Dynamic regulation of guard cell anion channels by cytosolic free Ca2+concentration and protein phosphorylation

In guard cells, activation of anion channels (I(anion)) is an early event leading to stomatal closure. Activation of I(anion) has been associated with abscisic acid (ABA) and its elevation of the cytosolic free Ca(2+) concentration (Ca(2+)(i)). However, the dynamics of the action of Ca(2+)(i) on I(anion) has never been established, despite its importance for understanding the mechanics of stomatal adaptation to stress. We have quantified the Ca(2+)(i) dynamics of I(anion) in Vicia faba guard cells, measuring channel current under a voltage clamp while manipulating and recording Ca(2+)(i) using Fura-2 fluorescence imaging. We found that I(anion) rises with Ca(2+)(i) only at concentrations substantially above the mean resting value of 125 +/- 13 nm, yielding an apparent K(d) of 720 +/- 65 nm and a Hill coefficient consistent with the binding of three to four Ca(2+) ions to activate the channels. Approximately 30% of guard cells exhibited a baseline of I(anion) activity, but without a dependence of the current on Ca(2+)(i). The protein phosphatase antagonist okadaic acid increased this current baseline over twofold. Additionally, okadaic acid altered the Ca(2+)(i) sensitivity of I(anion), displacing the apparent K(d) for Ca(2+)(i) to 573 +/- 38 nm. These findings support previous evidence for different modes of regulation for I(anion), only one of which depends on Ca(2+)(i), and they underscore an independence of Ca(2+)(i) from protein (de-)phosphorylation in controlling I(anion). Most importantly, our results demonstrate a significant displacement of I(anion) sensitivity to higher Ca(2+)(i) compared with that of the guard cell K(+) channels, implying a capacity for variable dynamics between net osmotic solute uptake and loss.