Naturally Occurring Missense Mutations Lead to Loss of Function of the Canine P2X7 Receptor — ASN Events
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  2. Session 7: Poster Session B - Including Happy Hour & Trade Display
  3. Naturally Occurring Missense Mutations Lead to Loss of Function of the Canine P2X7 Receptor

Naturally Occurring Missense Mutations Lead to Loss of Function of the Canine P2X7 Receptor (#220)

Ronald Sluyter 1 , Mari Spildrejorde 1 , Rachael Bartlett 1 , Iman Jalilian 1 , Michelle Peranec 1 , Vanessa Sluyter 1 , Belinda Curtis 2 , Aine Seavers 3 , Kristen Skarratt 4 , Jason McArthur 1 , Mark Dowton 1 , Leanne Stokes 5
  1. University of Wollongong, Wollongong, NSW, Australia
  2. Albion Park Veterinary Hospital, Albion Park, NSW, Australia
  3. Oak Flats Veterinary Clinic, Oak Flats, NSW, Australia
  4. University of Sydney, Penrith, NSW, Australia
  5. RMIT University, Bundoora, Victoria, Australia

In humans the damage-associated molecular pattern receptor P2X7 is a highly polymorphic ligand-gated cation channel, which stimulates the NALP3 inflammasome to cause IL-1β release from monocytes. This study aimed to determine if the canine P2RX7 gene encodes mutations that alter P2X7 function. Flow cytometric measurements showed that the P2X7 ligand ATP induces cation uptake into canine monocytes, as well as T and B cells. ELISA measurements demonstrated that ATP also induces IL-1β release from LPS-primed canine monocytes. The P2X7 antagonist A438079 impaired both ATP-induced cation uptake and IL-1β release. Relative monocyte P2X7 function however varied between the 52 dogs studied. P2RX7 gene sequencing of these animals identified two common mutations, Leu103Phe and Pro452Ser, and two rare mutations Arg270Cys and Arg365Gln. Of these, the Arg270Cys mutation corresponded with near absent P2X7 function in monocytes, while the Leu103Phe and Arg365Gln mutations corresponded with partial loss of P2X7 function in monocytes. Canine P2X7 was cloned and flow cytometric cation uptake measurements of transfected HEK-293 cells revealed that this receptor has pharmacological characteristics similar to that of human P2X7. Mutagenesis studies of this cloned receptor showed that the Arg270Cys mutation resulted in a near complete loss of P2X7 function despite similar amounts of receptor expression compared to control (wild-type) P2X7. Furthermore, mutagenesis of cloned P2X7 showed that the Leu103Phe and Arg365Gln mutations result in a partial loss of P2X7 function, but this corresponded with reduced receptor expression compared to control (wild-type) P2X7. Thus, these results demonstrate that canine P2X7 activation induces cation uptake and IL-1β release. However, the relative function of P2X7 varies between dogs, which in part can be explained by a rare Arg270Cys missense mutation, as well as Leu103Phe and Arg365Gln mutations possibly as a result of reduced receptor expression. These novel missense mutations will provide future insights into the expression, structure and function of P2X7 in mammals. Finally, P2X7 and the mutations identified may have implications for canine health.