Leucine 332 at the Boundary Between the Fourth Transmembrane Segment and the Cytoplasmic Domain of Na+,K+-ATPase Plays a Pivotal Role in the Ion Translocating Conformational Changes
- 1 October 1997
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 36 (43) , 13312-13324
- https://doi.org/10.1021/bi971030q
Abstract
Mutants Gly330→Ala, Leu332→Ala, Leu332→Pro, and Pro780→Ala of the α1-isoform of rat kidney Na+,K+-ATPase were expressed in COS-1 cells to active site concentrations between 20 and 70 pmol per mg of membrane protein. The functional properties of the mutants were characterized by titrations of Na+-, K+-, and ATP-dependencies of Na+,K+-ATPase activity as well as by a series of assays measuring the K+-dependence of the steady-state phosphoenzyme level, the kinetics of dephosphorylation under a variety of conditions, and the ADP−ATP exchange activity. In mutants Gly330→Ala, Leu332→Ala, and Leu332→Pro, the molecular turnover number was reduced relative to that of the wild-type Na+,K+-ATPase, and the steady-state phosphoenzyme level was high even in the presence of several millimolar K+. At a low Na+ concentration in the absence of K+, mutants Leu332→Pro and Gly330→Ala displayed high ADP−ATP exchange activity and formed a high level of the ADP-sensitive phosphoenzyme (E1P). The phosphoenzyme decayed slowly following a jump in salt concentration and chase with ATP and K+. Hence, the conversion of E1P to the K+-sensitive phosphoenzyme (E2P) was inhibited in mutants Leu332→Pro and Gly330→Ala. In the Leu332→Ala mutant, a high level of E2P was accumulated in the absence of K+, and the ADP−ATP exchange activity was low at low Na+ concentration in the absence of K+, but rose sharply on addition of K+. Dephosphorylation experiments indicated that in the Leu332→Ala mutant K+ induced reversal of the phosphoenzyme interconversion, forming E1P from E2P. Leu332 is therefore a pivotal residue in the conformational change. Mutants Gly330→Ala and Pro780→Ala displayed reduced K+ affinities relative to the wild type, determined in three independent assays.Keywords
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