ASP(804) AND ASP(808) IN THE TRANSMEMBRANE DOMAIN OF THE NA,K-ATPASE ALPHA-SUBUNIT ARE CATION COORDINATING RESIDUES

The functional roles of Asp(804) and Asp(808), located in the sixth tr ansmembrane segment of the Na,R-ATPase alpha subunit, were examined. N onconservative 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 Huys se, 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 i n 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, b ound ouabain and nucleotides, and adopted E(1)Na, E(1)ATP, and E(2)P c onformations. K+ competition of ouabain binding to sheep alpha 1 and A sp(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 pr oteins lacked this K+-ouabain antagonism, e.g. 150 min KCl did not inh ibit ouabain binding. Na+ antagonized ouabain binding to all the expre ssed isoforms, however, the proteins carrying nonconservative substitu tions 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), T herefore, Asp(804) and Asp(808) of the Na,K-ATPase are required for no rmal Na+ and K+ transport, possibly coordinating these cations during transport.