Nicotine exerts its oncogenic effects through the binding to nicotinic acetylcholine receptors (nAChRs) and the activation of downstream pathways that block apoptosis and promote neo-angiogenesis. The nAChRs of the α7 subtype are present on a wide variety of cancer cells and their inhibition by cobra venom neurotoxins has been proposed in several articles and reviews as a potential innovative lung cancer therapy. However, since part of the published results was recently retracted, we believe that the antitumoral activity of cobra venom neurotoxins needs to be independently re-evaluated.
We determined the activity of α-neurotoxins from Naja atra (short-chain neurotoxin, α-cobrotoxin) and Naja kaouthia (long-chain neurotoxin, α-cobratoxin) in vitro by cytotoxicity measurements in 5 lung cancer cell lines, by colony formation assay with α7nAChRs expressing and non-expressing cell lines and in vivo by assessing tumor growth in an orthotopic Non-Obese Diabetic/Severe Combined Immunodeficient (NOD/SCID) mouse model system utilizing different treatment schedules and dosages.
No statistically significant reduction in tumor growth was observed in the treatment arms in comparison to the control for both toxins. Paradoxically α-cobrotoxin from Naja atra showed the tendency to enhance tumor growth although, even in this case, the statistical significance was not reached.
In conclusion our results show that, in contrast with other reports, the nAChR inhibitors α-cobratoxin from N. kaouthia and α-cobrotoxin from N. atra neither suppressed tumor growth nor prolonged the survival of the treated animals.
The nicotinic acetylcholine receptor (nAChR) is a member of the ligand-gated ion channel family and is implicated in many neurological events. Yet, the receptor is difficult to target without high-resolution structures. In contrast, the structure of the acetylcholine binding protein (AChBP) has been solved to high resolution, and it serves as a surrogate structure of the extra-cellular domain in nAChR. Here we conduct a virtual screening study of the AChBP using the relaxed-complex method, which involves a combination of molecular dynamics simulations (to achieve receptor structures) and ligand docking. The library screened through comes from the National Cancer Institute, and its ligands show great potential for binding AChBP in various manners. These ligands mimic the known binders of AChBP; a significant subset docks well against all species of the protein and some distinguish between the various structures. These novel ligands could serve as potential pharmaceuticals in the AChBP/nAChR systems.
acetylcholine binding protein; nicotinic acetylcholine receptor; relaxed-complex; molecular dynamics; docking; virtual screening
Small recombinant antibody fragments (e.g. scFvs and VHHs), which are highly tissue permeable, are being investigated for antivenom production as conventional antivenoms consisting of IgG or F(ab’)2 antibody fragments do not effectively neutralize venom toxins located in deep tissues. However, antivenoms composed entirely of small antibody fragments may have poor therapeutic efficacy due to their short serum half-lives. To increase serum persistence and maintain tissue penetration, we prepared low and high molecular mass antivenom antibodies. Four llama VHHs were isolated from an immune VHH-displayed phage library and were shown to have high affinity, in the low nM range, for α-cobratoxin (α–Cbtx), the most lethal component of Naja kaouthia venom. Subsequently, our highest affinity VHH (C2) was fused to a human Fc fragment to create a VHH2-Fc antibody that would offer prolonged serum persistence. After in planta (Nicotiana benthamiana) expression and purification, we show that our VHH2-Fc antibody retained high affinity binding to α–Cbtx. Mouse α–Cbtx challenge studies showed that our highest affinity VHHs (C2 and C20) and the VHH2-Fc antibody effectively neutralized lethality induced by α–Cbtx at an antibody:toxin molar ratio as low as ca. 0.75×:1. Further research towards the development of an antivenom therapeutic involving these anti-α-Cbtx VHHs and VHH2-Fc antibody molecules should involve testing them as a combination, to determine whether they maintain tissue penetration capability and low immunogenicity, and whether they exhibit improved serum persistence and therapeutic efficacy.
Neonicotinoid insecticides, which act on nicotinic acetylcholine receptors (nAChRs) in a variety of ways, have extremely low mammalian toxicity, yet the molecular basis of such actions is poorly understood. To elucidate the molecular basis for nAChR–neonicotinoid interactions, a surrogate protein, acetylcholine binding protein from Lymnaea stagnalis (Ls-AChBP) was crystallized in complex with neonicotinoid insecticides imidacloprid (IMI) or clothianidin (CTD). The crystal structures suggested that the guanidine moiety of IMI and CTD stacks with Tyr185, while the nitro group of IMI but not of CTD makes a hydrogen bond with Gln55. IMI showed higher binding affinity for Ls-AChBP than that of CTD, consistent with weaker CH–π interactions in the Ls-AChBP–CTD complex than in the Ls-AChBP–IMI complex and the lack of the nitro group-Gln55 hydrogen bond in CTD. Yet, the NH at position 1 of CTD makes a hydrogen bond with the backbone carbonyl of Trp143, offering an explanation for the diverse actions of neonicotinoids on nAChRs.
Acetylcholine binding protein (Lymnaea stagnalis); Crystal structures; Neonicotinoids; Nicotinic acetylcholine receptors; Ion channels
Neuronal nAChRs are a diverse family of pentameric ion channels with wide distribution throughout cells of the nervous and immune systems. However, the role of specific subtypes in normal and pathological states remains poorly understood due to the lack of selective probes. Here, we used a binding assay based on acetylcholine-binding protein (AChBP), a homolog of the nicotinic acetylcholine ligand-binding domain, to discover a novel α-conotoxin (α-TxIA) in the venom of Conus textile. α-TxIA bound with high affinity to AChBPs from different species and selectively targeted the α3β2 nAChR subtype. A co-crystal structure of Ac-AChBP with the enhanced potency analog TxIA(A10L), revealed a 20° backbone tilt compared to other AChBP–conotoxin complexes. This reorientation was coordinated by a key salt bridge formed between Arg5 (TxIA) and Asp195 (Ac-AChBP). Mutagenesis studies, biochemical assays and electrophysiological recordings directly correlated the interactions observed in the co-crystal structure to binding affinity at AChBP and different nAChR subtypes. Together, these results establish a new pharmacophore for the design of novel subtype-selective ligands with therapeutic potential in nAChR-related diseases.
acetylcholine binding protein; conotoxin; cys-loop receptor; ion channel; nicotinic acetylcholine receptors
The electron diffraction structure of nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata and the X-ray crystallographic structure of acetylcholine binding protein (AChBP) are providing new answers to persistent questions about how nAChRs function as biophysical machines and as participants in cellular and systems physiology. New high-resolution information about nAChR structures might come from advances in crystallography and NMR, from extracellular domain nAChRs as high fidelity models, and from prokaryotic nicotinoid proteins. At the level of biophysics, structures of different nAChRs with different pharmacological profiles and kinetics will help describe how agonists and antagonists bind to orthosteric binding sites, how allosteric modulators affect function by binding outside these sites, how nAChRs control ion flow, and how large cytoplasmic domains affect function. At the level of cellular and systems physiology, structures of nAChRs will help characterize interactions with other cellular components, including lipids and trafficking and signaling proteins, and contribute to understanding the roles of nAChRs in addiction, neurodegeneration, and mental illness. Understanding nAChRs at an atomic level will be important for designing interventions for these pathologies.
Acetylcholine; Addiction; Neurodegeneration; Nicotine; Protein Design; Protein Folding; Protein Structure; Cys-Loop Receptors; Review
Background: Cytisine and varenicline are smoking cessation drugs binding to nicotinic receptors (nAChRs).
Results: We studied crystal structures of cytisine and varenicline with AChBP and analyzed binding of α4β2-like or α7-like AChBP mutants to cytisine.
Conclusion: Ligand selectivity relies on residues beyond the binding site primary shell.
Significance: These structures will contribute to designing novel compounds targeting specific nAChR subtypes.
Smoking cessation is an important aim in public health worldwide as tobacco smoking causes many preventable deaths. Addiction to tobacco smoking results from the binding of nicotine to nicotinic acetylcholine receptors (nAChRs) in the brain, in particular the α4β2 receptor. One way to aid smoking cessation is by the use of nicotine replacement therapies or partial nAChR agonists like cytisine or varenicline. Here we present the co-crystal structures of cytisine and varenicline in complex with Aplysia californica acetylcholine-binding protein and use these as models to investigate binding of these ligands binding to nAChRs. This analysis of the binding properties of these two partial agonists provides insight into differences with nicotine binding to nAChRs. A mutational analysis reveals that the residues conveying subtype selectivity in nAChRs reside on the binding site complementary face and include features extending beyond the first shell of contacting residues.
Crystal Structure; Cys-loop Receptors; Ion Channels; Ligand-binding Protein; Nicotinic Acetylcholine Receptors; Acetylcholine-binding Protein; α4β2-selective Ligands; Cytisine; Varenicline
Nicotinic acetylcholine receptors (nAChR) play important neurophysiological roles and are of considerable medical relevance. They have been studied extensively, greatly facilitated by the gastropod acetylcholine-binding proteins (AChBP) which represent soluble structural and functional homologues of the ligand-binding domain of nAChR. All these proteins are ring-like pentamers. Here we report that AChBP exists in the hemolymph of the planorbid snail Biomphalaria glabrata (vector of the schistosomiasis parasite) as a regular pentagonal dodecahedron, 22 nm in diameter (12 pentamers, 60 active sites). We sequenced and recombinantly expressed two ∼25 kDa polypeptides (BgAChBP1 and BgAChBP2) with a specific active site, N-glycan site and disulfide bridge variation. We also provide the exon/intron structures. Recombinant BgAChBP1 formed pentamers and dodecahedra, recombinant BgAChBP2 formed pentamers and probably disulfide-bridged di-pentamers, but not dodecahedra. Three-dimensional electron cryo-microscopy (3D-EM) yielded a 3D reconstruction of the dodecahedron with a resolution of 6 Å. Homology models of the pentamers docked to the 6 Å structure revealed opportunities for chemical bonding at the inter-pentamer interfaces. Definition of the ligand-binding pocket and the gating C-loop in the 6 Å structure suggests that 3D-EM might lead to the identification of functional states in the BgAChBP dodecahedron.
Nicotinic acetylcholine receptors (nAChRs), being responsible for mediating key physiological functions, are ubiquitous in the central and peripheral nervous systems. As members of the Cys loop ligand-gated ion channel family, neuronal nA-ChRs are pentameric, composed of various permutations of α (α2 to α10) and β (β2 to β4) subunits forming functional heteromeric or homomeric receptors. Diversity in nAChR subunit composition complicates development of selective ligands for specific subtypes, since the five binding sites reside at the subunit interfaces. The acetylcholine binding protein (AChBP), a soluble extracellular domain homologue secreted by mollusks, serves as a general structural surrogate for the nAChRs. In this work, homomeric AChBPs from Lymnaea and Aplysia snails were used as in situ templates for the generation of novel and potent ligands that selectively bind to these proteins. The cycloaddition reaction between building block azides and alkynes to form stable 1,2,3-triazoles generated the leads. The extent of triazole formation on the AChBP template correlated with the affinity of the triazole product at the nicotinic ligand binding site. Instead of the in situ protein-templated azide-alkyne cycloaddition reaction occurring at a localized, sequestered enzyme active center as previously shown, we demonstrate that the in situ reaction can take place at subunit interfaces of an oligomeric protein and can thus be used as a tool for identification of novel candidate nAChR ligands. The crystal structure of one of the in situ formed triazole–AChBP complexes shows binding poses and molecular determinants of interactions predicted from structures of known agonists and antagonists. Hence, the click chemistry approach with an in situ template of a receptor provides a novel synthetic avenue for generating candidate agonists and antagonists for ligand-gated ion channels.
Acetylcholine-binding protein (AChBP) recently emerged as a prototype for relating structure to function of the ligand binding domain of nicotinic acetylcholine receptors (AChRs). To understand interactions of competitive antagonists at the atomic structural level, we studied binding of the curare derivatives d-tubocurarine (d-TC) and metocurine to AChBP using computational methods, mutagenesis, and ligand binding measurements. To account for protein flexibility, we used a 2-ns molecular dynamics simulation of AChBP to generate multiple snapshots of the equilibrated dynamic structure to which optimal docking orientations were determined. Our results predict a predominant docking orientation for both d-TC and metocurine, but unexpectedly, the bound orientations differ fundamentally for each ligand. At one subunit interface of AChBP, the side chain of Tyr-89 closely approaches a positively charged nitrogen in d-TC but is farther away from the equivalent nitrogen in metocurine, whereas, at the opposing interface, side chains of Trp-53 and Gln-55 closely approach the metocurine scaffold but not that of d-TC. The different orientations correspond to ~170° rotation and ~30° degree tilt of the curare scaffold within the binding pocket. Mutagenesis of binding site residues in AChBP, combined with measurements of ligand binding, confirms the different docking orientations. Thus structurally similar ligands can adopt distinct orientations at receptor binding sites, posing challenges for interpreting structure-activity relationships for many drugs.
Structure-based drug design can potentially accelerate the development of new therapeutics. In this study, a co-crystal structure of the acetylcholine binding protein (AChBP) from Capitella teleta (Ct) in complex with a cyclopropane-containing, selective α4β2-nicotinic acetylcholine receptor (nAChR) partial agonist (compound 5) was acquired. The structural determinants required for ligand binding obtained from this AChBP X-ray structure were used to refine our previous model of the human α4β2-nAChR, thus possibly providing a better understanding of the structure of the human receptor. In order to validate the potential application of the structure of the Ct-AChBP in the engineering of new α4β2-nAChR ligands, homology modeling methods, combined with in silico ADME calculations, were used to design analogs of compound 5. The most promising compound 12, exhibited an improved metabolic stability in comparison to the parent compound 5 while retaining favorable pharmacological parameters together with appropriate behavioral endpoints in the rodent studies.
The initial coupling between ligand binding and channel gating in the human α7 nicotinic acetylcholine receptor (nAChR) has been investigated with targeted molecular dynamics (TMD) simulation. During the simulation, eight residues at the tip of the C-loop in two alternating subunits were forced to move toward a ligand-bound conformation as captured in the crystallographic structure of acetylcholine binding protein (AChBP) in complex with carbamoylcholine. Comparison of apo- and ligand-bound AChBP structures shows only minor rearrangements distal from the ligand-binding site. In contrast, comparison of apo and TMD simulation structures of the nAChR reveals significant changes toward the bottom of the ligand-binding domain. These structural rearrangements are subsequently translated to the pore domain, leading to a partly open channel within 4 ns of TMD simulation. Furthermore, we confirmed that two highly conserved residue pairs, one located near the ligand-binding pocket (Lys145 and Tyr188), and the other located toward the bottom of the ligand-binding domain (Arg206 and Glu45), are likely to play important roles in coupling agonist binding to channel gating. Overall, our simulations suggest that gating movements of the α7 receptor may involve relatively small structural changes within the ligand-binding domain, implying that the gating transition is energy-efficient and can be easily modulated by agonist binding/unbinding.
Nicotinic acetylcholine receptors are ligand-gated ion channels responsible for neurotransmitter-mediated signal transduction at synapses throughout the central and peripheral nervous systems. Binding of neurotransmitter molecules to subunit interfaces in the N-terminal extracellular domain induces structural rearrangements of the membrane-spanning domain permitting the influx of cations. A full understanding of how the conformational changes propagate from the ligand-binding site to the pore domain is of great interest to biologists, yet remains to be established. Using a special simulation technique known as targeted molecular dynamics, Cheng and colleagues probed the early stages of ligand-induced conformational rearrangements that may lead to channel opening. During the simulation, Cheng et al. observed a sequence of conformational changes that stem from the ligand-binding site to the transmembrane domain resulting in a wider channel. From these results, they suggest that gating movements may entail only small structural changes in the ligand-binding domain, implying that channel gating is energy-efficient and can readily be modulated by the binding/unbinding of agonist molecules.
The recent characterization of an acetylcholine binding protein (AChBP) from the fresh water snail, Lymnaea stagnalis, shows it to be a structural homolog of the extracellular domain of the nicotinic acetylcholine receptor (nAChR). To ascertain whether the AChBP exhibits the recognition properties and functional states of the nAChR, we have expressed the protein in milligram quantities from a synthetic cDNA transfected into human embryonic kidney (HEK) cells. The protein secreted into the medium shows a pentameric rosette structure with ligand stoichiometry approximating five sites per pentamer. Surprisingly, binding of acetylcholine, selective agonists, and antagonists ranging from small alkaloids to larger peptides results in substantial quenching of the intrinsic tryptophan fluorescence. Using stopped-flow techniques, we demonstrate rapid rates of association and dissociation of agonists and slow rates for the α-neurotoxins. Since agonist binding occurs in millisecond time frames, and the α-neurotoxins may induce a distinct conformational state for the AChBP-toxin complex, the snail protein shows many of the properties expected for receptor recognition of interacting ligands. Thus, the marked tryptophan quenching not only documents the importance of aromatic residues in ligand recognition, but establishes that the AChBP will be a useful functional as well as structural surrogate of the nicotinic receptor.
Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels that belong to the Cys-loop receptor superfamily. These receptors are allosteric proteins that exist in different conformational states, including resting (closed), activated (open), and desensitized (closed) states. The acetylcholine binding protein (AChBP) is a structural homologue of the extracellular ligand-binding domain of nAChRs. In previous studies, the degree of the C-loop radial extension of AChBP has been assigned to different conformational states of nAChRs. It has been suggested that a closed C-loop is preferred for the active conformation of nAChRs in complex with agonists whereas an open C-loop reflects an antagonist-bound (closed) state. In this work, we have determined the crystal structure of AChBP from the water snail Lymnaea stagnalis (Ls) in complex with dihydro-β-erythroidine (DHβE), which is a potent competitive antagonist of nAChRs. The structure reveals that binding of DHβE to AChBP imposes closure of the C-loop as agonists, but also a shift perpendicular to previously observed C-loop movements. These observations suggest that DHβE may antagonize the receptor via a different mechanism compared to prototypical antagonists and toxins.
Nicotinic Acetylcholine Receptors (nAChR) are cation-selective, ligand-gated ion channels of the Cys-loop gene superfamily. The recent crystal structure of a bacterial homologue from Erwinia chrysanthemi (ELIC) agrees with previous structures of the N-terminal domain of acetylcholine-binding protein (AChBP) and of the electronmicroscopy derived Torpedo nAChR structure. However, the ELIC transmembrane domain is significantly more tightly packed than the corresponding region of the Torpedo nAChR. We investigated the tightness of protein packing surrounding the extracellular end of the M2 transmembrane segment and around the loop connecting the M2 and M3 segments using the substituted cysteine accessibility method (SCAM). The M2 20′ to 27′ residues were highly water accessible and the variation in reaction rates were consistent with this region being α-helical. At all positions tested, the presence of ACh changed MTSEA modification rates by less than 10-fold. In the presence of ACh, reaction rates for residues in the last extracellular α-helical turn of M2 and in the M2M3 loop increased, whereas rates in M2's penultimate α-helical turn decreased. Only 3 out of 8 M2M3 loop residues were accessible to MTSEA in both the presence and absence of ACh. We infer that the protein packing around the M2M3 loop is tight, consistent with it's location at the interdomain interface where it is involved in the transduction of ligand binding in the extracellular domain to gating in the transmembrane domain. Our data indicate that the Torpedo nAChR transmembrane domain structure is a better model than the ELIC structure for eukaryotic Cys loop receptors.
acetylcholine; nicotine; serotonin; Cys-loop; ion-channel; gating
The details of interaction in a complex between potent antagonists such as long chain α-neurotoxins and α-conotoxins with nicotinic acetylcholine receptor (nAChR), and conformational changes induced by these antagonists, are not yet clear.
In order to uncover some of these critical structural features, we conducted a docking simulation and a molecular dynamics simulation (MD) of a model of the ligand binding domain of nAChR in complex with a long-chain α-neurotoxin and an α-conotoxin.
Our docking results confirm the claim that T.nAChR is in the basal or resting state, which favors binding to the alpha-neurotoxins. Moreover, more correct "hits" for the α/γ interface upon docking for conotoxin-nAChR confirm the preference of conotoxin GI for the α/γ interface. More importantly, upon binding of α-neurotoxin, ligand-bonded nAChR is less dynamic in certain domains than the apo form of the conotoxin-AChR complex. Some critical interactions in the binding site such as the salt bridge formed between K145/D200 in the neurotoxin-nAChR complex is further stabilized during the MD simulation, while it is obviously more labile in the apo form.
These observations could support the claim that alpha neurotoxins stabilize the nAChR resting state.
α-Conotoxins are peptide neurotoxins isolated from venomous marine cone snails that are potent and selective antagonists for different subtypes of nicotinic acetylcholine receptors (nAChRs). As such, they are valuable probes for dissecting the role that nAChRs play in nervous system function. In recent years, extensive insight into the binding mechanisms of α-conotoxins with nAChRs at the molecular level has aided in the design of synthetic analogs with improved pharmacological properties. This review examines the structure-activity relationship studies involving α-conotoxins as research tools for studying nAChRs in the central and peripheral nervous systems and their use towards the development of novel therapeutics.
α-conotoxin; nicotinic acetylcholine receptor; acetylcholine binding protein; structure-activity relationship studies; mutational analysis
A series of 14 new analogs of α-conotoxin PnIA Conus pennaceus was synthesized and tested for binding to the human α7 nicotinic acetylcholine receptor (nAChR) and acetylcholine-binding proteins (AChBP) Lymnaea stagnalis and Aplysia californica. Based on computer modeling and the X-ray structure of the A. californica AChBP complex with the PnIA[A10L, D14K] analog , single and multiple amino acid substitutions were introduced in α-conotoxin PnIA aimed at compounds of higher affinity and selectivity. Three analogs, PnIA[L5H], PnIA[A10L, D14K] and PnIA[L5R, A10L, D14R], have high affinities for AChBPs or α7 nAChR, as found in competition with radioiodinated α-bungarotoxin. That is why we prepared radioiodinated derivatives of these α-conotoxins, demonstrated their specific binding and found that among the tested synthetic analogs, most had almost 10-fold higher affinity in competition with radioactive α-conotoxins as compared to competition with radioactive α-bungarotoxin. Thus, radioiodinated α-conotoxins are a more sensitive tool for checking the activity of novel α-conotoxins and other compounds quickly dissociating from the receptor complexes.
α-conotoxin analogs; nicotinic acetylcholine receptors; acetylcholine-binding proteins; computer modeling; radioligand analysis
The discovery of the acetylcholine binding proteins (AChBPs) has provided critical soluble surrogates for examining structure and ligand interactions with nicotinic receptors and related pentameric ligand-gated ion channels. The multiple marine and freshwater sources of AChBP constitute a protein family with substantial sequence divergence and selectivity in ligand recognition for analyzing structure–activity relationships. The purification of AChBP in substantial quantities in the absence of a detergent enables one to conduct spectroscopic studies of the ligand–AChBP complexes. To this end, we have examined the interaction of a congeneric series of benzylidene-ring substituted anabaseines with AChBPs from Lymnaea, Aplysia, and Bulinus species and correlated their binding energetics with spectroscopic changes associated with ligand binding. The anabaseines display agonist activity on the α7 nicotinic receptor, a homomeric receptor with sequences similar to those of the AChBPs. Substituted anabaseines show absorbance and fluorescence properties sensitive to the protonation state, relative permittivity (dielectric constant), and the polarizability of the surrounding solvent or the proximal residues in the binding site. Absorbance difference spectra reveal that a single protonation state of the ligand binds to AChBP and that the bound ligand experiences a solvent environment with a high degree of polarizability. Changes in the fluorescence quantum yield of the bound ligand reflect the rigidification of the ring system of the bound ligand. Hence, the spectral properties of the bound ligand allow a description of the electronic character of the bound state of the ligand within its aromatic binding pocket and provide information complementary to that of crystal structures in defining the determinants of interaction.
The diversity of nicotinic acetylcholine receptor (nAChR) subtypes was explored by measuring the effects of gene deletion and pharmacological diversity of epibatidine binding sites in mouse brain. All epibatidine binding sites require expression of either the α7, β2, or β4 subunit. In agreement with general belief, the α4β2*-nAChR and α7-nAChR subtypes are major components of the epibatidine binding sites. α4β2*-nAChR sites account for approximately 70% of total high- and low-affinity epibatidine binding sites, while α7-nAChR accounts for 16% of the total sites all of which have lower affinity for epibatidine. The other subtypes are structurally diverse. Although these minor subtypes account for only 14% of total binding in whole brain, they are expressed at relatively high concentrations in specific brain areas indicating unique functional roles.
Neuronal nicotinic acetylcholine receptors; Epibatidine; Null mutant mice; α-Bungarotoxin; Cytisine; α-Conotoxin MII
Cys-loop receptors (CLR) are pentameric ligand-gated ion channels that mediate fast excitatory or inhibitory transmission in the nervous system. Strychnine and d-tubocurarine (d-TC) are neurotoxins that have been highly instrumental in decades of research on glycine receptors (GlyR) and nicotinic acetylcholine receptors (nAChR), respectively. In this study we addressed the question how the molecular recognition of strychnine and d-TC occurs with high affinity and yet low specificity towards diverse CLR family members. X-ray crystal structures of the complexes with AChBP, a well-described structural homolog of the extracellular domain of the nAChRs, revealed that strychnine and d-TC adopt multiple occupancies and different ligand orientations, stabilizing the homopentameric protein in an asymmetric state. This introduces a new level of structural diversity in CLRs. Unlike protein and peptide neurotoxins, strychnine and d-TC form a limited number of contacts in the binding pocket of AChBP, offering an explanation for their low selectivity. Based on the ligand interactions observed in strychnine- and d-TC-AChBP complexes we performed alanine-scanning mutagenesis in the binding pocket of the human α1 GlyR and α7 nAChR and showed the functional relevance of these residues in conferring high potency of strychnine and d-TC, respectively. Our results demonstrate that a limited number of ligand interactions in the binding pocket together with an energetic stabilization of the extracellular domain are key to the poor selective recognition of strychnine and d-TC by CLRs as diverse as the GlyR, nAChR, and 5-HT3R.
Ligand-gated ion channels play an important role in fast electrochemical signaling in the brain. Cys-loop receptors are a class of pentameric ligand-gated ion channels that are activated by specific neurotransmitters, including acetylcholine (ACh), serotonin (5-HT), glycine (Gly), and γ-aminobutyric acid (GABA). Each type of cys-loop receptor contains an extracellular domain that specifically recognizes only one of these four neurotransmitters and opens an ion-conducting channel pore upon ligand binding. In this study, we investigated the poor specificity with which two potent neurotoxic inhibitors, namely strychnine and d-tubocurarine, are recognized by different cys-loop receptors. Using X-ray crystallography we solved 3-dimensional structures of strychnine or d-tubocurarine in complex with ACh binding protein (AChBP), a well-recognized structural homolog of the nicotinic ACh receptor. Based on ligand-receptor interactions observed in AChBP structures we designed mutant GlyR and α7 nAChR to identify hot spots in the binding pocket of these receptors that define potent inhibition by strychnine and d-tubocurarine, respectively. Combined, our results offer detailed understanding of the molecular recognition of antagonists that have high affinity but poor specificity for different cys-loop receptors.
Neuronal nicotinic acetylcholine receptors (nAChRs)1 are pentamers composed of α and β subunits. Different molecular compositions of these subunits constitute various receptor subtypes that are implicated in the pathophysiology and/ or treatment of several disease states, but are difficult to distinguish among pharmacologically. α-Conotoxins are a group of small, structurally defined peptides that may be used to molecularly dissect the nAChR binding site. Heteromeric nAChRs generally contain either a β2 or β4 subunit in addition to an α subunit at the ligand-binding interface. α-Conotoxin BuIA kinetically distinguishes between β2- and β4-containing nAChRs with long off-times for the latter. Mutational studies were used to assess the influence of residues that line the putative acetylcholine- binding pocket, but differ between β2 and β4 subunits. Residues Thr/Lys59, Val/Ile111 and Phe/Gln119 of the respective β2- and β4 subunits are critical to off-rate differences. Among these residues, Thr59 of nAChR β2 may interfere with effective access to the binding site whereas Lys59 may facilitate this binding.
The agonist binding sensitivity and desensitization kinetics of nicotinic acetylcholine receptors (nAChRs) can be modulated by snake venom neurotoxins and related endogenous small proteins of the uPAR-Ly6 family. Here we identify Lypd6, a distantly related member of the u-PAR/Ly-6 family expressed in neurons as a novel modulator of nAChRs. Lypd6 overexpressed in trigeminal ganglia neurons selectively enhanced the Ca2+-component of nicotine-evoked currents through nAChRs, as evidenced by comparative whole-cell patch clamp recordings and Ca2+-imaging in wildtype and transgenic mice overexpressing Lypd6. In contrast, a knockdown of Lypd6 expression using siRNAs selectively reduced nicotine-evoked Ca2+-currents. Pharmacological experiments revealed that the nAChRs involved in this process are heteromers. Transgenic mice displayed behaviors that were indicative of an enhanced cholinergic tone, such as a higher locomotor arousal, increased prepulse-inhibition and hypoalgesia. These mice overexpressing Lypd6 mice were also more sensitive to the analgesic effects of nicotine. Transgenic mice expressing siRNAs directed against Lypd6 were unable to procreate, thus indicating a vital role for this protein. Taken together, Lypd6 seems to constitute a novel modulator of nAChRs that affects receptor function by selectively increasing Ca2+-influx through this ion channels.
transgenic mice; allosteric modulator; Ion selectivity; electrophysiology; behavior; receptor channel
A variety of molecular modeling, molecular docking, and first-principles electronic structure calculations were performed to study how the α4β2 nicotinic acetylcholine receptor (nAChR) binds with different species of two typical agonists, S-(−)-nicotine and R-(−)-deschloroepibatidine, each of which are distinguished by different free bases and protonation states. Based on these results, predictions were made regarding the corresponding microscopic binding free energies. Hydrogen bonding and cation-π interactions between the receptor and the respective ligands were found to be the dominant factors differentiating the binding strengths of different microscopic binding species. The calculated results and analyses demonstrate that for each agonist, all the species are interchangeable and can quickly achieve a thermodynamic equilibration in solution and at the nAChR binding site. This allows quantitation of the equilibrium concentration distributions of the free ligand species and the corresponding microscopic ligand-receptor binding species, their pH-dependence, and their contributions to the phenomenological binding affinity. The predicted equilibrium concentration distributions, pKa values, absolute phenomenological binding affinities and their pH-dependence are all in good agreement with available experimental data, suggesting that the computational strategy from the microscopic binding species and affinities to the phenomenological binding affinity is reliable for studying α4β2 nAChR-ligand binding. This should provide valuable information for future rational design of drugs targeting nAChRs. The general strategy of the “from-microscopic-to-phenomenological” approach for studying interactions of α4β2 nAChR with S-(−)-nicotine and R-(−)-deschloroepibatidine may also be useful in studying other types of ligand-protein interactions involving multiple molecular species of a ligand and in associated rational drug design.
Neuronal nicotinic acetylcholine receptors (nAChR), recognized targets for drug development in cognitive and neuro-degenerative disorders, are allosteric proteins with dynamic interconversions between multiple functional states. Activation of the nAChR ion channel is primarily controlled by the binding of ligands (agonists, partial agonists, competitive antagonists) at conventional agonist binding sites, but is also regulated in either negative or positive ways by the binding of ligands to other modulatory sites. In this review, we discuss models for the activation and desensitization of nAChR, and the discovery of multiple types of ligands that influence those processes in both heteromeric nAChR, such as the high affinity nicotine receptors of the brain, and homomeric α7-type receptors. In recent years, α7 nAChRs have been identified as a potential target for therapeutic indications leading to the development of α7-selective agonists and partial agonists. However, unique properties of α7 nAChR, including low probability of channel opening and rapid desensitization, may limit the therapeutic usefulness of ligands binding exclusively to conventional agonist binding sites. New enthusiasm for the therapeutic targeting of α7 has come from the identification of α7-selective positive allosteric modulators (PAMs) that work effectively on the intrinsic factors that limit α7 ion channel activation. While these new drugs appear promising for therapeutic development, we also consider potential caveats and possible limitations for their use, including PAM-insensitive forms of desensitization and cytotoxicity issues.
Alzheimer’s disease; schizophrenia; drug development; electrophysiology; modeling