Magnesium sensitizes slow vacuolar channels to physiological cytosolic calcium and inhibits fast vacuolar channels in fava bean guard cell vacuoles

Vacuolar ion channels in guard cells play important roles during stomatal movement and are regulated by many factors including Ca²⁺, calmodulin, protein kinases, and phosphatases. We report that physiological cytosolic and luminal Mg²⁺ levels strongly regulate vacuolar ion channels in fava bean (Vicia faba) guard cells. Luminal Mg²⁺ inhibited fast vacuolar (FV) currents with a K₁ of approximately 0.23 mM in a voltage-dependent manner at positive potentials on the cytoplasmic side. Cytosolic Mg²⁺ at 1 mM also inhibited FV currents. Furthermore, in the absence of cytosolic Mg²⁺, cytosolic Ca²⁺ at less than 10 μM did not activate slow vacuolar (SV) currents. However, when cytosolic Mg²⁺ was present, submicromolar concentrations of cytosolic Ca²⁺ activated SV currents with a K(d) of approximately 227 nM, suggesting a synergistic Mg²⁺-Ca²⁺ effect. The activation potential of SV currents was shifted toward physiological potentials in the presence of cytosolic Mg²⁺ concentrations. The direction of SV currents could also be changed from outward to both outward and inward currents. Our data predict a model for SV channel regulation, including a cytosolic binding site for Ca²⁺ with an affinity in the submicromolar range and a cytosolic low-affinity Mg²⁺-Ca²⁺ binding site. SV channels are predicted to contain a third binding site on the vacuolar luminal side, which binds Ca²⁺ and is inhibitory. In conclusion, cytosolic Mg²⁺ sensitizes SV channels to physiological cytosolic Ca²⁺ elevations. Furthermore, we propose that cytosolic and vacuolar Mg²⁺ concentrations ensure that FV channels do not function as a continuous vacuolar K⁺ leak, which would prohibit stomatal opening.