β Subunit M2–M3 Loop Conformational Changes Are Uncoupled from α1 β Glycine Receptor Channel Gating: Implications for Human Hereditary Hyperekplexia

Hereditary hyperekplexia, or startle disease, is a neuromotor disorder caused mainly by mutations that either prevent the surface expression of, or modify the function of, the human heteromeric α1 β glycine receptor (GlyR) chloride channel. There is as yet no explanation as to why hyperekplexia mutations that modify channel function are almost exclusively located in the α1 to the exclusion of β subunit. The majority of these mutations are identified in the M2–M3 loop of the α1 subunit. Here we demonstrate that α1 β GlyR channel function is less sensitive to hyperekplexia-mimicking mutations introduced into the M2–M3 loop of the β than into the α1 subunit. This suggests that the M2–M3 loop of the α subunit dominates the β subunit in gating the α1 β GlyR channel. A further attempt to determine the possible mechanism underlying this phenomenon by using the voltage-clamp fluorometry technique revealed that agonist-induced conformational changes in the β subunit M2–M3 loop were uncoupled from α1 β GlyR channel gating. This is in contrast to the α subunit, where the M2–M3 loop conformational changes were shown to be directly coupled to α1 β GlyR channel gating. Finally, based on analysis of α1 β chimeric receptors, we demonstrate that the structural components responsible for this are distributed throughout the β subunit, implying that the β subunit has evolved without the functional constraint of a normal gating pathway within it. Our study provides a possible explanation of why hereditary hyperekplexia-causing mutations that modify α1 β GlyR channel function are almost exclusively located in the α1 to the exclusion of the β subunit

Early Evolution of Ionotropic GABA Receptors and Selective Regimes Acting on the Mammalian-Specific Theta and Epsilon Subunits

BACKGROUND: The amino acid neurotransmitter GABA is abundant in the central nervous system (CNS) of both invertebrates and vertebrates. Receptors of this neurotransmitter play a key role in important processes such as learning and memory. Yet, little is known about the mode and tempo of evolution of the receptors of this neurotransmitter. Here, we investigate the phylogenetic relationships of GABA receptor subunits across the chordates and detail their mode of evolution among mammals. PRINCIPAL FINDINGS: Our analyses support two major monophyletic clades: one clade containing GABA(A) receptor alpha, gamma, and epsilon subunits, and another one containing GABA(A) receptor rho, beta, delta, theta, and pi subunits. The presence of GABA receptor subunits from each of the major clades in the Ciona intestinalis genome suggests that these ancestral duplication events occurred before the divergence of urochordates. However, while gene divergence proceeded at similar rates on most receptor subunits, we show that the mammalian-specific subunits theta and epsilon experienced an episode of positive selection and of relaxed constraints, respectively, after the duplication event. Sites putatively under positive selection are placed on a three-dimensional model obtained by homology-modeling. CONCLUSIONS: Our results suggest an early divergence of the GABA receptor subunits, before the split from urochordates. We show that functional changes occurred in the lineages leading to the mammalian-specific subunit theta, and we identify the amino acid sites putatively responsible for the functional divergence. We discuss potential consequences for the evolution of mammals and of their CNS

Interaction of (-)-reboxetine with nicotinic acetylcholine receptors in different conformational states

The interaction of (-)-reboxetine, a non-tricyclic norepinephrine selective reuptake inhibitor, with muscle-type nicotinic acetylcholine receptors (AChRs) in different conformational states was studied by functional and structural approaches. The results established that (-)-reboxetine: (a) inhibits (±)-epibatidine-induced Ca2+ influx in human (h) muscle embryonic (hα1β1γδ) and adult (hα1β1εδ) AChRs in a non-competitive manner and with potencies IC50 = 3.86 0.49 and 1.92 0.48 M, respectively, (b) binds with ~13-fold higher affinity to the luminal [3H]TCP site when the Torpedo AChR is in the desensitized state compared to the resting state, (c) enhances [3H]cytisine binding to the resting but activatable Torpedo AChR but not to the desensitized AChR, suggesting desensitizing properties. This desensitizing activity is produced in the same concentration range as that for tricyclic antidepressants (TCAs), and (d) interacts with the AChR, where it overlaps the PCP/TCA luminal sites in the resting and desensitized states, but also to non-luminal sites. The non-luminal sites are located at the top of the four transmembrane segments from the Torpedo AChR γ subunit, and whithin the / transmembrane interface on the adult muscle AChR. In conclusion, (-)-reboxetine non-competitively inhibits AChRs by binding to the PCP/TCA luminal site and by inducing receptor desensitization (maybe by interacting with non-luminal sites), a mechanism that is shared by TCAs

The International Journal of Biochemistry & Cell Biology

This work presents the design and synthesis of a series of novel 2-benzylquinuclidine derivatives, comprising 12 methiodide and 11 hydrochloride salts, and their structural and pharmacological characterization at the human (h) alpha 7 and alpha 4 beta 2 nicotinic receptors (nAChRs). The antagonistic potency of these compounds was tested by Ca2+ influx assays on cells expressing the h alpha 7 or h alpha 4 beta 2 nAChR subtype. To determine the inhibitory mechanisms, additional radioligand binding experiments were performed. The results indicate that the methiodides present the highest affinities for the flea nAChR agonist sites, while the same compounds bind preferably to the h alpha 4 beta 2 nAChR ion channel domain. These results indicate that the methiodides are competitive antagonists of the h alpha 7 nAChR but noncompetitive antagonists of the h alpha 4 beta 2 subtype. Docking and molecular dynamics simulations showed that the methiodide derivative 8d binds to the h alpha 7 orthosteric binding sites by forming stable cation-pi interactions between the quaternized quinulinuim moiety and the aromatic box in the receptor, whereas compounds 7j and 8j block the h alpha 4 beta 2 AChR ion channel by interacting with a luminal domain formed between the serine (position 6') and valine (position 13') rings that overlaps the imipramine binding site. (C) 2013 Elsevier Ltd. All rights reserve

Drimane sesquiterpenoids purified from Drimys winteri inhibit the human α4β2 nicotinic acetylcholine receptor by noncompetitive mechanisms

ABSTRACT Background and Purpose The aim of this study is to evaluate the pharmacological activity of four natural drimane sesquiterpenoids on human (h) α4β2 nicotinic acetylcholine receptors (AChRs), the most abundant receptor subtype in the brain. Experimental Approach The drimane sesquiterpenoids, drimenin, cinnamolide, dendocarbin A, and polygodial, were purified from the Canelo tree (Drimys winteri) and chemically characterized by spectroscopic methods. The pharmacological activity of these natural compounds was subsequently determined in vitro on hα4β2 AChRs by Ca2+ influx. To determine the structural components underlying the differences in inhibitory potency, structure-activity relationship, molecular docking and molecular dynamics experiments were performed on the hα4β2 AChR model. Key Results Drimane sesquiterpenoids, except dendocarbin A, inhibit hα4β2 AChRs with the following potency rank order: drimenin ~ cinnamolide > polygodial. The Ca2+ influx and structural results supported the view that these compounds inhibit hα4β2 AChRs in a noncompetitive manner, by interacting mainly with a non-luminal site located at the transmembrane region of the β2 subunit. Conclusion and Implications Drimenin sesquiterpenoids are novel hα4β2 AChR inhibitors. Drimenin could be used as a molecular scaffold for the development of more potent inhibitors with higher selectivity for the hα4β2 AChR. Drimenin derivatives might be developed as novel therapeutic approaches for the treatment of drug addictions, depression, and anxiety

Selectivity of (±)-citalopram at nicotinic acetylcholine receptors and different inhibitory mechanisms between habenular α3β4* and α9α10 subtypes

The inhibitory activity of (±)-citalopram on human (h) α3β4, α4β2, and α7 nicotinic acetylcholine receptors (AChRs) was determined by Ca2+ influx assays, whereas its effect on rat α9α10 and mouse habenular α3β4* AChRs by electrophysiological recordings. The Ca2+ influx results clearly establish that (±)-citalopram inhibits hα3β4 AChRs (5.1 ± 1.3) with higher potency (IC50's in µM) than that for hα7 (18.8 ± 1.1) and hα4β2 (19.1 ± 4.2) AChRs. This is in agreement with the [3H]imipramine competition binding results indicating that (±)-citalopram binds to imipramine sites at desensitized hα3β4 with >2-fold higher affinity than that for hα4β2. The electrophysiological results indicate that (±)-citalopram competitively inhibits rα9α10 AChRs (7.5 ± 0.9) in a voltage-independent manner, whereas it inhibits a homogeneous population of α3β4* AChRs at MHb (VI) neurons (7.6 ± 1.0) in a voltage-dependent manner. The results indicating that citalopram overlaps the imipramine luminal site and inhibits α3β4* AChRs in a voltage-dependent manner, suggest an ion channel blocking mechanism. Both results were in agreement with the conclusions of automatic molecular docking and molecular dynamics experiments. In conclusion, (±)-citalopram inhibits α3β4 and α9α10 AChRs with higher potency compared to other AChRs but by different mechanisms. (±)-Citalopram also inhibits α3β4*AChRs expressed in MHb (VI) neurons, supporting the notion that these receptors are important endogenous targets related to their anti-addictive activities

Tricyclic antidepressants inhibit hippocampal α7*and α9α10 nicotinic acetylcholine receptors by different mechanisms

The activity of tricyclic antidepressants (TCAs) at α7 and α9α10 nicotinic acetylcholine receptors (AChRs) as well as at hippocampal α7-containing (i.e., α7*) AChRs is determined by using Ca2+ influx and electrophysiological recordings. To determine the inhibitory mechanisms, additional functional tests and molecular docking experiments are performed. The results established that TCAs (a) inhibit Ca2+ influx in GH3-α7 cells with the following potency (IC50 in µM) rank: amitriptyline (2.7 ± 0.3) > doxepin (5.9 ± 1.1) ~ imipramine (6.6 ± 1.0). Interestingly, imipramine inhibits hippocampal α7* AChRs (42.2 ± 8.5 μM) in a noncompetitive and voltage-dependent manner, whereas it inhibits α9α10 AChRs (0.53 ± 0.05 µM) in a competitive and voltage-independent manner, and (b) inhibit [3H]imipramine binding to resting α7 AChRs with the following affinity rank (IC50 in μM): imipramine (1.6 ± 0.2) > amitriptyline (2.4 ± 0.3) > doxepin (4.9± 0.6), whereas imipramine’s affinity was no significantly different to that for the desensitized state. The molecular docking and functional results support the notion that imipramine noncompetitively inhibits α7 AChRs by interacting with two overlapping luminal sites, whereas it competitively inhibits α9α10 AChRs by interacting with the orthosteric sites. Collectively our data indicate that TCAs inhibit α7, α9α10, and hippocampal α7* AChRs at clinically relevant concentrations and by different mechanisms of action

The actions of chloride channel blockers, barbiturates and a benzodiazepine on Caenorhabditis elegans glutamate- and ivermectin-gated chloride channel subunits expressed in Xenopus oocytes

The pharmacology of Caenorhabditis elegans glutamate-gated chloride (GluCl) channels was determined by making intracellular voltage-clamp recordings from Xenopus oocytes expressing GluCl subunits. As previously reported (Cully et al. 1994), GluClalpha1beta responded to glutamate (in a picrotoxin sensitive manner) and ivermectin, while GluClbeta responded only to glutamate and GluClalpha1 only to ivermectin. This assay was used to further investigate the action of chloride channel compounds. The arylaminobenzoate, NPPB, reduced the action of glutamate on the heteromeric GluClalpha1beta channel (IC(50) 6.03 ± 0.81 µM). The disulphonate stilbene, DNDS, blocked the effect of both glutamate and ivermectin on GluClalpha1beta channels, the action of glutamate on GluClbeta subunits, and the effect of ivermectin on GluClalpha1 subunits (IC(50)s 1.58-3.83 µM). Surprisingly, amobarbital and pentobarbital, otherwise known as positive allosteric modulators of ligand-gated chloride channels, acted as antagonists. Both compounds reduced the action of glutamate on the GluClalpha1beta heteromer (IC(50)s of 2.04 ± 0.5 and 17.56 ± 2.16 µM, respectively). Pentobarbital reduced the action of glutamate on the GluClbeta homomeric subunit with an IC(50) of 0.59 ± 0.09 µM, while reducing the responses to ivermectin on both GluClalpha1beta and GluClalpha1 with IC(50)s of 8.7 ± 0.5 and 12.9 ± 2.5 µM, respectively. For all the antagonists, the mechanism is apparently non-competitive. The benzodiazepine, flurazepam had no apparent effect on these glutamate- and ivermectin-gated chloride channel subunits. Thus, arylaminobenzoates, disulphonate stilbenes, and barbiturates are non-competitive antagonists of C. elegans GluCl channels