title: Osmosensation in TRPV2 dominant negative expressing skeletal muscle fibres.
abstract: Increased plasma osmolarity induces intracellular water depletion and cell shrinkage (CS) followed by activation of a regulatory volume increase (RVI). In skeletal muscle, the hyperosmotic shock-induced CS is accompanied by a small membrane depolarization responsible for a release of Ca(2+) from intracellular pools. Hyperosmotic shock also induces phosphorylation of STE20/SPS1-related proline/alanine-rich kinase (SPAK). TRPV2 dominant negative expressing fibres challenged with hyperosmotic shock present a slower membrane depolarization, a diminished Ca(2+) response, a smaller RVI response, a decrease in SPAK phosphorylation and defective muscle function. We suggest that hyperosmotic shock induces TRPV2 activation, which accelerates muscle cell depolarization and allows the subsequent Ca(2+) release from the sarcoplasmic reticulum, activation of the Na(+) -K(+) -Cl(-) cotransporter by SPAK, and the RVI response. Increased plasma osmolarity induces intracellular water depletion and cell shrinkage followed by activation of a regulatory volume increase (RVI). In skeletal muscle, this is accompanied by transverse tubule (TT) dilatation and by a membrane depolarization responsible for a release of Ca(2+) from intracellular pools. We observed that both hyperosmotic shock-induced Ca(2+) transients and RVI were inhibited by Gd(3+) , ruthenium red and GsMTx4 toxin, three inhibitors of mechanosensitive ion channels. The response was also completely absent in muscle fibres overexpressing a non-permeant, dominant negative (DN) mutant of the transient receptor potential, V2 isoform (TRPV2) ion channel, suggesting the involvement of TRPV2 or of a TRP isoform susceptible to heterotetramerization with TRPV2. The release of Ca(2+) induced by hyperosmotic shock was increased by cannabidiol, an activator of TRPV2, and decreased by tranilast, an inhibitor of TRPV2, suggesting a role for the TRPV2 channel itself. Hyperosmotic shock-induced membrane depolarization was impaired in TRPV2-DN fibres, suggesting that TRPV2 activation triggers the release of Ca(2+) from the sarcoplasmic reticulum by depolarizing TTs. RVI requires the sequential activation of STE20/SPS1-related proline/alanine-rich kinase (SPAK) and NKCC1, a Na(+) -K(+) -Cl(-) cotransporter, allowing ion entry and driving osmotic water flow. In fibres overexpressing TRPV2-DN as well as in fibres in which Ca(2+) transients were abolished by the Ca(2+) chelator BAPTA, the level of P-SPAK(Ser373) in response to hyperosmotic shock was reduced, suggesting a modulation of SPAK phosphorylation by intracellular Ca(2+) . We conclude that TRPV2 is involved in osmosensation in skeletal muscle fibres, acting in concert with P-SPAK-activated NKCC1.
authors.affiliation: Laboratory of Cell Physiology, Institute of Neuroscience, Université catholique de Louvain, av. Mounier, B1.53.17, B-1200, Brussels, Belgium.
authors.forename: Nadège
authors.initials: N
authors.lastname: Zanou
authors.affiliation: Laboratory of Cell Physiology, Institute of Neuroscience, Université catholique de Louvain, av. Mounier, B1.53.17, B-1200, Brussels, Belgium.
authors.forename: Ludivine
authors.initials: L
authors.lastname: Mondin
authors.affiliation: Centre de Génétique et de Physiologie Cellulaire et Moléculaire, Université Claude Bernard Lyon 1, CNRS, UMR 5534, 69622, Villeurbanne, France.
authors.forename: Clarisse
authors.initials: C
authors.lastname: Fuster
authors.affiliation: Laboratory of Cell Physiology, Institute of Neuroscience, Université catholique de Louvain, av. Mounier, B1.53.17, B-1200, Brussels, Belgium.
authors.forename: François
authors.initials: F
authors.lastname: Seghers
authors.affiliation: Laboratory of Cell Physiology, Institute of Neuroscience, Université catholique de Louvain, av. Mounier, B1.53.17, B-1200, Brussels, Belgium.
authors.forename: Inès
authors.initials: I
authors.lastname: Dufour
authors.affiliation: Laboratory of Cell Physiology, Institute of Neuroscience, Université catholique de Louvain, av. Mounier, B1.53.17, B-1200, Brussels, Belgium.
authors.forename: Marie
authors.initials: M
authors.lastname: de Clippele
authors.affiliation: Laboratory of Cell Physiology, Institute of Neuroscience, Université catholique de Louvain, av. Mounier, B1.53.17, B-1200, Brussels, Belgium.
authors.forename: Olivier
authors.initials: O
authors.lastname: Schakman
authors.affiliation: Laboratory of Cell Physiology, Institute of Neuroscience, Université catholique de Louvain, av. Mounier, B1.53.17, B-1200, Brussels, Belgium.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Tajeddine
authors.affiliation: Department of Molecular Physiology, National Cardiovascular Center Research Institute Suita, Osaka, 565-8565, Japan.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Iwata
authors.affiliation: Department of Molecular Physiology, National Cardiovascular Center Research Institute Suita, Osaka, 565-8565, Japan.
authors.forename: Shigeo
authors.initials: S
authors.lastname: Wakabayashi
authors.affiliation: Laboratory of Ion Channel Research, Department of Cellular and Molecular Medicine, Katholiek Universiteit Leuven, B-3000, Leuven, Belgium.
authors.forename: Thomas
authors.initials: T
authors.lastname: Voets
authors.affiliation: Centre de Génétique et de Physiologie Cellulaire et Moléculaire, Université Claude Bernard Lyon 1, CNRS, UMR 5534, 69622, Villeurbanne, France.
authors.forename: Bruno
authors.initials: B
authors.lastname: Allard
authors.affiliation: Laboratory of Cell Physiology, Institute of Neuroscience, Université catholique de Louvain, av. Mounier, B1.53.17, B-1200, Brussels, Belgium.
authors.forename: Philippe
authors.initials: P
authors.lastname: Gailly
mesh_terms: D000818:Animals; D002118:Calcium; D015220:Calcium Channels / Q000502:physiology*; D048429:Cell Size; D008297:Male; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D018485:Muscle Fibers, Skeletal / Q000502:physiology*; D009994:Osmolar Concentration; D009997:Osmotic Pressure; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D064506:Solute Carrier Family 12, Member 2 / Q000502:physiology*; D050916:TRPV Cation Channels / Q000502:physiology*
chemical_list: D015220:Calcium Channels; C578878:Slc12a2 protein, mouse; D064506:Solute Carrier Family 12, Member 2; D050916:TRPV Cation Channels; C485717:Trpv2 protein, mouse; C486407:Stk39 protein, mouse; D017346:Protein Serine-Threonine Kinases; D002118:Calcium