Asp(804) and Asp(808) in the transmembrane domain of the Na,K-ATPase alpha subunit are cation coordinating residues

被引:96
作者
Kuntzweiler, TA [1 ]
Arguello, JM [1 ]
Lingrel, JB [1 ]
机构
[1] UNIV CINCINNATI, COLL MED, DEPT MOL GENET BIOCHEM & MICROBIOL, CINCINNATI, OH 45267 USA
关键词
D O I
10.1074/jbc.271.47.29682
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
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页码:29682 / 29687
页数:6
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