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

Abstract

To investigate effects of pH on the Na+,K+-ATPase, we used the Xenopus oocytes to measure transient charge movements in the absence of extracellular K+, and steady-state currents mediated by the pump as well as ATPase activity. The activity of purified Na+,K+-ATPase strongly depends on pH, which has been attributed to protonation of intracellular sites. The steady-state current reflects pump activity, the transient charge movement voltage-dependent interaction of external Na+ ions with the pump molecule and/or conformational changes during Na+/Na+ exchange. The steady-state current exhibits a characteristic voltage dependence with maximum at about 0 mV at low external K+ (≤2 mM) and with 50 Na+. This dependency is not significantly affected by changes in external pH in the range from pH 9 to pH 6. Only below pH 6, the voltage dependence of pump current becomes less steep, and may be attributed to a pH-dependent inhibition of the forward pump cycle by external Na+. External stimulation of the pump by K+ in the absence of Na+ can be described by a voltage-dependent Km value with an apparent valency zK. At higher external pH the zK value is reduced. The transient current signal in the absence of external K+ can be described by the sum of three exponentials with voltage-dependent time constants of about 50 ms, 700 μs and less than 100 μs during pulses to 0 mV. The charge distribution was calculated by integration of the transient current signals. The slowest component and the associated charge distributions do not significantly depend on external pH changes. The intermediate component of the transients is represented by a voltage-dependent rate constant which shows a minimum at about −120 mV and increases with decreasing pH. Nevertheless, the contribution to the charge movement is not altered by pH changes due to a simultaneous increase of the amplitude of this component. We conclude that reduction of external pH counteracts external K+ and Na+ binding.