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Originally published In Press as doi:10.1074/jbc.M211636200 on January 14, 2003

J. Biol. Chem., Vol. 278, Issue 11, 9027-9034, March 14, 2003
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Structural Basis for alpha 1 Versus alpha 2 Isoform-distinct Behavior of the Na,K-ATPase*

Laura SegallDagger , Zahid Z. Javaid§, Stephanie L. Carl§, Lois K. Lane§, and Rhoda BlosteinDagger

From the Dagger  Department of Biochemistry, McGill University, Montreal, Quebec H3G 1A4, Canada and § Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267

We showed earlier that the kinetic behavior of the alpha 2 isoform of the Na,K-ATPase differs from the ubiquitous alpha 1 isoform primarily by a shift in the steady-state E1/E2 equilibrium of alpha 2 in favor of E1 form(s). The aim of the present study was to identify regions of the alpha  chain that confer the alpha 1/alpha 2 distinct behavior using a mutagenesis and chimera approach. Criteria to assess shifts in conformational equilibrium included (i) K+ sensitivity of Na-ATPase measured at micromolar ATP, under which condition E2(K+) right-arrow E1 + K+ becomes rate-limiting, (ii) changes in K'ATP for low affinity ATP binding, (iii) vanadate sensitivity of Na,K-ATPase activity, and (iv) the rate of the partial reaction E1P right-arrow E2P. We first confirmed that interactions between the cytoplasmic domains of alpha 2 that modulate conformational shifts are fundamentally similar to those of alpha 1, suggesting that the predilection of alpha 2 for E1 state(s) is due to differences in primary structure of the two isoforms. Kinetic behavior of the alpha 1/alpha 2 chimeras indicates that the difference in E1/E2 poise of the two isoforms cannot be accounted for by their notably distinct N termini, but rather by the front segment extending from the cytoplasmic N terminus to the C-terminal end of the extracellular loop between transmembranes 3 and 4, with a lesser contribution of the alpha 1/alpha 2 divergent portion within the M4-M5 loop near the ATP binding domain. In addition, we show that the E1 shift of alpha 2 results primarily from differences in the conformational transition of the dephosphoenzyme, (E2(K+) right-arrow E1 + K+), rather than phosphoenzyme (E1P right-arrow E2P).

* This work was supported by Canadian Institutes of Health Research Grant MT-3876, an operating grant from the Quebec Heart and Stroke Foundation (to R. B.), National Institutes of Health Grant HL 49204 (to L. K.), and a predoctoral fellowship from the Heart and Stroke Foundation of Canada (to L. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Montreal General Hospital Research Institute, 1650 Cedar Ave., Montreal, Quebec H3G 1A4, Canada. Tel.: 514-934-1934 (ext. 44501); Fax: 514-934-8332; E-mail: rhoda.blostein@mcgill.ca.

Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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