Optical study of active ion transport in lipid vesicles containing reconstituted Na, K-ATPase

A fluorescence method is described for the measurement of ATP-driven ion fluxes in lipid vesicles containing purified Na,K-ATPase. The membrane voltage of enzyme containing vesicles was measured by using a voltage-sensitive indocyanine dye. By addition of valinomycin the vesicle membrane is made sel...

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Bibliographic Details
Published in:The Journal of membrane biology 1985-01, Vol.85 (1), p.49-63
Main Authors: APELL, H.-J, MARCUS, M. M, ANNER, B. M, OETLIKER, H, LÄUGER, P
Format: Article
Language:English
Subjects:
Animals
Biological and medical sciences
Calorimetry
Cell Membrane - enzymology
Cell Membrane - physiology
Cell physiology
Electric Conductivity
fluorescence
Fundamental and applied biological sciences. Psychology
ions
Kidney Medulla - enzymology
lipids
Liposomes
Mathematics
Membrane and intracellular transports
Membrane Lipids - physiology
Models, Biological
Molecular and cellular biology
Phosphatidylcholines - pharmacology
Potassium - blood
Rabbits
Sodium-Potassium-Exchanging ATPase - metabolism
Spectrometry, Fluorescence - methods
Valinomycin - pharmacology
vesicles
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Summary:A fluorescence method is described for the measurement of ATP-driven ion fluxes in lipid vesicles containing purified Na,K-ATPase. The membrane voltage of enzyme containing vesicles was measured by using a voltage-sensitive indocyanine dye. By addition of valinomycin the vesicle membrane is made selectively permeable to K+ so that the membrane voltage approaches the Nernst potential for K+. With constant external K+ concentration, the time course of internal K+ concentration can be continuously measured as change of the fluorescence signal after activation of the pump. The optical method has a higher time resolution than tracer-flux experiments and allows an accurate determination of initial flux rates. From the temperature dependence of active K+ transport its activation energy was determined to be 115 kJ/mol. ATP-stimulated electrogenic pumping can be measured as fast fluorescence change when the membrane conductance is low (i.e., at low or zero valinomycin concentration). In accordance with expectation, the amplitude of the fast signal change increases with decreasing passive ion permeability of the vesicle membrane. The resolution of the charge movement is so high that a few pump turnovers can be easily detected.
ISSN:0022-2631
1432-1424
DOI:10.1007/BF01872005