Desformylflustrabromine (dFBr; 1) and desformylflustrabromine-B (dFBr-B; 2) have been previously isolated from natural sources, and the former has been demonstrated to be a novel and selective positive allosteric modulator of α4β2 nicotinic acetylcholine (nACh) receptors. The present study describes the synthesis of water-soluble salts of 1 and 2, and confirms and further investigates the actions of 1 and 2 using two-electrode voltage clamp recordings.
Nicotinic cholinergic receptors; Allosteric modulators
Nicotinic acetylcholine receptors (nAChRs) are pentameric transmembrane proteins that belong to the cys-loop ligand-gated ion channel family. These receptors are widely expressed in the brain and implicated in the pathophysiology of many neurological conditions, including Alzheimer’s disease (AD), where typical symptoms include the loss of cognitive function and dementia. The presence of extracellular neuritic plaques composed of β amyloid (Aβ1–42) peptide is a characteristic feature of AD. Desformylflustrabromine (dFBr) is a positive allosteric modulator (PAM) for α4β2 nAChRs since it increases peak ACh responses without inducing a response on its own. Previously, the effect of dFBr on the α2β2 nAChR subtype was not known. The action of dFBr was tested on α2β2 receptors expressed in Xenopus oocytes. It was found that dFBr is also a PAM for the α2β2 receptor. Next we tested whether dFBr had any effect on the previously known block of both the α4β2 and α2β2 receptors by Aβ1–42. We found that the functional blockade of ACh-induced currents in oocytes expressing α4β2 and α2β2 receptors by Aβ1–42 was prevented by dFBr. We conclude that dFBr is a positive allosteric modulator for both α4β2 and α2β2 subtypes of nAChRs and that it also relieves the blockade of these receptors by Aβ1–42. This study demonstrates that PAMs for the non-α7 nAChRs have the potential to develop into clinically applicable drugs for AD and other disorders.
Alzheimer’s disease (AD); Beta amyloid (Aβ1–42); Desformylflustrabromine (dFBr); Electrophysiology; Nicotinic acetylcholine receptors (nAChRs); Positive allosteric modulators (PAMs)
Signaling through nicotinic acetylcholine (nACh) receptors underlies a diverse array of behaviors. In order for appropriate signaling to occur via nACh receptors, it is necessary for the genes encoding the receptor subunits to be expressed in a highly regulated temporal and spatial manner. Here we report a transgenic mouse approach to characterize the transcriptional regulation of the gene encoding the nACh receptor β4 subunit. nACh receptors containing this subunit play critical roles in both the central and peripheral nervous systems. We demonstrate that a 2.3-kilobase pair fragment of the β4 5′-flanking region is capable of directing reporter gene expression in transgenic animals. Importantly, the transcriptional activity of the promoter region is cell-type-specific and developmentally regulated and overlaps to a great extent with endogenous β4 mRNA expression. These data indicate that the 2.3-kilobase pair fragment contains transcriptional regulatory elements critical for appropriate β4 subunit gene expression.
nicotinic receptor; gene expression; transcription
The notion of functional interactions between the α7 nicotinic acetylcholine (α7 nACh) and the cannabinoid systems is emerging from recent in vitro and in vivo studies. Both the α7 nACh receptor and the cannabinoid receptor 1 (CB1) are highly expressed in the hippocampus. To begin addressing possible anatomical interactions between the α7 nACh and the cannabinoid systems in the rat hippocampus, we investigated the distribution of neurons expressing α7 nACh mRNA in relation to those containing CB1 mRNA. By in situ hybridization we found that the α7 nACh mRNA is diffusely expressed in principal neurons and is highly expressed in a subset of interneurons. We observed that the pattern of distribution of hippocampal interneurons co-expressing transcripts encoding α7 nACh and glutamate decarboxylase (GAD; synthesizing enzyme of GABA) closely resembles the one displayed by interneurons expressing CB1 mRNA. By double in situ hybridization we established that the majority of hippocampal interneurons expressing α7 nACh mRNA have high levels of CB1 mRNA. As CB1 interneurons contain cholecystokinin (CCK), we investigated the degree of cellular co-expression of α7 nACh mRNA and CCK, and found that the cellular co-existence of α7 nACh and CCK varies within the different layers of the hippocampus.
In summary, we established that most of the hippocampal α7 nACh expressing interneurons are endowed with CB1 mRNA. We found that these α7 nACh/CB1 interneurons are the major subpopulation of hippocampal interneurons expressing CB1 mRNA. The α7 nACh expressing interneurons represent half of the detected population of CCK containing neurons in the hippocampus. Since it is well established that the vast majority of hippocampal interneurons expressing CB1 mRNA have 5-HT type 3 (5-HT3) receptors, we conclude that these hippocampal α7 nACh/5HT3/CB1/CCK interneurons correspond to those previously postulated to relay inputs from diverse cortical and subcortical regions about emotional, motivational, and physiological states.
CB1; 5-HT3 receptors; 5-HT; CCK; Alzheimer’s disease; schizophrenia
Acetylcholine-evoked currents mediated by activation of nicotinic receptors in rat parasympathetic neurons were examined using whole-cell voltage clamp. The relative permeability of the neuronal nicotinic acetylcholine (nACh) receptor channel to monovalent and divalent inorganic and organic cations was determined from reversal potential measurements. The channel exhibited weak selectivity among the alkali metals with a selectivity sequence of Cs+ > K+ > Rb+ > Na+ > Li+, and permeability ratios relative to Na+ (Px/PNa) ranging from 1.27 to 0.75. The selectivity of the alkaline earths was also weak, with the sequence of Mg2+ > Sr2+ > Ba2+ > Ca2+, and relative permeabilities of 1.10 to 0.65. The relative Ca2+ permeability (PCa/PNa) of the neuronal nACh receptor channel is approximately fivefold higher than that of the motor endplate channel (Adams, D. J., T. M. Dwyer, and B. Hille. 1980. Journal of General Physiology. 75:493-510). The transition metal cation, Mn2+ was permeant (Px/PNa = 0.67), whereas Ni2+, Zn2+, and Cd2+ blocked ACh-evoked currents with half-maximal inhibition (IC50) occurring at approximately 500 microM, 5 microM and 1 mM, respectively. In contrast to the muscle endplate AChR channel, that at least 56 organic cations which are permeable to (Dwyer et al., 1980), the majority of organic cations tested were found to completely inhibit ACh- evoked currents in rat parasympathetic neurons. Concentration-response curves for guanidinium, ethylammonium, diethanolammonium and arginine inhibition of ACh-evoked currents yielded IC50's of approximately 2.5- 6.0 mM. The organic cations, hydrazinium, methylammonium, ethanolammonium and Tris, were measureably permeant, and permeability ratios varied inversely with the molecular size of the cation. Modeling suggests that the pore has a minimum diameter of 7.6 A. Thus, there are substantial differences in ion permeation and block between the nACh receptor channels of mammalian parasympathetic neurons and amphibian skeletal muscle which represent functional consequences of differences in the primary structure of the subunits of the ACh receptor channel.
Volatile aromatic compounds such as benzene are general anesthetics that cause amnesia, hypnosis, and immobility in response to noxious stimuli when inhaled. Although these compounds are not used clinically, they are frequently found in commercial items such as solvents and household cleaning products and are abused as inhalant drugs. Volatile aromatic anesthetics are useful pharmacological tools for probing the relationship between chemical structure and drug activity at putative general anesthetic targets. Neuronal nicotinic acetylcholine (nACh) receptors are ligand-gated ion channels widely expressed in the brain, which are thought to play important roles in learning and memory. In this study, we tested the hypothesis that aromatic anesthetics reversibly inhibit α4β2 neuronal nACh receptor function and sought to determine the structural correlates of receptor inhibition.
Electrophysiological techniques were used to quantify the effects of 8 volatile aromatic anesthetics on currents elicited by 1 mM ACh and mediated by human α4β2 nACh receptors expressed in Xenopus oocytes.
All of the volatile aromatic anesthetics used in this study reversibly inhibited α4β2 nACh receptors with IC50 values ranging from 0.00091 atm for 1,2-difluorobenzene to 0.045 atm for hexafluorobenzene. With the exception of hexafluorobenzene, all of the compounds had IC50 values less than minimum alveolar concentration. Inhibitory potency correlated poorly with the cation-π binding energies of the compounds (r2 = 0.48, P = 0.059). However, there was a good correlation between inhibitory potency and the octanol/gas partition coefficient (r2 = 0.87, P = 0.0008).
Volatile aromatic anesthetics potently and reversibly inhibit human α4β2 neuronal nACh receptors. This inhibition may play a role in producing amnesia. In contrast to N-methyl-D-aspartate receptors, the inhibitory potencies of aromatic anesthetics for α4β2 neubronal nACh receptors seem to be dependent on drug hydrophobicity rather than electrostatic properties. This implies that the volatile aromatic anesthetic binding site in the α4β2 neuronal nACh receptor is hydrophobic in character and differs from the nature of the binding site in N-methyl-D-aspartate receptors.
We synthesized 2,6-Diisopropyl-4-[3-(3-methyl-3H-diazirin-3-yl)-propyl]-phenol (p-(4-azipentyl)-propofol), or p-4-AziC5-Pro, a novel photoactivable derivative of the general anesthetic propofol. p-4-AziC5-Pro has an anesthetic potency similar to propofol. Like propofol, the compound potentiates inhibitory GABAA receptor current responses and allosterically modulates binding to both agonist and benzodiazepine sites, assayed on heterologously expressed GABAA receptors. p-4-AziC5-Pro inhibits excitatory current responses of nACh receptors expressed in Xenopus oocytes and photoincorporates into native nACh receptor-enriched Torpedo membranes. Thus p-4-AziC5-Pro is a functional general anesthetic that both modulates and photoincorporates into Cys-loop ligand-gated ion channels, making it an excellent candidate for use in identifying propofol binding sites.
Menthol is a common compound in pharmaceutical and commercial products and a popular additive to cigarettes. The molecular targets of menthol remain poorly defined. In this study we show an effect of menthol on the α7 subunit of the nicotinic acetylcholine (nACh) receptor function. Using a two-electrode voltage-clamp technique, menthol was found to reversibly inhibit α7-nACh receptors heterologously expressed in Xenopus oocytes. Inhibition by menthol was not dependent on the membrane potential and did not involve endogenous Ca2+-dependent Cl− channels, since menthol inhibition remained unchanged by intracellular injection of the Ca2+ chelator BAPTA and perfusion with Ca2+-free bathing solution containing Ba2+. Furthermore, increasing ACh concentrations did not reverse menthol inhibition and the specific binding of [125I] α-bungarotoxin was not attenuated by menthol. Studies of α7- nACh receptors endogenously expressed in neural cells demonstrate that menthol attenuates α7 mediated Ca2+ transients in the cell body and neurite. In conclusion, our results suggest that menthol inhibits α7-nACh receptors in a noncompetitive manner.
Subtype-selective ligands are important tools for the pharmacological characterisation of neurotransmitter receptors. This is particularly the case for nicotinic acetylcholine receptors (nAChRs), given the heterogeneity of their subunit composition. In addition to agonists and antagonists that interact with the extracellular orthosteric nAChR binding site, a series of nAChR allosteric modulators have been identified that interact with a distinct transmembrane site. Here we report studies conducted with three pharmacologically distinct nicotinic ligands, an orthosteric agonist (compound B), a positive allosteric modulator (TQS) and an allosteric agonist (4BP-TQS). The primary focus of the work described in this study is to examine the suitability of these compounds for the characterisation of native neuronal receptors (both rat and human). However, initial experiments were conducted on recombinant nAChRs demonstrating the selectivity of these three compounds for α7 nAChRs. In patch-clamp recordings on rat primary hippocampal neurons we found that all these compounds displayed pharmacological properties that mimicked closely those observed on recombinant α7 nAChRs. However, it was not possible to detect functional responses with compound B, an orthosteric agonist, using a fluorescent intracellular calcium assay on either rat hippocampal neurons or with human induced pluripotent stem cell-derived neurons (iCell neurons). This is, presumably, due to the rapid desensitisation of α7 nAChR that is induced by orthosteric agonists. In contrast, clear agonist-evoked responses were observed in fluorescence-based assays with the non-desensitising allosteric agonist 4BP-TQS and also when compound B was co-applied with the non-desensitising positive allosteric modulator TQS. In summary, we have demonstrated the suitability of subtype-selective orthosteric and allosteric ligands for the pharmacological identification and characterisation of native nAChRs and the usefulness of ligands that minimise receptor desensitisation for the characterisation of α7 nAChRs in fluorescence-based assays.
The α7 subunit of the nicotinic acetylcholine receptor (NAchRα7) is one of the principal brain receptors for nicotine and is thought to be a mediator of nicotine’s pro-cognitive effects. While nicotine is known to interact with the stress axis, little is known about the effect of stress or corticosteroids on the expression in the hippocampus, a brain region important to both cognition and stress reactivity. We examined the effects of chronic (21 day) restraint stress (CRS) and adrenalectomy with hormone replacement with the selective mineralocorticoid receptor (MR) agonist aldosterone, the selective glucocorticoid receptor (GR) agonist RU28,362 or corticosterone for 7 days, on the hippocampal expression of NAchRα7 mRNA and protein, as measured by 125I α-Bungarotoxin autoradiography. We found that CRS increase the levels of NAchRα7 mRNA in the CA1, CA3 and Dentate gyrus while levels of the protein were lowered by the same treatment. Corticosteroid replacement showed a GR specific increase in NAchRα7 mRNA, consistent with a corticosteroid mediated effect of CRS. While the mechanism behind these observations is as yet unclear, they may be neuroprotective against the damaging effects of CRS or an example of adaptation to the allostatic load produced by CRS.
Subtype selective molecules for α4β2 neuronal nicotinic acetylcholine receptors (nAChRs) have been sought as novel therapeutics for nicotine cessation. α4β2 nAChRs have been shown to be involved in mediating the addictive properties of nicotine while other subtypes (i.e., α3β4 and α7) are believed to mediate the undesired effects of potential CNS drugs. To obtain selective molecules, it is important to understand the physiochemical features of ligands that affect selectivity and potency on nAChR subtypes. Here we present novel QSAR/QSSR models for negative allosteric modulators of human α4β2 nAChRs and human α3β4 nAChRs. These models support previous homology model and site-directed mutagenesis studies that suggest a novel mechanism of antagonism. Additionally, information from the models presented in this work was used to synthesize novel molecules; which subsequently led to the discovery of a new selective antagonist of human α4β2 nAChRs.
Alpha7 nicotinic acetylcholine receptors (α7 nAChR) are widely distributed throughout the central nervous system and are found at particularly high levels in the hippocampus and cortex. Several lines of evidence indicate that pharmacological enhancement of α7 nAChRs function could be a potential therapeutic route to alleviate disease-related cognitive deficits. A recent pharmacological approach adopted to increase α7 nAChR activity has been to identify selective positive allosteric modulators (PAMs). α7 nAChR PAMs have been divided into two classes: type I PAMs increase agonist potency with only subtle effects on kinetics, whereas type II agents produce additional dramatic effects on desensitization and deactivation kinetics. Here we report novel observations concerning the pharmacology of the canonical type II PAM, PNU120596. Using patch clamp analysis of acetylcholine (ACh)-mediated currents through recombinant rat α7 nAChR we show that positive allosteric modulation measured in two different ways is greatly attenuated when the temperature is raised to near physiological levels. Furthermore, PNU120596 largely removes the strong inward rectification usually exhibited by α7 nAChR-mediated responses.
nicotinic receptors; electrophysiology; allosteric modulator; channel; patch clamp
We used the loose patch voltage clamp technique and rhodamine- conjugated alpha-bungarotoxin to study the regulation of Na channel (NaCh) and acetylcholine receptor (AChR) distribution on dissociated adult skeletal muscle fibers in culture. The aggregate of AChRs and NaChs normally found in the postsynaptic membrane of these cells gradually fragmented and dispersed from the synaptic region after several days in culture. This dispersal was the result of the collagenase treatment used to dissociate the cells, suggesting that a factor associated with the extracellular matrix was responsible for maintaining the high concentration of AchRs and NaChs at the neuromuscular junction. We tested whether the basal lamina protein agrin, which has been shown to induce the aggregation of AChRs on embryonic myotubes, could similarly influence the distribution of NaChs. By following identified fibers, we found that agrin accelerated both the fragmentation of the endplate AChR cluster into smaller patches as well as the appearance of new AChR clusters away from the endplate. AChR patches which were fragments of the original endplate retained a high density of NaChs, but no new NaCh hotspots were found elsewhere on the fiber, including sites of newly formed AChR clusters. The results are consistent with the hypothesis that extracellular signals regulate the distribution of AChRs and NaChs on skeletal muscle fibers. While agrin probably serves this function for the AChR, it does not appear to play a role in the regulation of the NaCh distribution.
An appropriate density of acetylcholine receptors (AChRs) and Na+ channels (NaChs) in the normal neuromuscular junction (NMJ) determines the magnitude of safety factor (SF) that guarantees fidelity of neuromuscular transmission. In myasthenia gravis (MG), an overall simplification of the postsynaptic folding secondary to NMJ destruction results in AChRs and NaChs depletion. Loss of AChRs and NaChs accounts respectively for 59% and 40% reduction of the SF at the endplate, which manifests as neuromuscular transmission failure. The extraocular muscles (EOM) have physiologically less developed postsynaptic folding, hence a lower baseline SF, which predisposes them to dysfunction in MG and development of fatigue during “high performance” eye movements, such as saccades. However, saccades in MG show stereotyped, conjugate initial components, similar to normal, which might reflect preserved neuromuscular transmission fidelity at the NMJ of the fast, pale global fibers, which have better developed postsynaptic folding than other extraocular fibers.
safety factor; extraocular muscles; saccades; neuromuscular junction
Neuronal nicotinic receptors have been implicated in several diseases and disorders such as: autism, Alzheimer’s disease, Parkinson’s disease, epilepsy, and various forms of addiction. To understand the role of nicotinic receptors in these conditions, it would be beneficial to have selective molecules that target specific nicotinic receptors in vitro and in vivo. Our laboratory has previously identified novel negative allosteric modulators of human α4β2 (Hα4β2) and human α3β4 (Hα3β4) nicotinic receptors. In the following studies, the effects of novel sulfonylpiperazine analogs that act as negative allosteric modulators on both Hα4β2 nAChRs and Hα3β4 nAChRs were investigated. This work, through structure-activity relationship (SAR) studies, describes the chemical features of these molecules that are important for both potency and selectivity on Hα4β2 nAChRs.
The level of expression of functional α7-containing nicotinic acetylcholine receptors (nAChRs) in hippocampal CA1 pyramidal neurons is believed to be very low compared to hippocampal CA1 interneurons, and for many years this expression was largely overlooked. However, high densities of expression of functional α7-containing nAChRs in CA1 pyramidal neurons may not be necessary for triggering important cellular and network functions, especially if activation of α7-containing nAChRs occurs in the presence of positive allosteric modulators such as PNU-120596.
An approach previously developed for α7-containing nAChRs expressed in tuberomammillary neurons was applied to investigate functional CA1 pyramidal α7-containing nAChRs using rat coronal hippocampal slices and patch-clamp electrophysiology. The majority (∼71%) of tested CA1 pyramidal neurons expressed low densities of functional α7-containing nAChRs as evidenced by small whole-cell responses to choline, a selective endogenous agonist of α7 nAChRs. These responses were potentiated by PNU-120596, a novel positive allosteric modulator of α7 nAChRs. The density of functional α7-containing nAChRs expressed in CA1 pyramidal neurons (and thus, the normalized net effect of activation, i.e., response net charge per unit of membrane capacitance per unit of time) was estimated to be ∼5% of the density observed in CA1 interneurons. The results of this study demonstrate that despite low levels of expression of functional pyramidal α7-containing nAChRs, physiological levels of choline (∼10 µM) are sufficient to activate these receptors and transiently depolarize and even excite CA1 pyramidal neurons in the presence of PNU-120596. The observed effects are possible because in the presence of 10 µM choline and 1–5 µM PNU-120596, a single opening of an individual pyramidal α7-containing nAChR ion channel appears to transiently depolarize (∼4 mV) the entire pyramidal neuron and occasionally trigger action potentials.
1) The majority of hippocampal CA1 pyramidal neurons express functional α7-containing nAChRs. In the absence of PNU-120596, a positive allosteric modulator of α7 nAChRs, a lack of responsiveness of some hippocampal CA1 pyramidal neurons to focal application of 0.5–1 mM choline does not imply a lack of expression of functional α7-containing nAChRs in these neurons. Rather, it may indicate a lack of detection of α7-containing nAChR-mediated currents by patch-clamp electrophysiology. 2) PNU-120596 can serve as a powerful tool for detection and enhancement of responsiveness of low densities of functional α7-containing nAChRs such as those present in hippocampal CA1 pyramidal neurons. 3) In the presence of PNU-120596, physiological concentrations of choline activate functional CA1 pyramidal α7-containing nAChRs and produce step-like currents that cause repetitive step-like depolarizations, occasionally triggering bursts of action potentials in CA1 pyramidal neurons. Therefore, the results of this study suggest that in the presence of PNU-120596 and possibly other positive allosteric modulators, endogenous choline may persistently activate CA1 pyramidal α7-containing nAChRs, enhance the excitability of CA1 pyramidal neurons and thus act as a potent therapeutic agent with potential neuroprotective and cognition-enhancing properties.
Muscarinic acetylcholine receptors (mAChRs) are drug targets for multiple neurodegenerative and neuropsychiatric disorders, but the full therapeutic potential of mAChR-targeted drugs has not been realized, mainly because of a lack of subtype-selective agonists. Recent advances have allowed the development of highly selective agonists that bind to an allosteric site on the M1 mAChR that is spatially distinct from the orthosteric acetylcholine binding site, but less is known about the profile of intracellular signals activated by orthosteric versus allosteric M1 mAChR agonists. We investigated the activation and regulatory mechanisms of two structurally distinct allosteric M1 mAChR agonists, AC260584 and TBPB. We show that allosteric agonists potently activate multiple signal transduction pathways linked to the M1 mAChR receptor but, compared to orthosteric agonists, much less efficiently recruit arrestin 3, a protein involved in regulation of G-protein coupled receptor signaling. Consistent with decreased arrestin recruitment, both allosteric agonists showed blunted responses in measurements of receptor desensitization, internalization, and downregulation. These results advance the understanding of mAChR biology and may shed light on unanticipated differences in the pharmacology of orthosteric vs. allosteric agonists that might be capitalized upon for drug development for the treatment of CNS diseases.
muscarinic; acetylcholine; receptor; allosteric; arrestin; internalization
Muscarinic acetylcholine receptors (mAChRs) are drug targets for multiple neurodegenerative and neuropsychiatric disorders, but the full therapeutic potential of mAChR-targeted drugs has not been realized, mainly because of a lack of subtype-selective agonists. Recent advances have allowed the development of highly selective agonists that bind to an allosteric site on the M1 mAChR that is spatially distinct from the orthosteric acetylcholine binding site, but less is known about the profile of intracellular signals activated by orthosteric versus allosteric M1 mAChR agonists. We investigated the activation and regulatory mechanisms of two structurally distinct allosteric M1 mAChR agonists, AC260584 and TBPB. We show that allosteric agonists potently activate multiple signal transduction pathways linked to the M1 mAChR receptor but, compared to orthosteric agonists, much less efficiently recruit arrestin 3, a protein involved in the regulation of G-protein coupled receptor signaling. Consistent with decreased arrestin recruitment, both allosteric agonists showed blunted responses in measurements of receptor desensitization, internalization, and downregulation. These results advance the understanding of mAChR biology and may shed light on unanticipated differences in the pharmacology of orthosteric versus allosteric agonists that might be capitalized upon for drug development for the treatment of CNS diseases.
Muscarinic; acetylcholine; receptor; allosteric; arrestin; internalization
We are interested in the positive allosteric modulation of neuronal nicotinic acetylcholine receptors (nAChRs), and have recently shown that the anthelmintic compound morantel potentiates by enhancing channel gating of the α3β2 subtype. Based on the demonstration that morantel-elicited currents were inhibited by the classic ACh-competitor dihydro-β-erythroidine in a noncompetitive manner and that morantel still potentiates at saturating concentrations of agonist (Wu et al., 2008), we hypothesized that morantel binds at the non-canonical β2(+)/α3(-) subunit interface. In the present study, we created seven cysteine-substituted subunits by site-directed mutagenesis, choosing residues in the putative morantel binding site with the aid of structural homology models. We co-expressed the mutant subunits and their respective wild type partners in Xenopus oocytes and characterized the morantel potentiation of ACh-evoked currents, as well as morantel-evoked currents, before and after treatment with a variety of methanethiosulfonate (MTS)-based compounds, using voltage-clamp recordings. The properties of four of the seven mutants, two residues on each side of the interface, were changed by MTS treatments. Co-application with ACh enhanced the extent of MTS modification for α3A106Cβ2 and α3β2S192C receptors. The activities of two mutants, α3T115Cβ2 and α3β2T150C, were dramatically altered by MTS modification. For α3β2T150C, while peak current amplitudes were reduced, potentiation was enhanced. For α3T115Cβ2, both current amplitudes and potentiation were reduced. MTS modification and morantel were mutually inhibitory: MTS treatment decreased morantel-evoked currents and morantel decreased the rate of MTS modification. We conclude that the four residues showing MTS effects contribute to the morantel binding site.
Nicotinic receptor; positive allosteric modulator; binding site; cysteine substitution; morantel; oocyte
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.
We studied allosteric potentiation of rat α3β2 neuronal nicotinic acetylcholine receptors (nAChRs) by the anthelmintic compound morantel. Macroscopic currents evoked by acetylcholine (ACh) from nAChRs expressed in Xenopus laevis oocytes increase up to 8-fold in the presence of low concentrations of morantel (≤10 μM); the magnitude of the potentiation depends on both agonist and modulator concentrations. It is noteworthy that the potentiated currents exceed the maximum currents achieved by saturating (millimolar) concentrations of agonist. Studies of macroscopic currents elicited by prolonged drug applications (100–300 s) indicate that morantel does not increase α3β2 receptor activity by reducing slow (≥1 s) desensitization. Instead, using outside-out patch-clamp recordings, we demonstrate that morantel increases the frequency of single-channel openings and alters the bursting characteristics of the openings in a manner consistent with enhanced channel gating; these results quantitatively explain the macroscopic current potentiation. Morantel is a very weak agonist alone, but we show that the classic competitive antagonist dihydro-β-erythroidine inhibits morantel-evoked currents noncompetitively, indicating that morantel does not bind to the canonical ACh binding sites.
Neuronal nicotinic acetylcholine receptors (nAChR) are ligand gated ion channels that mediate fast synaptic transmission. Methyllycaconitine (MLA) is a selective and potent antagonist of the α7 nAChR, and its anthranilate ester side-chain is important for its activity. Here we report the influence of structure on nAChR inhibition for a series of novel MLA analogs, incorporating either an alcohol or anthranilate ester side-chain to an azabicyclic or azatricyclic core against rat α7, α4β2, and α3β4 nAChRs expressed in Xenopus oocytes. The analogs inhibited ACh (EC50) within an IC50 range of 2.3−26.6 μM. Most displayed noncompetitive antagonism, but the anthranilate ester analogs exerted competitive behavior at the α7 nAChR. At α4β2 nAChRs, inhibition by the azabicyclic alcohol was voltage-dependent suggesting channel block. The channel-lining residues of α4 subunits were mutated to cysteine and the effect of azabicyclic alcohol was evaluated by competition with methanethiosulfonate ethylammonium (MTSEA) and a thiol-reactive probe in the open, closed, and desensitized states of α4β2 nAChRs. The azabicyclic alcohol was found to compete with MTSEA between residues 6′ and 13′ in a state-dependent manner, but the reactive probe only bonded with 13′ in the open state. The data suggest that the 13′ position is the dominant binding site. Ligand docking of the azabicyclic alcohol into a (α4)3(β2)2 homology model of the closed channel showed that the ligand can be accommodated at this location. Thus our data reveal distinct pharmacological differences between different nAChR subtypes and also identify a specific binding site for a noncompetitive channel blocker.
Ligand-gated ion channel (LGIC); nicotinic acetylcholine receptor (nAChR); noncompetitive antagonist (NCA); substituted cysteine accessibility method (SCAM); methyllycaconitine (MLA); homology model; reactive probe
Positive allosteric modulators (PAMs) of nicotinic acetylcholine receptors (nAChRs) have attracted considerable interest as a novel area of therapeutic drug discovery. Two types of α7-selective PAMs have been identified (type I and type II). Whilst both potentiate peak agonist-induced responses, they have different effects on the rate of agonist-induced receptor desensitization. Type I PAMs have little or no effect on the rapid rate of desensitization that is characteristic of α7 nAChRs, whereas type II PAMs cause dramatic slowing of receptor desensitization. Previously, we have obtained evidence indicating that PNU-120596, a type II PAM, causes potentiation by interacting with an allosteric transmembrane site. In contrast, other studies have demonstrated the importance of the ‘M2–M3 segment’ in modulating the effects of the type I PAM NS1738 and have led to the proposal that NS1738 may interact with the extracellular N-terminal domain. Here, our aim has been to compare the mechanism of allosteric potentiation of α7 nAChRs by NS1738 and PNU-120596. Functional characterization of a series of mutated α7 nAChRs indicates that mutation of amino acids within a proposed intrasubunit transmembrane cavity have a broadly similar effect on these two PAMs. In addition, we have employed a functional assay designed to examine the ability of ligands to act competitively at either the orthosteric or allosteric binding site of α7 nAChRs. These data, together with computer docking simulations, lead us to conclude that both the type I PAM NS1738 and the type II PAM PNU-120596 bind competitively at a mutually exclusive intrasubunit transmembrane site.
► We have examined two positive allosteric modulators of the α7 nicotinic receptor. ► The two modulators have differing effects on agonist-evoked desensitization. ► We provide evidence that they act at a common transmembrane binding site.
Acetylcholine receptor; Ligand-gated ion channel; Nicotinic receptor; Positive allosteric modulator
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
The α7 nicotinic acetylcholine receptor (nAChR) is a potential target for the treatment of cognitive deficits in patients with schizophrenia, ADHD and Alzheimer's disease. Here we test the hypothesis that upregulation of α7 nAChR levels underlies the enhanced and sustained procognitive effect of repeated administration of α7 nAChR agonists. We further compare the effect of agonists to that of α7 nAChR positive allosteric modulators (PAMs), which do not induce upregulation of the α7 nAChR. Using the social discrimination test as a measure of short-term memory, we show that the α7 nAChR agonist A-582941 improves short-term memory immediately after repeated (7× daily), but not a single administration. The α7 nAChR PAMs PNU-120596 and AVL-3288 do not affect short-term memory immediately after a single or repeated administration. This demonstrates a fundamental difference in the behavioral effects of agonists and PAMs that may be relevant for clinical development. Importantly, A-582941 and AVL-3288 increase short-term memory 24 hrs after repeated, but not a single, administration, suggesting that repeated administration of both agonists and PAMs may produce sustained effects on cognitive performance. Subsequent [125I]-bungarotoxin autoradiography revealed no direct correlation between α7 nAChR levels in frontal cortical or hippocampal brain regions and short-term memory with either compound. Additionally, repeated treatment with A-582941 did not affect mRNA expression of RIC-3 or the lynx-like gene products lynx1, lynx2, PSCA, or Ly6H, which are known to affect nAChR function. In conclusion, both α7 nAChR agonists and PAMs exhibit sustained pro-cognitive effects after repeated administration, and altered levels of the α7 nAChR per se, or that of endogenous regulators of nAChR function, are likely not the major cause of this effect.