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

Abstract

Summary 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.

Nucleotides Provide a Voltage-sensitive Gate for the Rapid Anion Channel of Arabidopsis Hypocotyl Cells

“…Taken together, these two initial results provide evidence that nucleotides play a key role in the gating mechanism of the channel. This nucleotide regulation is clearly distinct from the voltage-independent regulation reported for the guard cell anion channel GCAC1 (18).…”

“…The gating of the fast anion channel (GCAC1) of Vicia faba guard cells is affected by both extracellular factors such as chloride, malate (15,16), and auxins (17) and intracellular factors such as nucleotides, which have been proposed to be allosteric regulators of the channel protein (18). A different mode of regulation by ATP through cytosolic protein phosphorylation has been described for the voltage-dependent anion channel of tobacco cell suspension (19).…”

A transient outward-rectifying K + channel current down-regulated by cytosolic Ca 2+ in Arabidopsis thaliana guard cells

“…Rapid depolarization has been proposed to be initiated by anion channel activation, mediating anion efflux, in particular rapid anion channel activation (55). Interestingly, both rapid anion channel currents (56) and I AP (Fig. 6) show enhancement at more acidic pH i .…”

Studying guard cells in the intact plant: modulation of stomatal movement by apoplastic factors

“…These pH values are considerably lower than those used in most experiments with epidermal strips or guard cell protoplasts. The apoplastic pH has a direct effect on both inward K + channels (Blatt, 1992; and fast anion channels in the guard cell plasma membrane (Schulz-Lessdorf et al, 1996). Upon apoplastic acidification, the gating of fast anion channels is slowed down and the activation of the inward rectifying K + channels is shifted to less negative values.…”

“…Redrawn after Hedrich & Marten (1993) and Hedrich et al (1994) The pH of the cell wall also affects guard cells via more indirect pathways. Lowering the external pH may cause a decrease of the intracellular pH, which inhibits outward K + channels (Blatt & Armstrong, 1993;, activates inward K + channels (Grabov & Blatt, 1997) and enhances the activity of fast anion channels (Schulz-Lessdorf et al, 1996). Furthermore, cytoplasmic acidification stimulates the activity of H + ATPases in the plasma membrane of guard cells (Becker et al, 1993).…”