No abstract

The vacuole occupies 25‐95% of the plant cell volume and plays an essential role in maintaining cytoplasmic homeostasis of nutrients and ions. Recent patch‐clamp studies identified ion channels and electrogenic pumps as pathways for the movement of ions and metabolites across the vacuolar membrane (tonoplast). At high cytoplasmic Ca2+ (>10‐6 M) and negative potentials (inside the vacuole) non‐selective channels of the ‘slow‐vacuolar (SV)‐type’ were activated resulting in anion release or cation influx. In the present study these vacuolar channels were characterized pharmacologically by ion channel inhibitors. The cation‐transport inhibitors Ba2+, TEA+ and amiloride caused only partial and reversible block of the ‘SV‐type’channels, whereas anion‐transport inhibitors strongly affected the vacuolar channels. Pyridoxalphosphate and the dimethylaminecarboxylate derivates anthracene‐9‐carboxylic acid and C 144 reversibly blocked the channels up to 70% and Zncl2 up to 95%. DIDS and SITS inhibited this channel irreversibly up to 95%. The block developed under a variety of experimental conditions using solutions containing combinations of permanent cations and anions. The DIDS binding site is located on the cytoplasmic surface of the tonoplast, as intravacuolar DIDS did not block the channels. DIDS concentrations in the micromolar range, efficient in blocking 70—80% of the ‘SV‐type’ channels did not significantly affect ATP‐induced or pyrophosphate‐induced proton‐pumps. Stilbene derivatives may therefore be useful tools for studies on the substrate binding site on this vacuolar channel and for channel isolation.

No abstract

The electrical properties of the vacuolar-lysosomal H+ pumps were studied by direct measurement of the pump currents using the whole-cell configuration of the patchclamp technique. Both pumps, the proton-translocating ATPase and pyrophosphatase, when activated by MgATP or inorganic Mg pyrophosphate (MgPP;), transport protons into the vacuole and polarize the membrane potential (positive inside the vacuole). Accumulation of protons in the lumen of vacuole vesicles was monitored by absorbance changes of the pH probe, acridine orange. The electrochemical gradient provided by both the ATPase and pyrophosphatase stimulates effectively the uptake of various metabolites such as malate, citrate and sucrose. The maximal current density produced by the ATPase was -2.5 ,tA/cm2 and -0.5 /tA/cm2 for the pyrophosphatase. KmATP was 0.6 mM; Kmp'P was 15-20 ,OM with progressive inhibition above 150 ItM. At a cytoplasmic pH of 7.5 both enzymes were capable of pumping protons against a 10 000-fold concentration gradient (pH 3.5 inside the vacuole). Proton current produced by the ATPase was blocked reversibly by extravacuolar NO3 only.