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(+)/Na(+)-exchange conditions, we observed fluorescence changes that paralleled the transient currents originating from the E(1)P<-->E(2)P 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.
Geibel
Sven
S
Max-Planck-Institute of Biophysics, Kennedyallee 70, D-60596 Frankfurt/M., Germany.
Zimmermann
Dirk
D
Zifarelli
Giovanni
G
Becker
Anja
A
Koenderink
Jan B
JB
Hu
Yi-Kang
YK
Kaplan
Jack H
JH
Friedrich
Thomas
T
Bamberg
Ernst
E
D000595:Amino Acid Sequence; D000818:Animals; D001693:Biological Transport, Active; D017124:Conserved Sequence; D003545:Cysteine; D005260:Female; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014981:Xenopus
D011994:Recombinant Proteins; D017506:H(+)-K(+)-Exchanging ATPase; D000254:Sodium-Potassium-Exchanging ATPase; D003545:Cysteine