Calcium Fluxes across the Plasma Membrane of Commelina communis L. Assayed in a Cell-Free System

The inside-out fraction of plasma membrane-rich vesicles prepared from leaves of Commelina communis L. by aqueous twophase partitioning was loaded with (45)Ca(2+) through the action of the plasma membrane Ca(2+)-ATPase. While the Ca(2+)-loaded vesicles were tightly sealed, trifluoperazine (TFP) (effective concentration giving 50% of maximum effect [EC(50)] = 70 micromolar) and W-7 (EC(50) = 100 micromolar), but to a much lesser extent, W-5 (EC(50) = 500 micromolar) led to a rapid efflux of (45)Ca(2+) from the vesicles. This efflux could be blocked efficiently with low (<1 millimolar) concentrations of La(3+), but it remained unaffected by the addition of calmodulin (CM). Further experiments with vesicles incubated in (45)Ca(2+) in the absence of ATP, as well as experiments performed with control liposomes and nonloaded as well as Ca(2+)-loaded plasma membrane vesicles using the indicator dye arsenazo III showed, that TFP and W-7 and, again to a lesser extent, W-5 mobilized a pool of membrane-bound Ca(2+) from the vesicles. No indications for a detergent effect of TFP and W-7 were obtained. The EC(50)-values of these compounds for mobilizing membrane-associated Ca(2+) (TFP = 100 micromolar, W-7 = 100 micromolar, W-5 = 500 micromolar) or for the triggering of Ca(2+) release from Ca(2+)-loaded vesicles (see above) were very similar, suggesting a common basis of antagonist action on both processes. Our results suggest the presence of a Ca(2+) channel in the plasma membrane of C. communis. The channel is obtained in a Ca(2+)-inactivated state after preparation and Ca(2+)-loading of the vesicles. The inactivation is removed by TFP or W-7, presumably due to the Ca(2+)-mobilizing effect of these compounds. The activated Ca(2+) channel is La(3+) sensitive and, in the cell, would allow for passage of Ca(2+) into the cell. The possibility that TFP or W-7 act independent of CM, or through CM tightly associated with the plasma membrane, is discussed. The system described allows a cell free analysis of Ca(2+) influx, displaying channel properties, in a higher plant.