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Characterisation of C-terminal-truncated P2X7 receptor splice variants
journal contributionposted on 2023-06-08, 19:52 authored by Sara J Barnes, Marianela Masin, Viola Marschall, Elizabeth Mooney, Christopher Young, KoiNi Lim, Dariusz C Gorecki, Ruth Murrell-LagnadoRuth Murrell-Lagnado
Contributing to the diversity of human and rodent P2X7 receptors are alternatively spliced variants. Here, we describe two murine splice variants with C-terminal truncations (?C) that arise from the use of alternative exon 13 s. P2X7(13b) terminates at residue 430, whereas P2X7(13c) possesses an additional 11-amino acid stretch after residue 430. The tissue distribution and structural and functional characteristics of the ?C variants were investigated and compared to the fulllength variant, P2X7(a). RT-PCR analysis using tissue from the wild-type C57BL strain of mice indicated that the ?C variants are expressed in a range of tissues including brain, salivary gland and spleen. The recombinant receptors expressed in HEK293 cells were analysed by BN-PAGE and cross-linking studies and both ?C splice variants formed stable homotrimers. Whole-cell patch-clamp recordings revealed that BzATP-induced currents were at least an order of magnitude smaller for the ?C variants compared to P2X7(a) and the EC50 value was 1.3 mM. Co-expression of P2X7(13b) with P2X7 (a) reduced the amplitude of 1 mM BzATP-evoked currents by ~3-fold compared to P2X7(a) alone and shifted the EC50 value from 320 µM to 410 uM, suggesting that the ?C variant exerts a dominant negative effect. Expression of P2X7 receptor complexes in tissue from wild type and Pfizer P2X7-/- mice was analyzed by BN-PAGE and using cross-linkers. Blots using an antibody to the extracellular domain of P2X7 showed trimers of a size consistent with the ?C variants in spleen and salivary gland from Pfizer P2X7-/- mice, suggesting that these mice are not null for P2X7 expression.
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