R270C polymorphism leads to loss of function of the canine P2X7 receptor.
R270C polymorphism leads to loss of function of the canine P2X7 receptor.
复制标题
R270C 多态性导致犬 P2X7 受体功能丧失。
作者：
Mari Spildrejorde;R. Bartlett;L. Stokes;I. Jalilian;Michelle Peranec;V. Sluyter;Belinda L. Curtis;Kristen K Skarratt;A. Skora;T. Bakhsh;A. Seavers;J. Mcarthur;M. Dowton;R. Sluyter
由于P2 RX 7基因的多态性，P2 X7受体（一种ATP门控离子通道）的相对功能在人类之间存在差异。本研究旨在评估犬群随机样本中P2 X7变异的功能影响。血液和基因组DNA从69只狗中获得，这些狗被选为不同品种的代表。通过流式细胞术测量染料摄取和膜片钳测量内向电流来确定P2 X7功能。免疫印迹和免疫细胞化学检测P2 X7表达。采用测序法检测P2 RX 7基因多态性。P2 X7是从英国史宾格犬克隆的，并通过定点突变将点突变引入该受体。P2 X7对单核细胞的相对功能在个体犬之间变化。犬P2 RX 7基因编码4个错义多态性：F103 L和P452 S，发现在杂合和纯合剂量，和R270 C和R365 Q，只发现在杂合剂量。此外，R270 C和R365 Q分别与可卡犬和拉布拉多寻回犬相关。F103 L、R270 C和R365 Q而非P452 S对应于单核细胞中P2 X7功能降低，但不能解释犬间P2 X7功能的大多数差异，表明其他因素导致了这种变异性。P2 X7定点突变体在人胚肾-293细胞中的异源表达表明，R270 C突变体无功能，F103 L和R365 Q突变体功能部分降低，P452 S突变体功能正常。综上所述，这些数据强调了R270 C多态性对犬P2 X7具有重要的功能影响。
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.