Superfamily Of Pentameric Ligand-gated Ion Channels Research Articles

GABAA receptor-mediated seizure liabilities: a mixed-methods screening approach

GABAA receptors, members of the pentameric ligand-gated ion channel superfamily, are widely expressed in the central nervous system and mediate a broad range of pharmaco-toxicological effects including bidirectional changes to seizure threshold. Thus, detection of GABAA receptor-mediated seizure liabilities is a big, partly unmet need in early preclinical drug development. This is in part due to the plethora of allosteric binding sites that are present on different subtypes of GABAA receptors and the critical lack of screening methods that detect interactions with any of these sites. To improve in silico screening methods, we assembled an inventory of allosteric binding sites based on structural data. Pharmacophore models representing several of the binding sites were constructed. These models from the NeuroDeRisk IL Profiler were used for in silico screening of a compiled collection of drugs with known GABAA receptor interactions to generate testable hypotheses. Amoxapine was one of the hits identified and subjected to an array of in vitro assays to examine molecular and cellular effects on neuronal excitability and in vivo locomotor pattern changes in zebrafish larvae. An additional level of analysis for our compound collection is provided by pharmacovigilance alerts using FAERS data. Inspired by the Adverse Outcome Pathway framework, we postulate several candidate pathways leading from specific binding sites to acute seizure induction. The whole workflow can be utilized for any compound collection and should inform about GABAA receptor-mediated seizure risks more comprehensively compared to standard displacement screens, as it rests chiefly on functional data.

Duplicated binding site for RIC-3 chaperon protein in serotonin type 3A subunits.

Serotonin or 5-hydroxytryptamine type 3A receptors (5-HT3A) belong to the pentameric ligand-gated ion channel super-family, which have been long-standing therapeutic targets for psychiatric disorders and neurological diseases. Due to structural conservation and significant sequence similarities in the extracellular and transmembrane domains of this family, clinical trials for drug candidates targeting these two domains have been hampered by off-subunit modulation. The intracellular domain of this family, in contrast, exhibits significant diversity in length, amino acid composition, and function, and hence positions itself as a potential drug target. We therefore explored the interaction interface of the 5-HT3A intracellular domain (ICD) with its regulator, the resistance to inhibitors of choline esterase (RIC-3) protein. We have previously shown that RIC-3 interacts with the L1-MX segment of the ICD fused to maltose-binding protein. In this study, using synthetic L1-MX-based peptides, Ala-scanning, and a pull-down assay, we identified motif DWLRXX(X)VLDR, as a critical motif for interaction with RIC-3. This motif is repeated twice within the ICD. One site is within the MX-helix, another site is at the MAM4-helix transition. For both sites, triple-Ala substitutions (MX: W347, R349, and L353; MAM4: W447, R449, and L454) disrupt the interactions between 5-HT3A-ICD-peptide and RIC-3. Complementary studies using full-length 5-HT3A subunits confirmed that the Ala substitutions reduced the RIC-3 mediated modulation of 5-HT3A functional surface expression. We thus identified two binding sites for RIC-3 with a shared duplicated motif in 5-HT3A subunits, one in the MX-helix and one at the MAM4-helix transition.

Identification of the Binding Site of Bupropion on Serotonin Type 3A Receptors

Bupropion is an FDA approved medication marketed as an anti-depressant, smoking-cessation, and weight loss drug. Presently, the known effects of bupropion include inhibition of norepinephrine and dopamine reuptake, as well as inhibition of cation-selective Cys-loop ion channels like nicotinic acetylcholine and serotonin type 3 A (5-HT3A) receptors. The present study focuses on bupropion's inhibitory capacity with regard to the 5-HT3A receptor. 5-HT3R is a member of the pentameric ligand-gated ion channel superfamily, which also includes nicotinic acetylcholine, γ-aminobutyric acid type A (GABAA), and glycine receptors. These channels are made up of five homologous subunits around a central ion channel pore. Each subunit is divided into extracellular, transmembrane, and intracellular domain. Dysfunction within this superfamily has been linked to neurological disorders such as anxiety, depression, epilepsy, Alzheimer's, and Parkinson's Disease. Currently, the 5-HT3receptor is targeted clinically by anti-emetics and irritable bowel syndrome treatments, but the receptor could potentially become a target for the treatment of anxiety, psychosis, bipolar disorder, and several other neurological disorders. We engineered amino acids in the α-helical M2 and M3 transmembrane segments near the M2M3 loop that is located at the interface of transmembrane and extracellular domains. We used docking studies and site-directed mutagenesis to analyze potential binding site residues for bupropion. Two-electrode voltage-clamp recordings were used to examine the effect of mutations on bupropion inhibition of these engineered 5-HT3A channels expressed in Xenopus oocytes.

A Refined Open State of the Glycine Receptor Obtained via Molecular Dynamics Simulations

SummaryPentameric ligand-gated ion channels are key players in mediating fast neurotransmission. Glycine receptors are chloride-selective members of this receptor family that mediate inhibitory synaptic transmission and are implicated in neurological disorders including autism and hyperekplexia. They have been structurally characterized by both X-ray crystallography and cryoelectron microscopy (cryo-EM) studies, with the latter giving rise to what was proposed as a possible open state. However, recent work has questioned the physiological relevance of this open state structure, since it rapidly collapses in molecular dynamics simulations. Here, we show that the collapse can be avoided by a careful equilibration protocol that reconciles the more problematic regions of the original density map and gives a stable open state that shows frequent selective chloride permeation. The protocol developed in this work provides a means to refine open-like structures of the whole pentameric ligand-gated ion channel superfamily and reconciles the previous issues with the cryo-EM structure.

Enantiomeric barbiturates bind distinct inter- and intrasubunit binding sites in a nicotinic acetylcholine receptor (nAChR)

Nicotinic acetylcholine receptors (nAChRs) and γ-aminobutyric acid type A receptors (GABAARs) are members of the pentameric ligand-gated ion channel superfamily. Drugs acting as positive allosteric modulators of muscle-type α2βγδ nAChRs, of use in treatment of neuromuscular disorders, have been hard to identify. However, identification of nAChR allosteric modulator binding sites has been facilitated by using drugs developed as photoreactive GABAAR modulators. Recently, R-1-methyl-5-allyl-5-(m-trifluoromethyl-diazirinylphenyl) barbituric acid (R-mTFD-MPAB), an anesthetic and GABAAR potentiator, has been shown to inhibit Torpedo α2βγδ nAChRs, binding in the ion channel and to a γ+-α- subunit interface site similar to its GABAAR intersubunit binding site. In contrast, S-1-methyl-5-propyl-5-(m-trifluoromethyl-diazirinylphenyl) barbituric acid (S-mTFD-MPPB) acts as a convulsant and GABAAR inhibitor. Photolabeling studies established that S-mTFD-MPPB binds to the same GABAAR intersubunit binding site as R-mTFD-MPAB, but with negative rather than positive energetic coupling to GABA binding. We now show that S-mTFD-MPPB binds with the same state (agonist) dependence as R-mTFD-MPAB within the nAChR ion channel, but it does not bind to the intersubunit binding site. Rather, S-mTFD-MPPB binds to intrasubunit sites within the α and δ subunits, photolabeling αVal-218 (αM1), δPhe-232 (δM1), δThr-274 (δM2), and δIle-288 (δM3). Propofol, a general anesthetic that binds to GABAAR intersubunit sites, inhibited [3H]S-mTFD-MPPB photolabeling of these nAChR intrasubunit binding sites. These results demonstrate that in an nAChR, the subtle difference in structure between S-mTFD-MPPB and R-mTFD-MPAB (chirality; 5-propyl versus 5-allyl) determines selectivity for intra- versus intersubunit sites, in contrast to GABAARs, where this difference affects state dependence of binding to a common site.

Structural Studies of Nicotinic Acetylcholine Receptors: Using Acetylcholine-Binding Protein as a Structural Surrogate.

Nicotinic acetylcholine receptors (nAChRs) are members of the pentameric ligand-gated ion channel superfamily that play important roles in the control of neurotransmitter release in the central and peripheral nervous system. These receptors are important therapeutic targets for the development of drugs against a number of mental health disorders and for marketed smoking cessation aids. Unfortunately, drug discovery has been hampered by difficulties in obtaining sufficiently selective compounds. Together with functional complexity of the receptors, this has made it difficult to obtain drugs with sufficiently high-target to off-target affinity ratios. The recent and ongoing progress in structural studies holds promise to help understand structure-function relationships of nAChR drugs at the atomic level. This will undoubtedly lead to the design of more efficient drugs with fewer side effects. As a high-resolution structure of a nAChR is yet to be determined, structural studies are to a large extent based on acetylcholine-binding proteins (AChBPs) that despite low overall sequence identity display a high degree of conservation of overall structure and amino acids at the ligand-binding site. Further, AChBPs reproduce relative binding affinities of ligands at nAChRs. Over the past decade, AChBPs have been used extensively as models for nAChRs and have aided the understanding of drug receptor interactions at nAChRs significantly.

Crosslinking constraints and computational models as complementary tools in modeling the extracellular domain of the glycine receptor.

The glycine receptor (GlyR), a member of the pentameric ligand-gated ion channel superfamily, is the major inhibitory neurotransmitter-gated receptor in the spinal cord and brainstem. In these receptors, the extracellular domain binds agonists, antagonists and various other modulatory ligands that act allosterically to modulate receptor function. The structures of homologous receptors and binding proteins provide templates for modeling of the ligand-binding domain of GlyR, but limitations in sequence homology and structure resolution impact on modeling studies. The determination of distance constraints via chemical crosslinking studies coupled with mass spectrometry can provide additional structural information to aid in model refinement, however it is critical to be able to distinguish between intra- and inter-subunit constraints. In this report we model the structure of GlyBP, a structural and functional homolog of the extracellular domain of human homomeric α1 GlyR. We then show that intra- and intersubunit Lys-Lys crosslinks in trypsinized samples of purified monomeric and oligomeric protein bands from SDS-polyacrylamide gels may be identified and differentiated by MALDI-TOF MS studies of limited resolution. Thus, broadly available MS platforms are capable of providing distance constraints that may be utilized in characterizing large complexes that may be less amenable to NMR and crystallographic studies. Systematic studies of state-dependent chemical crosslinking and mass spectrometric identification of crosslinked sites has the potential to complement computational modeling efforts by providing constraints that can validate and refine allosteric models.

Interaction of the Glycine Receptor Alpha 1 Binding Site with Partial Agonists

The glycine receptor is a member of the pentameric ligand-gated ion channel superfamily. In vertebrates, it mediates fast inhibitory transmission, particularly in the brainstem and spinal cord. Several alpha subunits have been cloned in man, e.g. α1, α2 and α3, and are likely to form homomeric and heteromeric channels. Each subunit consists of a transmembrane domain, forming the channel pore, a large extracellular domain that contains the binding site and an intracellular domain (Lynch, Neuropharmacology, 56, 303, 2009).Our aim is to gain insight into the structure-function relation of the glycine receptor by characterising their activation mechanism and measuring agonist binding and the conformational changes that the channel undergoes as it activates. For this we use the patch-clamp technique in cell-attached configuration for low noise single-channel recordings. This is followed by an intensive analysis that includes time course fitting idealization and testing of postulated activation mechanisms by direct fitting to the recorded data. A further approach is the use of agonist concentration jumps applied to outside-out configuration patches to assess non equilibrium activation of the channel.Our functional analysis is complemented by computational homology modelling and molecular dynamics that allow us deeper interpretation of our experimental results in regard to the conformational changes of the protein during the initial stages of activation.Here we report a characterization of a range of partial agonists of the glycine receptor carried out to select the best agonist compounds with which to probe the receptor binding site. Promising results were obtained with the partial agonist D-alanine.

Pulsed Electron Spin Resonance Resolves the Coordination Site of Cu 2+ Ions in α1-Glycine Receptor

Herein, we identify the coordination environment of Cu 2+ in the human α1-glycine receptor (GlyR). GlyRs are members of the pentameric ligand-gated ion channel superfamily (pLGIC) that mediate fast signaling at synapses. Metal ions like Zn 2+ and Cu 2+ significantly modulate the activity of pLGICs, and metal ion coordination is essential for proper physiological postsynaptic inhibition by GlyR in vivo. Zn 2+ can either potentiate or inhibit GlyR activity depending on its concentration, while Cu 2+ is inhibitory. To better understand the molecular basis of the inhibitory effect we have used electron spin resonance to directly examine Cu 2+ coordination and stoichiometry. We show that Cu 2+ has one binding site per α1 subunit, and that five Cu 2+ can be coordinated per GlyR. Cu 2+ binds to E192 and H215 in each subunit of GlyR with a 40 μM apparent dissociation constant, consistent with earlier functional measurements. However, the coordination site does not include several residues of the agonist/antagonist binding site that were previously suggested to have roles in Cu 2+ coordination by functional measurements. Intriguingly, the E192/H215 site has been proposed as the potentiating Zn 2+ site. The opposing modulatory actions of these cations at a shared binding site highlight the sensitive allosteric nature of GlyR.