Canfield V A, Levenson R
Department of Pharmacology, Penn State College of Medicine, Milton S. Hershey Medical Center, Pennsylvania 17033, USA.
Biochemistry. 1998 May 19;37(20):7509-16. doi: 10.1021/bi980174h.
We have used expression of chimeras between the structurally related Na,K- and H,K-ATPase alpha subunits to localize regions that determine Na,K-ATPase activity. Segments of the rat Na,K-ATPase alpha1 subunit were replaced by the corresponding portions of the rat gastric H,K-ATPase alpha subunit, and the constructs were transfected into ouabain-sensitive human HEK 293 cells. Using the ability to transfer ouabain resistance as a measure of sodium pump activity, we identified segments within the sodium pump that could be replaced with proton pump sequences without the loss of biological activity. These functionally interchangeable segments encompassed approximately 75% of the amino acid differences between the two transporters. Segments that could not be exchanged mapped to three discrete regions. One region spans residues 63-117 and includes the first transmembrane (TM) segment and a portion of the amino-terminal cytoplasmic domain. The second, from residue 320 to residue 413, encompasses TM 4 and a portion of the third cytoplasmic domain, while the third region (encompassing residues 735-861 and 898-953) includes several TM domains in the carboxyl-terminal portion of the ATPase. Our results suggest that functional differences between Na,K- and H,K-ATPase, including differences in ion transport specificity, are likely to reside within these noninterchangeable segments.
我们利用结构相关的钠钾ATP酶和氢钾ATP酶α亚基之间的嵌合体表达来定位决定钠钾ATP酶活性的区域。将大鼠钠钾ATP酶α1亚基的片段替换为大鼠胃氢钾ATP酶α亚基的相应部分，并将构建体转染到对哇巴因敏感的人HEK 293细胞中。利用转移对哇巴因抗性的能力作为钠泵活性的衡量指标，我们确定了钠泵内可被质子泵序列替换而不丧失生物活性的片段。这些功能上可互换的片段涵盖了两种转运蛋白之间约75%的氨基酸差异。不能交换的片段定位于三个离散区域。一个区域跨越第63至117位残基，包括第一个跨膜（TM）片段和氨基末端胞质结构域的一部分。第二个区域从第320位残基到第413位残基，包括TM 4和第三个胞质结构域的一部分，而第三个区域（涵盖第735至861位残基和第898至953位残基）包括ATP酶羧基末端部分的几个TM结构域。我们的结果表明，钠钾ATP酶和氢钾ATP酶之间的功能差异，包括离子转运特异性的差异，可能存在于这些不可互换的片段中。
