The effects of the antihelmintic, ivermectin, were investigated in recombinantly expressed human ␣ 1 homomeric and ␣ 1 ␤ heteromeric glycine receptors (GlyRs). At low (0.03 M) concentrations ivermectin potentiated the response to sub-saturating glycine concentrations, and at higher (>0.03 M) concentrations it irreversibly activated both ␣ 1 homomeric and ␣ 1 ␤ heteromeric GlyRs. Relative to glycine-gated currents, ivermectin-gated currents exhibited a dramatically reduced sensitivity to inhibition by strychnine, picrotoxin, and zinc. The insensitivity to strychnine could not be explained by ivermectin preventing the access of strychnine to its binding site. Furthermore, the elimination of a known glycine-and strychnine-binding site by site-directed mutagenesis had little effect on ivermectin sensitivity, demonstrating that the ivermectin-and glycine-binding sites were not identical. Ivermectin strongly and irreversibly activated a fast-desensitizing mutant GlyR after it had been completely desensitized by a saturating concentration of glycine. Finally, a mutation known to impair dramatically the glycine signal transduction mechanism had little effect on the apparent affinity or efficacy of ivermectin. Together, these findings indicate that ivermectin activates the GlyR by a novel mechanism.

This study investigated the residues responsible for the reduced picrotoxin sensitivity of the ab heteromeric glycine receptor relative to the a homomeric receptor. By analogy with structurally related receptors, the b subunit M2 domain residues P278 and F282 were considered the most likely candidates for mediating this effect. These residues align with G254 and T258 of the a subunit. The T258A, T258C and T258F mutations dramatically reduced the picrotoxin sensitivity of the a homomeric receptor. Furthermore, the converse F282T mutation in the b subunit increased the picrotoxin sensitivity of the ab heteromeric receptor. The P278G mutation in the b subunit did not affect the picrotoxin sensitivity of the ab heteromer. Thus, a ring of ®ve threonines at the M2 domain depth corresponding to a subunit T258 is speci®cally required for picrotoxin sensitivity. Mutations to a subunit T258 also profoundly in¯uenced the apparent glycine af®nity. A substituted cysteine accessibility analysis revealed that the T258C sidechain increases its pore exposure in the channel open state. This provides further evidence for an allosteric mechanism of picrotoxin inhibition, but renders it unlikely that picrotoxin (as an allosterically acting`competitive' antagonist) binds to this residue.

Mutations in the extracellular M2-M3 loop of the glycine receptor (GlyR) ␣1 subunit have been shown previously to affect channel gating. In this study, the substituted cysteine accessibility method was used to investigate whether a structural rearrangement of the M2-M3 loop accompanies GlyR activation. All residues from R271C to V277C were covalently modified by both positively charged methanethiosulfonate ethyltrimethylammonium (MTSET) and negatively charged methanethiosulfonate ethylsulfonate (MTSES), implying that these residues form an irregular surface loop. The MTSET modification rate of all residues from R271C to K276C was faster in the glycine-bound state than in the unliganded state. MTSES modification of A272C, L274C, and V277C was also faster in the glycine-bound state. These results demonstrate that the surface accessibility of the M2-M3 loop is increased as the channel transitions from the closed to the open state, implying that either the loop itself or an overlying domain moves during channel activation.

Histidines 107 and 109 in the glycine receptor (GlyR) ␣ 1 subunit have previously been identified as determinants of the inhibitory zinc-binding site. Based on modeling of the GlyR ␣ 1 subunit extracellular domain by homology to the acetylcholine-binding protein crystal structure, we hypothesized that inhibitory zinc is bound within the vestibule lumen at subunit interfaces, where it is ligated by His 107 from one subunit and His 109 from an adjacent subunit. This was tested by co-expressing ␣ 1 subunits containing the H107A mutation with ␣ 1 subunits containing the H109A mutation. Although sensitivity to zinc inhibition is markedly reduced when either mutation is individually incorporated into all five subunits, the GlyRs formed by the co-expression of H107A mutant subunits with H109A mutant subunits exhibited an inhibitory zinc sensitivity similar to that of the wild type ␣ 1 homomeric GlyR. This constitutes strong evidence that inhibitory zinc is coordinated at the interface between adjacent ␣ 1 subunits. No evidence was found for ␤ subunit involvement in the coordination of inhibitory zinc, indicating that a maximum of two zincbinding sites per ␣ 1 ␤ receptor is sufficient for maximal zinc inhibition. Our data also show that two zinc-binding sites are sufficient for significant inhibition of ␣ 1 homomers. The binding of zinc at the interface between adjacent ␣ 1 subunits could restrict intersubunit movements, providing a feasible mechanism for the inhibition of channel activation by zinc.

The substituted cysteine accessibility method was used to probe the surface exposure of a pore-lining threonine residue (T6) common to both the glycine receptor (GlyR) and ␥-aminobutyric acid, type A receptor (GABA A R) chloride channels. This residue lies close to the channel activation gate, the ionic selectivity filter, and the main pore blocker binding site. Despite their high amino acid sequence homologies and common role in conducting chloride ions, recent studies have suggested that the GlyRs and GABA A Rs have divergent open state pore structures at the 6 position. When both the human ␣1 T6C homomeric GlyR and the rat ␣1 T6C ␤1 T6C heteromeric GABA A R were expressed in human embryonic kidney 293 cells, their 6 residue surface accessibilities differed significantly in the closed state. However, when a soluble cysteine-modifying compound was applied in the presence of saturating agonist concentrations, both receptors were locked into the open state. This action was not induced by oxidizing agents in either receptor. These results provide evidence for a conserved pore opening mechanism in anion-selective members of the ligand-gated ion channel family. The results also indicate that the GABA A R pore structure at the 6 level may vary between different expression systems. The ligand-gated ion channel (LGIC)1 superfamily includes the nicotinic acetylcholine receptor (nAChR), serotonin type 3 receptor (5HT 3 R), GABA A receptor (GABA A R), and glycine receptor (GlyR), as well as invertebrate glutamate and histidine receptors (1). Functional receptors of this family comprise five homologous subunits arranged in a ring to form a central ion-conducting pore. Each subunit is composed of a large extracellular ligand-binding N-terminal domain, four membranespanning segments (M1-M4), and a large intracellular domain between M3 and M4.The pore-lining, second transmembrane (M2) domain has an ␣-helical secondary structure that undergoes a conformational change as the channel is opened (2). To investigate this process in detail, state-dependent differences in the surface exposure of M2 domain residues can be assayed using the substituted cysteine accessibility method (3). In this technique, residues are mutated individually to cysteines, and changes in their reactivity rates with soluble cysteine-reactive reagents can identify structural changes between different functional states. As expected for receptors belonging to the same family, this technique has generally yielded a good correlation between the open state M2 domain secondary structures of the nAChR (4 -7), GABA A R (8), and 5HT 3 R (9, 10).The M2 domain 6Ј residue, which is a threonine in the GlyR ␣1 subunit and the GABA A R ␣1 and ␤1 subunits (see Fig. 1A), lines a critical part of the pore. It is close to the activation gate (6,11,12) and the ionic selectivity filter (13-15) and forms the main pore blocker binding site (reviewed in Ref. 16). Therefore, structural differences at this level may be expected to have significant functional consequences. In the homo...