Rate of Na+/Ca2+ exchange across the plasma membrane of synaptosomes measured using the fluorescence of chlorotetracycline. Implications to calcium homeostasis in synaptic terminals

摘要

It is shown that the fluorescence of chlorotetracycline (CTC) can be used to be used to continuously monitor Ca2+ fluxes mediated by the Na+/Ca2+-exchanger of the plasma membrane of synaptosomes. The kinetics of Ca2+ uptake can be followed from the kinetics of the increase of CTC fluorescence with external Ca2+ concentrations in the micromolar range. Since the fluorescence of CTC is not sensitive to Ca2+ concentration below 20 μM this avoids any significant contribution of Ca2+ flux through Ca2+ channels to CTC fluorescence. By replacing KC1 by choline chloride in the buffer to avoid plasma membrane depolarization it is shown that the amplitude of the CTC fluorescence change is dependent upon the Na+-gradient preimposed across the plasma membrane, and the rate constant of the kinetic process is dependent upon the Ca2+ concentration. The rate constant of the Ca2+ influx measured with depolarized and non-depolarized synaptic plasma membrane vesicles at 37°C and pH 7.4 were 0.55 ± 0.10 and 0.25 ± 0.02 min?1, respectively. The overall rate of Na+/Ca2+ exchange calculated under conditions close to physiological Na+ and Ca2+ gradients and membrane resting potential ranged from 15 to 25% of the activity of the plasma membrane Ca2+ pump under these experimental conditions. The results also point out that membrane depolarization increases approx. 2-fold the rate of Na+/Ca2+ exchange in synaptic plasma membrane vesicles.