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Functional consequences of mutation Asn 326 →Leu in the 4th transmembrane segment of the a‐subunit of the rat kidney Na + ,K + ‐ATPase
Author(s) -
Vilsen Bente
Publication year - 1995
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(95)00313-x
Subject(s) - ouabain , mutant , dephosphorylation , chemistry , enzyme , atpase , oligomycin , atp hydrolysis , biochemistry , microbiology and biotechnology , stereochemistry , phosphatase , biology , sodium , organic chemistry , gene
Site‐specific mutagenesis was used to replace Asn 326 in transmembrane segment M4 of the ouabain‐insensitive α 1 ‐isoform of rat kidney Na + ,K + ‐ATPase. Mutant Asn 326 → 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 Asn 326 → 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 Asn 326 → 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.