title: Membrane topology of an ATP-gated ion channel (P2X receptor).
abstract: Western blots of Xenopus oocyte membrane preparations showed that the apparent molecular mass of the wild type P2X2 receptor (about 65 kDa) was reduced by pretreatment with endoglycosidase H. Mutagenesis of one or more of three potential asparagines (N182S, N239S, and N298S) followed by Western blots showed that each of the sites was glycosylated in the wild type receptor. Functional channels were formed by receptors lacking any single asparagine, but not by channels mutated in two or three positions. Artificial consensus sequences (N-X-S/T) introduced into the N-terminal region (asparagine at position 9, 16, or 26) were not glycosylated. Asparagines were glycosylated when introduced at the C-terminal end of the first hydrophobic domain (positions 62 and 66) and at the N-terminal end of the second hydrophobic domain (position 324). A protein in which the C terminus of one P2X2 subunit was joined to the N terminus of a second P2X2 subunit (from a concatenated cDNA) had twice the molecular mass of the P2X2 receptor subunit, and formed fully functional channels. The experiments provide direct evidence for the topology originally proposed for the P2X receptor, with intracellular N and C termini, two membrane-spanning domains, and a large extracellular loop.
authors.affiliation: Geneva Biomedical Research Institute, Glaxo Wellcome Research and Development, Plan-les-Ouates, 1228 Geneva, Switzerland.
authors.forename: A
authors.initials: A
authors.lastname: Newbolt
authors.forename: R
authors.initials: R
authors.lastname: Stoop
authors.forename: C
authors.initials: C
authors.lastname: Virginio
authors.forename: A
authors.initials: A
authors.lastname: Surprenant
authors.forename: R A
authors.initials: RA
authors.lastname: North
authors.forename: G
authors.initials: G
authors.lastname: Buell
authors.forename: F
authors.initials: F
authors.lastname: Rassendren
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D019281:Dimerization; D004594:Electrophysiology; D006031:Glycosylation; D006596:Hexosaminidases / Q000378:metabolism; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D008565:Membrane Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009865:Oocytes / Q000502:physiology; D018048:Receptors, Purinergic P2 / Q000737:chemistry*; D058476:Receptors, Purinergic P2X2; D014162:Transfection / Q000235:genetics; D014982:Xenopus laevis
chemical_list: D008565:Membrane Proteins; C546401:P2RX2 protein, human; D018048:Receptors, Purinergic P2; D058476:Receptors, Purinergic P2X2; D000255:Adenosine Triphosphate; D006596:Hexosaminidases