Abstract
Substitution of alanine for Ser775 in a ouabain-resistant alpha1 sheep isoform causes a 30-fold decrease in apparent affinity for K+ as an activator of the Na,K-ATPase, as well as an increase in apparent affinity for ATP (Arguello, J. M., and Lingrel, J. B (1995) J. Biol. Chem. 270, 22764-22771). This study was carried out to determine whether Ser775 is a direct cation-ligating residue or whether the change in apparent affinity for K+ is secondary to a conformational alteration as evidenced in the change in ATP affinity, with the following results. Kinetics of K+(Rb+) influx into intact cells show that the change is due to a change in K+ interaction at the extracellular surface. The K+ dependence of formation of K+-occluded enzyme (E2(K)) and of the rate of formation of deoccluded enzyme from E2(K) indicate that the Ser775 --> Ala mutation results in a marked increase (>/=30-fold) in rate of release of K+ from E2(K). The high affinity Na+-like competitive antagonist 1,3-dibromo2,4,6-tris-(methylisothiouronium)benzene (Br2TITU), which interacts with the E1 conformation and blocks cytoplasmic cation binding (Hoving, S., Bar-Shimon, M., Tijmes, J. J. , Tal, D. M., and Karlish, S. J. D. (1995) J. Biol. Chem. 270, 29788-29793), inhibits Na+-ATPase of the mutant less than the control enzyme. With intact cells, Br2TITU acts as a competitive inhibitor of extracellular K+ activation of both the mutant and control enzymes. In this case, the mutant was more sensitive to inhibition. With vanadate as a probe of conformation, a difference in conformational equilibrium between the mutant and control enzymes could not be detected under turnover conditions (Na+- ATPase) in the absence of K+. These results indicate that the increase in apparent affinity for ATP effected by the Ser775 --> Ala mutation is secondary to a change in intrinsic cation affinity/selectivity. The large change in affinity for extracellular K+ compared with cytoplasmic Na+ and to Br2TITU binding supports the conclusion that the serine hydroxyl is either part of the K+-gate structure or a direct cation-ligating residue that is shared by at least one Na+ ion, albeit with less consequence on rate constants for Na+ binding or release compared with K+.
Highlights
The Na,K-ATPase is a heterodimeric protein comprised of a catalytic ␣ subunit and a smaller heavily glycosylated  subunit
Mutagenesis, Cloning, Tissue Culture, and Transfection—HeLa cells were transfected with sheep Na,K-ATPase ␣1 subunit cDNA modified by two mutations (Gln111 3 Arg and Asn122 3 Asp) to encode an ␣1 form with low affinity for ouabain (RD)2 and with a Ser775 3 Ala mutant (S775A) of RD
Mutation of Ser775 3 Ala in this enzyme results in suppression of growth unless the Kϩ concentration is raised to at least 20 mM [16]. This behavior is associated with a marked decrease in apparent affinity of the mutant enzyme (S775A) for Kϩ, as evidenced in studies of Kϩ-activation of Na,K-ATPase activity [16]
Summary
Mutagenesis, Cloning, Tissue Culture, and Transfection—HeLa cells were transfected with sheep Na,K-ATPase ␣1 subunit cDNA modified by two mutations (Gln111 Arg and Asn122 Asp) to encode an ␣1 form with low affinity for ouabain (RD) and with a Ser775 Ala mutant (S775A) of RD. Membrane Preparations and Enzyme Assays—Membranes were isolated, and assays of Na,K-ATPase and Na-ATPase were carried out as described previously [17] in medium containing 5 mM EGTA, 1 mM MgCl2, and the indicated concentrations of ATP, Naϩ, and Kϩ. Ouabainsensitive Kϩ(86Rbϩ) influx into transfected HeLa cells was measured as described by Munzer et al [19] using 24-well Falcon plates and with medium containing 10 mM NaCl, the indicated concentrations of KCl, and choline chloride to maintain a constant (150 mM) chloride concentration, 5 mM glucose, and 10 mM PO4-Tris, pH 7.4, with 12 M monensin to equilibrate Naϩ, and 10 M bumetanide. Br2TITU was synthesized as described by Tal and Karlish [20], dissolved in 2 mM Tris-HCl, pH 7.4 or pH 8.0, and stored at Ϫ20 °C as a 1 mM stock solution. [␥-32P]ATP was synthesized by a modification [21] of the method of Glynn and Chappell [22] and stored at Ϫ20 °C. 86RbCl was from Amersham Life Science, Inc
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