Timothy I. Webb

Molecular Pharmacology of the Glycine Receptor Chloride Channel

The glycine receptor (GlyR) Cl(-) channel belongs to the cysteine-loop family of ligand-gated ion channel receptors. It is best known for mediating inhibitory neurotransmission in motor and sensory reflex circuits of the spinal cord, although glycinergic synapses are also present in the brain stem, cerebellum and retina. Extrasynaptic GlyRs are widely distributed throughout the central nervous system and they are also found in sperm and macrophages. A total of 5 GlyR subunits (alpha1-4 and beta) have been identified. Embryonic receptors comprise alpha2 homomers whereas adult receptors comprise predominantly alpha beta heteromers in a 2:3 stoichiometry. Notably, the alpha3 subunit is present in synaptic GlyRs that mediate inhibitory neurotransmission onto spinal nociceptive neurons. These receptors are specifically inhibited by inflammatory mediators, implying a role for alpha3-containing GlyRs in inflammatory pain sensitisation. Because molecules that increase GlyR current may have clinical potential as muscle relaxant and peripheral analgesic drugs, this review focuses on the molecular pharmacology of GlyR potentiating substances. Of all GlyR potentiating substances identified to date, we conclude that 5HT(3)R antagonists such as tropisetron offer the most promise as therapeutic lead compounds. However, one problem is that that virtually all known GlyR potentiating compounds, including tropisetron analogues, lack specificity for the GlyR. Another is that almost nothing is known about the pharmacological properties of alpha3-containing GlyRs, which is the subtype of choice for targeting by novel antinociceptive agents. These issues need to be addressed before GlyR-specific therapeutics can be developed.

Molecular Determinants of Ivermectin Sensitivity at the Glycine Receptor Chloride Channel

Background: The ivermectin-binding site on the glutamate-gated chloride channel was recently resolved by crystallography. Results: Ivermectin binds in a similar orientation to the structurally related glycine receptor, although two H-bonds apparent in the crystal structure proved unimportant for binding to glycine receptors. Conclusion: Ivermectin-binding mechanisms vary among Cys-loop receptors. Significance: Understanding ivermectin-binding mechanisms may help in designing new drugs. Ivermectin is an anthelmintic drug that works by activating glutamate-gated chloride channel receptors (GluClRs) in nematode parasites. GluClRs belong to the Cys-loop receptor family that also includes glycine receptor (GlyR) chloride channels. GluClRs and A288G mutant GlyRs are both activated by low nanomolar ivermectin concentrations. The crystal structure of the Caenorhabditis elegans α GluClR complexed with ivermectin has recently been published. Here, we probed ivermectin sensitivity determinants on the α1 GlyR using site-directed mutagenesis and electrophysiology. Based on a mutagenesis screen of transmembrane residues, we identified Ala288 and Pro230 as crucial sensitivity determinants. A comparison of the actions of selamectin and ivermectin suggested the benzofuran C05-OH was required for high efficacy. When taken together with docking simulations, these results supported a GlyR ivermectin binding orientation similar to that seen in the GluClR crystal structure. However, whereas the crystal structure shows that ivermectin interacts with the α GluClR via H-bonds with Leu218, Ser260, and Thr285 (α GluClR numbering), our data indicate that H-bonds with residues homologous to Ser260 and Thr285 are not important for high ivermectin sensitivity or direct agonist efficacy in A288G α1 GlyRs or three other GluClRs. Our data also suggest that van der Waals interactions between the ivermectin disaccharide and GlyR M2–M3 loop residues are unimportant for high ivermectin sensitivity. Thus, although our results corroborate the ivermectin binding orientation as revealed by the crystal structure, they demonstrate that some of the binding interactions revealed by this structure do not pertain to other highly ivermectin-sensitive Cys-loop receptors.

Ircinialactams: subunit-selective glycine receptor modulators from Australian sponges of the family Irciniidae.

Screening an extract library of >2500 southern Australian and Antarctic marine invertebrates and algae for modulators of glycine receptor (GlyR) chloride channels identified three Irciniidae sponges that yielded new examples of a rare class of glycinyl lactam sesterterpene, ircinialactam A, 8-hydroxyircinialactam A, 8-hydroxyircinialactam B, ircinialactam C, ent-ircinialactam C and ircinialactam D. Structure-activity relationship (SAR) investigations revealed a new pharmacophore with potent and subunit selective modulatory properties against alpha1 and alpha3 GlyR isoforms. Such GlyR modulators have potential application as pharmacological tools, and as leads for the development of GlyR targeting therapeutics to treat chronic inflammatory pain, epilepsy, spasticity and hyperekplexia.

High throughput techniques for discovering new glycine receptor modulators and their binding sites

The inhibitory glycine receptor (GlyR) is a member of the Cys-loop receptor family that mediates inhibitory neurotransmission in the central nervous system. These receptors are emerging as potential drug targets for inflammatory pain, immunomodulation, spasticity and epilepsy. Antagonists that specifically inhibit particular GlyR isoforms are also required as pharmacological probes for elucidating the roles of particular GlyR isoforms in health and disease. Although a substantial number of both positive and negative GlyR modulators have been identified, very few of these are specific for the GlyR over other receptor types. Thus, the potential of known compounds as either therapeutic leads or pharmacological probes is limited. It is therefore surprising that there have been few published studies describing attempts to discover novel GlyR isoform-specific modulators. The first aim of this review is to consider various methods for efficiently screening compounds against these receptors. We conclude that an anion sensitive yellow fluorescent protein is optimal for primary screening and that automated electrophysiology of cells stably expressing GlyRs is useful for confirming hits and quantitating the actions of identified compounds. The second aim of this review is to demonstrate how these techniques are used in our laboratory for the purpose of both discovering novel GlyR-active compounds and characterizing their binding sites. We also describe a reliable, cost effective method for transfecting HEK293 cells in single wells of a 384-well plate using nanogram quantities of plasmid DNA.

Sesterterpene glycinyl-lactams: a new class of glycine receptor modulator from Australian marine sponges of the genus Psammocinia

Bioassay guided fractionation of three southern Australian marine sponges of the genus Psammocinia, selected for their ability to modulate glycine-gated chloride channel receptors (GlyRs), yielded the rare marine sesterterpenes (-)-ircinianin (1) and (-)-ircinianin sulfate (2), along with the new biosynthetically related metabolites (-)-ircinianin lactam A (3), (-)-ircinianin lactam A sulfate (4), (-)-oxoircinianin (5), (-)-oxoircinianin lactam A (6) and (-)-ircinianin lactone A (7). Acetylation of 1 returned (-)-ircinianin acetate (8). Whole cell patch-clamp electrophysiology on 1-8 established 3 as an exceptionally potent and selective α3 GlyR potentiator, and 6 as a selective α1 GlyR potentiator. The discovery and characterization of sesterterpenes 1-8, and in particular the glycinyl-lactams 3 and 6, provide valuable new insights into GlyR pharmacology. These insights have the potential to inform and inspire the development of new molecular tools to probe GlyR distribution and function, and therapeutics to treat a wide array of GlyR mediated diseases and disorders.

The M4 transmembrane segment contributes to agonist efficacy differences between alpha1 and alpha3 glycine receptors

To date there are few compounds known to pharmacologically discriminate between α1 and α3 subunit-containing glycine receptors (GlyRs). The present study stemmed from an observation that the glycinergic agonists, taurine and β-alanine, act with much lower agonist efficacy at α3 GlyRs than at α1 GlyRs. We sought to understand the structural basis of this difference to provide insights relevant to the development of α3-specific modulators as leads for the development of new anti-inflammatory analgesics. Using chimeras of α1 and α3 subunits, we identified the structurally divergent M4 transmembrane segment and C-terminal tail as a specific determinant of the efficacy difference. Because mutation of individual non-conserved M4 residues had little influence on agonist efficacies, the reduced agonist efficacy at α3 GlyRs is most likely a distributed effect of all non-conserved M4 residues. Given the lack of contact between M4 and other transmembrane segments, the efficacy differences are probably mediated by differential interactions with the surrounding lipid environment. This may explain why GlyR agonist efficacies differ among expression systems where membrane lipid composition is not conserved. It may also explain why GlyR agonist efficacy increases at high expression densities, as this would increase the propensity of receptors to cluster thereby inducing M4 segments of neighboring receptors to interact. This strong influence of M4 primary structure on partial agonist efficacy suggests that the relatively poorly conserved α3 GlyR M4 segment may be a promising domain to target in the search for α3 GlyR-specific modulators.

Closed-state crosslinking of adjacent β1 subunits in α1β1 GABA-A receptors via 6' introduced cysteines

The pore structural changes associated with Cys-loop receptor gating are currently the subject of considerable interest. Several functional approaches have shown that surface exposure of pore-lining side chains does not change significantly during activation. However, a disulfide trapping study on α1T6′Cβ1T6′C γ-aminobutyric acid type A (GABAA) receptors (GABAARs), which showed that adjacent β subunits cross-link in the open state only, concluded that channel gating is mediated by β subunits contra-rotating through a summed angle of ∼120°. Such a large rotation is not easily reconciled with other evidence. The present study initially sought to investigate an observation that appeared inconsistent with the rotation model: namely that α1T6′Cβ1T6′C GABAARs expressed in HEK293 cells form 6′ cysteine-mediated disulfide bonds in the closed state. On the basis of electrophysiological and Western blotting experiments, we conclude that adjacent βT6′C subunits dimerise efficiently in the closed state via cross-links between their respective 6′ cysteines and that this locks the channels closed. This questions the β subunit contra-rotation model of activation and raises the question of how the closed state cross-links form. We propose that β subunit 6′ cysteines move into sufficiently close proximity for disulfide formation via relatively large amplitude random thermal motions that appear to be a unique feature of β subunits. Because dimerized channels are locked closed, we conclude either that the spontaneous β subunit movements or asymmetries in the movements of adjacent β subunits during activation are essential for pore opening. Our results identify a novel feature of GABAAR gating that may be important for understanding its activation mechanism.

Closed-state Cross-linking of Adjacent β1 Subunits in α1β1 GABAa Receptors via Introduced 6′ Cysteines

The pore structural changes associated with Cys-loop receptor gating are currently the subject of considerable interest. Several functional approaches have shown that surface exposure of pore-lining side chains does not change significantly during activation. However, a disulfide trapping study on α1T6′Cβ1T6′C γ-aminobutyric acid type A (GABAA) receptors (GABAARs), which showed that adjacent β subunits cross-link in the open state only, concluded that channel gating is mediated by β subunits contra-rotating through a summed angle of ∼120°. Such a large rotation is not easily reconciled with other evidence. The present study initially sought to investigate an observation that appeared inconsistent with the rotation model: namely that α1T6′Cβ1T6′C GABAARs expressed in HEK293 cells form 6′ cysteine-mediated disulfide bonds in the closed state. On the basis of electrophysiological and Western blotting experiments, we conclude that adjacent βT6′C subunits dimerise efficiently in the closed state via cross-links between their respective 6′ cysteines and that this locks the channels closed. This questions the β subunit contra-rotation model of activation and raises the question of how the closed state cross-links form. We propose that β subunit 6′ cysteines move into sufficiently close proximity for disulfide formation via relatively large amplitude random thermal motions that appear to be a unique feature of β subunits. Because dimerized channels are locked closed, we conclude either that the spontaneous β subunit movements or asymmetries in the movements of adjacent β subunits during activation are essential for pore opening. Our results identify a novel feature of GABAAR gating that may be important for understanding its activation mechanism.