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Volume 272, Number 40, Issue of October 3, 1997 pp. 24987-24993
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Evidence That Ser⁷⁷⁵ in the  Subunit of the Na,K-ATPase Is a Residue in the Cation Binding Pocket

(Received for publication, May 9, 1997, and in revised form, July 4, 1997)

Rhoda Blostein , Ania Wilczynska , Steven J. D. Karlish ^¶ , Jose M. Argüello and Jerry B Lingrel

From the  Department of Medicine, McGill University, Montreal, Quebec, Canada H3G 1A4, the ^¶ Weizmann Institute of Science, Rehovot 76100, Israel, and the  Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-9524

Substitution of alanine for Ser⁷⁷⁵ in a ouabain-resistant 1 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⁺.

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