1. The effect of the divalent cation Zn2+ on inhibitory glycine receptor (GlyR) currents was investigated in rat embryonic spinal cord neurons and Xenopus oocytes expressing recombinant GlyRs.2. In cultured spinal neurons, Zn2+ potentiated glycine-induced whole-cell currents about 3-fold when applied extracellularly at concentrations of 0-5-10 /LM. In contrast, higher concentrations (> 100 /M) of Zn2+ decreased the glycine response. 3. A similar biphasic modulation of glycine-induced currents by Zn2+ was also found with recombinant homo-and hetero-oligomeric GlyRs generated in Xenopus oocytes. Doseresponse analysis showed that both the potentiating and inhibitory effects of Zn2+ result from changes in apparent agonist affinity. 4. Analysis of chimeric constructs of the GlyR al-and /-subunits revealed that the positive and negative modulatory effects of Zn2+ are mediated by different regions of the al -subunit.5. Our data indicate the existence of distinct high-and low-affinity Zn2+ binding sites on the ligand-binding ca-subunits of the GlyR. These sites may be implicated in the regulation of synaptic efficacy within glycinergic pathways.

The inhibitory glycine receptor (GlyR) is a pentameric chloride channel protein which mediates postsynaptic inhibition in the mammalian central nervous system. In spinal cord, different GlyR isoforms originate from the sequential expression of developmentally regulated variants of the ligand binding alpha subunit. Here, neonatal alpha 2 and adult alpha 1 subunits are shown to generate GlyRs with distinct agonist activation profiles upon heterologous expression in Xenopus oocytes. Whereas alpha 1 receptors are efficiently gated by beta‐alanine and taurine, alpha 2 GlyRs show only a low relative response to these agonists, which also display a reduced sensitivity to inhibition by the glycinergic antagonist strychnine. Construction of an alpha 2/alpha 1 subunit chimera and site‐directed mutagenesis of the extracellular region of the alpha 1 sequence identified amino acid positions 111 and 212 as important determinants of taurine activation. Our results indicate the existence of distinct subsites for agonists on alpha 1 and alpha 2 GlyRs and suggest that the ligand binding pocket of these receptor proteins is formed from discontinuous domains of their extracellular region.

Two cDNAs encoding variants (alpha 1 and alpha 2) of the strychnine binding subunit of the inhibitory glycine receptor (GlyR) were isolated from a human fetal brain cDNA library. The predicted amino acid sequences exhibit approximately 99% and approximately 76% identity to the previously characterized rat 48 kd polypeptide. Heterologous expression of the human alpha 1 and alpha 2 subunits in Xenopus oocytes resulted in the formation of glycine‐gated strychnine‐sensitive chloride channels, indicating that both polypeptides can form functional GlyRs. Using a panel of rodent‐human hybrid cell lines, the gene encoding alpha 2 was mapped to the short arm (Xp21.2‐p22.1) of the human X chromosome. In contrast, the alpha 1 subunit gene is autosomally located. These data indicate molecular heterogeneity of the human GlyR at the level of alpha subunit genes.

Hereditary hyperekplexia is a dominant neurological disorder associated with point mutations at the channel‐forming segment M2 of the glycine receptor alpha 1 subunit. Voltage‐clamp recordings from the heterologously expressed mutants (alpha 1R271L or alpha 1R271Q) revealed 146‐ to 183‐fold decreased potencies of glycine to activate the chloride channel, and significantly reduced maximal whole‐cell currents as compared with wild‐type receptors. In contrast, the ability of the competitive antagonist strychnine to block glycine‐induced currents was similar in all cases. Radioligand binding assays showed a 90‐ to 1365‐fold reduction in the ability of glycine to displace [3H]strychnine from its binding site on the mutant receptors. Paralleling the reductions in whole‐cell current, the elementary main‐state conductances of the mutants (alpha 1R271L, 64 pS; alpha 1R271Q, 14 pS) were lower than that of the wild‐type receptor (86 pS). The decreased agonist affinities and chloride conductances of the mutants are likely to cause neural hyperexcitability of affected patients by impairing glycinergic inhibition. In addition, our data reveal that structural modifications of the ion‐channel region can affect agonist binding to the glycine receptor.

The inhibitory postsynaptic glycine receptor (GlyR) of rat spinal cord is an oligomeric transmembrane protein which forms an agonist‐gated anion channel. Expression in Xenopus oocytes of its mol. wt 48,000 subunit generated glycine‐gated chloride channels which were analysed by voltage clamp. The agonist and antagonist response properties as well as the desensitization characteristics of these 48 kd subunit receptors resembled GlyRs expressed from spinal cord poly(A)+ RNA. These data indicate that the 48 kd subunit is capable of assembling into a functional receptor homo‐oligomer which displays the pharmacology characteristic of the spinal cord GlyR.