GCAC1 RECOGNIZES THE PH GRADIENT ACROSS THE PLASMA-MEMBRANE - A PH-SENSITIVE AND ATP-DEPENDENT ANION CHANNEL LINKS GUARD-CELL MEMBRANE-POTENTIAL TO ACID AND ENERGY-METABOLISM

Ion channels in the plasma membrane of guard cells provide key mechani sms in signal perception and volume regulation during stomatal movemen t. Recent studies have suggested that the strongly voltage-dependent, inactivating guard cell onion 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 c ytosolic pH dropped step-wise from 7.8 to 5.6, the single-channel acti vity increased as a function of proton concentration. This pH-sensitiv ity is characterized by a titratable site with an apparent pK value ar ound 6.9. While the steepness and direction of the transmembrane pH gr adient did not affect the kinetics of activation, deactivation and fas t inactivation of the whole cell anion current, the kinetics of slow i nactivation and reactivation were strongly influenced. When at a const ant intracellular proton concentration of pH 7.2 the external pH decre ased from 7.2 to 5.6 the time constants of slow inactivation and the h alf-times of reactivation increased two- and sevenfold, respectively. The mechanism of nucleotide activation of GCAC1 was quantitatively ana lysed on the level of single-channel events. Using inside-out, cell-fr ee membrane patches, GCAC1 half-activated around 0.4 mM ATP. The sigmo idal dose dependence of anion channel activation could be well fitted with an apparent Hill coefficient of 3.6. This behaviour might indicat e that the activation process involves a strongly cooperative interact ion of four ATP binding sites. Neither ATP nor its non-hydrolysable an alogue AMP-PMP, which also activated GCAC1, altered the voltage-depend ent gating. AMP-PMP stimulation and the insensitivity of GCAC1 towards the phosphatase inhibitor, okadaic acid, and the kinase inhibitors, s taurosporine and H-7, provided evidence that nucleotide binding rather than phosphorylation caused channel activation. Since the time- and v oltage-dependent activity of GCAC1 is strongly modulated by ATP and pr otons, this channel is capable of sensing changes in the energy status , acid metabolism and the Hf ATPase activity of guard cells.