Electrogenic sulfate/chloride exchange in Xenopus oocytes mediated by murine AE1 E699Q.
Electrogenic sulfate/chloride exchange in Xenopus oocytes mediated by murine AE1 E699Q.
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DOI：
10.1085/jgp.109.3.345
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发表时间：
1997-03
期刊：
影响因子：
--
通讯作者：
Alper SL
中科院分区：
文献类型：
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作者：
Chernova MN;Jiang L;Crest M;Hand M;Vandorpe DH;Strange K;Alper SL
对化学修饰的人红细胞的功能评估表明，带3阴离子交换剂AE 1的氨基酸残基E681位于阴离子转运途径上，是H+/SO 4 2−共转运所需的质子载体。我们已经测试了在爪蟾卵母细胞的功能性后果的突变在相应的残基E699小鼠AE 1。大多数检测的突变消除了AE 1介导的Cl−流入和流出。只有E699 Q突变增加了二苯乙烯二磺酸盐敏感的流出和SO 4 2−的流入。E699 Q介导的Cl−内流被细胞内SO 4 2−升高激活，但E699 Q介导的Cl−外流检测不到。E699 Q介导的SO 4 2−外排的DNDS（4，4 ′-二硝基芪-2，2 ′-二磺酸）敏感性与wt AE 1介导的Cl−外排的DNDS敏感性没有区别。E699 Q介导的SO 4 2−外排的细胞外阴离子选择性与wt AE 1介导的Cl−外排相似。E699 Q介导的细胞外Cl−与细胞内SO 4 2−交换的化学计量比为1：1。向表达野生型AE 1的卵母细胞注射SO 4 2-，膜电位或电阻几乎没有变化，而向表达E699 Q的卵母细胞注射SO 4 2-，而不是Cl-或葡萄糖酸盐，使膜去极化17 mV，膜电阻降低66%。用羟乙基磺酸盐代替Cl-浴导致表达E699 Q的SO 4 2-负载卵母细胞的28 mV超极化，但对表达wt AE 1的卵母细胞没有影响。SO 4 2−预加载卵母细胞的细胞外Cl−依赖性去极化被DNDS阻断。在存在细胞外Cl−的情况下测量的AE 1 E699 Q介导的内向电流的大小足以解释测量的35 SO 4 2−流出。因此，AE 1 E699 Q介导的SO 4 2−/ Cl−交换在很大程度上（如果不是唯一的话）是作为一种产电的、不对称的1：1阴离子交换进行的。这些数据证实了E699存在于AE 1的阴离子转运途径上或有助于AE 1的阴离子转运途径的完整性的提议。AE 1序列中的一个氨基酸变化将电中性阴离子交换转化为产电阴离子交换，而不会改变SO 4 2−/Cl−交换的化学计量。
Functional evaluation of chemically modified human erythrocytes has led to the proposal that amino acid residue E681 of the band 3 anion exchanger AE1 lies on the anion translocation pathway and is a proton carrier required for H+/SO4 2− cotransport. We have tested in Xenopus oocytes the functional consequences of mutations in the corresponding residue E699 of mouse AE1. Most mutations tested abolished AE1-mediated Cl− influx and efflux. Only the E699Q mutation increased stilbene disulfonate-sensitive efflux and influx of SO4 2−. E699Q-mediated Cl− influx was activated by elevation of intracellular SO4 2−, but E699Q-mediated Cl− efflux was undetectable. The DNDS (4,4′-dinitrostilbene-2,2′-disulfonic acid) sensitivity of E699Q-mediated SO4 2− efflux was indistinguishable from that of wt AE1-mediated Cl− efflux. The extracellular anion selectivity of E699Q-mediated SO4 2− efflux was similar to that of wt AE1-mediated Cl− efflux. The stoichiometry of E699Q-mediated exchange of extracellular Cl− with intracellular SO4 2− was 1:1. Whereas SO4 2− injection into oocytes expressing wt AE1 produced little change in membrane potential or resistance, injection of SO4 2−, but not of Cl− or gluconate, into oocytes expressing E699Q depolarized the membrane by 17 mV and decreased membrane resistance by 66%. Replacement of bath Cl− with isethionate caused a 28-mV hyperpolarization in SO4 2−-loaded oocytes expressing E699Q, but had no effect on oocytes expressing wt AE1. Extracellular Cl−-dependent depolarization of SO4 2−-preloaded oocytes was blocked by DNDS. AE1 E699Q-mediated inward current measured in the presence of extracellular Cl− was of magnitude sufficient to account for measured 35SO4 2− efflux. Thus, AE1 E699Q-mediated SO4 2−/ Cl− exchange operated largely, if not exclusively, as an electrogenic, asymmetric, 1:1 anion exchange. The data confirm the proposal that E699 resides on or contributes to the integrity of the anion translocation pathway of AE1. A single amino acid change in the sequence of AE1 converted electroneutral to electrogenic anion exchange without alteration of SO4 2−/Cl− exchange stoichiometry.