Role of K⁺-dependent Na⁺/Ca²⁺ exchangers in Ca²⁺-homeostasis at axon terminals of mammalian central neurons.

Suk-Ho Lee

National Research Lab. for Cell Physiology, Department of Physiology, Seoul National University College of Medicine

Two different families of Na⁺/Ca²⁺ exchangers, K⁺-independent NCX and K⁺-dependent NCKX, are known. Exploiting the outward K⁺ gradient, NCKX is able to extrude Ca²⁺ more efficiently than NCX even when the Na⁺ gradient is reduced. The NCKX, which was originally thought to be limited to the retinal photoreceptor, is recently shown to be widely distributed in the brain. The lack of specific inhibitors has hampered the differentiation between the activities of NCX and NCKX in central neurons. The internal dialysis of axon terminals with K⁺-free solution via patch pipette allowed us to study the role of NCKX in the Ca²⁺ clearance in two kinds of mammalian central neurons which have giant axon terminals amenable to patch-recordings: neurohypophysial axon terminals (NHP) and fast glutamatergic presynaptic terminals in central auditory pathway (calyx of Held). Using patch-clamp and microfluorometry techniques, we recorded the Ca²⁺ transients evoked by a depolarizing pulse, and estimated the relative contribution of Na/Ca exchanger and other Ca²⁺-clearance mechanisms.

In NHP axon terminals, Na⁺/Ca²⁺ exchange was the most important mechanism for the Ca²⁺ clearance in the rat NHP axon terminals, and about 90% of Na⁺/Ca²⁺ exchanger activity was dependent on intracellular [K⁺]. In contrast, Na⁺/Ca²⁺ exchanger activity was not observed at the somata of supraoptic magnocellular neurons, where the sequestration of Ca²⁺ into ER and mitochondria was the major Ca²⁺ clearance mechanism. Ca²⁺ clearance rate in axon terminals, which were calculated from time derivative of Ca²⁺ decay and the endogenous Ca²⁺-binding ratio (k_S) values, were about 3 folds higher than in somata. These results suggest that neurohypophysial axon terminals have greater Ca²⁺ clearance power than somata due to the specific localization of NCKX2.

The role of NCKX in presynaptic terminals was studied at the calyx of Held. We investigated the contribution of NCKX in conjunction with other Ca2+ clearance mechanisms at the calyx of Held. 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⁺ or Li⁺ decreased the Ca²⁺-decay rate by 42%, and the additional inhibition of NCX by reducing external [Na⁺] caused a further decrease by 26%. Inhibition of PMCA decreased the Ca²⁺-decay rate by 23%, whereas inhibition of SERCA had no effect. The contribution of mitochondria was negligible for small Ca²⁺-transients, but became apparent at [Ca²⁺]_i > 2.5 mM, when Na⁺/Ca²⁺ exchange became saturated. Mitochondrial contribution was also observed when the duration of Ca2+ transients was prolonged by inhibiting Na⁺/Ca²⁺ exchangers. These results suggest that in response to small Ca²⁺ transients (< 2 mM), 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.