title: Molecular determinants of permeation through the cation channel TRPV4.
abstract: We have studied the molecular determinants of ion permeation through the TRPV4 channel (VRL-2, TRP12, VR-OAC, and OTRPC4). TRPV4 is characterized by both inward and outward rectification, voltage-dependent block by Ruthenium Red, a moderate selectivity for divalent versus monovalent cations, and an Eisenman IV permeability sequence. We identify two aspartate residues, Asp(672) and Asp(682), as important determinants of the Ca(2+) sensitivity of the TRPV4 pore. Neutralization of either aspartate to alanine caused a moderate reduction of the relative permeability for divalent cations and of the degree of outward rectification. Neutralizing both aspartates simultaneously caused a much stronger reduction of Ca(2+) permeability and channel rectification and additionally altered the permeability order for monovalent cations toward Eisenman sequence II or I. Moreover, neutralizing Asp(682) but not Asp(672) strongly reduces the affinity of the channel for Ruthenium Red. Mutations to Met(680), which is located at the center of a putative selectivity filter, strongly reduced whole cell current amplitude and impaired Ca(2+) permeation. In contrast, neutralizing the only positively charged residue in the putative pore region, Lys(675), had no obvious effects on the properties of the TRPV4 channel pore. Our findings delineate the pore region of TRPV4 and give a first insight into the possible architecture of its permeation pathway.
authors.affiliation: Department of Physiology, Campus Gasthuisberg, KU Leuven, Herestraat 49, B-3000 Leuven, Belgium.
authors.forename: Thomas
authors.initials: T
authors.lastname: Voets
authors.forename: Jean
authors.initials: J
authors.lastname: Prenen
authors.forename: Joris
authors.initials: J
authors.lastname: Vriens
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Watanabe
authors.forename: Annelies
authors.initials: A
authors.lastname: Janssens
authors.forename: Ulrich
authors.initials: U
authors.lastname: Wissenbach
authors.forename: Matthias
authors.initials: M
authors.lastname: Bödding
authors.forename: Guy
authors.initials: G
authors.lastname: Droogmans
authors.forename: Bernd
authors.initials: B
authors.lastname: Nilius
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D027682:Cation Transport Proteins; D002478:Cells, Cultured; D006801:Humans; D007473:Ion Channels / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000502:physiology*; D008274:Magnesium / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010539:Permeability; D012430:Ruthenium Red / Q000494:pharmacology; D013329:Structure-Activity Relationship; D050916:TRPV Cation Channels
chemical_list: D027682:Cation Transport Proteins; D007473:Ion Channels; D050916:TRPV Cation Channels; C416986:TRPV4 protein, human; C486576:Trpv4 protein, mouse; D012430:Ruthenium Red; D008274:Magnesium; D002118:Calcium