Exploring ligand recognition and ion flow in comparative models of the human GABA type A receptor

doi:10.1016/j.jmgm.2007.04.012

Journal of Molecular Graphics and Modelling

Volume 26, Issue 4, November 2007, Pages 760-774

https://doi.org/10.1016/j.jmgm.2007.04.012 Get rights and content

Abstract

We present two comparative models of the GABA_A receptor. Model 1 is based on the 4-Å resolution structure of the nicotinic acetylcholine receptor from Torpedo marmorata and represents the unliganded receptor. Two agonists, GABA and muscimol, two benzodiazepines, flunitrazepam and alprazolam, together with the general anaesthetic halothane, have been docked to this model. The ion flow is also explored in model 1 by evaluating the interaction energy of a chloride ion as it traverses the extracellular, transmembrane and intracellular domains of the protein. Model 2 differs from model 1 only in the extracellular domain and represents the liganded receptor. Comparison between the two models not only allows us to explore commonalities and differences with comparative models of the nicotinic acetylcholine receptor, but also suggests possible protein sub-domain interactions with the GABA_A receptor not previously addressed.

Introduction

The family of γ-aminobutyric acid type A receptors (GABA_A receptors) is responsible for the majority of fast neuronal inhibition in the mammalian central nervous system. These oligomeric proteins belong to the cys-loop family of ligand-gated ion channels that includes the nicotinic acetylcholine (nACh), glycine and 5HT₃ receptors. The GABA_A receptors are composed of five subunits arranged pseudosymmetrically around the integral anion channel [63]. The subunits, of which 19 have thus far been identified, are separated into classes based on their sequence similarity: there are six α-subunits, three β, three γ, three ρ and single representatives of δ, ɛ, θ and π. The precise subunit isoform composition of the oligomer defines the recognition and biophysical characteristics of the particular receptor subtype. The most ubiquitous subtype, which accounts for approximately 30% of GABA_A receptors in the mammalian brain [95], contains two α₁-, two β₂- and a single γ₂-subunit [30].

The GABA_A receptors can be divided into three structural domains. First, the large extracellular domain carries the recognition sites for both the natural agonist GABA and the clinically important benzodiazepines [3], [27], [96]. Secondly, the transmembrane domain forms the ion channel and contains the channel gate [4], in addition to an intra-helical hydrophobic pocket that is important for the interaction of these receptors with intravenous and volatile anaesthetics [39]. Finally, the intracellular domain contains portals through which ions access the cell cytoplasm. These portals appear to have a significant impact on channel conductance in other members of this family, i.e., the 5HT₃ receptors [47] and the neuronal nAChRs [37].

The three-dimensional structure of the GABA_A receptor has not yet been determined experimentally. A number of homology models have been constructed recently and these have been based on both the low resolution structure of the homologous Torpedo nAChR, obtained by cryoelectron microscopy [60], [90] and the X-ray structures of related acetylcholine binding proteins (AChBP) [11], [16], [17], [18], [38], which are homologous to the extracellular domain of this receptor family. Here we have generated one such model using the most recently published structural information of the nAChR at 4 Å resolution [90]. This has allowed us to produce a homology model of the most common form of the GABA_A receptor ((α₁)₂(β₂)₂γ₂) that includes the extracellular, membrane-spanning and intracellular domains.

We have used this model (referred to here as model 1) to explore, for the first time, the ion passage through the complete receptor from the extracellular to the intracellular space. While recognising the restricted resolution of the side chains in several segments of the total structure, we have used this model to dock a number of ligands, having well-characterised recognition properties, to this particular GABA_A receptor subtype. The recognition domains have been explored further by refining the docking results using either calculations of site electrostatic potentials or studies of the interactions of alternative conformers of the ligand that have known differences in their activity.

In addition, we have produced a second model (model 2) from more recently available data in which other homologous AChBPs have been crystallised in the presence of different ligands [16], [17], [18], [38]. It is clear from these reports that the conformation of the receptor is distinct when occupied with agonist. Our data suggest that our model 1 is the apo-form of the receptor, while model 2 may represent an agonist-bound receptor complex that favours the open-channel conformation of the receptor. We have used model 2 to explore potential interactions that may occur only in the activated receptor.

Comparison of our models with hypotheses that have been developed for the nAChR in relation to the link between agonist recognition and channel gating [82], [99] suggests that analogous interactions within the GABA_A receptors are quite distinct. The purpose of this work has been to generate GABA_A receptor models which can be tested experimentally and thus allow their further refinement.

Section snippets

Comparative modelling

The subunit composition we used for the GABA_A receptor model has the stoichiometry (α₁)₂(β₂)₂γ₂. This is the most common receptor subtype in the human central nervous system [5], [70].

The amino acid sequences of the α₁- (P14867), β₂- (P47870) and γ₂- (P18507) subunits of the human GABA_A receptor were taken from the UniProt [98]. A multiple alignment of these sequences, together with homologues collected by BLAST [1], was generated by MAFFT [46]. Putative transmembrane helices were predicted by

Structure of the two models

Model 1 is shown in Fig. 4, Fig. 5. As it was based on the unliganded form of nAChR (2BG9), it is expected to present an apo-form. Since the majority of the templates for model 2 had the agonist binding sites occupied, they are expected to represent the agonist-bound form. For the different extracellular subunits, the MaxSub scores are follows:

Subunit	MaxSub score	Residues with RMS = 0
β₂ (chain A)	0.995	218/319 (56%)
γ₂ (chain B)	1.000	189/189 (100%)
α₁ (chain C)	0.986	210/213 (99%)
β₂ (chain D)	0.991	217/319

Discussion

There have been a number of attempts to create a model for the GABA_A receptor. The first attempt to model the GABA_A receptor was carried out by Yamakura et al. [100] using low-resolution structural data. O’Mara et al. [67] used the structure of the L. stagnalis acetylcholine-binding protein to model the extracellular domain of a GABA_A receptor model, and the structure of the nAChR transmembrane domain to model its transmembrane domain. They used this model for Brownian dynamics simulations.

Acknowledgements

The authors thank Erika Palin for help with processing data from the Cambridge Structural Database. YM is a recipient of a PhD scholarship from the Algerian Ministry of Higher Education and Scientific Research, MESRS. VNB holds a European Commission Marie Curie Fellowship. This work was partly supported by grants from the Canadian Institutes of Health Research (SMJD and ILM) and the Wellcome Trust (ILM).

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¹: These authors contributed equally to this work.

²: Present address: National Institute of Biomedical Innovation, 7-6-8 Saito Asagi, Ibaraki, Osaka 567-0085, Japan.

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Exploring ligand recognition and ion flow in comparative models of the human GABA type A receptor

Abstract

Introduction

Section snippets

Comparative modelling

Structure of the two models

Discussion

Acknowledgements

J. Mol. Biol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Neuron

J. Mol. Struct. (THEOCHEM)

Neuropharmacology

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Trends Pharmacol. Sci.

J. Mol. Biol.

J. Mol. Biol.

Drug Discov. Today

J. Mol. Biol.

Chem. Biol.

J. Biol. Chem.

Biophys. J.

Neuroscience

Adv. Protein Chem.

J. Mol. Biol.

J. Mol. Biol.

Eur. J. Pharmacol.

Chem. Phys. Lett.

J. Mol. Biol.

J. Biol. Chem.

Neuropharmacology

J. Theor. Biol.

Chem. Phys. Lett.

J. Mol. Biol.

Eur. J. Pharmacol.

Drug Discov. Today

J. Biol. Chem.

J. Biol. Chem.

Two tyrosine residues on the α subunit are crucial for benzodiazepine binding and allosteric modulation of γ-aminobutyric acidA receptor

Mol. Pharmacol.

GABAA receptor needs two homologous domains of the β-subunit for activation by GABA but not by pentobarbital

Nature

The α7 nicotinic acetylcholine receptor: molecular modelling, electrostatics and energetics

Mol. Membr. Biol.

International Union of Pharmacology. XV. Subtypes of γ-aminobutyric acidA receptors: classification on the basis of subunit structure and receptor function

Pharmacol. Rev.

Cation-π interactions in ligand recognition by serotonergic (5-HT3A) and nicotinic acetylcholine receptors: the anomalous binding properties of nicotine

Biochemistry

Quinazolines and 1,4-benzodiazepines. 92. Conformational recognition of the receptor by 1,4-benzodiazepines

Mol. Pharmacol.

Mapping the agonist binding site of the GABAA receptor: evidence for a β-strand

J. Neurosci.

Structural features of muscimol, a potent GABAA receptor agonist. Crystal structure and quantum chemical ab initio calculations

Struct. Chem.

Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors

Nature

Residues at positions 206 and 209 of the α1-subunit of γ-aminobutyric acidA receptors influence affinities for benzodiazepine binding site ligands

Mol. Pharmacol.

A point mutation in the γ2-subunit of the γ-aminobutyric acid type A receptors result in altered benzodiazepine binding specificity

Proc. Natl. Acad. Sci. USA

Structures of 7-bromo-1,3-dihydro-5-(2-pyridyl)-2H-1,4-benzodiazepin-2-one (bromazepam, C14H10BrN3O) and 5-(2-fluorophenyl)-1,3-dihydro-1-methyl-7-nitro-2H-1,4-benzodiazepin-2-one (flunitrazepam, C16H12FN3O3)

Acta Crystallogr.

Crystal structure of nicotinic acetylcholine receptor homolog AChBP in complex with an alpha-conotoxin PnIA variant

Nat. Struct. Mol. Biol.

Molecular recognition: 3D surface structure comparison by gnomonic projection

J. Mol. Graph.

Electrostatic complementarity between proteins and ligands. 1. Charge disposition, dielectric and interface effects

J. Comput-Aided Mol. Des.

Electrostatic complementarity between proteins and ligands. 2. Ligand moieties

J. Comput.-Aided Mol. Des.

Two tyrosine residues on the α subunit are crucial for benzodiazepine binding and allosteric modulation of γ-aminobutyric acid_A receptor

GABA_A receptor needs two homologous domains of the β-subunit for activation by GABA but not by pentobarbital

International Union of Pharmacology. XV. Subtypes of γ-aminobutyric acid_A receptors: classification on the basis of subunit structure and receptor function

Cation-π interactions in ligand recognition by serotonergic (5-HT_3A) and nicotinic acetylcholine receptors: the anomalous binding properties of nicotine

Mapping the agonist binding site of the GABA_A receptor: evidence for a β-strand

Structural features of muscimol, a potent GABA_A receptor agonist. Crystal structure and quantum chemical ab initio calculations

Residues at positions 206 and 209 of the α₁-subunit of γ-aminobutyric acid_A receptors influence affinities for benzodiazepine binding site ligands

A point mutation in the γ₂-subunit of the γ-aminobutyric acid type A receptors result in altered benzodiazepine binding specificity

Structures of 7-bromo-1,3-dihydro-5-(2-pyridyl)-2H-1,4-benzodiazepin-2-one (bromazepam, C₁₄H₁₀BrN₃O) and 5-(2-fluorophenyl)-1,3-dihydro-1-methyl-7-nitro-2H-1,4-benzodiazepin-2-one (flunitrazepam, C₁₆H₁₂FN₃O₃)