title: A 3.5-nm structure of rat TRPV4 cation channel revealed by Zernike phase-contrast cryoelectron microscopy.
abstract: The transient receptor potential vanilloid 4 (TRPV4) is a non-selective cation channel responsive to various stimuli including cell swelling, warm temperatures (27-35 degrees C), and chemical compounds such as phorbol ester derivatives. Here we report the three-dimensional structure of full-length rat TRPV4 purified from baculovirus-infected Sf9 cells. Hexahistidine-tagged rat TRPV4 (His-rTRPV4) was solubilized with detergent and purified through affinity chromatography and size-exclusion chromatography. Chemical cross-linking analysis revealed that detergent-solubilized His-rTRPV4 was a tetramer. The 3.5-nm structure of rat TRPV4 was determined by cryoelectron microscopy using single-particle reconstruction from Zernike phase-contrast images. The overall structure comprises two distinct regions; a larger dense component, likely corresponding to the cytoplasmic N- and C-terminal regions, and a smaller component corresponding to the transmembrane region.
authors.affiliation: Division of Nano-Structure Physiology, Okazaki Institute for Integrative Bioscience, Higashiyama, Myodaiji, Okazaki 444-8787 Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Shigematsu
authors.forename: Takaaki
authors.initials: T
authors.lastname: Sokabe
authors.forename: Radostin
authors.initials: R
authors.lastname: Danev
authors.forename: Makoto
authors.initials: M
authors.lastname: Tominaga
authors.forename: Kuniaki
authors.initials: K
authors.lastname: Nagayama
mesh_terms: D000818:Animals; D002118:Calcium / Q000737:chemistry; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods; D003432:Cross-Linking Reagents / Q000737:chemistry; D020285:Cryoelectron Microscopy / Q000379:methods*; D003902:Detergents / Q000494:pharmacology; D007091:Image Processing, Computer-Assisted; D007313:Insecta; D046529:Microscopy, Electron, Transmission / Q000379:methods; D008858:Microscopy, Phase-Contrast / Q000379:methods*; D008968:Molecular Conformation; D011487:Protein Conformation; D051381:Rats; D050916:TRPV Cation Channels / Q000737:chemistry*
chemical_list: D003432:Cross-Linking Reagents; D003902:Detergents; D050916:TRPV Cation Channels; C486577:Trpv4 protein, rat; D002118:Calcium