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.

Rhythms in Plants

“…3.2.3). Similar effect could be observed in wheat root protoplasts, where the H + influx was measured by the MIFE system (Tyerman et al 2001). In Fig.…”

An electrogenic pump in the xylem parenchyma of barley roots

“…For the identification of the electrogenic pump the H + ‐pump stimulator FC and the inhibitor DCCD were applied. FC and DCCD are commonly used to assign measured currents to H + ‐pump activity (Findlay et al 1994, Tyerman et al 2001). After an external application of 1 μ M FC, both the current at 0 mV increased and the membrane potential hyperpolarised indicating an increase in pump activity (Fig.…”

“…Here we report results of this work and address the function of the electrogenic pump in combination with anion conductances. So far, mostly the concerted action of the H + ‐pump and cation conductances has been considered (De Boer and Volkov 2003, Tyerman et al 2001) but not its importance for anion and acid release.…”

Mechanisms of Cl‐ transport contributing to salt tolerance

“…Recently, Beilby & Al Khazaaly (2009) have identified OH ‐ permeable channels as the most likely component of the ‘leak’ conductance activated by salinity, a surprising observation given that previously these channels were only observed at high pH (Beilby & Bisson 1992). This is highly significant for higher plant roots because there are similarities with Charophytes in H + /OH ‐ circulation at the root tip (Raven 1991), and there is evidence for their existence from combined patch clamp and microelectrode ion flux estimation (Tyerman et al. 2001).…”