Site-directed mutagenesis of amino acids in the cytoplasmic loop 6/7 of Na,K-ATPase.
The loop between transmembrane helices 6 and 7 (L6/7) of P-type ATPases has been suggested to be important for the functional linkage of ion binding and enzyme phosphorylation or to be a site of initial cation binding. To investigate the role of L6/7 in Na,K-ATPase, alanine substitutions were made for charged and conserved residues in L6/7 of the human alpha1 subunit and the proteins were expressed in yeast for analysis. All mutants except the triple mutant E825A/E828A/D830A bound ouabain. Although the equilibrium dissociation constant for ouabain binding by most mutants was similar to the wild-type value, the K(d) of R837A for ouabain binding was approximately 15-fold higher than the wild-type K(d). (18)O exchange measurements indicated that the apparent affinity of this mutant for Pi was reduced about 3-fold. The concentration dependence of KCl inhibition of ouabain binding or of NaCl inhibition of ouabain binding revealed 2-4-fold changes in the apparent affinity for cations in the E825A, E828A, and R837A mutants. The E825A and E828A mutants lost the ability to bind ouabain after extraction with 0.1% SDS or after brief heating, indicating that these mutations affected the stability of the enzyme. The ATPase activity of the other mutants was measured after extraction of crude yeast membranes with 0.1% SDS. For all mutants except R834A, R837A, and R848A, the activity was at least 50% of wild-type activity.
Xu
G
G
University of Southern California School of Medicine, Los Angeles 90033, USA.
Farley
R A
RA
Kane
D J
DJ
Faller
L D
LD
D000409:Alanine; D019943:Amino Acid Substitution; D017124:Conserved Sequence; D003593:Cytoplasm / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010042:Ouabain / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism*
D010710:Phosphates; D011994:Recombinant Proteins; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase; D000409:Alanine