title: Splice variants of the P2X7 receptor reveal differential agonist dependence and functional coupling with pannexin-1.
abstract: P2X7 receptors function as ATP-gated cation channels but also interact with other proteins as part of a larger signalling complex to mediate a variety of downstream responses that are dependent upon the cell type in which they are expressed. Receptor-mediated membrane permeabilization to large molecules precedes the induction of cell death, but remains poorly understood. The mechanisms that underlie differential sensitivity to NAD are also unknown. By studying alternative variants of the mouse P2X7 receptor we show that sensitivity to NAD is mediated through the P2X7k variant, which has a much more restricted distribution than the P2X7a receptor, but is expressed in T lymphocytes. The altered N-terminus and TM1 of the P2X7k receptor enhances the stability of the active state of this variant compared with P2X7a, thereby increasing the efficacy of NAD-dependent ADP ribosylation as measured by ethidium uptake, a rise in intracellular Ca(2+) and the activation of inward currents. Co-expression of P2X7k and P2X7a receptors reduced NAD sensitivity. P2X7k-receptor-mediated ethidium uptake was also triggered by much lower BzATP concentrations and was insensitive to the P451L single nucleotide polymorphism. P2X7k-receptor-mediated ethidium uptake occurred independently of pannexin-1 suggesting a pathway intrinsic to the receptor. Only for the P2X7aL451 receptor could we resolve a component of dye uptake dependent upon pannexin-1. Signalling occurred downstream of the activation of caspases rather than involving direct cross talk between the channels. However, an in situ proximity assay showed close association between P2X7 receptors and pannexin-1, which would facilitate ATP efflux through pannexin-1 acting in an autocrine manner.
authors.affiliation: Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD,  UK.
authors.forename: Xing Jian
authors.initials: XJ
authors.lastname: Xu
authors.forename: Miyyada
authors.initials: M
authors.lastname: Boumechache
authors.forename: Lucy E
authors.initials: LE
authors.lastname: Robinson
authors.forename: Viola
authors.initials: V
authors.lastname: Marschall
authors.forename: Dariusz C
authors.initials: DC
authors.lastname: Gorecki
authors.forename: Marianela
authors.initials: M
authors.lastname: Masin
authors.forename: Ruth D
authors.initials: RD
authors.lastname: Murrell-Lagnado
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D016923:Cell Death / Q000235:genetics; D002460:Cell Line; D017630:Connexins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D004996:Ethidium / Q000493:pharmacokinetics; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009243:NAD / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D020641:Polymorphism, Single Nucleotide; D020033:Protein Isoforms; D058911:Purinergic P2X Receptor Agonists / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D058486:Receptors, Purinergic P2X7 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000378:metabolism; D014162:Transfection
chemical_list: D017630:Connexins; D009419:Nerve Tissue Proteins; C503631:Panx1 protein, mouse; D020033:Protein Isoforms; D058911:Purinergic P2X Receptor Agonists; D034741:RNA, Small Interfering; D058486:Receptors, Purinergic P2X7; D009243:NAD; D000244:Adenosine Diphosphate; D004996:Ethidium