Cloning and characterization of an electrogenic Na/HCO3- cotransporter from the squid giant fiber lobe.
Cloning and characterization of an electrogenic Na/HCO3- cotransporter from the squid giant fiber lobe.
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鱿鱼巨纤维叶产电 Na/HCO3- 协同转运蛋白的克隆和表征。
DOI：
10.1152/ajpcell.00544.2006
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发表时间：
2007
期刊：
影响因子：
--
通讯作者：
Boron,WalterF
中科院分区：
文献类型：
--
作者：
Piermarini,PeterM;Choi,Inyeong;Boron,WalterF
鱿鱼巨轴突是了解可兴奋膜和离子运输的经典模型系统。迄今为止，Na+驱动的Cl-HCO 3 −交换器sqNDCBE与SLC 4超家族相关，并从巨纤维叶cDNA中克隆，是唯一从鱿鱼中克隆和表征的HCO 3 −转运蛋白。本研究的目的是克隆并鉴定另一个SLC 4样cDNA。我们利用简并PCR技术获得了鱿鱼纤维克隆3（squid fiber clone 3，SF 3）的部分cDNA克隆，并对其进行了5′和3′两个方向的延伸，获得了全长开放阅读框。预测的SF 3的氨基酸序列与sqNDCBE相似，膜结构域的系统发育分析表明SF 3与电中性Na+偶联的SLC 4转运蛋白簇合。然而，当我们测量表达SF 3的异种卵母细胞的pH值和膜电位时，或者使用双电极电压箝位来测量电流时，卵母细胞表现出产电Na/HCO 3 −协同转运蛋白NBCe的特征。也就是说，暴露于细胞外CO2/HCO 3 −不仅会导致pHi下降，随后出现强劲的恢复，而且还会导致快速超极化。电流-电压关系也是生电NBC的特征。pHi恢复和电流需要HCO 3 −和Na+，并被DIDS阻断。此外，K+和Li+都不能完全取代Na+来支持pH恢复。细胞外的Cl−对于转运蛋白的运行来说不是必需的。因此，SF 3是NBCe，代表第一个以无脊椎动物为特征的NBCe。
The squid giant axon is a classic model system for understanding both excitable membranes and ion transport. To date, a Na+-driven Cl-HCO3−exchanger, sqNDCBE—related to the SLC4 superfamily and cloned from giant fiber lobe cDNA—is the only HCO3−-transporting protein cloned and characterized from a squid. The goal of our study was to clone and characterize another SLC4-like cDNA. We used degenerate PCR to obtain a partial cDNA clone (squid fiber clone 3, SF3), which we extended in both the 5′ and 3′ directions to obtain the full-length open-reading frame. The predicted amino-acid sequence of SF3 is similar to sqNDCBE, and a phylogenetic analysis of the membrane domains indicates that SF3 clusters with electroneutral Na+-coupled SLC4 transporters. However, when we measure pHiand membrane potential—or use two-electrode voltage clamping to measure currents—onXenopusoocytes expressing SF3, the oocytes exhibit the characteristics of an electrogenic Na/HCO3−cotransporter, NBCe. That is, exposure to extracellular CO2/HCO3−not only causes a fall in pHi, followed by a robust recovery, but also causes a rapid hyperpolarization. The current-voltage relationship is also characteristic of an electrogenic NBC. The pHirecovery and current require HCO3−and Na+, and are blocked by DIDS. Furthermore, neither K+nor Li+can fully replace Na+in supporting the pHirecovery. Extracellular Cl−is not necessary for the transporter to operate. Therefore, SF3 is an NBCe, representing the first NBCe characterized from an invertebrate.
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