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 cyanidem-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+]iyielded the supralinear [Ca2+]idependence 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.