SummaryThe cellular mechanisms that regulate potassium (K +) channels in guard calls have been the subject of recent research, as K + channel modulation has been suggested to contribute to stomatal movements. Patch clamp studies have been pursued on guard call protoplests of Vicia faba to analyze the effects of physiological cytosolic free Ca 2+ concentrations, Ca 2+ buffers and GTP-binding protein modulators on inward-rectifying K + channels. Ca 2+ inhibition of inward-rectifying K + currents depended strongly on the concentration and effectiveness of the Ca 2+ buffer used, indicating a large Ca 2+ buffering capacity and pH increases in guard calls. When the cytosolic Ca 2+ concentration was buffered to micromolar levels using BAPTA, inward-rectifying K + channels were strongly inhibited. However, when EGTA was used as the Ca 2+ buffer, much less inhibition was observed, even when pipette solutions contained 1 pM free Ca 2+. Under the imposed conditions, GTPTS did not significantly inhibit inward-rectifying K + channel currents when cytosolic Ca z+ was buffered to low levels or when using EGTA a~the Ca 2+ buffer. Furthermore, GDP~S reduced inward K + currents at low cytosolic Ca 2+, indicating a novel mode of inward K + channel regulation by G-protein modulators, which is opposite in effect to that from previous reports. On the other hand, when Ca 2+ was effectively elevated in the cytosol to 1 pM using BAPTA, GTP~S produced an additional inhibition of the inward-rectifying K + channel currents in a population of cells, indicating possible Ca2+-dependent action of GTPbinding protein modulators in K + channel inhibition. Assays of stomatal opening show that 90% inhibition of inward K + currents does not prohibit, but slows, stomatal opening and reduces stomatal apertures by only 34% after 2 h light exposure. These data suggest that limited K + channel down-regulation alone may not be rate-limiting, and it is proposed that the concerted action of protonpump inhibition and additional anion channel activation is likely required for inhibition of stomatal opening.

In the present study abscisic acid-induced stomatal closing, and malate effects on stomatal apertures were analysed in the presence of guard cell ion channel regulators. A recent study has suggested that abscisic acid (ABA) activation of protein kinases and/or inhibition of protein phosphatases may be central to activation of guard cell slow anion channels and mediation of stomatal closing in Vicia faba (Schmidt et al., 1995). These findings were confirmed and extended in the present study showing that both in Vicia faba and in Commelina communis ABA-induced stomatal closings were abolished by kinase inhibitors and enhanced by the protein phosphatase inhibitor okadaic acid. Further detailed studies demonstrate that very high 40 mM extracellular malate concentrations are required to close stomata only partially and that okadaic acid also enhances malate-induced stomatal closing. In addition, when stomata are widely opened, even at 40 mM malate concentrations, no malate effect on stomatal apertures was observed. This finding may be explained by a complete inactivation of guard cell anion channels when stomatal apertures are opened very widely and suggests that extracellular malate cannot function as a primary CO(2) signal in stomatal regulation. The G-protein regulators mastoparan and mas7 as well as neomycin showed no significant effects on light-induced stomatal opening and ABA-induced stomatal closing. Findings reported here correlate closely to recent findings on slow anion channel regulation in guard cells and support the hypothesis that activation of these anion channels by phosphorylation events and complete inactivation by dephosphorylation events is a rate-limiting component in guard cell signal transduction. Furthermore, the presented data support a model in which ABA-activation of protein kinases and/or inhibition of okadaic acidsensitive protein phosphatases is central to ABA regulation of stomatal movements in Vicia faba and Commelina communis.