Mutation of an arginine residue in the human glycine receptor transforms β-alanine and taurine from agonists into competitive antagonists

Rajendra, Sundran; Lynch, Joseph W.; Pierce, Kerrie D.; French, Chris; Barry, Peter H.; Schofield, Peter R.

doi:10.1016/0896-6273(95)90251-1

Cited by 149 publications

(164 citation statements)

References 37 publications

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“…This confirmed that at least a fraction of the channel protein was correctly inserted into the plasma membrane. The affinity for glycine (ECs0 = 18.6 pM) and the degree of agonist cooperativity as expressed by the Hill coefficient of channel activation (h = 4.2) were similar to values previously found for GlyR ~1 receptors expressed in HEK-293 cells (ECs0 = 29-100/zM, h = 1.6~4.2; see [4,5,18]). Preliminary single channel analysis in the outside-out patch clamp configuration revealed a main elementary conductance of 88 pS accompanied by subconductance states of 70 pS, 54 pS and 35 pS (N. Rundstr6m, unpublished results).…”

Section: Discussionsupporting

confidence: 55%

“…A dissociation constant (Kd) for [3H]strychnine of 38 nM is within the range of values found for native spinal cord membranes and HEK-293 ceils transiently expressing ~1 GIyR (2-64 riM; see [19][20][21]18,15]). The maximal number of strychnine binding sites (Bma x = 71 pmol/mg) obtained with the Sf9 cell system, however, significantly exceeds the values found for spinal cord membranes and transfected HEK-293 cells (2.4-17 pmol/mg) by 4-to 30-fold (compare: [19,20,18,15]). Thus, infected Sf9 cells express the GlyR to much higher levels than native neurons or even transiently expressing mammalian cells.…”

Section: Discussionmentioning

confidence: 99%

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Baculovirus‐driven expression and purification of glycine receptor α1 homo‐oligomers

et al. 1995

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The glycine receptor is a figand-gated anion channel protein of postsynaptic membranes. We expressed a homo-oligomeric receptor composed of human al subunits in Spodoptera frugiperda cells by infection with a recombinant Autographa californica nuclear polyhedrosis virus. A substantial fraction of the recombinant receptor was incorporated as a functional channel protein into the cell's plasma membrane at expression levels 4-to 30-fold higher than in other eukaryotic heterologous expression systems or native rat spinal cord membranes, respectively. Upon detergent solubilization, the al receptor was found to exist in a predominantly monodisperse state and could be affinitypurified to near homogeneity. This preparation is a potential starting point for future crystallisation studies.Key words: Glycine receptor; Baculovirus; Affinity purification; Heterologous expression transmembrane topology characterized by an extended N-terminal extracellular domain followed by four transmembrane segments (M1-M4; see [1]).Here, we expressed al homo-oligomeric GlyR in Spodoptera frugiperda (Sf9) insect cells infected with a recombinant Autographa californica baculovirus, in which the GlyR al cDNA was placed under the control of the polyhedrin promoter. The baculovirus Sf9 cell system was previously used to overexpress the c~l GlyR [7] in order to produce preparative amounts of GlyR protein. Receptor purification described in that study involved solubilisation with digitonin/desoxycholate and resulted in a partially proteolysed protein. Here we used solubilisation of the receptor with the more economical conventional detergent Triton X-100 and affinity-purified it to a nearly homogenous and monodisperse state, which appears suitable for crystallization studies.

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Section: Discussionsupporting

confidence: 55%

Section: Discussionmentioning

confidence: 99%

Baculovirus‐driven expression and purification of glycine receptor α1 homo‐oligomers

et al. 1995

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“…At both locations, the mutations result in the substitution of a positive charged residue. The effect of these mutations is to disrupt signal transduction, as inferred from the increases in EC 50 for glycine and the conversion of taurine from an agonist to an antagonist (8). This was also demonstrated at the single channel level for the K276E mutation (9).…”

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confidence: 70%

“…We infer from these results that these two loop 2 charged residues (Glu-53 and Asp-57) are involved in channel gating or the signal transduction process leading to channel gating. The conversion of taurine from an agonist to a partial agonist or an antagonist has previously been demonstrated by mutations within the M2-M3 linker, establishing this region as being involved in channel gating (8). The two charged residues in loop 7 (Lys-143 and Asp-148) were also shown to be involved in channel gating.…”

Section: Role Of Charged Residues In Loops 2 and 7 Of The Extracellulmentioning

confidence: 94%

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

Absalom

Lewis

Kaplan

et al. 2003

Journal of Biological Chemistry

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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...

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“…In recombinantly expressed receptors, substitution of this residue weakens both the preference of the channel for states of high conductance 4,5 and the transition from closed to open states subsequent to glycine binding. 5,6 Also in recombinant receptors, the fluorescent signal of a thiol-reactive fluorophore bound to an introduced cysteine at this position is shifted to lower wavelengths, suggesting that during activation by glycine, the residue at position 271 enters a more hydrophobic environment. 7 In vivo, the decreased level of activation of R271-mutated α1 GlyRs results in neuronal hyperexcitability and the condition known as hyperekplexia or startle disease.…”

mentioning

confidence: 99%

Propofol Modulation of α1 Glycine Receptors Does Not Require a Structural Transition at Adjacent Subunits That Is Crucial to Agonist-Induced Activation

Lynagh

Kunz

Laube

2013

ACS Chem. Neurosci.

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Pentameric glycine receptors (GlyRs) couple agonist binding to activation of an intrinsic ion channel. Substitution of the R271 residue impairs agonist-induced activation and is associated with the human disease hyperekplexia. On the basis of a homology model of the α1 GlyR, we substituted residues in the vicinity of R271 with cysteines, generating R271C, Q226C, and D284C single-mutant GlyRs and R271C/Q226C and R271C/D284C double-mutant GlyRs. We then examined the impact of interactions between these positions on receptor activation by glycine and modulation by the anesthetic propofol, as measured by electrophysiological experiments. Upon expression in Xenopus laevis oocytes, D284C-containing receptors were nonfunctional, despite biochemical evidence of successful cell surface expression. At R271C/Q226C GlyRs, glycine-activated whole-cell currents were increased 3-fold in the presence of the thiol reductant dithiothreitol, whereas the ability of propofol to enhance glycine-activated currents was not affected by dithiothreitol. Biochemical experiments showed that mutant R271C/Q226C subunits form covalently linked pentamers, showing that intersubunit disulfide cross-links are formed. These data indicate that intersubunit disulfide links in the transmembrane domain prevent a structural transition that is crucial to agonist-induced activation of GlyRs but not to modulation by the anesthetic propofol and implicate D284 in the functional integrity of GlyRs.

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Mutation of an arginine residue in the human glycine receptor transforms β-alanine and taurine from agonists into competitive antagonists

Cited by 149 publications

References 37 publications

Baculovirus‐driven expression and purification of glycine receptor α1 homo‐oligomers

Baculovirus‐driven expression and purification of glycine receptor α1 homo‐oligomers

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

Propofol Modulation of α1 Glycine Receptors Does Not Require a Structural Transition at Adjacent Subunits That Is Crucial to Agonist-Induced Activation

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