title: Subunit dissociation of TRPC3 ion channel under high-salt condition.
abstract: Single particle analysis is a computer-aided method for determining protein structure using particle images obtained by electron microscopy. This technique has great advantages, especially for analyzing fragile membrane-integrated proteins including ion channels, transporters, and receptors, and for analyzing large complexes difficult to crystallize. It is also useful in the analysis of dynamic conformational changes. We previously determined the structure of TRPC3 (canonical transient receptor potential-3) from negatively stained specimens and from ice-embedded specimens using single particle analysis (BBRC 333: 768-777, 2005; JMB 367: 373-383, 2007). These analyses revealed TRPC3's unique structural features, as well as demonstrating the first visual evidence of the tetramer structure. In establishing the purification procedure, we noticed that the stability of the tetrameric assembly of TRPC3 subunits is largely dependent on the cation concentration in the solution. Here, we report that the TRPC3 tetramer dissociates to monomers under high-salt conditions. It was demonstrated as a delay of elution in size exclusion chromatography, or as a loss of tetrameric protein bands in cross-linking experiments. Electron microscopy of the negatively stained specimens demonstrated that the large tetrameric TRPC3 (200 A in width and 240 A in height) dissociates to round-shaped monomer particles (100 A in diameter) in an ion-strength-dependent manner. These results also suggested that electron microscopy is highly effective when used in the "quality check" of the specimen in each purification step.
authors.affiliation: Neuroscience Research Institute, National Institute of Advanced Industrial Science and Technology, Umezono 1-1-4, Tsukuba, Ibaraki 305-8568, Japan.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Mio
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Ogura
authors.forename: Shigeki
authors.initials: S
authors.lastname: Kiyonaka
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Mori
authors.forename: Chikara
authors.initials: C
authors.lastname: Sato
mesh_terms: D006801:Humans; D007091:Image Processing, Computer-Assisted; D015636:Magnesium Chloride; D046529:Microscopy, Electron, Transmission; D009994:Osmolar Concentration; D010316:Particle Size; D020836:Protein Structure, Quaternary; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000648:ultrastructure; D050052:TRPC Cation Channels / Q000737:chemistry* / Q000302:isolation & purification / Q000648:ultrastructure*
chemical_list: D021122:Protein Subunits; D011994:Recombinant Proteins; D050052:TRPC Cation Channels; C104603:TRPC3 cation channel; D015636:Magnesium Chloride