Functional consequences of amino-terminal diversity of the catalytic subunit of the Na,K-ATPase.
One region of marked sequence diversity among the highly homologous alpha isoforms of the Na,K-ATPase is the lysine-rich NH2 terminus. Expression of a mutant cDNA encoding an alpha 1 protein, minus the 32 NH2-terminal residues, results in a modified enzyme (alpha 1M32), which behaves similarly to alpha 1 in overall Na/K exchange activity (Vmax) and apparent affinities for intracellular Na+ and extracellular K+. However, with membranes isolated from HeLa cells expressing the rat alpha 1M32 mutant, as well as membranes from cells expressing the rat alpha 1 and the ouabain-resistant mutated forms of rat alpha 2 (alpha 2*) and alpha 3 (alpha 3*) developed by Jewell and Lingrel (Jewell, E. A., and Lingrel, J. B. (1991) J. Biol. Chem. 266, 16925-16930), distinct Na,K-ATPase kinetics are observed. Thus, at 1 microM ATP, the effects of K+ on the Na-ATPase activity of alpha 2* and alpha 1M32 are similar; both are activated, whereas alpha 1 and alpha 3 are inhibited by the addition of K+ at low (0.1 mM) concentration. These effects are attributed to different rates of a step involved in K+ deocclusion (E2(K)<-->E1K<-->E1 + K+) and are consistent with our earlier evidence (Wierzbicki, W., and Blostein, R. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 70-74) for a role of the NH2 terminus in the K+ deocclusion pathway of the Na,K-ATPase reaction. These differences are not directly related to differences in apparent affinities for ATP, since alpha 3* has alpha 1-like high affinity K+ inhibition but resembles alpha 2* and alpha 1M32 with respect to a lower K'ATP. Na-ATPase activities of alpha 2*, alpha 3*, and alpha 1M32, but not alpha 1, are activated by Li+ but not Rb+, consistent with a relatively faster rate of Li+ deocclusion (Post, R. L., Hegyvary, C., and Kume, S. (1972) J. Biol. Chem. 247, 6530-6540), as well as higher affinity of alpha 3 for extracellular K+ (Li+) activation of dephosphorylation (E2P + K+<-->E2(K) + Pi). Inhibition of Na-ATPase by higher concentrations (> or = 1 mM) K+ is observed with all isoforms and is attributed to K+ acting at inhibitory cytoplasmic sites.(ABSTRACT TRUNCATED AT 400 WORDS)
Daly
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SE
Department of Medicine, McGill University, Montreal, Quebec, Canada.
Lane
L K
LK
Blostein
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R
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002384:Catalysis / Q000187:drug effects; D002478:Cells, Cultured; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008094:Lithium / Q000494:pharmacology; D016296:Mutagenesis; D011188:Potassium / Q000494:pharmacology; D051381:Rats; D012413:Rubidium / Q000494:pharmacology; D012964:Sodium / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism*
D000255:Adenosine Triphosphate; D008094:Lithium; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium; D011188:Potassium