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Volume 271, Number 47, Issue of November 22, 1996 pp. 29682-29687
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Asp⁸⁰⁴ and Asp⁸⁰⁸ in the Transmembrane Domain of the Na,K-ATPase  Subunit Are Cation Coordinating Residues

(Received for publication, July 10, 1996, and in revised form, August 16, 1996)

From the University of Cincinnati College of Medicine, Department of Molecular Genetics, Biochemistry and Microbiology, Cincinnati, Ohio 45267-0524

The functional roles of Asp⁸⁰⁴ and Asp⁸⁰⁸, located in the sixth transmembrane segment of the Na,K-ATPase  subunit, were examined. Nonconservative replacement of these residues yielded enzymes unable to support cell viability. Only the conservative substitution, Ala⁸⁰⁸  Glu, was able to maintain the essential cation gradients (Van Huysse, J. W., Kuntzweiler, T. A., and Lingrel, J. B (1996) FEBS Lett. 389, 179-185). Asp⁸⁰⁴ and Asp⁸⁰⁸ were replaced by Ala, Asn, and Glu in the sheep 1 subunit and expressed in a mouse cell line where [³H]ouabain binding was utilized to probe the exogenous proteins. All of the heterologous proteins were targeted into the plasma membrane, bound ouabain and nucleotides, and adopted E₁Na, E₁ATP, and E₂P conformations. K⁺ competition of ouabain binding to sheep 1 and Asp⁸⁰⁸  Glu enzymes displayed IC₅₀ values of 4.11 mM (n_Hill = 1.4) and 23.8 mM (n_Hill = 1.6), respectively. All other substituted proteins lacked this K⁺-ouabain antagonism, e.g. 150 mM KCl did not inhibit ouabain binding. Na⁺ antagonized ouabain binding to all the expressed isoforms, however, the proteins carrying nonconservative substitutions displayed reduced Hill coefficients (n_Hill  2.0) compared to the control (n_Hill  2.8). Therefore, Asp⁸⁰⁴ and Asp⁸⁰⁸ of the Na,K-ATPase are required for normal Na⁺ and K⁺ transport, possibly coordinating these cations during transport.

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