Interplay between Na+/Ca2+ exchangers and mitochondria in Ca2+ clearance at the calyx of Held

The clearance of Ca2+ from nerve terminals is critical for determining the build-up of residual Ca2+ after repetitive presynaptic activity. We found previously that K+- dependent Na+/Ca2(+) exchangers (NCKXs) show polarized distributions in axon terminals of supraoptic magnocellular neurons and play a major role in Ca2+ clearance. The role of NCKXs in presynaptic terminals, however, has not been studied. We investigated the contribution of NCKX in conjunction with other Ca2+ clearance mechanisms at the calyx of Held by analyzing the decay of Ca2+ transients evoked by depolarizing pulses. Inhibition of Na+/ Ca2+ exchange by replacing external Na+ with Li+ decreased the Ca2+ decay rate by 68%. Selective inhibition of NCKX by replacing internal K+ with TEA(+) ( tetraethylammonium) or Li+ decreased the Ca2+ decay rate by 42%, and the additional inhibition of the K+- independent form of Na+/ Ca2+ exchanger (NCX) by reducing external [Na+] caused an additional decrease by 26%. Inhibition of plasma membrane Ca2(+)- ATPase (PMCA) decreased the Ca2+ decay rate by 23%, whereas inhibition of SERCA (smooth endoplasmic reticulum Ca2+- ATPase) had no effect. The contribution of mitochondria was negligible for small Ca2+ transients but became apparent at [Ca2(+)](i) > 2.5 mu M, when Na+/ Ca2+ exchange became saturated. Mitochondrial contribution was also observed when the duration of Ca2+ transients was prolonged by inhibiting Na+/ Ca2+ exchangers or by increasing Ca2+ buffers. These results suggest that, in response to small Ca2+ transients (< 2 mu M), Ca2+ loads are cleared from the calyx of Held by NCKX ( 42%), NCX ( 26%), and PMCA ( 23%), and that mitochondria participate when the Ca2+ load is larger or prolonged.