Palasis M, Kuntzweiler T A, Argüello J M, Lingrel J B
University of Cincinnati College of Medicine, Department of Molecular Genetics, Biochemistry and Microbiology, Cincinnati, Ohio 45267-0524, USA.
J Biol Chem. 1996 Jun 14;271(24):14176-82. doi: 10.1074/jbc.271.24.14176.
Cardiac glycosides such as ouabain and digoxin specifically inhibit the Na,K-ATPase. Three new residues in the carboxyl half of the Na, K-ATPase, Phe-786, Leu-793 (PFLIF786IIANIPL793PLGT797), and Phe-863 (FTYF863VIM) have been identified as ouabain sensitivity determinants using random mutagenesis. Polymerase chain reaction was utilized to randomly mutate the DNA sequence encoding the amino acids between Lys-691 and Lys-945 in the alpha subunit of the Na, K-ATPase. This region contains four transmembrane segments (H5, H6, H7, and H8) and the connecting extracellular and cytoplasmic loops. Diverse substitutions of these three residues resulted in proteins displaying 2.8-48-fold increases in the I50 of different cardiac glycosides for inhibition of the Na,K-ATPase activity. By locating these residues, in conjunction with Thr-797 (Feng, J., and Lingrel, J. B (1994) Biochemistry 33, 4218-4224), a new region of the protein containing the H5-H6 hairpin and the H7 transmembrane segment emerges as a major determinant of ouabain inhibition. Thus, a link between the cardiac glycoside binding site and the cation transport sites of the Na,K-ATPase transpires giving a structural base to the cation antagonism of ouabain binding. Furthermore, this link suggests a possible mechanism for cardiac glycoside inhibition of the Na,K-ATPase, such that ouabain binding to the implicated region blocks the movement of the H5 and H6 transmembrane domains which may be required for energy transduction and cation transport.
哇巴因和地高辛等强心苷能特异性抑制钠钾ATP酶。利用随机诱变技术，已确定钠钾ATP酶羧基端的三个新残基，即苯丙氨酸-786、亮氨酸-793（PFLIF786IIANIPL793PLGT797）和苯丙氨酸-863（FTYF863VIM）为哇巴因敏感性决定因素。采用聚合酶链反应对编码钠钾ATP酶α亚基中赖氨酸-691和赖氨酸-945之间氨基酸的DNA序列进行随机诱变。该区域包含四个跨膜片段（H5、H6、H7和H8）以及连接细胞外和细胞质的环。这三个残基的不同取代导致蛋白质对不同强心苷抑制钠钾ATP酶活性的I50增加2.8至48倍。通过定位这些残基，结合苏氨酸-797（Feng, J., and Lingrel, J. B (1994) Biochemistry 33, 4218 - 4224），一个包含H5 - H6发夹结构和H7跨膜片段的蛋白质新区域成为哇巴因抑制作用的主要决定因素。因此，强心苷结合位点与钠钾ATP酶的阳离子转运位点之间建立了联系，为哇巴因结合的阳离子拮抗作用提供了结构基础。此外，这种联系提示了强心苷抑制钠钾ATP酶的一种可能机制，即哇巴因与相关区域结合会阻断H5和H6跨膜结构域的移动，而这可能是能量转导和阳离子转运所必需的。
