High-affinity zinc potentiation of inhibitory postsynaptic glycinergic currents in the zebrafish hindbrain

Zinc has been reported to potentiate glycine receptors (GlyR), but the phys iological significance of this observation has been put in doubt by the rel atively high values of the EC50, 0.5-1 muM, since such concentrations may n ot be attained in the synaptic cleft of glycinergic synapses. We have re-ev aluated this observation in the frame of the hypothesis that contaminant he avy metals present in usual solutions may have lead to underestimate the af finity of the zinc binding site, and therefore to underestimate the potenti al physiological role of zinc. Using chelators either to complex heavy meta ls or to apply zinc at controlled concentrations, we have examined the acti on of zinc on GlyR kinetics in outside-out patches from 50-h-old zebrafish Mauthner cells. Chelating contaminating heavy metals with tricine or N,N,N' ,N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN) decreased the duratio n of the currents evoked by glycine, confirming that traces of heavy metals alter the GlyR response in control conditions. Using tricine- (10 mM) buff ered zinc solution, we then showed that zinc increases the amplitude of out side-out responses evoked by 0.1-0.5 mM glycine with an EC50 of 15 nM. In c ontrast zinc had no effect on the amplitude of currents evoked by a saturat ing concentration (3-10 mM) of glycine. This suggests that zinc enhances Gl yR apparent affinity for glycine. The study of the effects of zinc on the k inetics of the response indicates that this increase of apparent affinity i s due to a decrease of the glycine dissociation rate constant. We then anal yzed the effects of zinc on postsynaptic GlyRs in whole cell recordings of glycinergic miniature inhibitory postsynaptic currents (mIPSCs). Chelation of contaminant heavy metals decreased the amplitude and the duration of the mIPSCs; inverse effects were observed by adding zinc in buffered solutions containing nanomolar free zinc concentrations. Zinc plus tricine or tricin e alone did not change the coefficient of variation (approximate to0.85) of the mIPSC amplitude distributions. These results suggest that postsynaptic GlyRs are not saturated after the release of one vesicle.