title: Determinants of TRPV4 activity following selective activation by small molecule agonist GSK1016790A.
abstract: TRPV4 (Transient Receptor Potential Vanilloid 4) channels are activated by a wide range of stimuli, including hypotonic stress, non-noxious heat and mechanical stress and some small molecule agonists (e.g. phorbol ester 4α-PDD). GSK1016790A (GSK101) is a recently discovered specific small molecule agonist of TRPV4. Its effects on physical determinants of TRPV4 activity were evaluated in HeLa cells transiently transfected with TRPV4 (HeLa-TRPV4). GSK101 (10 nM) causes a TRPV4 specific Ca(2+) influx in HeLa-TRPV4 cells, but not in control transfected cells, which can be inhibited by ruthenium red and Ca(2+)-free medium more significantly at the early stage of the activation rather than the late stage, reflecting apparent partial desensitization. Western blot analysis showed that GSK101 activation did not induce an increase in TRPV4 expression at the plasma membrane, but caused an immediate and sustained downregulation of TRPV4 on the plasma membrane in HeLa-TRPV4 cells. Patch clamp analysis also revealed an early partial desensitization of the channel which was Ca(2+)-independent. FRET analysis of TRPV4 subunit assembly demonstrated that the GSK101-induced TRPV4 channel activation/desensitization was not due to alterations in homotetrameric channel formation on the plasma membrane. It is concluded that GSK101 specifically activates TRPV4 channels, leading to a rapid partial desensitization and downregulation of the channel expression on the plasma membrane. TRPV4 subunit assembly appears to occur during trafficking from the ER/Golgi to the plasma membrane and is not altered by agonist stimulation.
authors.affiliation: Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center, Houston, Texas, United States of America.
authors.forename: Min
authors.initials: M
authors.lastname: Jin
authors.forename: Zizhen
authors.initials: Z
authors.lastname: Wu
authors.forename: Ling
authors.initials: L
authors.lastname: Chen
authors.forename: Jose
authors.initials: J
authors.lastname: Jaimes
authors.forename: Diana
authors.initials: D
authors.lastname: Collins
authors.forename: Edgar T
authors.initials: ET
authors.lastname: Walters
authors.forename: Roger G
authors.initials: RG
authors.lastname: O'Neil
mesh_terms: D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004396:Coloring Agents / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical; D006367:HeLa Cells; D006801:Humans; D007930:Leucine / Q000031:analogs & derivatives* / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D055503:Protein Multimerization / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects / Q000235:genetics; D012430:Ruthenium Red / Q000494:pharmacology; D013379:Substrate Specificity; D013449:Sulfonamides / Q000494:pharmacology*; D050916:TRPV Cation Channels / Q000819:agonists* / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: D004396:Coloring Agents; C530602:N-(1-((4-(2-(((2,4-dichlorophenyl)sulfonyl)amino)-3-hydroxypropanoyl)-1-piperazinyl)carbonyl)-3-methylbutyl)-1-benzothiophene-2-carboxamide; D013449:Sulfonamides; D050916:TRPV Cation Channels; C416986:TRPV4 protein, human; D012430:Ruthenium Red; D007930:Leucine; D002118:Calcium