TRPM4 is a calcium-activated non-selective cation channel that is widely expressed and proposed to be involved in cell depolarization. In excitable cells, TRPM4 may regulate calcium influx by causing the depolarization that drives the activation of voltage-dependent calcium channels. We here report that insulin-secreting cells of the rat pancreatic beta-cell line INS-1 natively express TRPM4 proteins and generate large depolarizing membrane currents in response to increased intracellular calcium. These currents exhibit the characteristics of TRPM4 and can be suppressed by expressing a dominant negative TRPM4 construct, resulting in significantly decreased insulin secretion in response to a glucose stimulus. Reduced insulin secretion was also observed with arginine vasopressin stimulation, a Gq-coupled receptor agonist in beta-cells. Moreover, the recruitment of TRPM4 currents was biphasic in both INS-1 cells as well as HEK-293 cells overexpressing TRPM4. The first phase is due to activation of TRPM4 channels localized within the plasma membrane followed by a slower secondary phase, which is caused by the recruitment of TRPM4-containing vesicles to the plasma membrane during exocytosis. The secondary phase can be observed during perfusion of cells with increasing [Ca(2+)](i), replicated with agonist stimulation, and coincides with an increase in cell capacitance, loss of FM1-43 dye, and vesicle fusion. Our data suggest that TRPM4 may play a key role in the control of membrane potential and electrical activity of electrically excitable secretory cells and the dynamic translocation of TRPM4 from a vesicular pool to the plasma membrane via Ca(2+)-dependent exocytosis may represent a key short- and midterm regulatory mechanism by which cells regulate electrical activity.

The transient receptor potential (TRP) 2 channel proteins comprise six transmembrane domains and multimerize to form ion channel complexes. Among the known families of ion channels, TRPs are unique in displaying an impressive diversity of cation selectivities, activation mechanisms, and functions (1). Specifically, members of the "canonical" TRP (TRPC) subfamily are non-selective cation channels that cause eventually Ca 2ϩ entry and collapse of the cell membrane potential (2). TRPCs are readily activated after stimulation of receptor-tyrosine kinases or G-protein-coupled receptors that activate the phospholipase C signaling pathway (3). It is believed that diacylglycerol, a product of the phospholipase C signaling pathway, activates ion channels formed by TRPC3, TRPC6, and TRPC7 (4), although the activation mechanisms of TRPC1, TRPC4, and TRPC5 appear to be more complex (5, 6).TRPC5 is enriched in the brain, where it is believed to control neurite extension and growth cone morphology (7,8). TRPC5-deficient mice exhibit diminished innate fear levels, suggesting an essential role for TRPC5 in the function of the amygdala (9). Furthermore, TRPC5 has been implicated in endothelial and mast cell function as well as in rheumatoid arthritis (2). In growth cones TRPC5 forms homomeric channels, but it forms heteromultimers with TRPC1 to build TRPC1/TRPC5 ion channels in the soma of neurones (8). Numerous stimuli are apparently able to control the activity of TRPC5 channels. By interacting with extracellularly located binding sites, thioredoxin, protons, and lanthanides enhance TRPC5 channel currents (10 -13). Acting intracellularly, nitric oxide enhances TRPC5 channel currents as well (14). Lysophospholipids and hypoosmotic-and pressure-induced membrane stretch act also as activators of TRPC5 channels (15,16). Under some circumstances, the externalization of TRPC5 contributes substantially to the overall enhancement of TRPC5 channel currents (17,18). All in all, TRPC5 are apparently target molecules of both extracellular and intracellular signals (5). As for other TRPC channels (e.g. Ref. 19), however, a central question is whether TRPC5 participates in store-operated Ca 2ϩ entry. Previous studies have shown that maneuvers that activate store-operated Ca 2ϩ entry also activate TRPC5 channels (20,21). In mast cells the Ca 2ϩ entry is apparently dependent on the presence of TRPC5 as well as on STIM1 and ORAI1, the key components of storeoperated Ca 2ϩ entry (22). Our understanding of the role of TRPC5 in store-operated Ca 2ϩ entry was advanced by the finding that STIM1 binds to TRPC5 and is obligatory for TRPC5 channel activation via membrane receptor stimulation (23). Thus, it is likely that TRPC5 is part of the protein complex responsible for store-operated Ca 2ϩ entry. However, it has

The stimulation of membrane receptors coupled to the phopholipase C pathway leads to activation of the Ca 2؉ release-activated Ca 2؉ (CRAC) channels. Recent evidence indicates that ORAI1 is an essential pore subunit of CRAC channels. STIM1 is additionally required for CRAC channel activation. The present study focuses on the genomic organization, tissue expression pattern, and functional properties of the murine ORAI2. Additionally, we report the cloning of the murine ORAI1, ORAI3, and STIM1. Two chromosomal loci were identified for the murine orai2 gene, one containing an intronless gene and a second locus that gives rise to the splice variants ORAI2 long (ORAI2L) and ORAI2 short (ORAI2S). Northern blots revealed a prominent expression of the ORAI2 variants in the brain, lung, spleen, and intestine, while ORAI1, ORAI3, and STIM1 appeared to be near ubiquitously expressed in mice tissues. Specific antibodies detected ORAI2 in RBL 2H3 but not in HEK 293 cells, whereas both cell lines appeared to express ORAI1 and STIM1 proteins. Co-expression experiments with STIM1 and either ORAI1 or ORAI2 variants showed that ORAI2L and ORAI2S enhanced substantially CRAC current densities in HEK 293 but were ineffective in RBL 2H3 cells, whereas ORAI1 strongly amplified CRAC currents in both cell lines. Thus, the capability of ORAI2 variants to form CRAC channels depends strongly on the cell background. Additionally, CRAC channels formed by ORAI2S were strongly sensitive to inactivation by internal Ca 2؉ . When co-expressed with STIM1 and ORAI1, ORAI2S apparently plays a negative dominant role in the formation of CRAC channels.