Szerencsei R T, Prinsen C F, Schnetkamp P P
Department of Physiology and Biophysics and The CIHR Ion Channel and Transporter Group, Faculty of Medicine, University of Calgary, 3330 Hospital Drive, Northwest Calgary, Alberta T2N 4N1, Canada.
Biochemistry. 2001 May 22;40(20):6009-15. doi: 10.1021/bi0102353.
The transport stoichiometry is an essential property of antiporter and symporter transport proteins. In this study, we determined the transport stoichiometry of the retinal cone potassium-dependent Na/Ca exchanger (NCKX) expressed in sodium-loaded cultured insect cells. The Na/Ca and Rb/Ca coupling ratios were obtained by direct measurements of the levels of (86)Rb and (45)Ca uptake and sodium release associated with reverse Na/Ca exchange. Rb/Ca coupling ratios of 0.98 [standard deviation (SD) of 0.12, 15 observations] and 0.92 (SD of 0.12, 13 observations) were obtained for the chicken and human retinal cone NCKX, respectively. Na/Ca coupling ratios of 4.11 (SD of 0.24, 10 observations) and 3.98 (SD of 0.34, 15 observations) were obtained for the chicken and human retinal cone NCKX, respectively, whereas a lower average coupling ratio of 3.11 (SD of 0.34, 10 observations) was obtained with cells expressing the bovine Na/Ca exchanger (NCX1). These results are consistent with a 4Na/1Ca + 1K stoichiometry for retinal cone NCKX. High Five cells expressing full-length dolphin rod NCKX, Caenorhabditis elegans NCKX, or bovine rod NCKX from which the two large hydrophilic loops were removed all showed a significant calcium-dependent (86)Rb uptake, whereas no calcium-dependent (86)Rb uptake was observed in cells expressing bovine NCX1. The calcium dependence of (45)Ca uptake yielded values between 1 and 2.5 microM for the external calcium dissociation constant of the different NCKX proteins studied here.
转运化学计量是反向转运蛋白和同向转运蛋白的一个基本特性。在本研究中，我们测定了在钠负载的培养昆虫细胞中表达的视网膜视锥细胞钾依赖性钠/钙交换体（NCKX）的转运化学计量。通过直接测量与反向钠/钙交换相关的（86）Rb摄取、（45）Ca摄取和钠释放水平，获得了钠/钙和铷/钙耦合比。鸡和人类视网膜视锥细胞NCKX的铷/钙耦合比分别为0.98[标准差（SD）为0.12，15次观察]和0.92（SD为0.12，13次观察）。鸡和人类视网膜视锥细胞NCKX的钠/钙耦合比分别为4.11（SD为0.24，10次观察）和3.98（SD为0.34，15次观察），而表达牛钠/钙交换体（NCX1）的细胞获得的平均耦合比更低，为3.11（SD为0.34，10次观察）。这些结果与视网膜视锥细胞NCKX的4Na/1Ca + 1K化学计量一致。表达全长海豚视杆NCKX、秀丽隐杆线虫NCKX或去除了两个大亲水环的牛视杆NCKX的High Five细胞均显示出显著的钙依赖性（86）Rb摄取，而在表达牛NCX1的细胞中未观察到钙依赖性（86）Rb摄取。对于此处研究的不同NCKX蛋白，（45）Ca摄取的钙依赖性产生的外部钙解离常数在1至2.5微摩尔之间。
