Na+,K(+)-ATPase pump currents in giant excised patches activated by an ATP concentration jump.
The giant-patch technique was used to study the Na+,K(+)-ATPase in excised patches from rat or guinea pig ventricular myocytes. Na+,K(+)-pump currents showed a saturable ATP dependence with aK(m) of approximately 150 microM at 24 degrees C. The pump current can be completely abolished by ortho-vanadate. Dissociation of vanadate from the enzyme in the absence of extracellular Na+ was slow, with a Koff of 3.10(-4) S-1 (K1 approximately 0.5 microM, at 24 degrees C). Stationary currents were markedly dependent on intracellular pH, with a maximum at pH 7.9. Temperature-dependence measurements of the stationary pump current yielded an activation energy of approximately 100 kJ mol-1. Partial reactions in the transport cycle were investigated by generating ATP concentration jumps through photolytic release of ATP from caged ATP at pH 7.4 and 6.3. Transient outward currents were obtained at pH 6.3 with a fast rising phase followed by a slower decay to a stationary current. It was concluded that the fast rate constant of approximately 200 s-1 at 24 degrees C (pH 6.3) reflects a step rate-limiting the electrogenic Na+ release. Simulating the data with a simple three-state model enabled us to estimate the turnover rate under saturating substrate concentrations, yielding rates (at pH 7.4) of approximately 60 s-1 and 200 s-1 at 24 degrees C and 36 degrees C, respectively.
Max-Planck-Institut für Biophysik, Frankfurt am Main, Germany.
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T
Friedrich
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Bamberg
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Nagel
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D004553:Electric Conductivity; D006168:Guinea Pigs; D006321:Heart / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008564:Membrane Potentials; D008954:Models, Biological; D009206:Myocardium / Q000166:cytology; D018408:Patch-Clamp Techniques; D011487:Protein Conformation; D051381:Rats; D012964:Sodium / Q000502:physiology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013696:Temperature; D014638:Vanadates / Q000494:pharmacology
D014638:Vanadates; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase