Functional interaction of the cation channel transient receptor potential vanilloid 4 (TRPV4) and actin in volume regulation.
Many vertebrate cells react to hypotonic conditions with swelling, followed by an active downregulation of the cell volume; a progress called regulatory volume decrease (RVD). While the actual process of volume decrease by loss of osmotically active molecules like K(+) and Cl(-), followed by water efflux has been extensively investigated, the signal for activation of RVD still remains obscure. Studies with different cell lines demonstrated a participation of the cation channel transient receptor potential vanilloid 4 (TRPV4) as well as the actin cytoskeleton in volume regulation. Therefore, we analyzed putative links between TRPV4 and F-actin in RVD in HaCaT keratinocytes and CHO cells. Laser scanning microscopy studies revealed a distinct colocalization of TRPV4 and actin in highly dynamic membrane structures, such as microvilli, filopodia and lamellipodia edges. After treatment of cells with the actin-destabilizing reagent latrunculin A, TRPV4 and F-actin no longer colocalized within the membrane. In accordance with these data, close interaction between TRPV4 and F-actin was revealed by FRAP and FRET studies. For functional analysis, CHO cells that endogenously do not express TRPV4, were transfected with recombinant TRPV4, which rendered them RVD-competent. Treatment with latrunculin A abolished both, RVD and the accompanying rise of [Ca(2+)](i) after hypotonic stress in TRPV4-transfected CHO cells. Taken together, our data demonstrate a functional interaction between TRPV4 and F-actin in sensing hypotonicity and the onset of RVD.
Becker
Daniel
D
Institute for Cell Biology and Neuroscience, Center of Excellence Frankfurt: Macromolecular Complexes, JW Goethe University, Frankfurt/Main, Germany.
Bereiter-Hahn
Jürgen
J
Jendrach
Marina
M
D000199:Actins / Q000378:metabolism*; D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D016466:CHO Cells; D020013:Calcium Signaling / Q000187:drug effects; D048429:Cell Size / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007038:Hypotonic Solutions / Q000494:pharmacology; D015603:Keratinocytes / Q000166:cytology* / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D050916:TRPV Cation Channels / Q000378:metabolism*; D053778:Thiazolidines / Q000494:pharmacology
D000199:Actins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D007038:Hypotonic Solutions; D011993:Recombinant Fusion Proteins; D050916:TRPV Cation Channels; C416986:TRPV4 protein, human; D053778:Thiazolidines; D049452:Green Fluorescent Proteins; C037067:latrunculin A