The role of Na,K-ATPase alpha subunit serine 775 and glutamate 779 in determining the extracellular K+ and membrane potential-dependent properties of the Na,K-pump.
The role of Na,K-ATPase alpha subunit serine 775 and glutamate 779 in determining the extracellular K+ and membrane potential-dependent properties of the Na,K-pump.
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Na,K-ATP 酶 α 亚基丝氨酸 775 和谷氨酸 779 在确定 Na,K 泵的细胞外 K 和膜电位依赖性特性中的作用。
DOI：
10.1085/jgp.116.1.47
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发表时间：
2000
期刊：
影响因子：
--
通讯作者：
Berlin,JR
中科院分区：
文献类型：
--
作者：
Peluffo,RD;Argüello,JM;Berlin,JR
本研究探讨了Na，K-ATP酶α亚基第五跨膜段中的两个氨基酸Ser 775和Glu 779在酶介导的离子转运的电压依赖性和细胞外K+（K+o）依赖性中的作用。通过含有115 mM Na+溶液的贴片电极对表达绵羊Na，K-ATP酶α1亚基的HeLa细胞进行电压钳位（37 ℃）。由哇巴因抗性对照酶（RD）产生的Na，K -泵电流（含有Gln 111 Arg和Asn 122 Asp的氨基酸取代）在用0和148 mM含Na+的盐溶液灌流期间显示出与野生型Na，K-ATP酶相似的膜电位和K+依赖性。在Ser 775或Glu 779处的丙氨酸的额外取代分别产生了K+浓度的155倍和15倍的增加，在细胞外无Na+溶液中，在0 mV时，K+浓度达到最大激活Na，K -泵电流的一半。然而，在RD和丙氨酸取代的酶中，Na，K -泵电流的电压依赖性不变。因此，Na，K-ATP酶第五跨膜段的突变可以引起表观K+ o亲和力的大变化，而对K+转运的电压依赖性影响不大。对这些结果的一种解释是，负责K+结合和/或封闭动力学的蛋白质结构可能至少部分不同于负责Na，K-ATP酶的K+结合的电压依赖性的蛋白质结构。
The roles of Ser775 and Glu779, two amino acids in the putative fifth transmembrane segment of the Na,K -ATPase α subunit, in determining the voltage and extracellular K+(K+o) dependence of enzyme-mediated ion transport, were examined in this study. HeLa cells expressing the α1 subunit of sheep Na,K -ATPase were voltage clamped via patch electrodes containing solutions with 115 mM Na+(37°C). Na,K -pump current produced by the ouabain-resistant control enzyme (RD), containing amino acid substitutions Gln111Arg and Asn122Asp, displayed a membrane potential and K+odependence similar to wild-type Na,K -ATPase during superfusion with 0 and 148 mM Na+-containing salt solutions. Additional substitution of alanine at Ser775 or Glu779 produced 155- and 15-fold increases, respectively, in the K+oconcentration that half-maximally activated Na,K -pump current at 0 mV in extracellular Na+-free solutions. However, the voltage dependence of Na,K -pump current was unchanged in RD and alanine-substituted enzymes. Thus, large changes in apparent K+oaffinity could be produced by mutations in the fifth transmembrane segment of the Na,K -ATPase with little effect on voltage-dependent properties of K+transport. One interpretation of these results is that protein structures responsible for the kinetics of K+obinding and/or occlusion may be distinct, at least in part, from those that are responsible for the voltage dependence of K+obinding to the Na,K -ATPase.