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

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.

Pharmacological and genetic characterisation of the canine P2X4 receptor

“…The current study determined that the coding sequence of P2RX4 is highly conserved amongst 101 dogs. The lack of genetic variation in canine P2RX4 contrasts that of canine P2RX7 (Sophocleous et al, 2019; Spildrejorde et al, 2014) which encodes four characterised missense variants. The difference between canine P2RX4 and P2RX7 parallels the frequencies of missense variants between human P2RX4 (Stokes et al, 2011) and P2RX7 (Stokes et al, 2010).…”

The P2X7 receptor antagonist Brilliant Blue G reduces serum human interferon-γ in a humanized mouse model of graft-versus-host disease

“…ATP-induced YO-PRO-1 21 uptake assay P2X7 pore formation was quantified by measuring ATPinduced YO-PRO-1 21 uptake, as described previously [19]. Briefly, cells in NaCl medium (145 mM NaCl, 5 mM KCl, 5 mM glucose and 10 mM HEPES, pH 7Á4) at 378C were incubated in the absence or presence of BBG for 15 min, and then incubated with 1 lM YO-PRO-1 iodide (Molecular Probes, Eugene, OR, USA) in the absence or presence of 1 mM ATP (Sigma-Aldrich) for 10 min.…”

Probenecid directly impairs activation of the canine P2X7 receptor

“…[19] Moreover, the P2X7 antagonist profile of canine P2X7 is similar to that of human P2X7. [8,20,21] Thus, given the similar pharmacological profiles between these two receptors and the potential use of probenecid in dogs, the current study aimed to determine if probenecid could also directly impair canine P2X7. were from Sigma-Aldrich.…”