Torpedo nAChR Research Articles

New insights into the effects of organometallic ruthenium complexes on nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChRs) are expressed in excitable and non-excitable cells of the organism. Extensive studies suggest that nAChR ligands have therapeutic potential, notably for neurological and psychiatric disorders. Organometallic ruthenium complexes are known to inhibit several medically important enzymes such as cholinesterases. In addition, they can also interact with muscle- and neuronal-subtype nAChRs. The present study aimed to investigate the direct effects of three organometallic ruthenium complexes, [(η6-p-cymene)Ru(II)(5-nitro-1,10-phenanthroline)Cl]Cl (C1–Cl), [(η6-p-cymene)Ru(II)(1-hydroxypyridine-2(1H)-thionato)Cl] (C1a) and [(η6-p-cymene)Ru(II)(1-hydroxy-3-methoxypyridine-2(1H)-thionato)pta]PF6 (C1), on muscle-subtype (Torpedo) nAChRs and on the two most abundant human neuronal-subtype nAChRs in the CNS (α4β2 and α7) expressed in Xenopus laevis oocytes, using the two-electrode voltage-clamp. The results show that none of the three compounds had agonistic activity on any of the nAChR subtypes studied. In contrast, C1–Cl reversibly blocked Torpedo nAChR (half-reduction of ACh-evoked peak current amplitude by 332 nM of compound). When tested at 10 μM, C1–Cl was statistically more potent to inhibit TorpedonAChR than α4β2 and α7 nAChRs. Similar results of C1 effects were obtained on Torpedo and α4β2 nAChRs, while no action of the compound was detected on α7 nAChRs. Finally, the effects of C1a were statistically similar on the three nAChR subtypes but, in contrast to C1–Cl and C1, the inhibition was hardly reversible. These results, together with our previous studies on isolated mouse neuromuscular preparations, strongly suggest that C1–Cl is, among the three compounds studied, the only molecule that could be used as a potential myorelaxant drug.

From Peptides to Receptors

—In the 1960s and 1970s, the Institute of Chemistry of Natural Compounds developed a topochemical approach for designing new biologically active peptide compounds, the applicability of which to the creation of inhibitors and effective substrates of proteolytic enzymes was shown by the author of this review under the direct supervision of V.T. Ivanov. The next task was to establish the conformation of protein neurotoxins from snake venoms and to study the topography of their binding to the target, the nicotinic acetylcholine receptor (nAChR) from the electric organ of the Torpedo marmorata ray. With selectively labeled derivatives containing one fluorescent or spin label on established amino acid residues, neurotoxin residues in contact with nAChR were identified for the first time. Later, in collaboration with the laboratory of V.T. Ivanov, new analogs of α-conotoxins (peptide neurotoxins from venomous Conus mollusks), were synthesized including their photoactivated derivatives, which showed the participation of all Torpedo nAChR subunits in the binding of α-conotoxins. In the final part, the review briefly presents the recent achievements of the Department of Molecular Neuroimmune Signaling (headed by V.I. Tsetlin) concerning the isolation and synthesis of new peptide and protein neurotoxins and the study of how they work.

From Peptides to Receptors

In the 1960s and 1970s, the Institute of Chemistry of Natural Compounds developed a topochemical approach for designing new biologically active peptide compounds, the applicability of which to the creation of inhibitors and effective substrates of proteolytic enzymes was shown by the author of this review under the direct supervision of V.T. Ivanov. The next task was to establish the conformation of protein neurotoxins from snake venoms and to study the topography of their binding to the target, the nicotinic acetylcholine receptor (nAChR) from the electric organ of the Torpedo marmorata ray. With selectively labeled derivatives containing one fluorescent or spin label on established amino acid residues, neurotoxin residues in contact with nAChR were identified for the first time. Later, in collaboration with the laboratory of V.T. Ivanov, new analogues of α-conotoxins (peptide neurotoxins from poisonous mollusks Conus), were synthesized including their photoactivated derivatives, which showed the participation of all Torpedo nAChR subunits in the binding of α‑conotoxins. The final part of the review briefly presents the recent achievements of the Department of Molecular Neuroimmune Signaling (headed by V.I. Tsetlin) concerning the isolation and synthesis of new peptide and protein neurotoxins and the study of their mechanism of action.

Benocyclidine (BTCP) as Non-labelled Reporter Ligand for MS Binding Assays for the PCP Ion Channel Binding Site of the Desensitized Torpedo Nicotinic Acetylcholine Receptor (nAChR).

In this study we present MS Binding Assays for the PCP ion channel binding site of Torpedo californica nicotinic acetylcholine receptor (nAChR) as an alternative to radioligand binding assays. As MS Marker Benocyclidine (BTCP) was employed, found to be more affine (Kd of 84.2 nM) than the radioligands, e. g. [3 H]PCP, used so far in respective binding assays. Based on a highly sensitive and fast LC-ESI-MS/MS method for quantification of BTCP samples, BTCP MS Binding Assays for the PCP ion channel binding site of Torpedo nAChR could be established comprising saturation, kinetic and competition experiments. The affinities obtained in competitive BTCP MS Binding Assays for ligands addressing the PCP ion channel binding site of Torpedo nAChR were in excellent accord with those reported from radioligand experiments. Thus, the new BTCP MS Binding Assays represent a potent and reliable alternative to radioligand binding assays used so far for the characterization of ligand binding to the PCP ion channel binding site of the nAChR.

Interactions of Nereistoxin and Its Analogs with Vertebrate Nicotinic Acetylcholine Receptors and Molluscan ACh Binding Proteins.

Nereistoxin (NTX) is a marine toxin isolated from an annelid worm that lives along the coasts of Japan. Its insecticidal properties were discovered decades ago and this stimulated the development of a variety of insecticides such as Cartap that are readily transformed into NTX. One unusual feature of NTX is that it is a small cyclic molecule that contains a disulfide bond. In spite of its size, it acts as an antagonist at insect and mammalian nicotinic acetylcholine receptors (nAChRs). The functional importance of the disulfide bond was assessed by determining the effects of inserting a methylene group between the two sulfur atoms, creating dimethylaminodithiane (DMA-DT). We also assessed the effect of methylating the NTX and DMA-DT dimethylamino groups on binding to three vertebrate nAChRs. Radioligand receptor binding experiments were carried out using washed membranes from rat brain and fish (Torpedo) electric organ; [3H]-cytisine displacement was used to assess binding to the predominantly high affinity alpha4beta2 nAChRs and [125I]-alpha-bungarotoxin displacement was used to measure binding of NTX and analogs to the alpha7 and skeletal muscle type nAChRs. While the two quaternary nitrogen analogs, relative to their respective tertiary amines, displayed lower α4β2 nAChR binding affinities, both displayed much higher affinities for the Torpedo muscle nAChR and rat alpha7 brain receptors than their respective tertiary amine forms. The binding affinities of DMA-DT for the three nAChRs were lower than those of NTX and MeNTX. An AChBP mutant lacking the C loop disulfide bond that would potentially react with the NTX disulfide bond displayed an NTX affinity very similar to the parent AChBP. Inhibition of [3H]-epibatidine binding to the AChBPs was not affected by exposure to NTX or MeNTX for up to 24 hr prior to addition of the radioligand. Thus, the disulfide bond of NTX is not required to react with the vicinal disulfide in the AChBP C loop for inhibition of [3H]-epibatidine binding. However, a reversible disulfide interchange reaction of NTX with nAChRs might still occur, especially under reducing conditions. Labeled MeNTX, because it can be readily prepared with high specific radioactivity and possesses relatively high affinity for the nAChR-rich Torpedo nAChR, would be a useful probe to detect and identify any nereistoxin adducts.

Spatial Structure and Activity of Synthetic Fragments of Lynx1 and of Nicotinic Receptor Loop C Models.

Lynx1, membrane-bound protein co-localized with the nicotinic acetylcholine receptors (nAChRs) and regulates their function, is a three-finger protein (TFP) made of three β-structural loops, similarly to snake venom α-neurotoxin TFPs. Since the central loop II of α-neurotoxins is involved in binding to nAChRs, we have recently synthesized the fragments of Lynx1 central loop, including those with the disulfide between Cys residues introduced at N- and C-termini, some of them inhibiting muscle-type nAChR similarly to the whole-size water-soluble Lynx1 (ws-Lynx1). Literature shows that the main fragment interacting with TFPs is the C-loop of both nAChRs and acetylcholine binding proteins (AChBPs) while some ligand-binding capacity is preserved by analogs of this loop, for example, by high-affinity peptide HAP. Here we analyzed the structural organization of these peptide models of ligands and receptors and its role in binding. Thus, fragments of Lynx1 loop II, loop C from the Lymnaea stagnalis AChBP and HAP were synthesized in linear and Cys-cyclized forms and structurally (CD and NMR) and functionally (radioligand assay on Torpedo nAChR) characterized. Connecting the C- and N-termini by disulfide in the ws-Lynx1 fragment stabilized its conformation which became similar to the loop II within the 1H-NMR structure of ws-Lynx1, the activity being higher than for starting linear fragment but lower than for peptide with free cysteines. Introduced disulfides did not considerably change the structure of HAP and of loop C fragments, the former preserving high affinity for α-bungarotoxin, while, surprisingly, no binding was detected with loop C and its analogs.

Sequential purification and characterization of Torpedo californica nAChR-DC supplemented with CHS for high-resolution crystallization studies

Over the past 10 years we have been developing a multi-attribute analytical platform that allows for the preparation of milligram amounts of functional, high-pure, and stable Torpedo (muscle-type) nAChR detergent complexes for crystallization purpose. In the present work, we have been able to significantly improve and optimize the purity and yield of nicotinic acetylcholine receptors in detergent complexes (nAChR-DC) without compromising stability and functionality. We implemented new methods in the process, such as analysis and rapid production of samples for future crystallization preparations. Native nAChR was extracted from the electric organ of Torpedo californica using the lipid-like detergent LysoFos Choline 16 (LFC-16), followed by three consecutive steps of chromatography purification.We evaluated the effect of cholesteryl hemisuccinate (CHS) supplementation during the affinity purification steps of nAChR-LFC-16 in terms of receptor secondary structure, stability and functionality. CHS produced significant changes in the degree of β-secondary structure, these changes compromise the diffusion of the nAChR-LFC-16 in lipid cubic phase. The behavior was reversed by Methyl-β-Cyclodextrin treatment. Also, CHS decreased acetylcholine evoked currents of Xenopus leavis oocyte injected with nAChR-LFC-16 in a concentration-dependent manner. Methyl-β-Cyclodextrin treatment do not reverse functionality, however column delipidation produced a functional protein similar to nAChR-LFC-16 without CHS treatment.

Three-finger proteins from snakes and humans acting on nicotinic receptors: Old and new.

The first toxin to give rise to the three-finger protein (TFP) family was α-bungarotoxin (α-Bgt) from Bungarus multicinctus krait venom. α-Bgt was crucial for research on nicotinic acetylcholine receptors (nAChRs), and in this Review article we focus on present data for snake venom TFPs and those of the Ly6/uPAR family from mammalians (membrane-bound Lynx1 and secreted SLURP-1) interacting with nAChRs. Recently isolated from Bungarus candidus venom, αδ-bungarotoxins differ from α-Bgt: they bind more reversibly and distinguish two binding sites in Torpedo californica nAChR. Naja kaouthia α-cobratoxin, classical blocker of nAChRs, was shown to inhibit certain GABA-A receptor subtypes, whereas α-cobratoxin dimer with 2 intermolecular disulfides has a novel type of 3D structure. Non-conventional toxin WTX has additional 5th disulfide not in the central loop, as α-Bgt, but in the N-terminal loop, like all Ly6/uPAR proteins, and inhibits α7 and Torpedo nAChRs. A water-soluble form of Lynx1, ws-Lynx1, was expressed in E. coli, its 1 H-NMR structure and binding to several nAChRs determined. For SLURP-1, similar information was obtained with its recombinant analogue rSLURP-1. A common feature of ws-Lynx1, rSLURP-1, and WTX is their activity against nAChRs and muscarinic acetylcholine receptors. Synthetic SLURP-1, identical to the natural protein, demonstrated some differences from rSLURP-1 in distinguishing nAChR subtypes. The loop II fragment of the Lynx1 was synthesized having the same µM affinity for the Torpedo nAChR as ws-Lynx1. This review illustrates the productivity of parallel research of nAChR interactions with the two TFP groups.

Molecular interaction studies of endosulfan with the cholinergic pathway targets – An insilico approach

Endosulfan (ED) is a broad-spectrum organochlorine insecticide used around the world in agriculture to control insect pest. ED leaches to soil become persistent in environment. The main ED derivatives found in environmental sources is endosulfan sulfate, which leads both acute and chronic toxicity. The principal target of ED is nervous system. In the present study, the interaction of α-ED with cholinergic pathway proteins and receptors were accessed by insilico methods. Homology modeling of human adult and fetal muscle-type nicotinic acetylcholine receptor (nAChRs) was performed using SWISS-MODEL molecular modeling server with known crystal structure of Torpedo nAChR as a template. Both embryonic and adult nAChR that differ in a single subunit were modeled. Docking studies were carried out on α-ε and α-δ subunit interfaces of human adult nAChR and α-γ and α-δ subunit interfaces of human fetal nAChR with ED. The structure of acetylcholine (ACh), a natural ligand of the modeled receptor was used as a reference compound for docking studies. Docking of ACh and ED with the key enzymes in the pathway acetylcholine esterase (AChE) and docking of choline and ED with choline acetyltransferase (ChAT) were also performed using available molecular modeling software. The results showed that ED binding to all the selected targets, but in the case of modeled human nAChR, ED showed higher affinity towards the modeled fetal nAChR subunit interface than the modeled adult receptor. The ED showed higher binding affinity towards the ChAT. Our results implicated that more toxic effect of ED during the developmental stages.

Photoaffinity Labeling of A4B2 Nicotinic Acetylcholine Receptor using [3H]-Labeled Positive Allosteric Modulators

The α4β2 nicotinic acetylcholine receptor (nAChR) is a potential drug target for treating neuropathological and tobacco use disorders. Positive allosteric modulators (PAMs), which bind o site(s) distinct from the ACh (agonist) binding sites, may provide the required specificity by binding to unique site(s) present only in the α4β2 nAChR. However, the molecular pharmacology of this class of compounds is unclear, and the diversity of PAM binding site(s) have not been determined. In previous work, two radiolabeled α4β2 nAChR PAMs, desformylflustrabromine; ([3H]dFBr, 76 Ci/mmol) and 3-(2-chlorophenyl)-5-(5-methyl-1-(piperidin-4-yl)-1H- pyrrazol-4-yl) isoxazole ([3H]CMPI, 16 Ci/mmol) have been prepared and their merits as photoprobes established using Torpedo nAChR as a model (Hamouda et al. 2015, Mol. Pharmacol. 88:1; Hamouda et al. 2016, Mol. Pharmacol. 89: 575). Here we expand these studies to α4β2 nAChR. Using membrane fractions isolated from a HEK-293 cell line stably expressing α4β2 nAChR (HEK-α4β2 nAChR), we are examining the effects of orthosteric ligands, ion channel blockers and other PAMs on the equilibrium binding of [3H]dFBr and [3H]CMPI. In addition, we purified α4β2 nAChRs from HEK-α4β2 nAChR membranes using a bromo-acetylcholine bromide affinity column and performed initial [3H]dFBr and [3H]CMPI analytical photolabelings. When ∼100 μg of affinity-purified α4β2 nAChR and 20 μCi of [3H]dFBr (76 Ci/mmol) or [3H]CMPI (16 Ci/mmol) were used, photolysis at 312 nm for 5 min ([3H]dFBr) or 3 min ([3H]CMPI) resulted in photolabeling efficiency of (pmol of 3H incorporated/pmol of α4β2 nAChR) of ∼0.05% and 0.7%, respectively. Additional analytical [3H]dFBr and [3H]CMPI photolabeling in the presence of other nAChR ligands and preparative photolabeling experiments are in progress to identify amino acids contributing to dFBr and CMPI binding sites.

Heterogeneous Inhibition in Macroscopic Current Responses of Four Nicotinic Acetylcholine Receptor Subtypes by Cholesterol Enrichment.

The nicotinic acetylcholine receptor (nAChR), located in the cell membranes of neurons and muscle cells, mediates the transmission of nerve impulses across cholinergic synapses. In addition, the nAChR is also found in the electric organs of electric rays (e.g., the genus Torpedo). Cholesterol, which is a key lipid for maintaining the correct functionality of membrane proteins, has been found to alter the nAChR function. We were thus interested to probe the changes in the functionality of different nAChRs expressed in a model membrane with modified cholesterol to phospholipid ratios (C/P). In this study, we examined the effect of increasing the C/P ratio in Xenopus laevis oocytes expressing the neuronal α7, α4β2, muscle-type, and Torpedo californica nAChRs in their macroscopic current responses. Using the two-electrode voltage clamp technique, it was found that the neuronal α7 and Torpedo nAChRs are significantly more sensitive to small increases in C/P than the muscle-type nAChR. The peak current versus C/P profiles during enrichment display different behaviors; α7 and Torpedo nAChRs display a hyperbolic decay with two clear components, whereas muscle-type and α4β2 nAChRs display simple monophasic decays with different slopes. This study clearly illustrates that a physiologically relevant increase in membrane cholesterol concentration produces a remarkable reduction in the macroscopic current responses of the neuronal α7 and Torpedo nAChRs functionality, whereas the muscle nAChR appears to be the most resistant to cholesterol inhibition among all four nAChR subtypes. Overall, the present study demonstrates differential profiles for cholesterol inhibition among the different types of nAChR to physiological cholesterol increments in the plasmatic membrane. This is the first study to report a cross-correlation analysis of cholesterol sensitivity among different nAChR subtypes in a model membrane.

Realtime Single Molecular Motion Analysis of Nicotinic Acetylcholine Receptor Alpha 7 by Diffracted X-Ray Tracking Method

The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel. Binding of ligands (e.g. acetylcholine, nicotine) to nAChR induces ion flux through the channel formed by pentameric subunits, which leads to the changes of cell excitability, and then triggers the following signaling cascades in the peripheral and the central nervous system. In the previous study we applied Diffracted X-ray Tracking (DXT) method to acetylcholine binding protein (AChBP) and Torpedo nAChR. DXT is a method to track the X-ray diffraction spots from the gold nanocrystal labeled on an individual single protein in real time and real space. We observed molecular fluctuations of the proteins even without ligand, and enhancement of tilting and twisting motions with ligand [Sekiguchi, et al. (2014) Sci. Rep. 4: 6384].Here, to acquire more precise information on molecular motions of nAChR in the three sub-states (close, open, and desensitization) and their sate-to-state transitions, we applied the DXT for the neuronal nAChR alpha 7. The alpha 7 receptor in the central nervous system plays essential roles in the cholinergic neuro-signal transduction, and it is one of the drug targets for therapeutics of Alzheimer diseases and cognitive impairment associated with schizophrenia (CIAS). Alpha 7 receptor was recombinantly prepared from the cDNA expressed in Xenopus oocytes. To orderly configure the receptors defined tags were introduced at the defined positions; e.g. “Met tag” for labeling with gold nanocrystal in the N-terminal, and “His tag” for absorption on mica substrate in the second cytoplasmic loop of the receptor. Characteristic molecular motion(s) during the three states of the nAChR alpha 7 were revealed by the DXT analysis. Possible transition modeling will be discussed.

Desformylflustrabromine (dFBr) and [3H]dFBr-Labeled Binding Sites in a Nicotinic Acetylcholine Receptor.

Desformylflustrabromine (dFBr) is a positive allosteric modulator (PAM) of α4β2 and α2β2 nAChRs that, at concentrations >1 µM, also inhibits these receptors and α7 nAChRs. However, its interactions with muscle-type nAChRs have not been characterized, and the locations of its binding site(s) in any nAChR are not known. We report here that dFBr inhibits human muscle (αβεδ) and Torpedo (αβγδ) nAChR expressed in Xenopus oocytes with IC50 values of ∼ 1 μM. dFBr also inhibited the equilibrium binding of ion channel blockers to Torpedo nAChRs with higher affinity in the nAChR desensitized state ([(3)H]phencyclidine; IC50 = 4 μM) than in the resting state ([(3)H]tetracaine; IC50 = 60 μM), whereas it bound with only very low affinity to the ACh binding sites ([(3)H]ACh, IC50 = 1 mM). Upon irradiation at 312 nm, [(3)H]dFBr photoincorporated into amino acids within the Torpedo nAChR ion channel with the efficiency of photoincorporation enhanced in the presence of agonist and the agonist-enhanced photolabeling inhibitable by phencyclidine. In the presence of agonist, [(3)H]dFBr also photolabeled amino acids in the nAChR extracellular domain within binding pockets identified previously for the nonselective nAChR PAMs galantamine and physostigmine at the canonical α-γ interface containing the transmitter binding sites and at the noncanonical δ-β subunit interface. These results establish that dFBr inhibits muscle-type nAChR by binding in the ion channel and that [(3)H]dFBr is a photoaffinity probe with broad amino acid side chain reactivity.

Non-competitive Inhibition of Nicotinic Acetylcholine Receptors by Ladybird Beetle Alkaloids.

Ladybird beetles (Family Coccinellidae) secrete an alkaloid rich venom from their leg joints that protects them from predators. Coccinellines, the major venom constituents, are alkaloids composed of three fused piperidine rings that share a common nitrogen atom. Although many coccinellines have been isolated and chemically characterized, their pharmacological properties are essentially unknown. Using radioligand binding and functional assays we investigated the actions of several coccinellines on skeletal muscle and α7 nicotinic acetylcholine receptors (nAChRs). The alkaloids were shown to displace the specific binding of tritiated piperidyl-N-(1-(2-thienyl)cyclohexyl)-3,4-piperidine ([(3)H]-TCP), which has been shown to bind deep within the ion channel of the electric fish (Torpedo) muscle nAChR. The stereoisomers precoccinelline and hippodamine (whose nitrogens are predicted to be ionized at physiological pH) and their respective analogs N-methyl-precoccinelline and N-methyl-hippodamine (whose quaternary nitrogens are permanently charged) displayed similar IC50s for inhibition of [(3)H]-TCP binding. However, the corresponding precoccinelline and hippodamine N-oxides, coccinelline and convergine (which have an electronegative oxygen bonded to an electropositive nitrogen) displayed significantly higher binding IC50s. Finally, exochomine, a dimeric coccinelline containing the hippodamine structure, displayed the highest IC50 (lowest affinity) for displacing specific [(3)H]-TCP binding. The presence of a desensitizing concentration (10(-3) M) of carbachol (CCh) had little or no effect on the affinity of the Torpedo nAChR for the three coccinellines tested. High concentrations of the coccinellid alkaloids did not affect binding of [(3)H]-cytisine to Torpedo receptor ACh binding sites. Inhibition of the alpha7 nAChR with pre-equilibrated precoccinelline was insurmountable with respect to ACh concentration. We conclude that the coccinellines bind to one or more allosteric sites rather than to the ACh binding sites, and inhibit nAChR responses to ACh through a non-competitive mechanism. Future chemical and pharmacological investigations of other ladybird beetle alkaloids are likely to reveal other interesting alkaloids affecting ligand-gated receptors.

Identifying Barbiturate Binding Sites in a Nicotinic Acetylcholine Receptor with [3H]Allyl m-Trifluoromethyldiazirine Mephobarbital, a Photoreactive Barbiturate

At concentrations that produce anesthesia, many barbituric acid derivatives act as positive allosteric modulators of inhibitory GABAA receptors (GABAARs) and inhibitors of excitatory nicotinic acetylcholine receptors (nAChRs). Recent research on [(3)H]R-mTFD-MPAB ([(3)H]R-5-allyl-1-methyl-5-(m-trifluoromethyldiazirinylphenyl)barbituric acid), a photoreactive barbiturate that is a potent and stereoselective anesthetic and GABAAR potentiator, has identified a second class of intersubunit binding sites for general anesthetics in the α1β3γ2 GABAAR transmembrane domain. We now characterize mTFD-MPAB interactions with the Torpedo (muscle-type) nAChR. For nAChRs expressed in Xenopus oocytes, S- and R-mTFD-MPAB inhibited ACh-induced currents with IC50 values of 5 and 10 µM, respectively. Racemic mTFD-MPAB enhanced the equilibrium binding of [(3)H]ACh to nAChR-rich membranes (EC50 = 9 µM) and inhibited binding of the ion channel blocker [(3)H]tenocyclidine in the nAChR desensitized and resting states with IC50 values of 2 and 170 µM, respectively. Photoaffinity labeling identified two binding sites for [(3)H]R-mTFD-MPAB in the nAChR transmembrane domain: 1) a site within the ion channel, identified by photolabeling in the nAChR desensitized state of amino acids within the M2 helices of each nAChR subunit; and 2) a site at the γ-α subunit interface, identified by photolabeling of γMet299 within the γM3 helix at similar efficiency in the resting and desensitized states. These results establish that mTFD-MPAB is a potent nAChR inhibitor that binds in the ion channel preferentially in the desensitized state and binds with lower affinity to a site at the γ-α subunit interface where etomidate analogs bind that act as positive and negative nAChR modulators.

Assessment of Homology Templates and an Anesthetic Binding Site within the γ-Aminobutyric Acid Receptor

Anesthetics mediate portions of their activity via modulation of the γ-aminobutyric acid receptor (GABAaR). Although its molecular structure remains unknown, significant progress has been made toward understanding its interactions with anesthetics via molecular modeling. The structure of the torpedo acetylcholine receptor (nAChRα), the structures of the α4 and β2 subunits of the human nAChR, the structures of the eukaryotic glutamate-gated chloride channel (GluCl), and the prokaryotic pH-sensing channels, from Gloeobacter violaceus and Erwinia chrysanthemi, were aligned with the SAlign and 3DMA algorithms. A multiple sequence alignment from these structures and those of the GABAaR was performed with ClustalW. The Modeler and Rosetta algorithms independently created three-dimensional constructs of the GABAaR from the GluCl template. The CDocker algorithm docked a congeneric series of propofol derivatives into the binding pocket and scored calculated binding affinities for correlation with known GABAaR potentiation EC50s. Multiple structure alignments of templates revealed a clear consensus of residue locations relevant to anesthetic effects except for torpedo nAChR. Within the GABAaR models generated from GluCl, the residues notable for modulating anesthetic action within transmembrane segments 1, 2, and 3 converged on the intersubunit interface between α and β subunits. Docking scores of a propofol derivative series into this binding site showed strong linear correlation with GABAaR potentiation EC50. Consensus structural alignment based on homologous templates revealed an intersubunit anesthetic binding cavity within the transmembrane domain of the GABAaR, which showed a correlation of ligand docking scores with experimentally measured GABAaR potentiation.

Experimental determination of the vertical alignment between the second and third transmembrane segments of muscle nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChR) are members of the Cys-loop ligand-gated ion channel superfamily. Muscle nAChR are heteropentamers that assemble from two α, and one each of β, γ, and δ subunits. Each subunit is composed of three domains, extracellular, transmembrane and intracellular. The transmembrane domain consists of four α-helical segments (M1-M4). Pioneering structural information was obtained using electronmicroscopy of Torpedo nAChR. The recently solved X-ray structure of the first eukaryotic Cys-loop receptor, a truncated (intracellular domain missing) glutamate-gated chloride channel α (GluClα) showed the same overall architecture. However, a significant difference with regard to the vertical alignment between the channel-lining segment M2 and segment M3 was observed. Here, we used functional studies utilizing disulfide trapping experiments in muscle nAChR to determine the spatial orientation between M2 and M3. Our results are in agreement with the vertical alignment as obtained when using the GluClα structure as a template to homology model muscle nAChR, however, they cannot be reconciled with the current Torpedo nAChR model. The vertical M2-M3 alignments as observed in X-ray structures of prokaryotic Gloeobacter violaceus ligand-gated ion channel and GluClα are in agreement. Our results further confirm that this alignment in Cys-loop receptors is conserved between prokaryotes and eukaryotes.

Identification of Propofol Binding Sites in a Nicotinic Acetylcholine Receptor with a Photoreactive Propofol Analog*

Propofol, a widely used intravenous general anesthetic, acts at anesthetic concentrations as a positive allosteric modulator of γ-aminobutyric acid type A receptors and at higher concentration as an inhibitor of nicotinic acetylcholine receptors (nAChRs). Here, we characterize propofol binding sites in a muscle-type nAChR by use of a photoreactive analog of propofol, 2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol (AziPm). Based upon radioligand binding assays, AziPm stabilized the Torpedo nAChR in the resting state, whereas propofol stabilized the desensitized state. nAChR-rich membranes were photolabeled with [(3)H]AziPm, and labeled amino acids were identified by Edman degradation. [(3)H]AziPm binds at three sites within the nAChR transmembrane domain: (i) an intrasubunit site in the δ subunit helix bundle, photolabeling in the nAChR desensitized state (+agonist) δM2-18' and two residues in δM1 (δPhe-232 and δCys-236); (ii) in the ion channel, photolabeling in the nAChR resting, closed channel state (-agonist) amino acids in the M2 helices (αM2-6', βM2-6' and -13', and δM2-13') that line the channel lumen (with photolabeling reduced by >90% in the desensitized state); and (iii) at the γ-α interface, photolabeling αM2-10'. Propofol enhanced [(3)H]AziPm photolabeling at αM2-10'. Propofol inhibited [(3)H]AziPm photolabeling within the δ subunit helix bundle at lower concentrations (IC50 = 40 μm) than it inhibited ion channel photolabeling (IC50 = 125 μm). These results identify for the first time a single intrasubunit propofol binding site in the nAChR transmembrane domain and suggest that this is the functionally relevant inhibitory binding site.

Lidocaine effects on acetylcholine-elicited currents from mouse superior cervical ganglion neurons

Lidocaine is a commonly used local anaesthetic that, besides blocking voltage-dependent Na+ channels, has multiple inhibitory effects on muscle-type nicotinic acetylcholine (ACh) receptors (nAChRs). In the present study, we have investigated the effects of lidocaine on ACh-elicited currents (IAChs) from cultured mouse superior cervical ganglion (SCG) neurons, which mainly express heteromeric α3β4 nAChRs. Neurons were voltage-clamped by using the perforated-patch method and IAChs were elicited by fast application of ACh (100-300μM), either alone or in presence of lidocaine at different concentrations.IAChs were reversibly blocked by lidocaine in a concentration-dependent way (IC50=41μM; nH close to 1) and the inhibition was, at least partially, voltage-dependent, indicating an open-channel blockade. Besides, lidocaine blocked resting (closed) nAChRs, as evidenced by the increased inhibition caused by a 12s lidocaine application just before its co-application with the agonist, and also enhanced IAChs desensitisation, at concentrations close to the IC50.These results indicate that lidocaine has diverse inhibitory actions on neuronal heteromeric nAChRs resembling those previously reported for Torpedo (muscle-type) nAChRs (Alberola-Die et al., 2011). The similarity of lidocaine actions on different subtypes of heteromeric nAChRs differs with the specific effects of other compounds, restricted to particular subtypes of nAChRs.

Effects of Lipids on the Structure and Function of GLIC and ELIC

The prokaryotic pentameric ligand-gated ion channels (pLGICs), GLIC and ELIC, are excellent models for probing the mechanisms of pLGIC function. We are interested in the mechanisms by which lipids act as allosteric modulators of the prototypic pLGIC, the nicotinic acetylcholine receptor (nAChR) from Torpedo. In mixed phosphatidylcholine (PC) membranes such as those formed from soybean asolectin or those containing neutral and/or anionic lipids, the nAChR adopts an activatable resting conformation. In PC membranes lacking neutral and anionic lipids, the nAChR is stabilized in an uncoupled conformation that binds agonist with resting-state like affinity, but does not undergo conformational transitions. To test whether GLIC and ELIC exhibit similar lipid dependencies, we developed a protocol for reconstituting both GLIC and ELIC into model membranes composed of either soybean asolectin or PC. Both reconstituted pLGICs exhibit secondary structures similar to that of the nAChR, but both have substantially enhanced thermal stabilities, a likely requirement for the formation of diffraction quality crystals. Whereas incorporation of the nAChR into PC membranes leads to an increase in peptide hydrogen exchange relative to the nAChR in asolectin, this was not observed with GLIC and ELIC. In addition, the functional properties of both were explored by two-voltage electrode clamp electrophysiology. Our results suggest that although both GLIC and ELIC exhibit a functional sensitivity to their lipid environments, these sensitivities differ substantially from those of the Torpedo nAChR. These functional differences will be discussed in light of the current structures of these three pLGICs.