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

Abstract

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.