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 m ovement and are regulated by many factors including Ca2+, calmodulin, prote in kinases, and phosphatases. We report that physiological cytosolic and lu minal Mg2+ levels strongly regulate vacuolar ion channels in fava bean (Vic ia faba) guard cells. Luminal Mg2+ inhibited fast vacuolar (FV) currents wi th a K-i 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 Ca2at less than 10 mu M 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 Mg2concentrations. The direction of SV currents could also be changed from out ward to both outward and inward currents. Our data predict a model for SV c hannel regulation, including a cytosolic binding site for Ca2+ with an affi nity in the submicromolar range and a cytosolic low-affinity Mg2+-Ca2+ bind ing site. SV channels are predicted to contain a third binding site on the vacuolar luminal side, which binds Ca2+ and is inhibitory. In conclusion, c ytosolic Mg2+ sensitizes SV channels to physiological cytosolic Ca2+ elevat ions. Furthermore, we propose that cytosolic and vacuolar Mg2+ concentratio ns ensure that FV channels do not function as a continuous vacuolar K+ leak , which would prohibit stomatal opening.