DISPLACEMENT OF MEMBRANE CALCIUM BY A LOCAL ANESTHETIC (CHLORPROMAZINE)

1. 1. The effect of chlorpromazine on the membrane concentration of Ca2+ was examined. Erythrocyte ghosts, which were free of hemoglobin and Ca2+, were mixed with 45Ca2+ in the presence of different concentrations of Ca2+ and chlorpromazine. The amount of Ca2+ adsorbed to the membrane was determined from the amount of 45Ca2+ remaining in the supernatant. 2. 2. It was found that [3H]sorbitol rapidly equilibrated over the entire water space of the ghost suspension. [14C]Inulin, however, completely permeated only about 80% of the ghosts. The water space for 45Ca2+ was found to be similar to that for inulin; this was obtained by saturating the membrane sites for Ca2+ (which occurred between 10 and 50 mM Ca2+), measuring the radioactivity in the supernatant, and comparing it with the control value. 3. 3. The membrane concentration of Ca2+ was determined as a function of the extracellular Ca2+ concentration. At free concentrations of Ca2+ below 1 mM the adsorption isotherm indicated one set of binding sites with a saturation capacity of 0.081 mole Ca2+ per 1 of solid (and 30% hydrated) membrane, and with an affinity constant of 1940 l membrane solid per mole Ca2+. 4. 4. The addition of saponin (2 mg/mg dry ghost) greatly increased the inulin space and the adsorption of Ca2+ to the membranes. Rehemolysis of the ghosts also increased the binding of Ca2+ to the mebranes. Saponin and rehemolysis presumably exposed intracellular sites by disrupting the membrane. 5. 5. Chlorpromazine, at free concentrations of 1-10 μM (or total concentrations 0f 0.1-1 mM) competitively displaced the membrane-bound Ca2+. The maximum amount of membrane Ca2+ displaced when the membrane was saturated with chlorpromazine (0.066 mole chlorpromazine per 1 solid membrane) was 0.021 mole Ca2+ per 1 membrane solid. It was concluded that 1 Ca2+ was displaced from the membrane by about 2 chlorpromazine molecules. © 1969.