To study the structure of the pathway of cations across the Na,K-ATPase, we applied the substituted cysteine accessibility method to the putative 5th transmembrane segment of the K K subunit of the Na,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,K-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 K K-helical model but the pattern of reactivity would rather suggest a L L-sheet structure for the inner half of the putative transmembrane segment. These results demonstrate the contribution of the 5th transmembrane segment to the palytoxininduced channel and indicate which amino acid positions are exposed to the pore of this channel. ß

The Na,K-ATPase is a major ion-motive ATPase of the P-type family responsible for many aspects of cellular homeostasis. To determine the structure of the pathway for cations across the transmembrane portion of the Na,K-ATPase, we mutated 24 residues of the fourth transmembrane segment into cysteine and studied their function and accessibility by exposure to the sulfhydryl reagent 2-aminoethyl-methanethiosulfonate. Accessibility was also examined after treatment with palytoxin, which transforms the Na,K-pump into a cation channel. Of the 24 tested cysteine mutants, seven had no or a much reduced transport function. In particular cysteine mutants of the highly conserved "PEG" motif had a strongly reduced activity. However, most of the non-functional mutants could still be transformed by palytoxin as well as all of the functional mutants. Accessibility, determined as a 2-aminoethyl-methanethiosulfonate-induced reduction of the transport activity or as inhibition of the membrane conductance after palytoxin treatment, was observed for the following positions: Phe

The accessibility of the residues of the sixth transmembrane segment (TM) of the Bufo marinus Na,KATPase K K subunit was explored by cysteine scanning mutagenesis. Methanethiosulfonate reagents reached only the two most extracellular positions (T803, D804) in the native conformation of the Na,K-pump. Palytoxin induced a conductance in all mutants, including D811C, T814C and D815C which showed no active electrogenic transport. After palytoxin treatment, four additional positions (V805, L808, D811 and M816) became accessible to the sulfhydryl reagent. We conclude that one side of the sixth TM helix forms a wall of the palytoxin-induced channel pore and, probably, of the cation pathway from the extracellular side to one of their binding sites. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

The present work establishes a unique framework for the simulation study of ion-motive pumps in general and the Na + /K + -ATPase, or sodium pump, in particular. We shall discuss the implications of electrostatic analysis, valence calculations, and protein cavity data, each carried over data extracted from molecular dynamics simulations, on the structure-function relationship of Na + /K + -ATPase. These diverse set of tools will be used to investigate atomic-level characteristics that remain undetermined such as ion binding and accessibility.

The mechanism of cation translocation by the Na,K-ATPase was investigated by cysteine scanning mutagenesis and measurements of accessibility through exposure to cysteine reagents. In the native protein, accessible residues were found only at the most extracellular residues of the 5th and 6th transmembrane segments (TMS) and the short loop between them. However, after modification by palytoxin a number of residues became accessible along the whole length of the 5th TMS and in the outer half of the 6th TMS, showing the contribution of each of these segments to the "channel" formed by the palytoxin-transformed Na,K-pump. Assuming that this structure is similar in the native and the palytoxin-transformed pump, our data allow us to determine the residues lining the cation pathway from the extracellular solution to their binding sites. A critical position in the 5th TMS contains a lysine conserved in all known nonelectrogenic H,K-ATPases, and a serine in all known electrogenic Na,K-ATPase sequences. Wild-type or mutant Na,K-or H,K-ATPase a subunits were coinjected with the Bufo beta2 subunit in Xenopus oocytes and Rb(86) uptake and electrophysiological measurements were performed. An electrogenic activity was recorded for the H,K-ATPase mutants in which the positively charged lysine had been replaced by neutral or negatively charged residues, while nonelectrogenic transport was observed with the S(782)R mutant of the Na,K-ATPase. The presence or the absence of a positively charged residue at the S(782) position appears to be critical for the stoichiometry of cation exchange.