Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.
Methods. 2010 May;51(1):134-45. doi: 10.1016/j.ymeth.2009.12.012. Epub 2010 Jan 4.
The Xenopus laevis oocyte is a model system for the electrophysiological study of exogenous ion transporters. Three main reasons make the oocyte suitable for this purpose: (a) it has a large cell size (approximately 1mm diameter), (b) it has an established capacity to produce-from microinjected mRNAs or cRNAs-exogenous ion transporters with close-to-physiological post-translational modifications and actions, and (c) its membranes contain endogenous ion-transport activities which are usually smaller in magnitude than the activities of exogenously-expressed ion transporters. The expression of ion transporters as green fluorescent protein fusions allows the fluorometric assay of transporter yield in living oocytes. Monitoring of transporter-mediated movement of ions such as Cl(-), H(+) (and hence base equivalents like OH(-) and HCO(3)(-)), K(+), and Na(+) is achieved by positioning the tips of ion-sensitive microelectrodes inside the oocyte and/or at the surface of the oocyte plasma membrane. The use of ion-sensitive electrodes is critical for studying net ion-movements mediated by electroneutral transporters. The combined use of fluorometry and electrophysiology expedites transporter study by allowing measurement of transporter yield prior to electrophysiological study and correlation of relative transporter yield with transport rates.
非洲爪蟾卵母细胞是研究外源离子转运体的电生理学模型系统。有三个主要原因使卵母细胞适合于该目的：（a）它具有较大的细胞尺寸（约 1mm 直径），（b）它具有从微注射的 mRNA 或 cRNA 产生具有接近生理的翻译后修饰和功能的外源离子转运体的既定能力，以及（c）其膜含有内源性离子转运活性，其通常比表达的离子转运体的活性小。离子转运体作为绿色荧光蛋白融合体的表达允许在活卵母细胞中进行转运体产量的荧光测定。通过将离子敏微电极的尖端定位在卵母细胞内和/或卵母细胞膜表面，可以监测离子（如 Cl(-)、H(+)（因此碱当量如 OH(-)和 HCO(3)(-))、K(+)和 Na(+)）介导的转运体介导的运动。离子敏电极的使用对于研究电中性转运体介导的净离子运动至关重要。荧光法和电生理学的联合使用通过允许在电生理学研究之前测量转运体产量并将相对转运体产量与转运速率相关联，从而加速转运体研究。
