Changes in steady-state conformational equilibrium resulting from cytoplasmic mutations of the Na,K-ATPase alpha-subunit.
Mutations comprising either deletion of 32 amino acids from the NH2 terminus (alpha1M32) or a Glu233 --> Lys substitution in the first M2-M3 cytoplasmic loop (E233K) of the alpha1-subunit of the Na, K-ATPase result in a shift in the steady-state E1 left arrow over right arrow E2 conformational equilibrium toward E1 form(s). In the present study, the functional consequences of both NH2-terminal deletion and Glu233 substitution provide evidence for mutual interactions of these cytoplasmic regions. Following transfection and selection of HeLa cells expressing the ouabain-resistant alpha1M32E233K double mutant, growth was markedly reduced unless the K+ concentration in the culture medium was increased to at least 10 mM. Marked changes effected by this double mutation included 1) a 15-fold reduction in catalytic turnover (Vmax/EPmax), 2) a 70-fold increase in apparent affinity for ATP, 3) a marked decrease in vanadate sensitivity, and 4) marked (approximately 10-fold) K+ activation of the Na-ATPase activity measured at micromolar ATP under which condition the E2(K) --> --> E1 pathway is normally (alpha1) rate-limiting and K+ is inhibitory. The decrease in catalytic turnover was associated with a 5-fold decrease in Vmax and a compensatory approximately 3-fold increase in expressed alpha1M32E233K protein. In contrast to the behavior of either alpha1M32 or E233K, alpha1M32E233K also showed alterations in apparent cation affinities. K'Na was decreased approximately 2-fold and K'K was increased approximately 2-fold. The importance of the charge at residue 233 is underscored by the consequences of single and double mutations comprising either a conservative change (E233D) or neutral substitution (E233Q). Thus, whereas mutation to a positively charged residue (E233K) causes a drastic change in enzymatic behavior, a conservative change causes only a minor change and the neutral substitution, an intermediate effect. Overall, the combined effects of the NH2-terminal deletion and the Glu233 substitutions are synergistic rather than additive, consistent with an interaction between the NH2-terminal region, the first cytoplasmic loop, and possibly the large M4-M5 cytoplasmic loop bearing the nucleotide binding and phosphorylation sites.
Boxenbaum
N
N
Departments of Biochemistry and Experimental Medicine, McGill University, Montreal, Quebec, Canada H3G 1A4.
Daly
S E
SE
Javaid
Z Z
ZZ
Lane
L K
LK
Blostein
R
R
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D016764:Cell Polarity; D018698:Glutamic Acid / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008024:Ligands; D009154:Mutation; D011188:Potassium / Q000378:metabolism; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D017384:Sequence Deletion; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology
D008024:Ligands; D011994:Recombinant Proteins; D018698:Glutamic Acid; D014638:Vanadates; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium