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 te rminus (alpha 1M32) or a Glu(233) --> Lys substitution in the first M2 -M3 cytoplasmic loop (E233K) of the alpha 1-subunit of the Na,K-ATPase result in a shift in the steady-state E-1 <-> E-2 conformational equi librium toward E-1 form(s), In the present study, the functional conse quences of both NH2-terminal deletion and Glu(233) substitution provid e evidence for mutual interactions of these cytoplasmic regions. Follo wing transfection and selection of HeLa cells expressing the ouabain-r esistant alpha 1M32E233K double mutant, growth was markedly reduced un less the K+ concentration in the culture medium was increased to at le ast 10 mM. Marked changes effected by this double mutation included 1) a 15-fold reduction in catalytic turnover (V-max/EPmax) a 70-fold inc rease in apparent affinity for ATP, 3) a marked decrease in vanadate s ensitivity, and 4) marked (approximate to 10-fold) K+ activation of th e Na-ATPase activity measured at micromolar ATP under which condition the E-2(K) --> --> E-1 pathway is normally (alpha 1) rate-limiting and K+ is inhibitory. The decrease in catalytic turnover was associated w ith a 5-fold decrease in V-max and a compensatory approximate to 3-fol d increase in expressed alpha 1M32E233K protein. In contrast to the be havior of either alpha 1M32 or E233K, alpha 1M32E233K also showed alte rations in apparent cation affinities. K-Na' was decreased approximate to 2-fold and K-K' was increased approximate to 2-fold. The importanc e of the charge at residue 233 is underscored by the consequences of s ingle and double mutations comprising either a conservative change (E2 33D) or neutral substitution (E233Q). Thus, whereas mutation to a posi tively charged residue (E233K) causes a drastic change in enzymatic be havior, a conservative change causes only a minor change and the neutr al substitution, an intermediate effect. Overall, the combined effects of the NH2-terminal deletion and the Glu(233) substitutions are syner gistic rather than additive, consistent with an interaction between th e NH2-terminal region, the first cytoplasmic loop, and possibly the la rge M4-M5 cytoplasmic loop bearing the nucleotide binding and phosphor ylation sites.