TRPC5 as well as TRPC1.arteriole; smooth muscle cell; calcium channel; store-operated channel; transient receptor potential; functional antibody ARTERIOLES ARE small precapillary arteries containing a single smooth muscle layer. Because of the small diameter and number of arterioles, they have a major impact on peripheral resistance and provide the primary structure regulating local blood flow according to metabolic needs of surrounding tissue.

In this study, we determined a pharmacological profile of store‐operated channels (SOCs) in smooth muscle cells of rabbit pial arterioles. Ca2+‐indicator dyes, fura‐PE3 or fluo‐4, were used to track [Ca2+]i and 10 μM methoxyverapamil (D600) was present in all experiments on SOCs to prevent voltage‐dependent Ca2+ entry. Store depletion was induced using thapsigargin or cyclopiazonic acid. SOC‐mediated Ca2+ entry was inhibited concentration dependently by Gd3+ (IC50 101 nM). It was also inhibited by 10 μM La3+ (70% inhibition, N=5), 100 μM Ni2+ (57% inhibition, N=5), 75 μM 2‐aminoethoxydiphenylborate (66% inhibition, N=4), 100 μM capsaicin (12% inhibition, N=3) or preincubation with 10 μM wortmannin (76% inhibition, N=4). It was completely resistant to 1 μM nifedipine (N=5), 10 μM SKF96365 (N=6), 10 μM LOE908 (N=14), 10–100 μM ruthenium red (N=1+2), 100 μM sulindac (N=4), 0.5 mM streptomycin (N=3) or 1 : 10,000 dilution Grammostolla spatulata venom (N=4). RT–PCR experiments on isolated arteriolar fragments showed expression of mRNA species for TRPC1, 3, 4, 5 and 6. The pharmacological profile of SOC‐mediated Ca2+ entry in arterioles supports the hypothesis that these SOCs are distinct from tonically active background channels and several store‐operated and other nonselective cation channels described in other cells. Similarities with the pharmacology of TRPC1 support the hypothesis that TRPC1 is a subunit of the arteriolar smooth muscle SOC.> British Journal of Pharmacology (2003) 139, 955–965. doi: