The Free Zinc Concentration in the Synaptic Cleft of Artificial Glycinergic Synapses Rises to At least 1 μM

Zn(2+) is concentrated into presynaptic vesicles at many central synapses and is released into the synaptic cleft by nerve terminal stimulation. There is strong evidence that synaptically released Zn(2+) modulates glutamatergic neurotransmission, although there is debate concerning the peak concentration it reaches in the synaptic cleft. Glycine receptors (GlyRs), which mediate inhibitory neurotransmission in the spinal cord and brainstem, are potentiated by low nanomolar Zn(2+) and inhibited by micromolar Zn(2+). Mutations that selectively ablate Zn(2+) potentiation result in hyperekplexia phenotypes suggesting that Zn(2+) is a physiological regulator of glycinergic neurotransmission. There is, however, little evidence that Zn(2+) is stored presynaptically at glycinergic terminals and an alternate possibility is that GlyRs are modulated by constitutively bound Zn(2+). We sought to estimate the peak Zn(2+) concentration in the glycinergic synaptic cleft as a means of evaluating whether it is likely to be synaptically released. We employed ‘artificial’ synapses because they permit the insertion of engineered α1β GlyRs with defined Zn(2+) sensitivities into synapses. By comparing the effect of Zn(2+) chelation on glycinergic IPSCs with the effects of defined Zn(2+) and glycine concentrations applied rapidly to the same recombinant GlyRs in outside-out patches, we inferred that synaptic Zn(2+) rises to at least 1 μM following a single presynaptic stimulation. Moreover, using the fast, high-affinity chelator, ZX1, we found no evidence for tonic Zn(2+) bound constitutively to high affinity GlyR binding sites. We conclude that diffusible Zn(2+) reaches 1 μM or higher and is therefore likely to be phasically released in artificial glycinergic synapses.