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

Abstract

The inside-out fraction of plasma membrane-rich vesicles prepared from leaves of Commelina communis L. by aqueous two-phase partitioning was loaded with 45Ca2+ through the action of the plasma membrane Ca2+-ATPase. While the Ca2+-loaded vesicles were tightly sealed, trifluoperazine (TFP) (effective concentration giving 50% of maximum effect [EC50] = 70 micromolar) and W-7 (EC50 = 100 micromolar), but to a much lesser extent, W-5 (EC50 = 500 micromolar) led to a rapid efflux of 45Ca2+ from the vesicles. This efflux could be blocked efficiently with low (< 1 millimolar) concentrations of La3+, but it remained unaffected by the addition of calmodulin (CM). Further experiments with vesicles incubated in 45Ca2+ in the absence of ATP, as well as experiments performed with control liposomes and nonloaded as well as Ca2+-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 Ca2+ from the vesicles. No indications for a detergent effect of TFP and W-7 were obtained. The EC50-values of these compounds for mobilizing membrane-associated Ca2+ (TFP = 100 micromolar, W-7 = 100 micromolar, W-5 = 500 micromolar) or for the triggering of Ca2+ release from Ca2+ loaded vesicles (see above) were very similar, suggesting a common basis of antagonist action on both processes. Our results suggest the presence of a Ca2+ channel in the plasma membrane of C. communis. The channel is obtained in a Ca2+-inactivated state after preparation and Ca2+-loading of the vesicles. The inactivation is removed by TFP or W-7, presumably due to the Ca2+-mobilizing effect of these compounds. The activated Ca2+ channel is La3+ sensitive and, in the cell, would allow for passage of Ca2+ 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 Ca2+ influx, displaying channel properties, in a higher plant.