Abstract
The glycine receptor is a member of the ligand-gated ion channel receptor superfamily that mediates fast synaptic transmission in the brainstem and spinal cord. Following ligand binding, the receptor undergoes a conformational change that is conveyed to the transmembrane regions of the receptor resulting in the opening of the channel pore. Using the acetylcholine-binding protein structure as a template, we modeled the extracellular domain of the glycine receptor alpha1-subunit and identified the location of charged residues within loops 2 and 7 (the conserved Cys-loop). These loops have been postulated to interact with the M2-M3 linker region between the transmembrane domains 2 and 3 as part of the receptor activation mechanism. Charged residues were substituted with cysteine, resulting in a shift in the concentration-response curves to the right in each case. Covalent modification with 2-(trimethylammonium) ethyl methanethiosulfonate was demonstrated only for K143C, which was more accessible in the open state than the closed state, and resulted in a shift in the EC50 toward wild-type values. Charge reversal mutations (E53K, D57K, and D148K) also impaired channel activation, as inferred from increases in EC50 values and the conversion of taurine from an agonist to an antagonist in E53K and D57K. Thus, each of the residues Glu-53, Asp-57, Lys-143, and Asp-148 are implicated in channel gating. However, the double reverse charge mutations E53K:K276E, D57K:K276E, and D148K:K276E did not restore glycine receptor function. These results indicate that loops 2 and 7 in the extracellular domain play an important role in the mechanism of activation of the glycine receptor although not by a direct electrostatic mechanism.
Highlights
Fast synaptic transmission in the central nervous system is mediated by members of the ligand-gated ion channel (LGIC)1 receptor superfamily
Modeling of the glycine receptor (GlyR)—By threading the amino acid sequence of the GlyR ␣1-subunit extracellular domain over the acetylcholine-binding protein (AChBP) structure, we were able to identify the location of residues that may be involved in various functions of the receptor
The spasmodic loop and the conserved Cys-loop of the GlyR correspond to loop 2 and loop 7, respectively, of the AChBP structure. These are located in regions that are postulated to be close to the ion channel [15, 18] and to the M2–M3 linker (Fig. 1)
Summary
D148C 0.012 Ϯ 0.003 0.97 Ϯ 0.03b 0.88 Ϯ 0.02b 4 a All values are presented as mean Ϯ S.E. b p Ͻ 0.01 compared with C41A. The location of loop 7 in the AChBP suggests that the conserved Cysloop of LGIC receptors is in a position to interact with the transmembrane domains of the receptor, and so may be involved in gating [15, 18]. Alignment of the AChBP and GlyR ␣1-subunit sequences shows that loop 2 corresponds to the location of the A52S mutation in spasmodic mice [19, 20]. The effect of this mutation is to impair the GlyR activation [19, 20], resulting in an exaggerated startle phenotype, which suggests that loop 2 might be involved in gating of the GlyR. We demonstrate that residues in these loops play an important role in receptor activation but not by a simple electrostatic interaction as has been observed in the GABAAR [21]
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