Ion channels in the plasma membrane of guard cells provide key mechanisms in signal perception and volume regulation during stomatal movement. Recent studies have suggested that the strongly voltage-dependent, inactivating guard cell anion channel (GCAC1) acts as a sensor of the ambient extracellular CO2 concentration and as a target of modulation by nucleotides and Ca2+ ions. Applying the patch-clamp technique it is demonstrated here that GCAC1 is activated by cytoplasmic ATP in a pH-dependent manner.
When the apoplastic pH was buffered to 5.6 and the cytosolic pH dropped step-wise from 7.8 to 5.6, the single-channel activity increased as a function of proton concentration. This pH-sensitivity is characterized by a titratable site with an apparent pK value around 6.9. While the steepness and direction of the transmembrane pH gradient did not affect the kinetics of activation, deactivation and fast inactivation of the whole-cell anion current, the kinetics of slow inactivation and reactivation were strongly influenced. When at a constant intracellular proton concentration of pH 7.2 the external pH decreased from 7.2 to 5.6 the time constants of slow inactivation and the half-times of reactivation increased two- and sevenfold, respectively.
The mechanism of nucleotide activation of GCAC1 was quantitatively analysed on the level of single-channel events. Using inside-out, cell-free membrane patches, GCAC1 half-activated around 0.4 mM ATP. The sigmoidal dose-dependence of anion channel activation could be well fitted with an apparent Hill coefficient of 3.6. This behaviour might indicate that the activation process involves a strongly cooperative interaction of four ATP-binding sites. Neither ATP nor its non-hydrolysable analogue AMP-PMP, which also activated GCAC1, altered the voltage-dependent gating. AMP-PMP stimulation and the insensitivity of GCAC1 towards the phosphatase inhibitor, okadaic acid, and the kinase inhibitors, staurosporine and H-7, provided evidence that nucleotide binding rather than phosphorylation caused channel activation.
Since the time- and voltage-dependent activity of GCAC1 is strongly modulated by ATP and protons, this channel is capable of sensing changes in the energy status, acid metabolism and the H+ ATPase activity of guard cells.