The influence of cytosolic pH (pH(i)) in controlling K+-channel activity and its interaction with cytosolic-free Ca2+ concentration ([Ca2+](i)) was examined in stomatal guard cells of Vicia faba L. Intact guard cells were impaled with multibarrelled microelectrodes and K+-channel currents were recorded under voltage clamp while pH(i) or [Ca2+](i) was monitored concurrently by fluorescence ratio photometry using the fluorescent dyes 2',7'-bis (2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and Fura-2. In 10 mM external K+ concentration, current through inward-rectifying K+ channels (I-K.in) was evoked on stepping the membrane from a holding potential of -100 mV to voltages from -120 to -250 mV. Challenge with 0.3-30 mM Na+-butyrate and Na+-acetate outside imposed acid loads, lowering pH(i) from a mean resting value of 7.64 +/- 0.03 (n = 25) to values from 7.5 to 6.7. The effect on pH(i) was independent of the weak acid used, and indicated a H+-buffering capacity which rose from 90 mM H+/pH unit near 7.5 to 160 mM H+/pH unit near pH(i) 7.0. With acid-going pH(i), I-K.in was promoted in scalar fashion, the current increasing in magnitude with the acid load, but without significant effect on the current relaxation kinetics at voltages negative of -150 mV or the voltage-dependence for channel gating, Washout of the weak acid was followed by transient rise in pH(i) lasting 3-5 min and was accompanied by a reduction in I-K.in before recovery of the initial resting pH(i) and current amplitude, The pH(i)-sensitivity of the current was consistent with a single titratable site for H+ binding with a pK(a) near 6.3. Acid pH(i) loads also affected current through the outward-rectifying K+ channels (I-K.out) in a manner antiparallel to I-K,I-in. The effect on I-K,I-out was also scalar, but showed an apparent pK(a) of 7.4 and was best accommodated by a cooperative binding of two H+. Parallel measurements showed that Na-+-butyrate loads were generally without significant effect on [Ca2+](i), except when pH(i) was reduced to 7.0 and below. Extreme acid loads evoked reversible increases in [Ca2+](i) in roughly half the cells measured, although the effect was generally delayed with respect to the time course of pH(i) changes and K+-channel responses, The action on [Ca2+](i) coincided with a greater variability in I-K,I-in stimulation evident at pH(i) values around 7.0 and below, and with negative displacements in the voltage-dependence of I-K,I-in gating. These results distinguish the actions of pH(i) and [Ca2+](i) in modulating I-K,I-in; they delimit the effect of pH(i) to changes in current amplitude without influence on the voltage-dependence of channel Eating: and they support a role for pH(i) as a second messenger capable of acting in parallel with, but independent of [Ca2+](i) in controlling the K+ channels.
