We have compared activation by heat of TRPV4 channels, heterogeneously expressed in HEK293 cells, and endogenous channels in mouse aorta endothelium (MAEC). Increasing the temperature above 25°C activated currents and increased [Ca 2؉ ] i in HEK293 cells transfected with TRPV4 and in MAEC. When compared with activation of TRPV4 currents by the selective ligand 4␣PDD (␣-phorbol 12,13-didecanoate), heat-activated currents in both systems showed the typical biophysical properties of currents through TRPV4, including their single channel conductance. Deletion of the three N-terminal ankyrin binding domains of TRPV4 abolished current activation cells by heat in HEK293. In inside-out patches, TRPV4 could not be activated by heat but still responded to the ligand 4␣PDD. In MAEC, the same channel is activated under identical conditions as in the HEK expression system. Our data indicate that TRPV4 is a functional temperature-sensing channel in native endothelium, that is likely involved in temperature-dependent Ca 2؉ signaling. The failure to activate TRPV4 channels by heat in inside-out patches, which responded to 4␣PDD, may indicate that heat activation depends on the presence of an endogenous ligand, which is missing in inside-out patches.Sensing of temperature in the body and the environment is one of the most essential mechanisms for controlling the homeostasis of several regulatory pathways in the mammalian body (1). In recent years, unraveling of thermosensing mechanisms has been very successful, because at least four members of the TRPV subfamily of transient receptor potential cation channels, TRPV 1 1, -2, -3, and -4 and a more distantly related protein TRPM8 have been identified (for a unified nomenclature see Refs. 2 and 3) as sensors of temperature. Proteins of this subfamily typically contain 3-6 ankyrin repeats in the N terminus, and six transmembrane segments with a pore region between segments 5 and 6. The first identified non-mammalian member of this subfamily, the Caenorhabditis elegans OSM-9 channel, is activated by changes in osmolarity (4). The second protein of the TRPV family that has been identified is the mammalian vanilloid receptor channel VR1 (TRPV1), which is activated by vanilloid compounds such as capsaicin, pepper, hot chili, moderate heat, or protons (5). Unlike TRPV1, another close relative of this channel, TRPV2, is constitutively activated by growth factors (6) or by noxious heat (7). TRPV1 and TRPV2 are activated by temperatures above 43 and 52°C, respectively (5, 7). Currents through TRPV3 exponentially increase at temperatures above 35°C (8 -10). It has also been shown in current measurements on oocytes and by cytoplasmic Ca 2ϩ ([Ca 2ϩ ] i) measurements in HEK cells that TRPV4 is activated in both expression systems at temperature above 30°C (11). TRPM8 is activated by temperatures below 22°C and is therefore a candidate for cold reception (12, 13).So far, the molecular mechanism of channel activation by heat for any of these channels is not known, and functional measurements of ...

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.

Background & Aims The cAMP and Ca2+ signaling pathways synergize to regulate many physiological functions. However, little is known about the mechanisms by which these pathways interact. We investigated the synergy between these signaling pathways in mouse pancreatic and salivary gland ducts. Methods We created mice with disruptions in genes encoding the solute carrier family 26, member 6 (Slc26a6−/− mice) and inositol 1,4,5-triphosphate (InsP3) receptor-binding protein released with InsP3 (Irbit−/− mice). We investigated fluid secretion by sealed pancreatic ducts and the function of Slc26a6 and the cystic fibrosis transmembrane conductance regulator (CFTR) in HeLa cells and in ducts isolated from mouse pancreatic and salivary glands. Slc26a6 activity was assayed by measuring intracellular pH, and CFTR activity by measuring Cl− current. Protein interactions were determined by immunoprecipitation analyses. Results Irbit mediated the synergistic activation of CFTR and Slc26a6 by Ca2+ and cAMP. In resting cells, Irbit was sequestered by InsP3 receptors (IP3Rs) in the endoplasmic reticulum. Stimulation of Gs-coupled receptors led to phosphorylation of IP3Rs, which increased their affinity for InsP3 and reduced their affinity for Irbit. Subsequent weak stimulation of Gq-coupled receptors, which led to production of low levels of IP3, caused dissociation of Irbit from IP3Rs and allowed translocation of Irbit to CFTR and Slc26a6 in the plasma membrane. These processes stimulated epithelial secretion of electrolytes and fluid. These pathways were not observed in pancreatic and salivary glands from Irbit−/− or Slc26a6−/− mice, or in salivary gland ducts expressing mutant forms of IP3Rs that could not undergo protein kinase A-mediated phosphorylation. Conclusions Irbit promotes synergy between the Ca2+ and cAMP signaling pathways in cultured cells and in pancreatic and salivary ducts from mice. Defects in this pathway could be involved in CF, pancreatitis, or Sjögren’s syndrome.

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Gb3 accumulation reduces K(Ca)3.1 channel expression by down-regulating ERK and AP-1 and up-regulating REST and the channel activity by decreasing intracellular levels of PI(3)P. Gb3 thereby evokes K(Ca)3.1 channel dysfunction, and the channel dysfunction in vascular endothelial cells may contribute to vasculopathy in Fabry disease.

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Objective— Globotriaosylceramide (Gb3) induces K Ca 3.1 downregulation in Fabry disease (FD). We investigated whether Gb3 induces K Ca 3.1 endocytosis and degradation. Approach and Results— K Ca 3.1, especially plasma membrane–localized K Ca 3.1, was downregulated in both Gb3-treated mouse aortic endothelial cells (MAECs) and human umbilical vein endothelial cells. Gb3-induced K Ca 3.1 downregulation was prevented by lysosomal inhibitors but not by a proteosomal inhibitor. Endoplasmic reticulum stress–inducing agents did not induce K Ca 3.1 downregulation. Gb3 upregulated the protein levels of early endosome antigen 1 and lysosomal-associated membrane protein 2 in MAECs. Compared with MAECs from age-matched wild-type mice, those from aged α-galactosidase A (Gla)-knockout mice, an animal model of FD, showed downregulated K Ca 3.1 expression and upregulated early endosome antigen 1 and lysosomal-associated membrane protein 2 expression. In contrast, no significant difference was found in early endosome antigen 1 and lysosomal-associated membrane protein 2 expression between young Gla-knockout and wild-type MAECs. In aged Gla-knockout MAECs, clathrin was translocated close to the cell border and clathrin knockdown recovered K Ca 3.1 expression. Rab5, an effector of early endosome antigen 1, was upregulated, and Rab5 knockdown restored K Ca 3.1 expression, the current, and endothelium-dependent relaxation. Conclusions —Gb3 accelerates the endocytosis and lysosomal degradation of endothelial K Ca 3.1 via a clathrin-dependent process, leading to endothelial dysfunction in FD.