Functional consequences of mutation Asn326→Leu in the 4th transmembrane segment of the a‐subunit of the rat kidney Na+,K+‐ATPase

FEBS Letters

Vilsen, Bente¹ 

[1] Danish Biomembrane Research Centre, Department of Physiology, University of Aarhus, Ole Worms AN 160, DK-8000 Aarhus C,Denmark

关键词: Asparagine;    Na+/K+-pump;    Na site;    Mutation;    Oligomycin;    E1;    conformation with high Na+ and ATP affinities;    E2;    K+-occluded conformation with low affinity for ATP;    K 0.5;    concentration giving half-maximal activation;    M1–M10;    the putative transmembrane helices numbered from the NH2- terminal end of the peptide;   

DOI  :  10.1016/0014-5793(95)00313-X

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Site-specific mutagenesis was used to replace Asn³²⁶ in transmembrane segment M4 of the ouabain-insensitive α ₁-isoform of rat kidney Na⁺,K⁺-ATPase. Mutant Asn³²⁶ → Leu was functional as demonstrated by the ability of COS cells expressing the mutant enzyme to grow in the presence of ouabain. In three independent assays encompassing Na⁺ titrations of Na⁺,K⁺-ATPase activity, Na⁺-ATPase activity, and phosphorylation from ATP, the Asn³²⁶ → Leu mutant displayed a reduced apparent affinity for Na⁺. By contrast, this mutant exhibited a slightly increased apparent affinity for K⁺ relative to the wild-type enzyme. In the presence of Na⁺. without K⁺, the Asn³²⁶ → Len mutant hydrolyzed ATP at a high rate corresponding to 32% of the maximal Na⁺,K⁺-ATPase activity, and the rate of dephosphorylation of the phosphoenzyme intermediate was enhanced in the mutant relative to that of the wild-type enzyme. Oligomycin, known to stabilize the Na⁺-occluded phosphoenzyme intermediate, reduced the dephosphorylation rate of the mutant and increased the steady-state phosphoenzyme level formed by the mutant at least 3-fold, whereas an increase in the steady-state phosphoenzyme level of only 10–15% was determined for the wild-type enzyme. The molecular turnover number for the Na⁺,K⁺-ATPase reaction, calculated when the steady-state phosphoenzyme level obtained in the presence of oligomycin was taken as a measure of the concentration of active sites, was slightly reduced relative to that of the wild-type enzyme. The data are discussed in terms of a role for Asn326 in binding of cytoplasmic Na⁺ and in mediation of inhibition of dephosphorylation.

【 授权许可】

Unknown