| Abstrakt: |
The functional roles of Asp804and Asp808, located in the sixth transmembrane segment of the Na,K-ATPase α 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). Asp804and Asp808were replaced by Ala, Asn, and Glu in the sheep α1 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 α1 and Asp808→ Glu enzymes displayed IC50values of 4.11 mM(nHill= 1.4) and 23.8 mM(nHill= 1.6), respectively. All other substituted proteins lacked this K+-ouabain antagonism, e.g.150 mMKCl did not inhibit ouabain binding. Na+antagonized ouabain binding to all the expressed isoforms, however, the proteins carrying nonconservative substitutions displayed reduced Hill coefficients (nHill≤ 2.0) compared to the control (nHill≤ 2.8). Therefore, Asp804and Asp808of the Na,K-ATPase are required for normal Na+and K+transport, possibly coordinating these cations during transport. |
