EFFECTS OF CYTOSOLIC CALCIUM AND LIMITED, POSSIBLE DUAL, EFFECTS OF G-PROTEIN MODULATORS ON GUARD-CELL INWARD POTASSIUM CHANNELS

The cellular mechanisms that regulate potassium (K+) channels in guard cells have been the subject of recent research, as K+ channel modulat ion has been suggested to contribute to stomatal movements. Patch clam p studies have been pursued on guard cell protoplasts of Vicia faba to analyze the effects of physiological cytosolic free Ca2+ concentratio ns, Ca2+ buffers and GTP-binding protein modulators on inward-rectifyi ng K+ channels. Ca2+ inhibition of inward-rectifying K+ currents depen ded strongly on the concentration and effectiveness of the Ca2+ buffer used, indicating a large Ca2+ buffering capacity and pH increases in guard cells. When the cytosolic Ca2+ concentration was buffered to mic romolar levels using BAPTA, inward-rectifying K+ channels were strongl y inhibited. However, when EGTA was used as the Ca2+ buffer, much less inhibition was observed, even when pipette solutions contained 1 mu M free Ca2+. Under the imposed conditions, GTP gamma S did not signific antly inhibit inward-rectifying K+ channel currents when cytosolic Ca2 + was buffered to low levels or when using EGTA as the Ca2+ buffer. Fu rthermore, GDP beta S reduced inward K+ currents at low cytosolic Ca2, 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 Ca2+ was effectively elevated in the cytosol to 1 mu M using BAPTA, GTP gamma S produced an additional inhibition of the inward-rectifying K+ channel currents in a population of cells, indicating possible Ca2+-dependent action of GTP-binding protein modu lators in K+ channel inhibition. Assays of stomatal opening show that 90% inhibition of inward K+ currents does not prohibit, but slows, sto matal opening and reduces stomatal apertures by only 34% after 2 h lig ht exposure. These data suggest that limited K+ channel down-regulatio n alone may not be rate-limiting, and it is proposed that the concerte d action of proton-pump inhibition and additional anion channel activa tion is likely required for inhibition of stomatal opening. Furthermor e, G-protein modulators regulate inward K+ channels in a more complex and limited, possibly Ca2+ dependent, manner than previously proposed.