Summary

Proton fluxes 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 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 flux of H⁺ occured 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 stimulatoey 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.