Agonist-induced Ca2+ entry into cells by both store-operated channels and channels activated independently of Ca2+-store depletion has been described in various cell types. The molecular structures of these channels are unknown as is, in most cases, their impact on various cellular functions. Here we describe a store-operated Ca2+ current in vascular endothelium and show that endothelial cells of mice deficient in TRP4 (also known as CCE1) lack this current. As a consequence, agonist-induced Ca2+ entry and vasorelaxation is reduced markedly, showing that TRP4 is an indispensable component of store-operated channels in native endothelial cells and that these channels directly provide an Ca2+-entry pathway essentially contributing to the regulation of blood vessel tone.

Lower vertebrates have an intrinsically-photosensitive iris and thus a local pupillary light reflex (PLR). In contrast, it has been a dogma that the PLR in mammals generally requires neuronal circuitry connecting the eye and the brain. We report here that an intrinsic component of the PLR is actually widespread in nocturnal and crepuscular mammals. In mouse, this intrinsic PLR requires the visual pigment, melanopsin. It also requires PLCβ4, the vertebrate homolog of the Drosophila NorpA phospholipase C mediating rhabdomeric phototransduction. The Plcβ4−/− genotype, besides removing the intrinsic PLR, also essentially eliminates the intrinsic light response of the M1-subtype of melanopsin-expressing, intrinsically-photosensitive retinal ganglion cells (M1-ipRGCs), by far the most photosensitive ipRGCs and with the largest responses. Ablating in mouse the expression of both TRPC6 and TRPC7, members of the TRP channel superfamily, likewise essentially eliminated the M1-ipRGC light response, but spared the intrinsic PLR. Thus, melanopsin signaling exists in both iris and retina, involving a PLCβ4-mediated pathway that nonetheless diverges in the two locations.

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...

Canonical transient receptor potential (TRPC) channels influence various neuronal functions. Using quantitative high-resolution mass spectrometry, we demonstrate that TRPC1, TRPC4, and TRPC5 assemble into heteromultimers with each other, but not with other TRP family members in the mouse brain and hippocampus. In hippocampal neurons from -triple-knockout () mice, lacking any TRPC1-, TRPC4-, or TRPC5-containing channels, action potential-triggered excitatory postsynaptic currents (EPSCs) were significantly reduced, whereas frequency, amplitude, and kinetics of quantal miniature EPSC signaling remained unchanged. Likewise, evoked postsynaptic responses in hippocampal slice recordings and transient potentiation after tetanic stimulation were decreased. , mice displayed impaired cross-frequency coupling in hippocampal networks and deficits in spatial working memory, while spatial reference memory was unaltered. animals also exhibited deficiencies in adapting to a new challenge in a relearning task. Our results indicate the contribution of heteromultimeric channels from TRPC1, TRPC4, and TRPC5 subunits to the regulation of mechanisms underlying spatial working memory and flexible relearning by facilitating proper synaptic transmission in hippocampal neurons.

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.

In mammalian cells, one-third of all polypeptides are integrated into the membrane or translocated into the lumen of the endoplasmic reticulum (ER) via the Sec61 channel. While the Sec61 complex facilitates ER import of most precursor polypeptides, the Sec61-associated Sec62/Sec63 complex supports ER import in a substrate-specific manner. So far, mainly posttranslationally imported precursors and the two cotranslationally imported precursors of ERj3 and prion protein were found to depend on the Sec62/ Sec63 complex in vitro. Therefore, we determined the rules for engagement of Sec62/Sec63 in ER import in intact human cells using a recently established unbiased proteomics approach. In addition to confirming ERj3, we identified 22 novel Sec62/Sec63 substrates under these in vivo-like conditions. As a common feature, those previously unknown substrates share signal peptides (SP) with comparatively longer but less hydrophobic hydrophobic region of SP and lower carboxy-terminal region of SP (C-region) polarity. Further analyses with four substrates, and ERj3 in particular, revealed the combination of a slowly gating SP and a downstream translocation-disruptive positively charged cluster of amino acid residues as decisive for the Sec62/Sec63 requirement. In the case of ERj3, these features were found to be responsible for an additional immunoglobulin heavy-chain binding protein (BiP) requirement and to correlate with sensitivity toward the Sec61-channel inhibitor CA7M741. Thus, the human Sec62/Sec63 complex may support Sec61-channel opening for precursor polypeptides with slowly gating SPs by direct interaction with the cytosolic amino-terminal peptide of Sec61a or via recruitment of BiP and its interaction with the ER-lumenal loop 7 of Sec61a. These novel insights into the mechanism of human ER protein import contribute to our understanding of the etiology of SEC63-linked polycystic liver disease.

An oscillatory increase in pancreatic beta cell cytoplasmic free Ca2+ concentration, [Ca2+]i, is a key feature in glucose-induced insulin release. The role of the voltage-gated Ca2+ channel beta3 subunit in the molecular regulation of these [Ca2+]i oscillations has now been clarified by using beta3 subunit-deficient beta cells. beta3 knockout mice showed a more efficient glucose homeostasis compared to wild-type mice due to increased glucose-stimulated insulin secretion. This resulted from an increased glucose-induced [Ca2+]i oscillation frequency in beta cells lacking the beta3 subunit, an effect accounted for by enhanced formation of inositol 1,4,5-trisphosphate (InsP3) and increased Ca2+ mobilization from intracellular stores. Hence, the beta3 subunit negatively modulated InsP3-induced Ca2+ release, which is not paralleled by any effect on the voltage-gated L type Ca2+ channel. Since the increase in insulin release was manifested only at high glucose concentrations, blocking the beta3 subunit in the beta cell may constitute the basis for a novel diabetes therapy.

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