Functional characterization of ivermectin binding sites in α1β2γ2L gaba(A) receptors

GABAA receptors (GABAARs) are the major inhibitory neurotransmitter receptors in the brain and are therapeutic targets for many indications including sedation, anesthesia and anxiolysis. There is, however, considerable scope for the development of new therapeutics with improved beneficial effects and reduced side-effect profiles. The anthelminthic drug, ivermectin, activates the GABAAR although its binding site is not known. The molecular site of action of ivermectin has, however, been defined by crystallography in the homologous glutamate-gated chloride channel. Resolving the molecular mechanisms of ivermectin binding to α1β2γ2L GABAARs may provide insights into the design of improved therapeutics. Given that ivermectin binds to subunit interfaces, we sought to define (1) which subunit interface sites it binds to, (2) whether these sites are equivalent in terms of ivermectin sensitivity or efficacy, and (3) how many must be occupied for maximal efficacy. Our approach involved precluding ivermectin from binding to particular interfaces by introducing bulky M3 domain 36'F sidechains to the "+" side of those interfaces. We thereby demonstrated that ivermectin produces irreversible channel activation only when it binds to the single γ2L-β2 interface site. When it binds to α1-β2 sites it elicits potentiation of GABA-gated currents but has no irreversible activating effect. Ivermectin cannot bind to the β2-α1 interface site due to its endogenous bulky 36' methionine. Replacing this with an alanine creates a functional site at this interface, but surprisingly it is inhibitory. Molecular docking simulations reveal that the γ2L-β2 interface forms more contacts with ivermectin than the other interfaces, possibly explaining why ivermectin appears to bind irreversibly at this interface. This study demonstrates unexpectedly stark pharmacological differences among GABAAR ivermectin binding sites

Glycine Receptor Drug Discovery

Postsynaptic glycine receptor (GlyR) chloride channels mediate inhibitory neurotransmission in the spinal cord and brain stem, although presynaptic and extrasynaptic GlyRs are expressed more widely throughout the brain. In humans, GlyRs are assembled as homo- or heteromeric pentamers of α1-3 and β subunits. GlyR malfunctions have been linked to a range of neurological disorders including hyperekplexia, temporal lobe epilepsy, autism, breathing disorders, and chronic inflammatory pain. Although it is possible that GlyRs may eventually be clinically targeted for a variety of neurological disorders, most research to date has focused on developing GlyR-targeted treatments for chronic pain. Inflammatory pain sensitization is caused by inflammatory mediators downregulating the magnitude of α3 GlyR-mediated inhibitory postsynaptic currents in spinal nociceptive neurons. Consistent with this paradigm, it is now well established that the selective enhancement of α3 GlyR current magnitude is effective in alleviating inflammatory pain. In this review, we briefly describe the physiological roles and pharmacological properties of GlyRs. We then outline the methods commonly used to discover new GlyR-active compounds and review recent progress, in our laboratory and elsewhere, in developing GlyR-targeted analgesics. We conclude that the eventual development of an α3 GlyR-targeted analgesic is an eminently feasible goal. However, in selecting or designing new therapeutic leads, we caution against the automatic exclusion of compounds with potentiating effects on α1 GlyRs. Also, as GlyRs are strongly potentiated by Zn at nanomolar concentrations, we also caution against the identification of false positives caused by contaminating Zn in otherwise pure compound samples

Dipicrylamine modulates GABAρ1 receptors through interactions with residues in the TM4 and Cys-loop domains

Dipicrylamine (DPA) is a commonly used acceptor agent in Förster resonance energy transfer experiments that allows the study of high-frequency neuronal activity in the optical monitoring of voltage in living cells. However, DPA potently antagonizes GABAA receptors that contain a1 and b2 subunits by a mechanism which is not clearly understood. In this work, we aimed to determine whether DPA modulation is a general phenomenon of Cys-loop ligand-gated ion channels (LGICs), and whether this modulation depends on particular amino acid residues. For this, we studied the effects of DPA on human homomeric GABAr1, a7 nicotinic, and 5-HT3A serotonin receptors expressed in Xenopus oocytes. Our results indicate that DPA is an allosteric modulator of GABAr1 receptors with an IC50 of 1.6 mM, an enhancer of a7 nicotinic receptors at relatively high concentrations of DPA, and has little, if any, effect on 5-HT3A receptors. DPA antagonism of GABAr1 was strongly enhanced by preincubation, was slightly voltage-dependent, and its washout was accelerated by bovine serum albumin. These results indicate that DPA modulation is not a general phenomenon of LGICs, and structural differences between receptors may account for disparities in DPA effects. In silico modeling of DPA docking to GABAr1, a7 nicotinic, and 5-HT3A receptors suggests that a hydrophobic pocket within the Cys-loop and the M4 segment in GABAr1, located at the extracellular/membrane interface, facilitates the interaction with DPA that leads to inhibition of the receptor. Functional examinations of mutant receptors support the involvement of the M4 segment in the allosteric modulation of GABAr1 by DPA

Rebuilding a macromolecular membrane complex at the atomic scale: case of the Kir6.2 potassium channel coupled to the muscarinic acetylcholine receptor M2

Ion channel-coupled receptors (ICCR) are artificial proteins built from a G protein-coupled receptor and an ion channel. Their use as molecular biosensors is promising in diagnosis and high-throughput drug screening. The concept of ICCR was initially validated with the combination of the muscarinic receptor M2 with the inwardly rectifying potassium channel Kir6.2. A long protein engineering phase has led to the biochemical characterization of the M2-Kir6.2 construct. However, its molecular mechanism remains to be elucidated. In particular, it is important to determine how the activation of M2 by its agonist acetylcholine triggers the modulation of the Kir6.2 channel via the M2-Kir6.2 linkage. In the present study, we have developed and validated a computational approach to rebuild models of the M2-Kir6.2 chimera from the molecular structure of M2 and Kir6.2. The protocol was first validated on the known protein complexes of the μ-opioid Receptor, the CXCR4 receptor and the Kv1.2 potassium channel. When applied to M2-Kir6.2, our protocol produced two possible models corresponding to two different orientations of M2. Both models highlights the role of the M2 helices I and VIII in the interaction with Kir6.2, as well as the role of the Kir6.2 N-terminus in the channel opening. Those two hypotheses will be explored in a future experimental study of the M2-Kir6.2 construct

Probing the structural mechanism of partial agonism in glycine receptors using the fluorescent artificial amino acid, ANAP

The efficacy of an agonist at a pentameric ligand-gated ion channel is determined by the rate at which it induces a conformational change from the resting closed state to a preopen ("flip") state. If the ability of an agonist to promote this isomerization is sufficiently low, then it becomes a partial agonist. As partial agonists at pentameric ligand-gated ion channels show considerable promise as therapeutics, understanding the structural basis of the resting-flip-state isomerization may provide insight into therapeutic design. Accordingly, we sought to identify structural correlates of the resting-flip conformational change in the glycine receptor chloride channel. We used nonsense suppression to introduce the small, fluorescent amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (ANAP), into specific sites in the extracellular and transmembrane domains. Then, under voltage-clamp conditions in Xenopus oocytes, we simultaneously quantified current and fluorescence responses induced by structurally similar agonists with high, medium, and low efficacies (glycine, β-alanine, and taurine, respectively). Analyzing results from nine ANAP-incorporated sites, we show that glycine receptor activation by agonists with graded efficacies manifests structurally as correspondingly graded movements of the β1β2 loop, the β8 β9 loop, and the Cys-loop from the extracellular domain and the TM2-TM3 linker in the transmembrane domain. We infer that the resting-flip transition involves an efficacy-dependent molecular reorganization at the extracellular-transmembrane domain interface that primes receptors for efficacious opening

Effects of glutamate and ivermectin on single glutamate-gated chloride channels of the parasitic nematode <i>H</i>. <i>contortus</i>

<div><p>Ivermectin (IVM) is a widely-used anthelmintic that works by binding to and activating glutamate-gated chloride channel receptors (GluClRs) in nematodes. The resulting chloride flux inhibits the pharyngeal muscle cells and motor neurons of nematodes, causing death by paralysis or starvation. IVM resistance is an emerging problem in many pest species, necessitating the development of novel drugs. However, drug optimisation requires a quantitative understanding of GluClR activation and modulation mechanisms. Here we investigated the biophysical properties of homomeric α (avr-14b) GluClRs from the parasitic nematode, <i>H</i>. <i>contortus</i>, in the presence of glutamate and IVM. The receptor proved to be highly responsive to low nanomolar concentrations of both compounds. Analysis of single receptor activations demonstrated that the GluClR oscillates between multiple functional states upon the binding of either ligand. The G36’A mutation in the third transmembrane domain, which was previously thought to hinder access of IVM to its binding site, was found to decrease the duration of active periods and increase receptor desensitisation. On an ensemble macropatch level the mutation gave rise to enhanced current decay and desensitisation rates. Because these responses were common to both glutamate and IVM, and were observed under conditions where agonist binding sites were likely saturated, we infer that G36’A affects the intrinsic properties of the receptor with no specific effect on IVM binding mechanisms. These unexpected results provide new insights into the activation and modulatory mechanisms of the <i>H</i>. <i>contortus</i> GluClRs and provide a mechanistic framework upon which the actions of drugs can be reliably interpreted.</p></div

Ensemble glutamate-induced activation properties of GluClRs.

<p><b>A)</b> Superimposed recordings revealing the effects of 50 ms (above) or 500 ms (below) applications of indicated glutamate concentrations onto macropatches expressing multiple GluClRs. <b>B)</b> Mean glutamate concentration-response relationship of peak currents as determined by fast agonist application. The curve represents a Hill equation fit with an EC₅₀ of 43 μM. <b>C)</b> Normalised currents showing the concentration dependence of the activation phase of the current. The activation phase of each current trace was fitted to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006663#ppat.1006663.e002" target="_blank">Eq 2</a>. <b>D)</b> Mean glutamate concentration-response relationship of the activation rate (k_act). The curve represents a Hill equation fit with an EC₅₀ of 0.95 μM. The numbers with arrows in A and C are the glutamate concentrations (in μM) that correspond to the currents. The arrows point to the peak current in A and the corresponding current onset in C. The data in B and D are means from 6–15 patches.</p

Comparison of the effect of 1 mM glutamate on wild-type and G36’A mutant GluClRs.

<p><b>A)</b> Examples of continuous single channel activity recorded from G36’A mutant GluClRs. Note the emergence of a ‘spiky’ activation mode (red boxes) that is not observed in wild-type GluClRs. Wild-type-like activations are termed ‘mode 1’ or ‘high Po’, whereas spiky activations are termed ‘mode 2’ or ‘low Po’. <b>B)</b> Examples of continuous single channel activity recorded from wild-type GluClRs included for comparison. <b>C)</b> Comparison of mean active durations (upper panel) and Po (lower panel) of low (LP_O) and high (HP_O) P_O events recorded from G36’A mutant GluClRs (n = 6 patches). <b>D)</b> Examples of activations demarcated by a grey bar in A and B. These activations are of the high P_O mode for the G36’A mutant (above) and normal mode for wild-type (below). The comparison indicates that there are more numerous open-shut events within the activations of G36’A compared to wild-type. <b>E)</b> Shut and open dwell histograms for data obtained from G36’A mutant GluClRs at 1 mM glutamate. This plot combined LP_O and HP_O activations of G36’A receptors at 1 mM glutamate. The histograms show that the mutant receptors have two shut and three open components. <b>F)</b> Shut and open dwell histograms for data obtained from wild-type GluClRs at 1 mM glutamate, revealing two shut and three open components.</p

Concentration-dependence of glutamate effects on GluClRs.

<p><b>A)</b> Examples of how intra-activation open probability (P_O) increased as glutamate concentration was increased. In the absence of glutamate single receptor activity was negligible. Activations of similar duration were selected to facilitate comparison. <b>B)</b> Effect of glutamate concentration on the time constants of the long (red symbols) and short (black symbols) shut-state dwell components. <b>C)</b> Effect of glutamate concentration on time constants of the open-state dwell components. The symbols denote the long (green symbols), intermediate (red symbols) and short (black symbols) time constants Note the disappearance of the longest open component and the reduction in length of the shorter open components at nanomolar glutamate. <b>D)</b> Mean intra-activation open probability (P_O) plotted as a function of glutamate concentration. The curve represents a Hill equation fit with an EC₅₀ of 70 nM. <b>E)</b> Mean active period duration plotted as a function of glutamate concentration. The curve represents a Hill equation fit with an EC₅₀ of 31.2 μM. The data in B-E are means from 3–12 patches (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006663#ppat.1006663.s002" target="_blank">S2 Table</a>).</p

Macropatch deactivation and desensitisation.

<p>Macropatch deactivation and desensitisation.</p