Sheng J Z, Prinsen C F, Clark R B, Giles W R, Schnetkamp P P
Department of Physiology and Biophysics and the MRC Group on Ion Channels/Transporters, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
Biophys J. 2000 Oct;79(4):1945-53. doi: 10.1016/S0006-3495(00)76443-5.
The recently cloned retinal cone Na(+)-Ca(2+)-K(+) exchanger (NCKX) was expressed in cultured insect cells, and whole-cell patch clamp was used to measure transmembrane currents generated by this transcript and compare them with currents generated by retinal rod NCKX or by a deletion mutant rod NCKX from which the two large hydrophilic loops were removed. We have characterized the ionic currents generated by both the forward (Ca(2+) extrusion) and reverse (Ca(2+) influx) modes of all three NCKX proteins. Reverse NCKX exchange generated outward current that required the simultaneous presence of both external Ca(2+) and external K(+). Forward NCKX exchange carried inward current with Na(+), but not with Li(+) in the bath solution. The cation dependencies of the three NCKX tested (external K(+), external Na(+), internal Ca(2+)) were very similar to each other and to those reported previously for the in situ rod NCKX. These findings provide the first electrophysiological characterization of cone NCKX and the first electrophysiological characterization of potassium-dependent Na(+)-Ca(+) exchangers in heterologous systems. Our results demonstrate the feasibility of combining heterologous expression and biophysical measurements for detailed NCKX structure/function studies.
最近克隆的视网膜视锥细胞钠钙钾交换体（NCKX）在培养的昆虫细胞中表达，采用全细胞膜片钳技术测量该转录本产生的跨膜电流，并将其与视网膜视杆细胞NCKX或去除两个大亲水环的视杆细胞NCKX缺失突变体产生的电流进行比较。我们已经对所有三种NCKX蛋白的正向（钙外排）和反向（钙内流）模式产生的离子电流进行了表征。反向NCKX交换产生外向电流，这需要胞外钙和胞外钾同时存在。正向NCKX交换携带的内向电流需要浴液中有钠离子，但不需要锂离子。所测试的三种NCKX的阳离子依赖性（胞外钾、胞外钠、胞内钙）彼此非常相似，且与先前报道的原位视杆细胞NCKX的阳离子依赖性相似。这些发现首次对视锥细胞NCKX进行了电生理表征，也是首次在异源系统中对钾依赖性钠钙交换体进行电生理表征。我们的结果证明了将异源表达和生物物理测量相结合用于详细的NCKX结构/功能研究的可行性。
