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

Abstract

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 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 (E _K ), 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 E _K , similar flux–V curves were observed. In the depolarised state where Vm was greater than E _K 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.