A lysine residue within the highly conserved center of the fifth transmembrane segment in PIIC-type ATPase α-subunits is uniquely found in Ha,K-ATPases instead of a serine in all Na,K-ATPase isoforms. Because previous studies suggested a prominent role of this residue in determining the electrogenicity of non-gastric H,K-ATPase and in PKa modulation of the proton-translocating residues in the gastric H,K-ATPases as well, we investigated its functional significance for ion trans- port by expressing several Lys-791 variants of the gastric H,K- ATPase in Xenopus oocytes. Although the mutant proteins were all detected at the cell surface, none of the investigated mutants displayed any measurable K+-induced stationary currents. In Rb+ uptake measurements, replacement of Lys-791 by Arg, Ala, Ser, and Glu substantially impaired transport activity and reduced the sensitivity toward the E2-specific inhibitor SCH28080. Furthermore, voltage clamp fluorometry using a reporter site in the TM5/TM6 loop for labeling with tetramethylrhodamine-6-maleimide revealed markedly changed fluorescence signals. All four investigated mutants exhibited a strong shift toward the E1P state, in agreement with their reduced SCH28080 sensitivity, and an about 5-10-fold decreased forward rate constant of the E1P 7#x2194; E2P conformational transition, thus explaining the E1P shift and the reduced Rb+ transport activity. When Glu-820 in TM6 adjacent to Lys- 791 was replaced by non-charged or positively charged amino acids, severe effects on fluorescence signals and Rb+ transport were also observed, whereas substitution by aspartate was less disturbing. These results suggest that formation of an E2P- stabilizing interhelical salt bridge is essential to prevent futile proton exchange cycles of H+ pumping P-type ATPases. [ABSTRACT FROM AUTHOR]