Extracellular pH modulates kinetics of the Na+,K+-ATPase

To investigate effects of pH on the Na+,K+-ATPase, we used the Xenopus oocy tes to measure transient charge movements in the absence of extracellular K +, and steady-state currents mediated by the pump as well as ATPase activit y. The activity of purified Na+,K+-ATPase strongly depends on pH, which has been attributed to protonation of intracellular sites. The steady-state cu rrent reflects pump activity, the transient charge movement voltage-depende nt interaction of external Na+ ions with the pump molecule and/or conformat ional changes during Na+/Na+ exchange. The steady-state current exhibits a characteristic voltage dependence with maximum at about 0 mV at low externa l K+ (less than or equal to 2 mM) and with 50 Na+. This dependency is not s ignificantly affected by changes in external pH in the range from pH 9 to p H 6. Only below pH 6, the voltage dependence of pump current becomes less s teep, and may be attributed to a pH-dependent inhibition of the forward pum p cycle by external Na+. External stimulation of the pump by K+ in the abse nce of Naf can be described by a voltage-dependent K-m value with an appare nt valency z(K). At higher external pH the z(K) value is reduced. The trans ient current signal in the absence of external K+ can be described by the s um of three exponentials with voltage-dependent time constants of about 50 ms, 700 mus and less than 100 mus during pulses to 0 mV. The charge distrib ution was calculated by integration of the transient current signals. The s lowest component and the associated charge distributions do not significant ly depend on external pH changes. The intermediate component of the transie nts is represented by a voltage-dependent rate constant which shows a minim um at about -120 mV and increases with decreasing pH. Nevertheless, the con tribution to the charge movement is not altered by pH changes due to a simu ltaneous increase of the amplitude of this component. We conclude that redu ction of external pH counteracts external K+ and Na+ binding. (C) 2000 Else vier Science B.V. All rights reserved.