Identification of Asp804 and Asp808 as Na+ and K+ coordinating residues in alpha-subunit of renal Na,K-ATPase.
Mutations to Asp804 and Asp808 in the alpha-subunit almost abolish Na,K-ATPase activity, but high-affinity binding of [3H]ATP or [3H]ouabain at equilibrium and E1-E2 transitions are preserved. Titration of K+-ion displacement of [3H]ATP or [3H]ouabain shows that the mutations interfere with occlusion of K+ in the E2[2K] conformation. Reduced phosphorylation levels or affinities for Na+ in presence of oligomycin indicate that Asp804 and Asp808 also contribute to coordination of Na+ in the E1P[3Na] form. Demonstration of alternate interactions of Na+ or K+ with Asp804 and Asp808 support the notion of cation binding in a ping-pong sequence in catalytic models of Na,K-pumping.
Pedersen
P A
PA
Biomembrane Research Centre, August Krogh Institute, Copenhagen University, Denmark.
Rasmussen
J H
JH
Nielsen
J M
JM
Jorgensen
P L
PL
D000255:Adenosine Triphosphate / Q000378:metabolism; D001224:Aspartic Acid; D016297:Mutagenesis, Site-Directed; D010042:Ouabain / Q000378:metabolism; D011188:Potassium / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism*; D015003:Yeasts
D001224:Aspartic Acid; D010042:Ouabain; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium