Ca2+-dependent Ca2+ clearance via mitochondrial uptake and plasmalemmal extrusion in frog motor nerve terminals

Ca2+ clearance in frog motor nerve terminals was studied by fluorometry of Ca2+ indicators. Rises in intracellular Ca2+ ([Ca2+](i)) in nerve terminals induced by tetanic nerve stimulation (100 Hz, 100 or 200 stimuli: Ca2+ transient) reached a peak or plateau within 6-20 stimuli and decayed at least in three phases with the time constants of 82-87 ms (81-85%), a few seconds (11-12%), and several tens of seconds (less than a few percentage). Blocking both Na/Ca exchangers and Ca2+ pumps at the cell membrane by external Li+ and high external pH (9.0), respectively, increased the time constants of the initial and second decay components with no change in their magnitudes. By contrast, similar effects by Li+ alone, but not by high alkaline alone, were seen only on 200 stimuli-induced Ca2+ transients. Blocking Ca2+ pumps at Ca2+ stores by thapsigargin did not affect 100 stimuli-induced Ca2+ transients but increased the initial decay time constant of 200 stimuli-induced Ca2+ transients with no change in other parameters. Inhibiting mitochondrial Ca2+ uptake by carbonyl cyanide m-chlorophenylhydrazone markedly increased the initial and second decay time constants of 100 stimuli-induced Ca2+ transients and the amplitudes of the second and the slowest components. Plotting the slopes of the decay of 100 stimuli-induced Ca2+ transients against [Ca2+](i) yielded the supralinear [Ca2+](i) dependence of Ca2+ efflux out of the cytosol. Blocking Ca2+ extrusion or mitochondrial Ca2+ uptake significantly reduced this [Ca2+](i)-dependent Ca2+ efflux. Thus Ca2+-dependent mitochondrial Ca2+ uptake and plasmalemmal Ca2+ extrusion clear out a small Ca2+ load in frog motor nerve terminals, while thapsigargin-sensitive Ca2+ pump boosts the clearance of a heavy Ca2+ load. Furthermore, the activity of plasmalemmal Ca2+ pump and Na/Ca exchanger is complementary to each other with the slight predominance of the latter.