Sequential potassium binding at the extracellular side of the Na,K-pump.

Ion binding at the extracellular face of the Na,K-ATPase is electrogenic and can be monitored by the styryl dye RH 421 in membrane fragments containing a high density of the Na,K-pumps. The fluorescent probe is noncovalently bound to the membrane and responds to changes of the local electric field generated by binding or release of cations inside the protein. Due to the fact that K+ binding from the extracellular side is an electrogenic reaction, it is possible to detect the amount of ions bound to the pump as function of the aqueous concentration. The results are in contradiction to a second order reaction, i.e., a simultaneous binding of two K+ ions. A mathematical model is presented to discuss the nature of the two step binding process. On the basis of this model the data allow a quantitative distinction between binding of the first and the second K+ ion. The temperature dependence of ion binding has been investigated. At low temperatures the apparent dissociation constants differ significantly. In the temperature range above 20 degrees C the resulting apparent dissociation constants for both K+ ions merge and have values between 0.2 and 0.3 mM, which is consistent with previous experiments. The activation energy for the half saturating concentration of K+ is 22 kJ/mol. Additional analysis of the titration curve of K+ binding to the state P-E2 by the Hill equation yields a Hill coefficient, nHill, of 1.33, which is in agreement with previously published data.