Structure of the 5th transmembrane segment of the Na,K-ATPase α subunit:: a cysteine-scanning mutagenesis study

To study the structure of the pathway of cations across the Ka,K-ATPase, we applied the substituted cysteine accessibility method to the putative 5th transmembrane segment of the alpha subunit of the Ka,K-ATPase of the toad Bufo marinus. Only the most extracellular amino acid position (A(796)) was accessible from the extracellular side in the native Na,g-pump. After treatment with palytoxin, six other positions (Y-778, L-780, S-782, p(785), E-786 and L-791), distributed along the whole length of the segment, became readily accessible to a small-size methanethiosulfonate compound (2-aminoethyl methanethiosulfonate), The accessible residues are not located on the same side of an ct-helical model but the pattern of reactivity would rather suggest a B-sheet structure for the inner half df the putative transmembrane segment. These results demonstrate the contribution of the 5th transmembrane segment to the palytoxin-induced channel and indicate which amino acid positions are exposed to the pore of this channel, (C) 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.