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Identification and characterization of a novel variant of the human P2X7 receptor resulting in gain of function

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Abstract

The P2X7 receptor exhibits significant allelic polymorphism in humans, with both loss and gain of function variants potentially impacting on a variety of infectious and inflammatory disorders. At least five loss-of-function polymorphisms (G150R, R307Q, T357S, E496A, and I568N) and two gain-of-function polymorphisms (H155Y and Q460R) have been identified and characterized to date. In this study, we used RT-PCR cloning to isolate and characterize P2X7 cDNA clones from human PBMCs and THP-1 cells. A previously unreported variant with substitutions of V80M and A166G was identified. When expressed in HEK293 cells, this variant exhibited heightened sensitivity to the P2X7 agonist (BzATP) relative to the most frequent allele, as shown by pore formation measured by fluorescent dye uptake into cells. Mutational analyses showed that A166G alteration was critical for the gain-of-function change, while V80M was not. Full-length variants with multiple previously identified nonsynonymous SNPs (H155Y, H270R, A348T, and E496A) were also identified. Distinct functional phenotypes of the P2X7 variants or mutants constructed with multiple polymorphisms were observed. Gain-of-function variations (A166G or H155Y) could not rescue the loss-of-function E496A polymorphism. Synergistic effects of the gain-of-function variations were also observed. We also identified the A348T alteration as a weak gain-of-function variant. Thus, these results identify the new gain-of-function variant A166G and demonstrate that multiple-gene polymorphisms contribute to functional phenotypes of the human P2X7 receptor. Furthermore, the results demonstrate that the C-terminal of the cysteine-rich domain 1 of P2X7 is critical for regulation of P2X7-mediated pore formation.

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Abbreviations

PI:

Propidium iodide

BzATP:

2′,3′-O-(4-benzoyl-benzoyl) ATP

SNP:

Single-nucleotide polymorphism

MFI:

Mean of fluorescence intensity

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Acknowledgements

This work was supported by NIH grants A157168 and CA73743.

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Authors and Affiliations

  1. Department of Immunology, University of Pittsburgh School of Medicine, E1052 Biomedical Science Tower, Pittsburgh, PA, 15261, USA

    Chengqun Sun, Jessica Chu, Sarita Singh & Russell D. Salter

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  1. Chengqun Sun
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  2. Jessica Chu
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  3. Sarita Singh
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  4. Russell D. Salter
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Corresponding author

Correspondence to Russell D. Salter.

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Footnotes

The sequence of the new gain-of-function P2X7 variant (Variant A) has been submitted to NCBI GenBank database (GeneBank accession number, GQ180122).

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Sun, C., Chu, J., Singh, S. et al. Identification and characterization of a novel variant of the human P2X7 receptor resulting in gain of function. Purinergic Signalling 6, 31–45 (2010). https://doi.org/10.1007/s11302-009-9168-9

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  • DOI: https://doi.org/10.1007/s11302-009-9168-9

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