Characterisation of C-terminal-truncated P2X7 receptor splice variants

Barnes, Sara J, Masin, Marianela, Marschall, Viola, Mooney, Elizabeth, Young, Christopher, Lim, KoiNi, Gorecki, Dariusz C and Murrell-Lagnado, Ruth (2010) Characterisation of C-terminal-truncated P2X7 receptor splice variants. Purinergic Signalling, 6 (1S). p. 57. ISSN 1573-9538

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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 (ΔC) 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 ΔC 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 ΔC 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 ΔC 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 ΔC 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 ΔC 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 ΔC variants in spleen and salivary gland from Pfizer P2X7−/− mice, suggesting that these mice are not null for P2X7 expression.

Item Type: Article
Schools and Departments: School of Life Sciences > Biochemistry
Subjects: Q Science > QH Natural history > QH0301 Biology
Depositing User: Tom Gittoes
Date Deposited: 25 Mar 2015 13:28
Last Modified: 25 Mar 2015 13:28
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