The influence of the plant water-stress hormone abscisic acid (ABA) on anion channel activity and its interaction with protein kinase and phosphatase antagonists was examined in stomatal guard cells of wild-type Nicotiana benthamiana L. and of transgenic plants expressing the dominant-negative (mutant) Arabidopsis abil-1 protein phosphatase. Intact guard cells were impaled with double-barrelled micro-electrodes and membrane current was recorded under voltage clamp in the presence of 15 mM CsCl and 15 mM tetraethylammonium chloride (TEA-CI) to eliminate Kf channel currents. Under these conditions, the free-running voltage was situated close to 0 mV (+9 +/- 6 mV, n = 18) and the membrane under voltage clamp was dominated by anion channel current (I-Cl) as indicated from tail current reversal near the expected chloride equilibrium potential, current sensitivity to the anion channel blockers g-anthracene carboxylic acid and niflumic acid, and by its voltage-dependent kinetics. Pronounced activation of I-Cl was recorded on stepping from a conditioning voltage of -250 mV to voltages between -30 and +50 mV, and the current deactivated with a voltage-dependent halftime at more negative voltages (tau similar or equal to 0.3 sec at -150 mV). Challenge with 20 mu M ABA increased the steady-state current conductance, g(Cl), near 0 mV by 1.2- to 2.8-fold and at -150 mV by 4.5- to sixfold with a time constant of 40 +/- 4 sec, and it slowed I-Cl deactivation as much as fourfold at voltages near -50 mV, introducing two additional voltage-sensitive kinetic components to these current relaxations. Neither the steady-state and kinetic characteristics of I-Cl, nor its sensitivity to ABA were influenced by H7 or staurosporine, both broad-range protein kinase antagonists. However, the protein phosphatase 1/2A antagonist calyculin A mimicked the effects of ABA on gel and current relaxations on its own and exhibited a synergistic interaction with ABA, enhancing Icl sensitivity to ABA three- to four-fold. Quantitatively similar current characteristics were recorded from guard cells of abi1-1 transgenic N. benthamiana, indicating that the abi1-1 protein phosphatase does not influence the anion current or its response to ABA directly. These results demonstrate that ABA stimulates I-Cl and modulates its voltage sensitivity. Furthermore, they show that ABA promotes I-Cl, either by introducing additional long-lived states of the channel or by activating a second anion channel with similar permeation characteristics but with a very long dwell time in the open state. Overall, the data are broadly consistent with the view that ABA action engenders coordinate control of I-Cl together with guard cell K+ channels to effect solute loss and stomatal closure.
