A P2RX7 single nucleotide polymorphism haplotype promotes exon 7 and 8 skipping and disrupts receptor function

Kristen K. Skarratt, Ben J. Gu, Michael D. Lovelace, Carol J. Milligan, Leanne Stokes, Rachel Glover, Steven Petrou, James S. Wiley, Stephen J. Fuller

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Abstract

P2X7 is an ATP-gated membrane ion channel that is expressed by multiple cell types. Brief exposure to ATP induces the opening of a nonselective cation channel; while repeated or prolonged exposure induces formation of a transmembrane pore. This process may be partially regulated by alternative splicing of full-length P2RX7A pre-mRNA, producing isoforms that delete or retain functional domains. Here, we report cloning and expression of a novel P2RX7 splice variant, P2RX7L, that is, characterized by skipping of exons 7 and 8. In HEK 293 cells, expression of P2RX7L produces a protein isoform, P2X7L, that forms a heteromer with P2X7A. A haplotype defined by six single nucleotide polymorphisms (SNPs) (rs208307, rs208306, rs36144485, rs208308, rs208309, and rs373655596) promotes allele-specific alternative splicing, increasing mRNA levels of P2RX7L and another isoform, P2RX7E, which in addition has a truncated C-terminus. Skipping of exons 7 and 8 is predicted to delete critical amino acids in the ATP-binding site. P2X7L-transfected HEK 293 cells have phagocytic but not channel, pore, or membrane-blebbing function, and double-transfected P2X7L and P2X7A cells have reduced pore function. Heteromeric receptor complexes of P2X7A and P2X7L are predicted to have reduced numbers of ATP-binding sites, which potentially alters receptor function compared to homomeric P2X7A complexes.

Original languageEnglish
Pages (from-to)3884-3901
Number of pages18
JournalThe FASEB Journal
Volume34
Issue number3
Early online date31 Jan 2020
DOIs
Publication statusPublished - Mar 2020

Keywords

  • P2X7
  • alternative splicing
  • heteromer
  • single nucleotide polymorphism
  • ALLELE
  • CANCER-CELLS
  • ACTIVATION
  • C-TERMINUS
  • QUANTITATIVE METHOD
  • PORE FORMATION
  • ATP-BINDING
  • P2X(7) RECEPTOR
  • P2X7 RECEPTOR
  • ION-CHANNEL

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