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Volume 271, Number 24, Issue of June 14, 1996 pp. 14176-14182
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Ouabain Interactions with the H5-H6 Hairpin of the Na,K-ATPase Reveal a Possible Inhibition Mechanism via the Cation Binding Domain

(Received for publication, January 18, 1996, and in revised form, March 25, 1996)

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

Cardiac glycosides such as ouabain and digoxin specifically inhibit the Na,K-ATPase. Three new residues in the carboxyl half of the Na,K-ATPase, Phe-786, Leu-793 (PFLIF⁷⁸⁶IIANIPL⁷⁹³PLGT⁷⁹⁷), and Phe-863 (FTYF⁸⁶³VIM) have been identified as ouabain sensitivity determinants using random mutagenesis. Polymerase chain reaction was utilized to randomly mutate the DNA sequence encoding the amino acids between Lys-691 and Lys-945 in the  subunit of the Na,K-ATPase. This region contains four transmembrane segments (H5, H6, H7, and H8) and the connecting extracellular and cytoplasmic loops. Diverse substitutions of these three residues resulted in proteins displaying 2.8-48-fold increases in the I₅₀ of different cardiac glycosides for inhibition of the Na,K-ATPase activity. By locating these residues, in conjunction with Thr-797 (Feng, J., and Lingrel, J. B (1994) Biochemistry 33, 4218-4224), a new region of the protein containing the H5-H6 hairpin and the H7 transmembrane segment emerges as a major determinant of ouabain inhibition. Thus, a link between the cardiac glycoside binding site and the cation transport sites of the Na,K-ATPase transpires giving a structural base to the cation antagonism of ouabain binding. Furthermore, this link suggests a possible mechanism for cardiac glycoside inhibition of the Na,K-ATPase, such that ouabain binding to the implicated region blocks the movement of the H5 and H6 transmembrane domains which may be required for energy transduction and cation transport.

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