Ca2+-dependent potentiation of the nonselective cation channel TRPV4 is mediated by a C-terminal calmodulin binding site.
Most Ca2+-permeable ion channels are inhibited by increases in the intracellular Ca2+ concentration ([Ca2+]i), thus preventing potentially deleterious rises in [Ca2+]i. In this study, we demonstrate that currents through the osmo-, heat- and phorbol ester-sensitive, Ca2+-permeable nonselective cation channel TRPV4 are potentiated by intracellular Ca2+. Spontaneous TRPV4 currents and currents stimulated by hypotonic solutions or phorbol esters were reduced strongly at all potentials in the absence of extracellular Ca2+. The other permeant divalent cations Ba2+ and Sr2+ were less effective than Ca2+ in supporting channel activity. An intracellular site of Ca2+ action was supported by the parallel decrease in spontaneous currents and [Ca2+]i on removal of extracellular Ca2+ and the ability of Ca2+ release from intracellular stores to restore TRPV4 activity in the absence of extracellular Ca2+. During TRPV4 activation by hypotonic solutions or phorbol esters, Ca2+ entry through the channel increased the rate and extent of channel activation. Currents were also potentiated by ionomycin in the presence of extracellular Ca2+. Ca2+-dependent potentiation of TRPV4 was often followed by inhibition. By mutagenesis, we localized the structural determinant of Ca2+-dependent potentiation to an intracellular, C-terminal calmodulin binding domain. This domain binds calmodulin in a Ca2+-dependent manner. TRPV4 mutants that did not bind calmodulin lacked Ca2+-dependent potentiation. We conclude that TRPV4 activity is tightly controlled by intracellular Ca2+. Ca2+ entry increases both the rate and extent of channel activation by a calmodulin-dependent mechanism. Excessive increases in [Ca2+]i via TRPV4 are prevented by a Ca2+-dependent negative feedback mechanism.
Strotmann
Rainer
R
Institut für Pharmakologie, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, Thielallee 67-73, 14195 Berlin, Germany.
Schultz
Gunter
G
Plant
Tim D
TD
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002147:Calmodulin / Q000378:metabolism*; D027682:Cation Transport Proteins; D002413:Cations, Divalent / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D005246:Feedback; D006801:Humans; D066298:In Vitro Techniques; D007473:Ion Channels / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D050916:TRPV Cation Channels
D002147:Calmodulin; D027682:Cation Transport Proteins; D002413:Cations, Divalent; D007473:Ion Channels; D007476:Ionophores; D011994:Recombinant Proteins; D050916:TRPV Cation Channels; C416986:TRPV4 protein, human; D015759:Ionomycin; D004247:DNA; D002118:Calcium