Mast cells are key effector cells in allergic reactions. Aggregation of the receptor FceRI in mast cells triggers the influx of calcium (Ca 2+ ) and the release of inflammatory mediators. Here we show that transient receptor potential TRPM4 proteins acted as calcium-activated nonselective cation channels and critically determined the driving force for Ca 2+ influx in mast cells. Trpm4 -/-bone marrow-derived mast cells had more Ca 2+ entry than did TRPM4 +/+ cells after FceRI stimulation. Consequently, Trpm4 -/-bone marrow-derived mast cells had augmented degranulation and released more histamine, leukotrienes and tumor necrosis factor. Trpm4 -/-mice had a more severe IgE-mediated acute passive cutaneous anaphylactic response, whereas late-phase passive cutaneous anaphylaxis was not affected. Our results establish the physiological function of TRPM4 channels as critical regulators of Ca 2+ entry in mast cells.Mast cells are bone marrow-derived hematopoietic cells localized near surfaces exposed to the environment, such as the skin, the airway epithelia and the intestine, where pathogens, allergens and other environmental agents are frequently encountered 1 . Activation and degranulation of mast cells is a key step in the pathogenesis of allergic diseases such as bronchial asthma and systemic anaphylaxis 2 . An allergic reaction develops when allergens encountered by antigen-presenting cells are processed and presented to T cells. Ensuing T helper type 2 responses cause B cells to produce allergen-specific immunoglobulin E (IgE). The IgE molecules bind to the receptor FceRI on the surfaces of mast cells. After re-exposure to the allergen, FceRI-associated IgE molecules bind allergen and aggregate, thereby activating mast cells. Activated mast cells secrete preformed mediators, including proteases and vasoactive amines, such as histamine, that are stored in cytoplasmic granules. In addition, mast cell activation results in the de novo synthesis of proinflammatory lipid mediators, cytokines and chemokines. The instant release of histamine is crucial for the development of immediate-type allergic reactions that result in vasodilatation, increased vascular permeability and smooth muscle contraction 1,2 . In addition, IgE-dependent mast cell activation may be complemented by signaling cascades triggered by several endogenous ligands, such as adenosine, resulting in the amplification and maintenance of FceRI-mediated degranulation 3,4 .FceRI crosslinking activates many signaling molecules 5,6 . A chief 'downstream' target is phospholipase C-g1, which catalyzes the hydrolysis of phosphatidylinositol-4,5-bisphosphate to diacylglycerol and inositol-1,4,5-trisphosphate 5 . In contrast, adenosine stimulation involves the activation of G ai protein-coupled A 3 adenosine receptors in mouse mast cells, which leads to the activation of phospholipase C and phospholipase D through G bg protein and phosphatidylinositol-3-OH kinase-g 7,8 . Inositol-1,4,5-trisphosphate and diacylglycerol promote the activation of protein kinase C an...

TRPV6 [transient receptor potential vanilloid 6] is a calcium ion (Ca²+)-selective channel originally identified in the duodenal epithelium and in placenta; replacement of a negatively charged aspartate in the pore-forming region with an uncharged alanine (D541A) renders heterologously expressed TRPV6 channels nonfunctional. We found that male, but not female, mice homozygous for this mutation (Trpv6(D541A/D541A)) showed severely impaired fertility. The motility and fertilization capacity of sperm were markedly reduced, despite intact spermatogenesis. Trpv6 was expressed in epididymal epithelium where the protein was detected in the apical membrane, whereas it was not expressed in spermatozoa or the germinal epithelium. The Ca²+ concentration of the fluid in the cauda epididymis of Trpv6(D541A/D541A) males was 10 times higher than that of wild-type mice, which was accompanied by a seven- to eightfold decrease in Ca²+ absorption through the epididymal epithelium and was associated with reduced sperm viability. Thus, appropriate Ca²+ absorption and a consequent TRPV6-mediated decrease in the extracellular Ca²+ concentration toward the distal segments of the epididymal duct are essential for the acquisition of basic functions and the survival of spermatozoa.

Cytotoxic T lymphocytes kill virus-infected and tumorigenic target cells through the release of perforin and granzymes via fusion of lytic granules at the contact site, the immunological synapse. It has been postulated that this fusion process is mediated by non-neuronal members of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex protein family. Here, using a synaptobrevin2-monomeric red fluorescence protein knock-in mouse we demonstrate that, surprisingly, the major neuronal v-SNARE synaptobrevin2 is expressed in cytotoxic T lymphocytes and exclusively localized on granzyme B-containing lytic granules. Cleavage of synaptobrevin2 by tetanus toxin or ablation of the synaptobrevin2 gene leads to a complete block of lytic granule exocytosis while leaving upstream events unaffected, identifying synaptobrevin2 as the v-SNARE responsible for the fusion of lytic granules at the immunological synapse.

Available data on transient receptor potential channel (TRPC) protein functions indicate that these proteins represent essential constituents of agonist-activated and phospholipase Cdependent cation entry pathways in primary cells which contribute to the elevation of cytosolic Ca 2+ . In addition, a striking number of biological functions have already been assigned to the various TRPC proteins, including mechanosensing activity (TRPC1), chemotropic axon guidance (TRPC1 and TRPC3), pheromone sensing and the regulation of sexual and social behaviour (TRPC2), endothelial-dependent regulation of vascular tone, endothelial permeability and neurotransmitter release (TRPC4), axonal growth (TRPC5), modulation of smooth muscle tone in blood vessels and lung and regulation of podocyte structure and function in the kidney (TRPC6). The lack of compounds which specifically block or activate TRPC proteins impairs the analysis of TRPC function in primary cells. We therefore concentrate in this contribution on (i) studies of TRPC-deficient mouse lines, (ii) data obtained by gene-silencing approaches using antisense oligonucleotides or RNA interference, (iii) expression experiments employing dominant negative TRPC constructs, and (iv) recent data correlating mutations of TRPC genes associated with human disease.

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