Poster Presentation Australasian Society for Immunology Annual Scientific Meeting 2014

A natural P452S mutation, equivalent in position to the loss-of-function P451L mutation in murine P2X7, does not alter canine P2X7 function (#213)

Ronald Sluyter 1 , Rachael Bartlett 1 , Mari Spildrejorde 1 , Michelle Peranec 1 , Iman Jalilian 1 , Belinda Curtis 2 , Vanessa Sluyter 1 , Mark Dowton 1 , Leanne Stokes 3
  1. University of Wollongong, Wollongong, NSW, Australia
  2. Albion Park Veterinary Hospital, Albion Park, NSW, Australia
  3. RMIT University, Bundoora, Victoria, Australia
The damage-associated molecular pattern receptor P2X7 is an ATP-gated ion channel, which stimulates the NLRP3 inflammasome to cause IL-1β maturation and release from human and murine monocytes. Mouse strains, such as C57BL/6 but not BALB/c, encode a natural P451L mutation in the cytoplasmic tail of P2X7 that impairs receptor function. Dogs naturally encode a P452S mutation in P2X7, a position equivalent to that of P451L in murine P2X7. Therefore, this study aimed to examine the functional impact of the P452S mutation and this proline residue in canine P2X7. P2RX7 gene sequencing of a random population of 66 dogs revealed that the P452S mutation has an allele frequency of 0.39. Flow cytometric measurements of ATP-induced cation uptake into monocytes from 51 dogs demonstrated that the P452S in either heterozygous or homozygous dosage did not alter relative P2X7 function compared to that of dogs encoding wild-type P2X7. ELISA measurements of ATP-induced IL-1β release from LPS-primed monocytes from 10 dogs also demonstrated similar P2X7 function between these different genotypes. Furthermore, relative P2X7 function was similar in HEK-293 cells expressing either heterologous wild-type or P452S mutant canine P2X7. Notably the relative function of P452L mutant canine P2X7 was also similar to that of wild-type or P452S mutant canine P2X7 in HEK-293 cells. Both immunoblotting and confocal microscopy confirmed that all three canine P2X7 variants were expressed in similar amounts in HEK-293 cells. In conclusion, these results indicate that the P452S mutation or mutation of this proline residue to a leucine does not alter the function of canine P2X7. Thus, these results indicate that this proline residue in the cytoplasmic tail of canine and mouse P2X7 contributes differently to the function of this receptor in these two species.