Conformational transitions and change translocation by the Na,K pump: comparison of optical and electrical transients elicited by ATP-concentration jumps.
The electrogenic properties of the Na,K-ATPase were studied by correlating transient electrical events in the pump molecule with conformational transitions elicited by an ATP-concentration jump. Flat membrane fragments containing a high density (approximately 8000 microm(-2)) of oriented Na,K-ATPase molecules were bound to a planar lipid bilayer acting as a capacitive electrode. ATP was released in the medium from a photolabile inactive ATP derivative ("caged" ATP) by a 40-microsec light flash. Electrical signals resulting from transient charge movements in the protein under single-turnover conditions were recorded in the external measuring circuit. In parallel experiments carried out under virtually identical conditions, the fluorescence of membrane fragments containing Na,K-ATPase with covalently-bound 5-iodoacetamido-fluorescein (5-IAF) was monitored after the ATP-concentration jump. When the medium contained Na+, but no K+, the fluorescence of the 5-IAF-labeled protein decreases monotonously after release of ATP. In the experiments with membrane fragments bound to a planar bilayer, a transient pump current was observed which exhibited virtually the same time behavior as the fluorescence decay. This indicates that optical and electrical transients are governed by the same rate-limiting reaction step. Experiments with chymotrypsin-modified Na,K-ATPase suggest that both the fluorescence change as well as the charge movement are associated with the deocclusion of Na+ and release to the extracellular side. In experiments with Na+-free K+ media, a large inverse fluorescence change is observed after the ATP-concentration jump, but no charge translocation can be detected. This indicates that deocclusion of K+ is an electrically silent process.
Stürmer
W
W
Department of Biology, University of Konstanz, Federal Republic of Germany.
Apell
H J
HJ
Wuddel
I
I
Läuger
P
P
D000818:Animals; D002918:Chymotrypsin / Q000378:metabolism; D004560:Electricity; D005453:Fluorescence; D005456:Fluorescent Dyes; D007679:Kidney Medulla / Q000201:enzymology; D007700:Kinetics; D011817:Rabbits; D000254:Sodium-Potassium-Exchanging ATPase / Q000502:physiology*; D013223:Statistics as Topic
D005456:Fluorescent Dyes; D002918:Chymotrypsin; D000254:Sodium-Potassium-Exchanging ATPase