Importance of intramembrane carboxylic acids for occlusion of K+ ions at equilibrium in renal Na,K-ATPase.
Site-directed mutagenesis and assay of Rb+ and Tl+ occlusion in recombinant Na,K-ATPase from yeast were combined to establish structure-function relationships of amino acid side chains involved in high-affinity occlusion of K+ in the E2[2K] form. The wild-type yeast enzyme was capable of occluding 2 Rb+ or Tl+ ions/ouabain binding site or alpha 1 beta 1 unit with high apparent affinity (Kd(Tl+) = 7 +/- 2 microM), like the purified Na,K-ATPase from pig kidney. Mutations of Glu327(Gln,Asp), Asp804(Asn, Glu), Asp808(Asn, Glu) and Glu779(Asp) abolished high-affinity occlusion of Rb+ or Tl+ ions. The substitution of Glu779 for Gln reduced the occlusion capacity to 1 Tl+ ion/alpha 1 beta 1-unit with a 3-fold decrease of the apparent affinity for the ion (Kd(Tl+) = 24 +/- 8 microM). These effects on occlusion were closely correlated to effects of the mutations on K0.5(K+) for K+ displacement of ATP binding. Each of the four carboxylate residues Glu327, Glu779, and Asp804 or Asp808 in transmembrane segments 4, 5, and 6 is therefore essential for high-affinity occlusion of K+ in the E2[2K] form. These residues either may engage directly in cation coordination or they may be important for formation or stability of the occlusion cavity.
Nielsen
J M
JM
Biomembrane Research Center, August Krogh Institute, Copenhagen University, Denmark.
Pedersen
P A
PA
Karlish
S J
SJ
Jorgensen
P L
PL
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002264:Carboxylic Acids / Q000378:metabolism*; D018698:Glutamic Acid / Q000235:genetics; D007425:Intracellular Membranes / Q000201:enzymology*; D007668:Kidney / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015221:Potassium Channels / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012413:Rubidium / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism*; D013552:Swine; D013793:Thallium / Q000378:metabolism
D002264:Carboxylic Acids; D015221:Potassium Channels; D011994:Recombinant Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D000255:Adenosine Triphosphate; D013793:Thallium; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium