Evidence that Ser775 in the alpha subunit of the Na,K-ATPase is a residue in the cation binding pocket.
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+.
Blostein
R
R
Department of Medicine, McGill University, Montreal, Quebec, Canada H3G 1A4.
Wilczynska
A
A
Karlish
S J
SJ
Argüello
J M
JM
Lingrel
J B
JB
D000255:Adenosine Triphosphate / Q000378:metabolism; D000409:Alanine; D000818:Animals; D001665:Binding Sites; D002414:Cations, Monovalent / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D007550:Isothiuronium / Q000031:analogs & derivatives / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D010042:Ouabain / Q000494:pharmacology; D017354:Point Mutation; D011188:Potassium / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012413:Rubidium / Q000378:metabolism; D012694:Serine; D012756:Sheep; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection
C108416:1,3-dibromo-2,4,6-tris-(methylisothiouronium)benzene; D002414:Cations, Monovalent; D004791:Enzyme Inhibitors; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D007550:Isothiuronium; D012694:Serine; D010042:Ouabain; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium; D000409:Alanine; D011188:Potassium