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R270C polymorphism leads to loss of function of the canine P2X7 receptor

Journal Article


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Abstract


  • 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.

Authors


  •   Spildrejorde, Mari (external author)
  •   Bartlett, Rachael (external author)
  •   Stokes, Leanne (external author)
  •   Jalilian, Iman (external author)
  •   Peranec, Michelle (external author)
  •   Sluyter, Vanessa (external author)
  •   Curtis, Belinda L. (external author)
  •   Skarratt, Kristen K. (external author)
  •   Skora, Amanda S. (external author)
  •   Bakhsh, Tahani (external author)
  •   Seavers, Aine (external author)
  •   McArthur, Jason D.
  •   Dowton, Mark P.
  •   Sluyter, Ronald

Publication Date


  • 2014

Citation


  • Spildrejorde, M., Bartlett, R., Stokes, L., Jalilian, I., Peranec, M., Sluyter, V., Curtis, B. L., Skarratt, K. K., Skora, A., Bakhsh, T., Seavers, A., McArthur, J. D., Dowton, M. & Sluyter, R. (2014). R270C polymorphism leads to loss of function of the canine P2X7 receptor. Physiological Genomics, 46 (14), 512-522.

Scopus Eid


  • 2-s2.0-84904279024

Ro Full-text Url


  • http://ro.uow.edu.au/cgi/viewcontent.cgi?article=3035&context=smhpapers

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers/2017

Number Of Pages


  • 10

Start Page


  • 512

End Page


  • 522

Volume


  • 46

Issue


  • 14

Abstract


  • 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.

Authors


  •   Spildrejorde, Mari (external author)
  •   Bartlett, Rachael (external author)
  •   Stokes, Leanne (external author)
  •   Jalilian, Iman (external author)
  •   Peranec, Michelle (external author)
  •   Sluyter, Vanessa (external author)
  •   Curtis, Belinda L. (external author)
  •   Skarratt, Kristen K. (external author)
  •   Skora, Amanda S. (external author)
  •   Bakhsh, Tahani (external author)
  •   Seavers, Aine (external author)
  •   McArthur, Jason D.
  •   Dowton, Mark P.
  •   Sluyter, Ronald

Publication Date


  • 2014

Citation


  • Spildrejorde, M., Bartlett, R., Stokes, L., Jalilian, I., Peranec, M., Sluyter, V., Curtis, B. L., Skarratt, K. K., Skora, A., Bakhsh, T., Seavers, A., McArthur, J. D., Dowton, M. & Sluyter, R. (2014). R270C polymorphism leads to loss of function of the canine P2X7 receptor. Physiological Genomics, 46 (14), 512-522.

Scopus Eid


  • 2-s2.0-84904279024

Ro Full-text Url


  • http://ro.uow.edu.au/cgi/viewcontent.cgi?article=3035&context=smhpapers

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers/2017

Number Of Pages


  • 10

Start Page


  • 512

End Page


  • 522

Volume


  • 46

Issue


  • 14