Department of Molecular and Cell Biology, University of Leicester, Leicester, LE1 7RH, UK.
Purinergic Signal. 2019 Sep;15(3):397-402. doi: 10.1007/s11302-019-09666-1. Epub 2019 Jul 8.
A P2X1-eYFP knock-in mouse was generated to study receptor expression and mobility in smooth muscle and blood cells. eYFP was added to the C-terminus of the P2X1R and replaced the native P2X1R. Fluorescence corresponding to P2X1-eYFPR was detected in urinary bladder smooth muscle, platelets and megakaryocytes. ATP-evoked currents from wild type and P2X1-eYFP isolated urinary bladder smooth muscle cells had the same peak current amplitude and time-course showing that the eYFP addition had no obvious effect on properties. Fluorescence recovery after photobleaching (FRAP) in bladder smooth muscle cells demonstrated that surface P2X1Rs are mobile and their movement is reduced following cholesterol depletion. Compared to the platelet and megakaryocyte, P2X1-eYFP fluorescence was negligible in red blood cells and the majority of smaller marrow cells. The spatial pattern of P2X1-eYFP fluorescence in the megakaryocyte along with FRAP assessment of mobility suggested that P2X1Rs are expressed extensively throughout the membrane invagination system of this cell type. The current study highlights that the spatiotemporal properties of P2X1R expression can be monitored in real time in smooth muscle cells and megakaryocytes/platelets using the eYFP knock-in mouse model.
一种 P2X1-eYFP 基因敲入小鼠被用于研究平滑肌和血细胞中受体的表达和迁移。eYFP 添加到 P2X1R 的 C 末端，取代了天然的 P2X1R。在膀胱平滑肌、血小板和巨核细胞中检测到与 P2X1-eYFPR 相对应的荧光。从野生型和 P2X1-eYFP 分离的膀胱平滑肌细胞中，ATP 诱导的电流具有相同的峰值电流幅度和时程，表明 eYFP 的添加对特性没有明显影响。膀胱平滑肌细胞中的荧光恢复后光漂白（FRAP）表明，表面 P2X1R 是可移动的，胆固醇耗竭后其运动减少。与血小板和巨核细胞相比，P2X1-eYFP 荧光在红细胞和大多数较小的骨髓细胞中可以忽略不计。在巨核细胞中 P2X1-eYFP 荧光的空间模式以及流动性的 FRAP 评估表明，P2X1R 在这种细胞类型的膜内陷系统中广泛表达。本研究强调了使用 eYFP 基因敲入小鼠模型，可以实时监测平滑肌细胞和巨核细胞/血小板中 P2X1R 表达的时空特性。
