The unique extracellular disulfide loop of the glycine receptor is a principal ligand binding element.

Rajendra, Sundran; Vandenberg, Robert J.; Pierce, Kerrie D.; Cunningham, Anne M.; French, Peter; Barry, Peter H.; Schofield, Peter R.

doi:10.1002/j.1460-2075.1995.tb07301.x

Cited by 82 publications

(86 citation statements)

References 33 publications

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“…The opening of the GlyR pore is initiated by the binding of the glycine molecule at interfaces between loops A, B, and C, on the "+" side of one subunit and β sheet segments D, E, and F on the "−" side of an adjacent subunit (3). Glycine binding is stabilized by its interactions with the side chains of several amino acids in the "+" and "−" sides of adjacent subunits, including R119 (3,(16)(17)(18). This residue is also implicated in GABA binding (19).…”

Section: Discussionmentioning

confidence: 99%

Disruption of an intersubunit electrostatic bond is a critical step in glycine receptor activation

Todorovic

Welsh

Bertaccini

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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Proper regulation of neurotransmission requires that ligandactivated ion channels remain closed until agonist binds. How channels then open remains poorly understood. Glycine receptor (GlyR) gating is initiated by agonist binding at interfaces between adjacent subunits in the extracellular domain. Aspartate-97, located at the α1 GlyR interface, is a conserved residue in the cys-loop receptor superfamily. The mutation of D97 to arginine (D97R) causes spontaneous channel opening, with open and closed dwell times similar to those of maximally activated WT GlyR. Using a model of the N-terminal domain of the α1 GlyR, we hypothesized that an arginine-119 residue was forming intersubunit electrostatic bonds with D97. The D97R/R119E charge reversal restored this interaction, stabilizing channels in their closed states. Cysteine substitution shows that this link occurs between adjacent subunits. This intersubunit electrostatic interaction among GlyR subunits thus contributes to the stabilization of the closed channel state, and its disruption represents a critical step in GlyR activation.cysteine substitution | electrophysiology | mutagenesis | Xenopus oocytes G lycine receptors (GlyR) are pentameric anion-conducting members of the cys-loop receptor superfamily, with their subunits arranged around a central ion pore. Each subunit consists of a large extracellular N-terminal ligand binding domain and four transmembrane segments (TM1-TM4); TM2 of each subunit lines the pore (1). When glycine binds to initiate channel opening it interacts with specific amino acids located at intersubunit interfaces, and spontaneous openings do not occur in the absence of neurotransmitter (2). Six loops of amino acids located on adjacent subunits constitute the known glycine binding site. On the plus (+) side of the interface on one subunit are loops A-C, whereas loops D-F are located on the minus (−) side of an adjacent subunit (3). In the related nicotinic acetylcholine receptor (nAChR), signal transduction after agonist binding is described as a "Brownian conformational wave" that travels down the interface between subunits (4). Auerbach and colleagues used ϕ analysis to show that the binding pocket region of the N-terminal domain is the first to move after ligand binds. Loops 2 and 7 (the cys-loop) of the Nterminal domain interact with the extracellular end of TM1 and the TM2-3 linker region to transmit binding signals to the channel gate (5-7). In the α1 GlyR subunit, D148 in loop 7 forms an electrostatic bridge with K276 in the TM2-3 linker (8).Our previous work demonstrated that mutation of D97 in loop A results in spontaneous channel opening (9). This D97 residue is conserved within the cys-loop receptor superfamily (Fig. 1), suggesting its critical role in channel function. In this article we report on an electrostatic interaction between specific charged amino acid residues at the interfaces of adjacent subunits that contribute to the stabilization of the closed channel state in the absence of neurotransmitter. Disruption of these ...

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Section: Discussionmentioning

confidence: 99%

Disruption of an intersubunit electrostatic bond is a critical step in glycine receptor activation

Todorovic

Welsh

Bertaccini

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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“…Residues mutated to cysteine are marked with a "C" above the residue. Residues located in the GABA binding site, ␤ 2 Y205 and ␤ 2 R207 (Amin and Weiss, 1993;Wagner and Czajkowski, 2001), and the binding sites of other LGICs (Kao et al, 1984;Mishina et al, 1985;Dennis et al, 1988;Ruiz-Gomez et al, 1990;Galzi et al, 1991a,b;Vandenberg et al, 1992a,b;Rajendra et al, 1995) are marked with an asterisk. Elements of secondary structure are indicated below the sequences by the arrow (␤-strand 10) and rectangle (␣ helix, M1 transmembrane region).…”

Section: Functional Characterization Of Pre-m1 Mutant Receptorsmentioning

confidence: 99%

Charged Residues in the α₁and β₂Pre-M1 Regions Involved in GABA_AReceptor Activation

Mercado¹,

Czajkowski²

2006

J. Neurosci.

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For Cys-loop ligand-gated ion channels (LGIC), the protein movements that couple neurotransmitter binding to channel gating are not well known. The pre-M1 region, which links the extracellular agonist-binding domain to the channel-containing transmembrane domain, is in an ideal position to transduce binding site movements to gating movements. A cluster of cationic residues in this region is observed in all LGIC subunits, and in particular, an arginine residue is absolutely conserved. We mutated charged pre-M1 residues in the GABA A receptor ␣ 1 (K219, R220, K221) and ␤ 2 (K213, K215, R216) subunits to cysteine and expressed the mutant subunits with wild-type ␤ 2 or ␣ 1 in Xenopus oocytes. Cysteine substitution of ␤ 2 R216 abolished channel gating by GABA without altering the binding of the GABA agonist [3 H]muscimol, indicating that this residue plays a key role in coupling GABA binding to gating. Tethering thiol-reactive methanethiosulfonate (MTS) reagents onto ␣ 1 K219C, ␤ 2 K213C, and ␤ 2 K215C increased maximal GABA-activated currents, suggesting that structural perturbations of the pre-M1 regions affect channel gating. GABA altered the rates of sulfhydryl modification of ␣ 1 K219C, ␤ 2 K213C, and ␤ 2 K215C, indicating that the pre-M1 regions move in response to channel activation. A positively charged MTS reagent modified ␤ 2 K213C and ␤ 2 K215C significantly faster than a negatively charged reagent, and GABA activation eliminated modification of ␤ 2 K215C by the negatively charged reagent. Overall, the data indicate that the pre-M1 region is part of the structural machinery coupling GABA binding to gating and that the transduction of binding site movements to channel movements is mediated, in part, by electrostatic interactions.

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“…Note that this RT loop insertion has no effect on the position of the disulfide bridge (198)(199)(200)(201)(202)(203)(204)(205)(206)(207)(208)(209) or ligand-binding residues (Lys 200, v r 202, Thr 204) (Rajendra et al, 1995b) (see Fig. I) compared with the original ID-3D alignment for domain B (Fig.…”

Section: Refinement Of Domain B (Sh3) For Glyrmentioning

confidence: 99%

“…The GlyR residues identified by site-directed mutagenesis to affect channel activation by agonist or blockade by antagonists are: Phe 159, Gly 160, Tyr 161, Lys 200, Tyr 202, and Thr 204 (Schmieden et al, 1992(Schmieden et al, , 1993Vandenberg et al, 1992aVandenberg et al, , 1992bVandenberg et al, , 1993Devillers-Thiery et al, 1993;Vandenberg & Schofield, 1994;Rajendra et al, 1995b), located in Changeux's ligand-binding loops B and C. Lys 200, Tyr 202, and Thr 204 are within a loop between strands 11 and 12 in the SH3 domain (Fig. 2D), and Phe 159, Gly 160, and Tyr 161 are part of the structure built between domains A and B.…”

Section: Proximity Of Ligand-binding Residuesmentioning

confidence: 99%

Predicted structure of the extracellular region of ligand‐gated ion‐channel receptors shows SH2‐like and SH3‐like domains forming the ligand‐binding site

Gready¹,

Ranganathan²,

Schofield³

et al. 1997

Protein Science

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Fast synaptic neurotransmission is mediated by ligand-gated ion-channel (LGIC) receptors, which include receptors for acetylcholine, serotonin, GABA, glycine, and glutamate.LGICs are pentamers with extracellular ligand-binding domains and form integral membrane ion channels that are selective for cations (acetylcholine and serotonin 5HT3 receptors) or anions (GABAA and glycine receptors and the invertebrate glutamate-binding chloride channel). They form a protein superfamily with no sequence similarity to any protein of known structure. Using a 1D-3D structure mapping approach, we have modeled the extracellular ligand-binding domain based on a significant match with the SH2 and SH3 domains of the biotin repressor structure. Refinement of the model based on knowledge of the large family of SH2 and SH3 structures, sequence alignments, and use of structure templates for loop building, allows the prediction of both monomer and pentamer models. These are consistent with medium-resolution electron microscopy structures and with experimental structure/function data from ligand-binding, antibody-binding, mutagenesis, protein-labeling and subunitlinking studies, and glycosylation sites. Also, the predicted polarity of the channel pore calculated from electrostatic potential maps of pentamer models of superfamily members is consistent with known ion selectivities. Using the glycine receptor a1 subunit, which forms homopentamers, the monomeric and pentameric models define the agonist and antagonist (strychnine) binding sites to a deep crevice formed by an extended loop, which includes the invariant disulfide bridge, between the SH2 and SH3 domains. A detailed binding site for strychnine is reported that is in strong agreement with known structure/function data. A site for interaction of the extracellular ligand-binding domain with the activation of the M2 transmembrane helix is also suggested.Keywords: acetylcholine receptor; biotin repressor; 1D-3D structure prediction; extracellular ligand-binding domain; glycine receptor; ligand-gated ion-channel receptors; M2 transmembrane helix; pentameric channels; SH2 and SH3 domainsThe ligand-gated ion-channel (LGIC) neurotransmitter receptor superfamily includes the acetylcholine, GABAA, serotonin 5-HT3, and glycine receptors from vertebrates (Barnard, 1992; Karlin & Akabas, 1995) and the recently characterized avermectin-sensitive glutamate-gated chloride channel from invertebrates (Cully et al., 1994). The binding of specific neurotransmitter molecules (mainly small amino acids) to these LGICs triggers a conformational change Reprint requests to: J.E. Gready, Computational Molecular Biology and Drug Design Group, John Curtin School of Medical Research, Australian National University, P.O. Box 334, Canberra ACT 2601, Australia; e-mail: jill.gready@anu.edu.au in the receptor and renders the channel transiently and selectively permeable to either small cations or small anions. The channels are characterized by a gate, which can permit or stop ion flux, located almost 50 8, ...

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The unique extracellular disulfide loop of the glycine receptor is a principal ligand binding element.

Cited by 82 publications

References 33 publications

Disruption of an intersubunit electrostatic bond is a critical step in glycine receptor activation

Disruption of an intersubunit electrostatic bond is a critical step in glycine receptor activation

Charged Residues in the α₁and β₂Pre-M1 Regions Involved in GABA_AReceptor Activation

Predicted structure of the extracellular region of ligand‐gated ion‐channel receptors shows SH2‐like and SH3‐like domains forming the ligand‐binding site

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The unique extracellular disulfide loop of the glycine receptor is a principal ligand binding element.

Cited by 82 publications

References 33 publications

Disruption of an intersubunit electrostatic bond is a critical step in glycine receptor activation

Disruption of an intersubunit electrostatic bond is a critical step in glycine receptor activation

Charged Residues in the α1and β2Pre-M1 Regions Involved in GABAAReceptor Activation

Predicted structure of the extracellular region of ligand‐gated ion‐channel receptors shows SH2‐like and SH3‐like domains forming the ligand‐binding site

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Charged Residues in the α₁and β₂Pre-M1 Regions Involved in GABA_AReceptor Activation