R270C polymorphism leads to loss of function of the canine P2X7 receptor.
The relative function of the P2X7 receptor, an ATP-gated ion channel, varies between humans due to polymorphisms in the P2RX7 gene. This study aimed to assess the functional impact of P2X7 variation in a random sample of the canine population. Blood and genomic DNA were obtained from 69 dogs selected as representatives of a cross section of different breeds. P2X7 function was determined by flow cytometric measurements of dye uptake and patch-clamp measurements of inward currents. P2X7 expression was determined by immunoblotting and immunocytochemistry. Sequencing was used to identify P2RX7 gene polymorphisms. P2X7 was cloned from an English springer spaniel, and point mutations were introduced into this receptor by site-directed mutagenesis. The relative function of P2X7 on monocytes varied between individual dogs. The canine P2RX7 gene encoded four missense polymorphisms: F103L and P452S, found in heterozygous and homozygous dosage, and R270C and R365Q, found only in heterozygous dosage. Moreover, R270C and R365Q were associated with the cocker spaniel and Labrador retriever, respectively. F103L, R270C, and R365Q but not P452S corresponded to decreased P2X7 function in monocytes but did not explain the majority of differences in P2X7 function between dogs, indicating that other factors contribute to this variability. Heterologous expression of site-directed mutants of P2X7 in human embryonic kidney-293 cells indicated that the R270C mutant was nonfunctional, the F103L and R365Q mutants had partly reduced function, and the P452S mutant functioned normally. Taken together, these data highlight that a R270C polymorphism has major functional impact on canine P2X7.
School of Biological Sciences, University of Wollongong, Wollongong, Australia; Illawarra Health and Medical Research Institute, Wollongong, Australia;
Mari
M
Spildrejorde
School of Biological Sciences, University of Wollongong, Wollongong, Australia; Illawarra Health and Medical Research Institute, Wollongong, Australia;
Rachael
R
Bartlett
Health Innovations Research Institute, School of Medical Sciences, RMIT University, Bundoora, Australia; Sydney Medical School Nepean, University of Sydney, Nepean Hospital, Penrith, Australia;
Leanne
L
Stokes
School of Biological Sciences, University of Wollongong, Wollongong, Australia; Illawarra Health and Medical Research Institute, Wollongong, Australia;
Iman
I
Jalilian
School of Biological Sciences, University of Wollongong, Wollongong, Australia; Illawarra Health and Medical Research Institute, Wollongong, Australia;
Michelle
M
Peranec
School of Biological Sciences, University of Wollongong, Wollongong, Australia; Illawarra Health and Medical Research Institute, Wollongong, Australia;
Vanessa
V
Sluyter
Albion Park Veterinary Hospital, Albion Park, Australia; and.
Belinda L
BL
Curtis
Sydney Medical School Nepean, University of Sydney, Nepean Hospital, Penrith, Australia;
Kristen K
KK
Skarratt
School of Biological Sciences, University of Wollongong, Wollongong, Australia; Illawarra Health and Medical Research Institute, Wollongong, Australia;
Amanda
A
Skora
School of Biological Sciences, University of Wollongong, Wollongong, Australia; Illawarra Health and Medical Research Institute, Wollongong, Australia;
Tahani
T
Bakhsh
Oak Flats Veterinary Clinic, Oak Flats, Australia.
Aine
A
Seavers
School of Biological Sciences, University of Wollongong, Wollongong, Australia; Illawarra Health and Medical Research Institute, Wollongong, Australia;
Jason D
JD
McArthur
School of Biological Sciences, University of Wollongong, Wollongong, Australia; Illawarra Health and Medical Research Institute, Wollongong, Australia;
Mark
M
Dowton
School of Biological Sciences, University of Wollongong, Wollongong, Australia; Illawarra Health and Medical Research Institute, Wollongong, Australia; rsluyter@uow.edu.au.
Ronald
R
Sluyter
D000818:Animals; D002460:Cell Line; D004285:Dogs; D057809:HEK293 Cells; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D009000:Monocytes / Q000378:metabolism; D020125:Mutation, Missense / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D058486:Receptors, Purinergic P2X7 / Q000235:genetics*
D058486:Receptors, Purinergic P2X7
dog; missense mutation; monocyte; purinergic receptor; single nucleotide polymorphism