Conformational. dynamics of Na+/K+- and H+/K+-ATPase probed by voltage clamp fluorometry

We used the method of site-directed fluorescence labeling in combination with voltage-clamp fluorometry for time-resolved recording of localized conformational transitions of the Na+/K+-and H+/K+-ATPase. Therefore, single cysteine mutations were introduced into the extracellular TM5-TM6 loop of the sheep Na+/K+-ATPase alpha(1)-subunit devoid of other extracellular cysteines. Upon expression in Xenopus oocytes and covalent attachment of tetramethylrhodamine-maleimide (TMRM) as a reporter fluorophore, Cys-mutant N790C showed large fluorescence changes of up to 5 % in response to extracellular K+ that were completely abolished by ouabain. When voltage jumps were applied under Na+/Ne-exchange conditions, we observed fluorescence changes that paralleled the transient currents originating from the E1P<---->E2P transition. These fluorescence changes were also completely inhibited by ouabain, as were the voltage jump-induced transient currents. Transient fluorescence changes could also be measured as a function of increasing K+ concentrations, that is, under turnover conditions. As a result, the distribution between E-1 and E-2 states can be determined at any time and membrane potential. Very similar fluorescence signals were obtained for rat gastric H+/K+-ATPase upon expression in oocytes, when a single cysteine was introduced at a position homologous to N790 in Na+/K+-ATPase for attachment of the fluorophore. As to the high sequence similarity among P-type ATPases within the TM5 helix and the TM5-TM6 loop region, our results enable new means of kinetic investigation for these pumps under physiological conditions in living cells.