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

P>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 Ca2+ concentration ([Ca2+](i)). However, the dynamics of the action of [Ca2+](i) on I-anion has never been established, despite its importance for understanding the mechanics of stomatal adaptation to stress. We have quantified the [Ca2+](i) dynamics of I-anion in Vicia faba guard cells, measuring channel current under a voltage clamp while manipulating and recording [Ca2+](i) using Fura-2 fluorescence imaging. We found that I-anion rises with [Ca2+](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 Ca2+ 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 [Ca2+](i). The protein phosphatase antagonist okadaic acid increased this current baseline over twofold. Additionally, okadaic acid altered the [Ca2+](i) sensitivity of I-anion, displacing the apparent K-d for [Ca2+](i) to 573 +/- 38 nm. These findings support previous evidence for different modes of regulation for I-anion, only one of which depends on [Ca2+](i), and they underscore an independence of [Ca2+](i) from protein (de-)phosphorylation in controlling I-anion. Most importantly, our results demonstrate a significant displacement of I-anion sensitivity to higher [Ca2+](i) compared with that of the guard cell K+ channels, implying a capacity for variable dynamics between net osmotic solute uptake and loss.