Deceleration of the E1P-E2P transition and ion transport by mutation of potentially salt bridge-forming residues Lys-791 and Glu-820 in gastric H+/K+-ATPase

A lysine residue within the highly conserved center of the fifth transmembrane segment in P_IIC-type ATPase α-subunits is uniquely found in H,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 pK_a modulation of the proton-translocating residues in the gastric H,K-ATPases as well, we investigated its functional significance for ion transport 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 {"type":"entrez-protein","attrs":{"text":"SCH28080","term_id":"1053015931","term_text":"SCH28080"}}SCH28080. Furthermore, voltage clamp fluorometry using a reporter site in the TM5/TM6 loop for labeling with tetra-methylrhodamine-6-maleimide revealed markedly changed fluorescence signals. All four investigated mutants exhibited a strong shift toward the E₁P state, in agreement with their reduced {"type":"entrez-protein","attrs":{"text":"SCH28080","term_id":"1053015931","term_text":"SCH28080"}}SCH28080 sensitivity, and an about 5–10-fold decreased forward rate constant of the E₁P ↔ 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.