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 cytosolic free Ca<sup>2+</sup> concentration ([Ca<sup>2+</sup>](i)). However, the dynamics of [Ca<sup>2+</sup>](i) action on I(anion) have never established, despite their importance for understanding the mechanics of stomatal adaptation to stress. We have quantified the [Ca<sup>2+</sup>](i) dynamics of I(anion) in Vicia faba guard cells, measuring channel current under voltage clamp while manipulating and recording [Ca<sup>2+</sup>](i) using Fura-2 fluorescence imaging. We found I(anion) to rise with [Ca<sup>2+</sup>](i) only at concentrations substantially above the mean resting value of 125+/-13 nM, yielding an apparent K(d) of 720+/-65 nM and Hill coefficient consistent with the binding of 3-4 Ca<sup>2+</sup> ions to activate the channels. Approximately 30% of guard cells exhibited a baseline of I(anion) activity, but without a [Ca<sup>2+</sup>](i) dependence to the current. The protein phosphatase antagonist okadaic acid increased this current baseline over 2-fold. Additionally, okadaic acid altered the [Ca<sup>2+</sup>](i)-sensitivity of I(anion), displacing the apparent K(d) for [Ca<sup>2+</sup>](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<sup>2+</sup>](i), and they underscore an independence of [Ca<sup>2+</sup>](i) from protein (de-)phosphorylation in controlling I(anion). Most important, our results demonstrate a significant displacement of I(anion) sensitivity to higher [Ca<sup>2+</sup>](i) compared with that of the guard cell K<sup>+</sup> channels, implicating a capacity for variable dynamics between net osmotic solute uptake and loss