title: Expression, assembly and function of novel C-terminal truncated variants of the mouse P2X7 receptor: re-evaluation of P2X7 knockouts.
abstract: OBJECTIVE
  Splice variants of P2X7 receptor transcripts contribute to the diversity of receptor-mediated responses. Here, we investigated expression and function of C-terminal truncated (ΔC) variants of the mP2X7 receptor, which are predicted to escape inactivation in one strain of P2X7(-/-) mice (Pfizer KO).
  
  
  METHODS
  Expression in wild-type (WT) and Pfizer KO tissue was investigated by reverse transcription (RT)-PCR and Western blot analysis. ΔC variants were also cloned and expressed in HEK293 cells to investigate their assembly, trafficking and function.
  
  
  RESULTS
  RT-PCR indicates expression of a ΔC splice variant in brain, salivary gland (SG) and spleen from WT and Pfizer KO mice. An additional ΔC hybrid transcript, containing sequences of P2X7 upstream of exon 12, part of exon 13 followed in-frame by the sequence of the vector used to disrupt the P2X7 gene, was also identified in the KO mice. By blue native (BN) PAGE analysis and the use of cross linking reagents followed by SDS-PAGE, P2X7 trimers, dimers and monomers were detected in the spleen and SG of Pfizer KO mice. The molecular mass was reduced compared with P2X7 in WT mice tissue, consistent with a ΔC variant. When expressed in HEK293 cells the ΔC variants were inefficiently trafficked to the cell surface and agonist-evoked whole cell currents were small. Co-expressed with P2X7A, the ΔC splice variant acted in a dominant negative fashion to inhibit function.
  
  
  CONCLUSIONS
  Pfizer KO mice are not null for P2X7 receptor expression but express ΔC variants with reduced function.
authors.affiliation: Department of Pharmacology, University of Cambridge, Cambridge, UK.
authors.forename: Marianela
authors.initials: M
authors.lastname: Masin
authors.forename: Christopher
authors.initials: C
authors.lastname: Young
authors.forename: Koini
authors.initials: K
authors.lastname: Lim
authors.forename: Sara J
authors.initials: SJ
authors.lastname: Barnes
authors.forename: Xing Jian
authors.initials: XJ
authors.lastname: Xu
authors.forename: Viola
authors.initials: V
authors.lastname: Marschall
authors.forename: Wojciech
authors.initials: W
authors.lastname: Brutkowski
authors.forename: Elizabeth R
authors.initials: ER
authors.lastname: Mooney
authors.forename: Dariusz C
authors.initials: DC
authors.lastname: Gorecki
authors.forename: Ruth
authors.initials: R
authors.lastname: Murrell-Lagnado
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D020033:Protein Isoforms / Q000235:genetics / Q000502:physiology; D012333:RNA, Messenger / Q000235:genetics; D058486:Receptors, Purinergic P2X7 / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012469:Salivary Glands / Q000378:metabolism; D016415:Sequence Alignment; D013154:Spleen / Q000378:metabolism
chemical_list: D020033:Protein Isoforms; D012333:RNA, Messenger; D058486:Receptors, Purinergic P2X7