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.
Air pollutants have been identified in terms of gases and particulate matter, which consists of small solid particles and liquid droplets. The diameter of particulate matter varies from several μm to more than 10 μm. Fine particles or PM 2.5 are particulate matters having a diameter less than 2.5 µm, while ultrafine particles or PM 0.1 have a diameter less than 0.1 µm. The constituent, concentration and size distribution depends on the source. Generally, particulate matter is derived from anthropogenic and natural sources. As an anthropogenic source, smoking activity generates particulate matter having a diameter between 0.02 μm to 1 μm. The measured concentrations are varied from 41,800 to 115,000 particles/cm 3 [1]. Numerous recent studies have investigated the other sources of PM related to size distribution and the constituent. As a key point, the primary PM source is human activity, including biomass burning [2], road traffic sector [3], daily
The ability to measure directly individual protoplast ion¯uxes is a valuable addition to patch clamp and other techniques when using protoplasts to study membrane transporters. Before interpreting observations on protoplasts in terms of behaviour of intact cells and tissues, some methodological questions should be addressed. These include eects of space and time variations of transporter activities over the membrane, the osmotic dependence of speci®c ion transporters and the eect of the regenerating cell wall. In this study net H + and Ca 2+¯u xes were measured from individual corn (Zea mays L.) coleoptile protoplasts using a noninvasive microelectrode technique for ion¯ux measurements. For Ca 2+ , the¯ux distribution was almost symmetrical, ranging 30 nmol á m A2 á s A1 around zero. For H + it was skewed towards eux ranging from A100 to +10 nmol á m A2 á s A1 . The distribution of H +¯u xes through the protoplast surface was a complex mosaic which changed with time, sometimes showing oscillations. These¯ux variations with time and position around the surface, apparently driven by endogenous mechanisms, may be relevant to protoplast pH homeostasis. When the new cell wall was partially regenerated on the next day, the correlation between H + and Ca 2+ uxes increased, which is consistent with the weak-acid Donnan-Manning model of cell wall ion exchange.Abbreviations: BSMM = basal salt medium containing mannitol; WADM = weak-acid Donnan-Manning
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