Electrogenicity of the sodium transport pathway in the Na,K-ATPase probed by charge-pulse experiments.
A charge-pulse technique was designed to measure charge movements in the Na-transport mode of the Na,K-ATPase in membrane fragments adsorbed to a planar lipid bilayer with high time resolution. 1) Na+ transport was measured as a function of membrane potential, and 2) voltage-dependent extracellular ion binding and release were analyzed as a function of Na+ concentration and membrane potential. The results could be fitted and explained on the basis of a Post-Albers cycle by simulations with a mathematical model. The minimal reaction sequence explaining the electrogenicity of the pump consists of the following steps: (Na3)E1-P <--> P-E2(Na3) <--> P-E2(Na2) <--> P-E2(Na) <--> P-E2. The conformational change, E1 to E2, is electrogenic (beta 0 < or = 0.1) and the rate-limiting step of forward Na+ transport with a rate constant of 25 s-1 (T = 20 degrees C). The first ion release step, P-E2(Na3) <--> P-E2(Na2), is the major charge translocating process (delta 0 = 0.65). It is probably accompanied by a protein relaxation in which the access structure between aqueous phase and binding site reduces the dielectric distance. The release of the subsequent Na+ ions has a significantly lower dielectric coefficient (delta1 = delta 2 = 0.2). Compared with other partial reactions, the ion release rates are fast (1400 s-1, 700 s-1, and 4000 s-1). On the basis of these findings, a refined electrostatic model of the transport cycle is proposed.
Wuddel
I
I
Department of Biology, University of Konstanz, Germany.
Apell
H J
HJ
D000818:Animals; D001692:Biological Transport; D004563:Electrochemistry; D004594:Electrophysiology / Q000295:instrumentation / Q000379:methods; D007679:Kidney Medulla / Q000201:enzymology; D007700:Kinetics; D008433:Mathematics; D008564:Membrane Potentials; D008954:Models, Biological; D008962:Models, Theoretical; D011487:Protein Conformation; D011817:Rabbits; D012964:Sodium / Q000737:chemistry / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase