title: Visualization of the trimeric P2X2 receptor with a crown-capped extracellular domain.
abstract: The P2X2 purinergic receptor permeates cationic ions in response to stimulation by ATP and mediates fast synaptic transmission. Here, we purified the P2X2 receptor using baculovirus-Sf9 cell expression system and observed its structure using electron microscopy. The FLAG-tagged P2X2 receptor, which has intact ion channel function, was purified to be a single peak by affinity purification and gel filtration chromatography. It was confirmed to be a trimer by introducing cross-linking. Negatively stained P2X2 protein images were homogeneous and picked up by automated pick-up programs, aligned, and classified using the modified growing neural gas network method. Similarly oriented projections were averaged to decrease the signal-to-noise ratio. These images demonstrate an inverted three-sided pyramid with the dimensions of 215 A in height and 200 A in side length. It is composed of a high-density trunk and a stain-permeable swollen extracellular domain of a crown-shaped structure. The internal cavities and constituent segments were clearly demonstrated in both the raw images and the averaged images. The threefold symmetrical top view demonstrates the first visual evidence of the trimeric composition of the P2X receptor family.
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: Yoshihiro
authors.initials: Y
authors.lastname: Kubo
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Ogura
authors.forename: Tomomi
authors.initials: T
authors.lastname: Yamamoto
authors.forename: Chikara
authors.initials: C
authors.lastname: Sato
mesh_terms: D000818:Animals; D019281:Dimerization; D005110:Extracellular Space / Q000737:chemistry*; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry / Q000648:ultrastructure; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000737:chemistry* / Q000648:ultrastructure*; D058476:Receptors, Purinergic P2X2; D013329:Structure-Activity Relationship
chemical_list: D046912:Multiprotein Complexes; D021122:Protein Subunits; D018048:Receptors, Purinergic P2; D058476:Receptors, Purinergic P2X2