title: Mechanical force and cytoplasmic Ca(2+) activate yeast TRPY1 in parallel.
abstract: The ability to sense mechanical and osmotic stimuli is vital to all organisms from mammals to bacteria. Members of the transient receptor potential (TRP) ion-channel family have attracted intense attention for their involvement in mechanosensation. The yeast homologue TRPY1 can clearly be activated by hypertonic shock in vivo and by stretch force under patch clamp. Like its animal counterparts, TRPY1 is polymodal, being gated by membrane stretch force and by cytoplasmic Ca(2+). Here, we investigated how these two gating principles interact. We found that stretch force can induce some channel activation without cytoplasmic Ca(2+). Tens of micromolar Ca(2+) greatly enhance the observed force-induced activities, with open probabilities following well the Boltzmann distribution, in which the two gating energies are summed as exponents. To map this formalism to structures, we found Ca(2+)-binding proteins such as calmodulin or calcineurin to be unnecessary. However, removing a dense cluster of negative charges in the C-terminal cytoplasmic domain of TRPY1 greatly diminishes the Ca(2+) activation as well as its influence on force activation. We also found a strategic point upstream of this charge cluster, at which insertion of amino acids weakens Ca(2+) activation considerably but leaves the mechanosensitivity nearly intact. These results led to a structure-function model in which Ca(2+) binding to the cytoplasmic domain and stretching of the membrane-embedded domain both generate gating force, reaching the gate in parallel.
authors.affiliation: University of Wisconsin-Madison, 53706, USA.
authors.forename: Zhenwei
authors.initials: Z
authors.lastname: Su
authors.forename: Xinliang
authors.initials: X
authors.lastname: Zhou
authors.forename: Stephen H
authors.initials: SH
authors.lastname: Loukin
authors.forename: Yoshiro
authors.initials: Y
authors.lastname: Saimi
authors.forename: Ching
authors.initials: C
authors.lastname: Kung
mesh_terms: D001696:Biomechanical Phenomena; D019703:Calcineurin / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism*; D004594:Electrophysiology; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D050051:Transient Receptor Potential Channels / Q000378:metabolism*
chemical_list: D002147:Calmodulin; D029701:Saccharomyces cerevisiae Proteins; D050051:Transient Receptor Potential Channels; D019703:Calcineurin; D002118:Calcium