Interplay between Na+/Ca2+ Exchangers and Mitochondria in Ca2+ Clearance at the Calyx of Held

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Abstract
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 μm, 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+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.