The functional roles of Asp(804) and Asp(808), located in the sixth transmembrane segment of the Na,R-ATPase alpha subunit, were examined. Nonconservative replacement of these residues yielded enzymes unable to support cell viability. Only the conservative substitution, Ala(808) --> Glu, was able to maintain the essential cation gradients (Van Huysse, J. W., Kuntzweiler, T. A., and Lingrel, J. B (1996) FEES Left. 389, 179-185). Asp(804) and Asp(808) were replaced by Ala, Asn, and Glu in the sheep alpha 1 subunit and expressed in a mouse cell line where [H-3]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 E(1)Na, E(1)ATP, and E(2)P conformations. K+ competition of ouabain binding to sheep alpha 1 and Asp(808) --> 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 min 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) less than or equal to 2.0) compared to the control (n(Hill) less than or equal to 2.8), Therefore, Asp(804) and Asp(808) of the Na,K-ATPase are required for normal Na+ and K+ transport, possibly coordinating these cations during transport.