Role of Charged Residues in Coupling Ligand Binding and Channel Activation in the Extracellular Domain of the Glycine Receptor

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 ␣1-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 EC 50 toward wild-type values. Charge reversal mutations (E53K, D57K, and D148K) also impaired channel activation, as inferred from increases in EC 50 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.Fast synaptic transmission in the central nervous system is mediated by members of the ligand-gated ion channel (LGIC) 1 receptor superfamily. The glycine receptor (GlyR) is a member of the nicotinic-like LGIC superfamily that includes the nicotinic acetylcholine (nAChR), serotonin type 3 (5-HT 3 R), and ␥-aminobutyric acid (GABA A R) receptors (1, 2). Each of these receptors are pentameric complexes arranged around a central ion conducting pore. Individual subunits share a similar membrane topology, with hydropathy analysis predicting a large extracellular domain at the N terminus and four putative transmembrane domains (M1-M4) (1). A key characteristic of these receptors is the integral ion channel that is opened following ligand binding. The extracellular domain contains the ligand binding site, which is spatially separate from the M2 domain that lines the ion channel pore of these receptors (3, 4). While there is an accumulated body of data on the structures involved in ligand binding (2) and the ion channel pore (2-4), relatively little is known about the structures that may link these two spatially separate domains to effect receptor activation.Inherited mutations located in the regions flanking the M2 domain of the GlyR ␣1-subunit are associated with human startle disease (hyperekplexia) (5) and start...

Comparison of Taurine- and Glycine-induced Conformational Changes in the M2-M3 Domain of the Glycine Receptor

“…from R271C to K276C) resulted in irreversible changes in the glycine EC 50 values without affecting the saturating current magnitudes (14,17). The same study also showed that the MTSET reaction rate of all these cysteines was relatively slow in the resting closed state but was increased dramatically in the glycine-bound state.…”

“…A structural study of the Torpedo nAChR has shown recently (4) that these residues form an extramembranous extension to the M2 ␣-helix. This domain is likely to interact closely with loops 2 and 5 of the ligand-binding domain (4,17,18,23,24). A particular advantage of investigating this domain is that it lies outside the ligand-binding pocket (6), so changes in methanethiosulfonate reaction rates are unlikely to be influenced by steric effects of ligand binding.…”

Molecular Determinants for G Protein βγ Modulation of Ionotropic Glycine Receptors

“…This model was first suggested by hydrophobicity plots of the ligand-gated channels and was recently confirmed by biochemical and crystallographic data from studies with nAChR (2, 28,29,32). Coincidentally, several recent studies showed similar channel gating mechanisms for the LGIC members; involving the ligand-binding domain and the extracellular loop between TM2 and TM3 (32)(33)(34).…”

Structural Basis for Allosteric Coupling at the Membrane-Protein Interface in Gloeobacter violaceus Ligand-gated Ion Channel (GLIC)

“…We propose that the transition to the desensitized conformation is accompanied by an outward movement of the M2-M3 linker disengaging from the intrasubunit interactions with the ␤1-␤2 and ␤6-␤7 loops to establish intersubunit contacts with the pre-M1 region of the adjacent subunit. Studies in GlyR have shown that the substituted Cys in the M2-M3 linker is more water exposed in the open state than when the channel is in the closed conformation (20). The outward movement of M2-M3 also releases the ␤6-␤7 linker to now move downward into the intrasubunit cavity, interacting with tightly associated membrane lipids.…”

“…15-17 and references therein), and it is known that this region contributes to both the lifetime of the open state and the kinetics of desensitization (18). The interface also contains several charged residues, and gating models have been proposed on the basis of the interactions between specific charged pairs (19,20). However, it is to be noted that these interactions are not conserved across the family (21).…”