title: Identification of P2X2/P2X4/P2X6 heterotrimeric receptors using atomic force microscopy (AFM) imaging.
abstract: Seven P2X purinergic receptor subunits have been identified: P2X1-P2X7. The overlapping expression of P2X2, P2X4 and P2X6 subunits has been shown in different cell types, and functional analysis of P2X receptors in Leydig cells suggests that the three subunits might interact. Here, His6-tagged P2X2, HA-tagged P2X4 and FLAG-tagged P2X6 subunits were co-expressed in tsA 201 cells. After sequential co-immunoprecipitation using anti-HA and anti-FLAG beads, all three subunits were present, demonstrating their interaction. Atomic force microscopy (AFM) imaging revealed receptors that were specifically decorated by both an anti-His6 antibody and an anti-HA Fab fragment, indicating the presence of a P2X2/4/6 heterotrimer. To our knowledge, this is the first report of a P2X receptor containing three different subunits.
authors.affiliation: Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, United Kingdom; Department of Physiology, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil.
authors.forename: Ligia S
authors.initials: LS
authors.lastname: Antonio
authors.affiliation: Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, United Kingdom.
authors.forename: Andrew P
authors.initials: AP
authors.lastname: Stewart
authors.affiliation: Department of Physiology, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil.
authors.forename: Wamberto A
authors.initials: WA
authors.lastname: Varanda
authors.affiliation: Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, United Kingdom. Electronic address: jme1000@cam.ac.uk.
authors.forename: J Michael
authors.initials: JM
authors.lastname: Edwardson
mesh_terms: D000818:Animals; D057809:HEK293 Cells; D006801:Humans; D018625:Microscopy, Atomic Force; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000737:chemistry; D058469:Receptors, Purinergic P2X / Q000737:chemistry*; D058476:Receptors, Purinergic P2X2 / Q000737:chemistry; D058848:Receptors, Purinergic P2X4 / Q000737:chemistry
chemical_list: D018048:Receptors, Purinergic P2; D058469:Receptors, Purinergic P2X; D058476:Receptors, Purinergic P2X2; D058848:Receptors, Purinergic P2X4; C110590:purinoceptor P2X6
keywords: Atomic force microscopy; P2X receptor; Receptor assembly