Abstract
The influence of cytosolic pH (pHi) in controlling K+-channel activity and its interaction with cytosolic-free Ca2+ concentration ([Ca2+]i) was examined in stomatal guard cells ofVicia faba L. Intact guard cells were impaled with multibarrelled microelectrodes and K+-channel currents were recorded under voltage clamp while pHi 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 (IK,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 pHi from a mean resting value of 7.64 ± 0.03 (n = 25) to values from 7.5 to 6.7. The effect on pHi 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 pHi 7.0. With acid-going pHi, (IK,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 pHi lasting 3–5 min and was accompanied by a reduction in (IK,in) before recovery of the initial resting pHi and current amplitude. The pHi-sensitivity of the current was consistent with a single, titratable site for H+ binding with a pKa near 6.3. Acid pHi loads also affected current through the outward-rectifying K+ channels (IK,out) in a manner antiparallel to (IK,in) The effect on IK, out was also scalar, but showed an apparent pKa 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 pHi 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 pHi changes and K+-channel responses. The action on [Ca2+]i coincided with a greater variability in (IK,in) stimulation evident at pHi values around 7.0 and below, and with negative displacements in the voltage-dependence of (IK,in) gating. These results distinguish the actions of pHi and [Ca2+]i in modulating (IK,in) they delimit the effect of pHi to changes in current amplitude without influence on the voltage-dependence of channel gating; and they support a role for pHi as a second messenger capable of acting in parallel with, but independent of [Ca2+]i in controlling the K+ channels.
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Abbreviations
- BCECF:
-
2′,7′-bis (2-carboxyethyl)-5(6)-carboxy fluorescein
- [Ca2+]i :
-
cytosolic free Ca2+ concentration
- gK :
-
ensemble (steady-state) K+-channel conductance
- IK,out, IK,in :
-
outward-, inward-rectifying K+ channel (current)
- IN:
-
current-voltage (relation)
- Mes:
-
2-(N-morpholinolethanesulfonic acid
- pHi :
-
cytosolic pH
- V:
-
membrane potential
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Grabov, A., Blatt, M.R. Parallel control of the inward-rectifier K+ channel by cytosolic free Ca2+ and pH inVicia guard cells. Planta 201, 84–95 (1997). https://doi.org/10.1007/BF01258684
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DOI: https://doi.org/10.1007/BF01258684