Oscillations in proton transport revealed from simultaneous measurements of net current and net proton fluxes from isolated root protoplasts: MIFE meets patch-clamp

Proton fuxes were measured non-invasively on patch-clamped protoplasts isolated from wheat roots using an external H⁺ electrode to measure the electrochemical gradient in the external solution. Under voltage clamp in the whole-cell configuration, the H⁺ fluxes across the plasma membrane could be measured as a function of voltage and time and correlated with the simultaneous measurements of membrane current. Protoplasts could exist in three states based on the current-voltage (I-V) curves and the flux-V curves. In the pump-state where the membrane voltage (Vm) was more negative than the electrochemical equilibrium potential for potassium (EK), a net efflux of H⁺ occurred that was voltage-dependent such that the efflux increased as Vm was clamped more positive. In the K-state, where Vm was close to EK, similar flux-V curves were observed. In the depolarised state where Vm was greater than EK the proton flux was characterised by a net influx of H⁺ (H⁺-influx state) that reversed direction at more positive values of Vm. The inhibitory effect of DCCD and stimulatory effect of fusicoccin were used to correlate current and H⁺ flux through the H⁺-ATPase for which there was reasonably good agreement within the limits of the flux measurements. Some protoplasts were kept in the whole-cell configuration for up to 3 h revealing slow sustained oscillations (period about 40 min) in H⁺ flux that were in phase with oscillations in free-running Vm. These oscillations were also observed under voltage clamp, with membrane current in phase with H⁺ flux, but which became damped out after a few cycles. The oscillations encompassed the pump-state, K⁺-state and H⁺-influx-state. The H⁺-flux-V curves and I-V curves were used to model the electrical characteristics of the plasma membrane with H⁺-ATPase, inward and outward K⁺ rectifiers, a linear conductance, and a passive H⁺ influx possibly through gated proton channels.