Ca²⁺ uptake to mitochondria accompanied by depolarization of mitochondrial membrane potential occured when [Ca²⁺]_i increased to a critical level at frog motor nerve terminal

Abstract

To investigate the Ca²⁺ clearance mechanisms in presynaptic terminals, we measured Ca²⁺ dynamics during nerve stimulation in frog neuro-muscular junctions with a low affinity Ca²⁺ dye, Oregon Green 488 BAPTA 6F (K_d=13μM, pH=7.2). During 100Hz tetanus for 4 sec in a normal Ringer's solution (1.8mM Ca²⁺, 0mM Mg²⁺), [Ca²⁺]_i increased in two phases, firstly steep rising (4 μM/sec for 0.2 sec) and secondly slow increasing (0.8 μM/sec). Whenmitochondria were inhibited by rotenone and oligomycin, slow phase disappeared and [Ca²⁺]_i increased monotony with rapid rate. Membrane voltage imaging of mitochondria with TMRE suggested that Ca²⁺ uptake to mitochondria accompanied by depolarization of mitochondrial membrane potential. When tetanus frequency was reduced to 20Hz, [Ca²⁺]_i dynamics did not affected by rotenone and oligomycin, however, CCCP increased [Ca²⁺]_i during tetanus. The effect of rotenone and oligomycin appeared when [Ca²⁺]_i increased more than 0.6 μM during 40Hz tetanus. These results suggest that Ca²⁺ uptake to mitochondria started when [Ca²⁺]_i increased more than 0.6 μM. CCCP had larger effect on [Ca²⁺]_i increase during 40 Hz tetanus than rotenone and oligomycin. These results suggest that CCCP sensitive some Ca²⁺ clearance mechanisms other than mitochondrial Ca²⁺ uptake contributed largely when [Ca²⁺]_i was lower than some μM. [J Physiol Sci. 2006;56 Suppl:S174]