
%0 Journal Article
%A Tyerman, Stephen D. 
%A Beilby, Mary 
%A Whittington, John 
%A Juswono, Unggul 
%A Newman, Ian 
%A Shabala, Sergey 
%T Oscillations in proton transport revealed from simultaneous measurements of net current and net proton fluxes from isolated root protoplasts: MIFE meets patch-clamp
%J Functional Plant Biology
%D 2001/07/27
%V 28
%N 7
%P 591-606
%K H + channels, MIFE, oscillations, patch-clamp, proton channels, proton fluxes, proton pump.
%R https://doi.org/10.1071/PP01030
%U https://www.publish.csiro.au/paper/PP01030 
%X 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&#8211;voltage (I&#8211;V) curves and the flux&#8211;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&#8211;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&#8211;V curves and I&#8211;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. 
