title: An uncharged region within the N terminus of the P2X6 receptor inhibits its assembly and exit from the endoplasmic reticulum.
abstract: ATP-gated P2X receptors are trimeric complexes formed by the homomeric or heteromeric assembly of seven different subunits. We have shown previously that, unlike all of the other P2X subunits, the P2X6 subunit cannot form homomeric receptors and when expressed alone is retained in the endoplasmic reticulum (ER) in monomeric form (J Biol Chem 280: 107591-10765, 2005). However, other studies have shown that P2X6 can form functional heteromeric receptors with P2X2 and P2X4 subunits. In this study, we used a combination of immunocytochemistry, surface biotinylation, and atomic force microscopy to investigate the assembly and trafficking of the P2X6 subunit, both alone and as part of a heteromer. We show that as a heteromer, it exits the ER and is either stably expressed at the cell surface or constitutively internalized, depending on its partner. Through the use of targeted mutation, we demonstrate that an uncharged region at the N terminus of P2X6 exerts an inhibitory effect on its assembly and export from the ER. When this region is removed, or when charge is added to it, P2X6 forms homotrimeric assemblies, undergoes complex glycosylation and is delivered to the plasma membrane, albeit less efficiently than the P2X2 receptor. The N-terminal mutants were, however, nonfunctional. Substituting the uncharged 14-amino acid N-terminal region for the equivalent region of P2X2 increased ER retention but was not sufficient to prevent the formation of functional homomeric receptors. We propose that the N terminus of the P2X6 subunit contributes to a mechanism that prevents the inappropriate export and plasma membrane expression of nonfunctional P2X receptors.
authors.affiliation: Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, United Kingdom.
authors.forename: Susan J
authors.initials: SJ
authors.lastname: Ormond
authors.forename: Nelson P
authors.initials: NP
authors.lastname: Barrera
authors.forename: Omar S
authors.initials: OS
authors.lastname: Qureshi
authors.forename: Robert M
authors.initials: RM
authors.lastname: Henderson
authors.forename: J Michael
authors.initials: JM
authors.lastname: Edwardson
authors.forename: Ruth D
authors.initials: RD
authors.lastname: Murrell-Lagnado
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000502:physiology*; D006801:Humans; D007668:Kidney; D046911:Macromolecular Substances; D018625:Microscopy, Atomic Force; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D020239:Receptor Cross-Talk; D018048:Receptors, Purinergic P2 / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D014162:Transfection
chemical_list: D046911:Macromolecular Substances; D010446:Peptide Fragments; D018048:Receptors, Purinergic P2; C110590:purinoceptor P2X6