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Expression, assembly and function of novel C-terminal truncated variants of the mouse P2X7 receptor: re-evaluation of P2X7 knockouts

journal contribution
posted on 2023-06-08, 19:51 authored by Marianela Masin, Christopher Young, KoiNi Lim, Sara J Barnes, Xing Jian Xu, Viola Marschall, Wojciech Brutkowski, Elizabeth R Mooney, Dariusz C Gorecki, Ruth Murrell-LagnadoRuth Murrell-Lagnado
BACKGROUND AND PURPOSE Splice variants of P2X7 receptor transcripts contribute to the diversity of receptor-mediated responses. Here, we investigated expression and function of C-terminal truncated (?C) variants of the mP2X7 receptor, which are predicted to escape inactivation in one strain of P2X7 -/- mice (Pfizer KO). EXPERIMENTAL APPROACH Expression in wild-type (WT) and Pfizer KO tissue was investigated by reverse transcription (RT)-PCR and Western blot analysis. ?C variants were also cloned and expressed in HEK293 cells to investigate their assembly, trafficking and function. KEY RESULTS RT-PCR indicates expression of a ?C splice variant in brain, salivary gland (SG) and spleen from WT and Pfizer KO mice. An additional ?C 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 ?C variant. When expressed in HEK293 cells the ?C variants were inefficiently trafficked to the cell surface and agonist-evoked whole cell currents were small. Co-expressed with P2X7A, the ?C splice variant acted in a dominant negative fashion to inhibit function. CONCLUSIONS AND IMPLICATIONS Pfizer KO mice are not null for P2X7 receptor expression but express ?C variants with reduced function. © 2011 The British Pharmacological Society.

History

Publication status

  • Published

Journal

British Journal of Pharmacology

ISSN

0007-1188

Publisher

Nature Publishing Group

Volume

165

Page range

978-993

Department affiliated with

  • Biochemistry Publications

Full text available

  • No

Peer reviewed?

  • Yes

Legacy Posted Date

2015-03-25

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