Evidence that Ser(775) in the alpha subunit of Na,K-ATPase is a residue in the cation binding pocket

Substitution of alanine for Ser(775) in a ouabain-resistant alpha 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 Ser(775) 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 (E-2(K)) and of the rate of formation of deoccluded enzyme from E-2(K) indicate that the Ser(775) --> Ala mutation results in a marked increase (greater than or equal to 30-fold) in rate of release of K+ from E-2(K), The high affinity Na+-like competitive antagonist 1,3-dibromo-2,4,6-tris-(methylisothiouronium)benzene (Br2TITU), which interacts with the E-1 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 Ser(775) --> 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+.