Calcium-activated K⁺ channels and calcium-induced calcium release by slow vacuolar ion channels in guard cell vacuoles implicated in the control of stomatal closure

Stomatal closing requires the efflux of K⁺ from the large vacuolar organelle into the cytosol and across the plasma membrane of guard cells. More than 90% of the K⁺ released from guard cells during stomatal closure originates from the guard cell vacuole. However, the corresponding molecular mechanisms for the release of K⁺ from guard cell vacuoles have remained unknown. Rises in the cytoplasmic Ca²⁺ concentration have been shown to trigger ion efflux from guard cells, resulting in stomatal closure. Here, we report a novel type of largely voltage-independent K⁺-selective ion channel in the vacuolar membrane of guard cells that is activated by physiological increases in the cytoplasmic Ca²⁺ concentration. These vacuolar K⁺ (VK) channels had a single channel conductance of 70 pS with 100 mM KCl on both sides of the membrane and were highly selective for K⁺ over NH₄⁺ and Rb⁺. Na⁺, Li⁺, and Cs⁺ were not measurably permeant. The Ca²⁺, voltage, and pH dependences, high selectivity for K⁺, and high density of VK channels in the vacuolar membrane of guard cells suggest a central role for these K⁺ channels in the initiation and control of K⁺ release from the vacuole to the cytoplasm required for stomatal closure. The activation of K⁺-selective VK channels can shift the vacuolar membrane to more positive potentials on the cytoplasmic side, sufficient to activate previously described slow vacuolar cation channels (SV-type). Analysis of the ionic selectivity of SV channels demonstrated a Ca²⁺ over K⁺ selectivity (permeability ratio for Ca²⁺ to K⁺ of ∼3:1) of these channels in broad bean guard cells and red beet vacuoles, suggesting that SV channels play an important role in Ca²⁺-induced Ca²⁺ release from the vacuole during stomatal closure. A model is presented suggesting that the interaction of VK and SV channel activities is crucial in regulating vacuolar K⁺ and Ca²⁺ release during stomatal closure. Furthermore, the possibility that the ubiquitous SV channels may represent a general mechanism for Ca²⁺-induced Ca²⁺ release from higher plant vacuoles is discussed.