Functional and structural interaction of (−)-lobeline with human α4β2 and α4β4 nicotinic acetylcholine receptor subtypes

To determine the pharmacologic activity of (−)-lobeline between human (h)α4β2 and hα4β4 nicotinic acetylcholine receptors (AChRs), functional and structural experiments were performed. The Ca²⁺ influx results established that (−)-lobeline neither actives nor enhances the function of the studied AChR subtypes, but competitively inhibits hα4β4 AChRs with potency ∼10-fold higher than that for hα4β2 AChRs. This difference is due to a higher binding affinity for the [³H]cytisine sites at hα4β4 compared to hα4β2 AChRs, which, in turn, can be explained by our molecular dynamics (MD) results: (1) higher stability of (−)-lobeline and its hydrogen bonds within the α4β4 pocket compared to the α4β2 pocket, (2) (−)-lobeline promotes Loop C to cap the binding site at the α4β4 pocket, but forces Loop C to get apart from the α4β2 pocket, precluding the gating process elicited by agonists, and (3) the orientation of (−)-lobeline within the α4β4, but not the α4β2, subpocket, promoted by the t− (or t+) rotameric state of α4-Tyr98, remains unchanged during the whole MD simulation. This study gives a detailed view of the molecular and dynamics events evoked by (−)-lobeline supporting the differential binding affinity and subsequent inhibitory potency between hα4β2 and hα4β4 AChRs, and supports the possibility that the latter subtype is also involved in its activity.     

The α7 nicotinic acetylcholine receptor in neuronal plasticity

A growing body of evidence indicates that neuronal nicotinic acetylcholine receptors (nAChRs), in addition to promoting fast cholinergic transmission, may modulate other neuronal activities within the central nervous system (CNS). In particular, the α7 nAChR is highly permeable to Ca²⁺ and may serve a distinct role in regulating neuronal plasticity. By elevating intracellular Ca²⁺ levels in discrete neuronal locations, these ligand-gated ion channels may influence numerous physiological processes in developing and adult CNS. In this article, we review evidence that both pre- and postsynaptic α7 nAChRs modulate transmitter release in the brain and periphery through Ca²⁺-dependent mechanisms. The possible role of α7 nAChRs in regulating neuronal growth and differentiation in developing CNS is also evaluated. We consider an interaction between cholinergic and glutamatergic transmission and propose a hypothesis on the possible coregulation of intracellular Ca²⁺ byN-methyl-d-aspartate (NMDA) receptors and α7 nAChRs. Finally, the clinical significance of alterations in the normal function of α7 nAChRs is discussed as it pertains to prenatal nicotine exposure, schizophrenia, and epilepsy.     

Novel positive allosteric modulators of the human α7 nicotinic acetylcholine receptor

The pharmacological activity of a series of novel amide derivatives was characterized on several nicotinic acetylcholine receptors (AChRs). Ca(2+) influx results indicate that these compounds are not agonists of the human (h) α4β2, α3β4, α7, and α1β1γδ AChRs; compounds 2-4 are specific positive allosteric modulators (PAMs) of hα7 AChRs, whereas compounds 1-4, 7, and 12 are noncompetitive antagonists of the other AChRs. Radioligand binding results indicate that PAMs do not inhibit binding of [(3)H]methyllycaconitine but enhance binding of [(3)H]epibatidine to hα7 AChRs, indicating that these compounds do not directly, but allosterically, interact with the hα7 agonist sites. Additional competition binding results indicate that the antagonistic action mediated by these compounds is produced by direct interaction with neither the phencyclidine site in the Torpedo AChR ion channel nor the imipramine and the agonist sites in the hα4β2 and hα3β4 AChRs. Molecular dynamics and docking results suggest that the binding site for compounds 2-4 is mainly located in the inner β-sheet of the hα7-α7 interface, ～12 ? from the agonist locus. Hydrogen bond interactions between the amide group of the PAMs and the hα7 AChR binding site are found to be critical for their activity. The dual PAM and antagonistic activities elicited by compounds 2-4 might be therapeutically important.     

Identification of a negative allosteric site on human α4β2 and α3β4 neuronal nicotinic acetylcholine receptors

Acetylcholine-based neurotransmission is regulated by cationic, ligand-gated ion channels called nicotinic acetylcholine receptors (nAChRs). These receptors have been linked to numerous neurological diseases and disorders such as Alzheimer's disease, Parkinson's disease, and nicotine addiction. Recently, a class of compounds has been discovered that antagonize nAChR function in an allosteric fashion. Models of human α4β2 and α3β4 nicotinic acetylcholine receptor (nAChR) extracellular domains have been developed to computationally explore the binding of these compounds, including the dynamics and free energy changes associated with ligand binding. Through a blind docking study to multiple receptor conformations, the models were used to determine a putative binding mode for the negative allosteric modulators. This mode, in close proximity to the agonist binding site, is presented in addition to a hypothetical mode of antagonism that involves obstruction of C loop closure. Molecular dynamics simulations and MM-PBSA free energy of binding calculations were used as computational validation of the predicted binding mode, while functional assays on wild-type and mutated receptors provided experimental support. Based on the proposed binding mode, two residues on the β2 subunit were independently mutated to the corresponding residues found on the β4 subunit. The T58K mutation resulted in an eight-fold decrease in the potency of KAB-18, a compound that exhibits preferential antagonism for human α4β2 over α3β4 nAChRs, while the F118L mutation resulted in a loss of inhibitory activity for KAB-18 at concentrations up to 100 μM. These results demonstrate the selectivity of KAB-18 for human α4β2 nAChRs and validate the methods used for identifying the nAChR modulator binding site. Exploitation of this site may lead to the development of more potent and subtype-selective nAChR antagonists which may be used in the treatment of a number of neurological diseases and disorders.     

Pharmacological and molecular studies on the interaction of varenicline with different nicotinic acetylcholine receptor subtypes. Potential mechanism underlying partial agonism at human α4β2 and α3β4 subtypes

To determine the structural components underlying differences in affinity, potency, and selectivity of varenicline for several human (h) nicotinic acetylcholine receptors (nAChRs), functional and structural experiments were performed. The Ca^2 + influx results established that: (a) varenicline activates (μM range) nAChR subtypes with the following rank sequence: hα7 > hα4β4 > hα4β2 > hα3β4 >>> hα1β1γδ; (b) varenicline binds to nAChR subtypes with the following affinity order (nM range): hα4β2 ~ hα4β4 > hα3β4 > hα7 >>> Torpedo α1β1γδ. The molecular docking results indicating that more hydrogen bond interactions are apparent for α4-containing nAChRs in comparison to other nAChRs may explain the observed higher affinity; and that (c) varenicline is a full agonist at hα7 (101%) and hα4β4 (93%), and a partial agonist at hα4β2 (20%) and hα3β4 (45%), relative to (±)-epibatidine. The allosteric sites found at the extracellular domain (EXD) of hα3β4 and hα4β2 nAChRs could explain the partial agonistic activity of varenicline on these nAChR subtypes. Molecular dynamics simulations show that the interaction of varenicline to each allosteric site decreases the capping of Loop C at the hα4β2 nAChR, suggesting that these allosteric interactions limit the initial step in the gating process. In conclusion, we propose that in addition to hα4β2 nAChRs, hα4β4 nAChRs can be considered as potential targets for the clinical activity of varenicline, and that the allosteric interactions at the hα3β4- and hα4β2-EXDs are alternative mechanisms underlying partial agonism at these nAChRs.     

Localization by site-directed mutagenesis of a galantamine binding site on α7 nicotinic acetylcholine receptor extracellular domain

Galantamine is an approved drug treatment for Alzheimer's disease. Initially identified as a weak cholinesterase inhibitor, we have established that galantamine mainly acts as an 'allosterically potentiating ligand (APL)' of nicotinic acetylcholine receptors (nAChR). Meanwhile other 'positive allosteric modulators (PAM)' of nAChR channel activity have been discovered, and for one of them a binding site within the transmembrane domain has been proposed. Here we show, by performing site-directed mutagenesis studies of ectopically expressed chimeric chicken α7/mouse 5-hydroxytryptamine 3 receptor-channel complex, in combination with whole-cell current measurements, in the presence and absence of galantamine, that the APL binding site is different from the proposed PAM binding site. We demonstrate that residues T197, I196, and F198 of ?-strand 10 represent major elements of the galantamine binding site. Residue K123, earlier suggested as being 'close to' the APL binding site, is not part of this site but rather appears to play a role in coupling of agonist binding to channel opening and closing. Our data confirm our earlier results that the galantamine binding site is different from the ACh binding site. Both sites are in close proximity and hence may influence each other in a synergistic fashion. Other interesting areas identified in the present study are a 'hinge' region around and containing residues F122, K123, and K143 possibly being involved in relaying the signal of agonist binding to gating of the transmembrane channel, and a 'folding centre', with P119 as the dominating residue, that crucially positions the agonist binding site with respect to the hinge region.     

Molecular modeling of the α9α10 nicotinic acetylcholine receptor subtype

This study reports the comparative molecular modeling, docking and dynamic simulations of human α9α10 nicotinic acetylcholine receptors complexed with acetylcholine, nicotine and α-conotoxin RgIA, using as templates the crystal structures of Aplysia californica and Lymnaea stagnalis acetylcholine binding proteins. The molecular dynamics simulations showed that Arg112 in the complementary α10(?) subunit, is a determinant for recognition in the site that binds small ligands. However, Glu195 in the principal α9(+), and Asp114 in the complementary α10(?) subunit, might confer the potency and selectivity to α-conotoxin RgIA when interacting with Arg7 and Arg9 of this ligand.     

Structural and dynamic studies of the γ-M4 trans-membrane domain of the nicotinic acetylcholine receptor

A structural characterization of a synthetic peptide corresponding to the fourth transmembrane domain (M4-TMD) of the γ-subunit of the nicotinic acetylcholine receptor from Torpedo californica has been undertaken. Solid-state NMR and CD spectroscopy studies indicate that upon reconstitution into lipid vesicles or magnetically aligned lipid bilayers, the synthetic M4-TMD adopts a linear α-helical conformation with the helix aligned within 15° of the membrane normal. Furthermore, analysis of the motional averaging of anisotropic interactions present in the solid-state NMR spectra of the reconstituted peptide, indicate that the dynamics of the peptide within the bilayer are highly sensitive to the phase adopted by the lipid bilayer, providing an insight into how the interaction of lipids with this domain may play a important role in the modulation of this receptor by its lipid environment.     

Functional expression of the α7 and α4-containing nicotinic acetylcholine receptors on the neonatal rat carotid body

The carotid bodies (CBs) are chemosensory organs that respond to hypoxemia with transmitter neurosecretion, leading to a respiratory reflex response. It has been proposed that acetylcholine is a key regulator of transmitter release through activation of presynaptic nicotinic acetylcholine receptors (nAChRs). In the present work, we studied the identity of such nAChRs and their contribution to catecholamine release from CBs. Neonatal rat CBs were placed in a recording chamber for electrochemical recordings or disassociated for voltage-clamp studies on isolated cells. Fast nicotine superfusion increases catecholamine release from intact CBs. This response was diminished reversibly by the non-selective nAChR blocker hexamethonium, by the selective α7 blocker α-bungarotoxin and by the α4-containing nAChR blocker erysodine. In isolated CB cells the nAChR agonists nicotine, acetylcholine and cytisine all evoke inward currents with similar potencies. The nicotine-evoked current was fully blocked by mecamylamine and partially inhibited by α-bungarotoxin or erysodine. However, the combination of both α-bungarotoxin an erysodine failed to suppress this response. Immunodetection studies confirm the presence of α7 and α4 subunits in isolated dopaminergic CB cells. Our results show that activation of α7 and/or α4-containing nAChR subtypes have the ability to regulate catecholamine release from intact CB due to activation of fast inward currents expressed in chemoreceptor cells. Therefore, our results suggest that both nAChR subtypes contribute to the cholinergic nicotinic regulation of catecholamine signaling in the carotid body system.     

Molecular docking study on the α3β2 neuronal nicotinic acetylcholine receptor complexed with α-conotoxin GIC

Nicotinic acetylcholine receptors (nAChRs) are a diverse family of homo- or heteropentameric ligand-gated ion channels. Understanding the physiological role of each nAChR subtype and the key residues responsible for normal and pathological states is important. α-Conotoxin neuropeptides are highly selective probes capable of discriminating different subtypes of nAChRs. In this study, we performed homology modeling to generate the neuronal α3, β2 and β4 subunits using the x-ray structure of the α1 subunit as a template. The structures of the extracellular domains containing ligand binding sites in the α3β2 and α3β4 nAChR subtypes were constructed using MD simulations and ligand docking processes in their free and ligand-bound states using α-conotoxin GIC, which exhibited the highest α3β2 vs. α3β4 discrimination ratio. The results provide a reasonable structural basis for such a discriminatory ability, supporting the idea that the present strategy can be used for future investigations on nAChR-ligand complexes.     

The structures of the human neuronal nicotinic acetylcholine receptor β2- and α3-subunit genes (CHRNB2 and CHRNA3)

The α4-subunit gene (CHRNA4) of the neuronal nicotinic acetylcholine receptor (nAChR) subunit family has recently been identified in two families as the gene responsible for autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), a rare monogenic idiopathic epilepsy. As a result of this finding, other subunits of the neuronal nAChR gene family are being considered as candidate genes for ADNFLE in families not linked to CHRNA4 and for other idiopathic epilepsies. α4-subunitsoften assemble together with β2-subunits (gene symbol CHRNB2) to build heteromeric nAChRs. The gene encoding another abundant AChR subunit, the α3-subunit gene (CHRNA3), is present with those encoding two other subunits, CHRNB4 and CHRNA5, in a gene cluster whose functional role is still unclear. Here we provide the information on the genomic structures of both the CHRNB2 and the CHRNA3 genes that is necessary for comprehensive mutational analyses, and we refine the genomic assignment of CHRNB2 on chromosome 1. Received: 5 August 1998 / Accepted: 13 October 1998     

Diazaspirocyclic compounds as selective ligands for the α4β2 nicotinic acetylcholine receptor

Diazaspirocyclic ligands have been synthesized in four steps as selective α4β2 nicotinic acetylcholine receptor antagonists. Structural assignment of 1-(pyridin-3-yl)-2-spiropyrrolidino-3,2'-1-azabiclo[2.2.1]heptane 2, was confirmed using a combination of NMR experiments on a key intermediate, spirolactam 9. All three target compounds synthesized in this diazaspirocyclic series exhibited high affinity (K(i)<35 nM) at the human α4β2 nAChR subtype, and very low affinity for the human α7, α3β4 (ganglion) and α1β1γδ (muscle) subtypes (K(i)>500 nM).     

Inhibition mechanism of rat α₃β₄ nicotinic acetylcholine receptor by the Alzheimer therapeutic tacrine

Nicotinic acetylcholine receptors (nAChRs) were studied in detail in the past regarding their interaction with therapeutic and drug addiction related compounds. Using fast kinetic whole-cell recording, we have now studied effects of tacrine, an agent used clinically to treat Alzheimer's disease, on currents elicited by activation of rat α(3)β(4) nAChR heterologously expressed in KXα3β4R2 cells. Characterization of receptor activation by nicotine used as agonist revealed a K(d) of 23 ± 0.2 μM and 4.3 ± 1.3 for the channel opening equilibrium constant, Φ(-1). Experiments were performed to investigate whether tacrine is able to activate the α(3)β(4) nAChR. Tacrine did not activate whole-cell currents in KXα3β4R2 cells but inhibited receptor activity at submicromolar concentration. Dose-response curves obtained with increasing agonist or inhibitor concentration revealed competitive inhibition of nAChRs by tacrine, with an apparent inhibition constant, K(I), of 0.8 μM. The increase of Φ(-1) in the presence of tacrine suggests that the drug stabilizes a nonconducting open channel form of the receptor. Binding studies with TCP and MK-801 ruled out tacrine binding to common allosteric sites of the receptor. Our study suggests a novel mechanism for action of tacrine on nAChRs besides inhibition of acetylcholine esterase.     

Photoaffinity labeling the agonist binding domain of α4β4 and α4β2 neuronal nicotinic acetylcholine receptors with [I]epibatidine and 5[I]A-85380

The development of nicotinic acetylcholine receptor (nAChR) agonists, particularly those that discriminate between neuronal nAChR subtypes, holds promise as potential therapeutic agents for many neurological diseases and disorders. To this end, we photoaffinity labeled human α4β2 and rat α4β4 nAChRs affinity-purified from stably transfected HEK-293 cells, with the agonists [¹²⁵I]epibatidine and 5[¹²⁵I]A-85380. Our results show that both agonists photoincorporated into the β4 subunit with little or no labeling of the β2 and α4 subunits respectively. [¹²⁵I]epibatidine labeling in the β4 subunit was mapped to two overlapping proteolytic fragments that begin at β4V102 and contain Loop E (β4I109-P120) of the agonist binding site. We were unable to identify labeled amino acid(s) in Loop E by protein sequencing, but we were able to demonstrate that β4Q117 in Loop E is the principal site of [¹²⁵I]epibatidine labeling. This was accomplished by substituting residues in the β2 subunit with the β4 homologs and finding [¹²⁵I]epibatidine labeling in β4 and β2F119Q subunits with little, if any, labeling in α4, β2, or β2S113R subunits. Finally, functional studies established that the β2F119/β4Q117 position is an important determinant of the receptor subtype-selectivity of the agonist 5I-A-85380, affecting both binding affinity and channel activation.     

Detection of a CfoI polymorphism within exon 5 of the human neuronal nicotinic acetylcholine receptor α4 subunit gene (CHRNA4)

The gene coding for the 4 subunit of the neuronal nicotinic acetylcholine receptor (CHRNA4) has been recently mapped in the candidate region for benign familial neonatal convulsions (BFNC) on chromosome 20q13.2–q13.3. The region is only partially covered with polymorphic markers, and so far no PCR-based polymorphisms have been described in the critical region for BFNC. We now report the first polymorphic marker in the coding region of CHRNA4. The new marker, which is detected by PCR, will be useful for evaluation of the role of CHRNA4 as a candidate gene for BFNC. It will further enable the investigation of this important brain-specific gene in association studies with different types of epileptic diseases and other neurological disorders.     

Development of spiroguanidine-derived α7 neuronal nicotinic receptor partial agonists

We describe the synthesis of quinuclidine-containing spiroguanidines and their utility as α7 neuronal nicotinic acetylcholine receptor (nAChR) partial agonists. The convergent synthetic route developed for this study allowed for rapid SAR investigation and provided access to a structurally diverse set of analogs. A potent and selective α7 nAChR partial agonist, N‐(6‐methyl‐1,3‐benzoxazol‐2‐yl)‐3′,5′‐dihydro‐4‐azaspiro[bicyclo[2.2.2]octane‐2,4′‐imidazole]‐2′‐amine (BMS-910731, 16), was identified. This compound induced immediate early genes c-fos and Arc in a preclinical rodent model of α7 nAChR-derived cellular activation and plasticity. Importantly, the ability to incorporate selectivity for the α7 nACh receptor over the 5-HT_3A receptor in this series suggested a significant difference in steric requirements between the two receptors.     

Multiple nicotinic acetylcholine receptor α-subunits are expressed in the arterial system of the rat

Neuronal nicotinic acetylcholine receptors (nAChRs) are hetero- and homopentamers built up by nine different alpha-subunits and three different beta-subunits. The subtype composition within the receptor determines ligand specificity, affinity and cation permeability. In this study we focused on the distribution of the ligand binding alpha-subunits in the rat arterial system by means of RT-PCR and immunohistochemistry. Subtypes alpha3, alpha5, alpha7 and alpha10 were found to be expressed by endothelial cells, suggesting that they are equipped both with calcium-preferring (alpha7 homopentamers) and monovalent cation-preferring (heteropentamers containing alpha3- and alpha5-subunits) nAChR channels. All alpha-subtypes except alpha9 were expressed by vascular smooth muscle cells with a highly specific distribution pattern along the vascular tree. While every alpha-subunit except alpha9 was detected in the thoracic aorta, intrapulmonary arterial branches contained only alpha7 immunoreactivity, and other vascular beds held intermediate positions with respect to the extent of alpha-subunit expression. Current knowledge does not allow to correlate these distribution patterns to specific functions, but it can be anticipated that at least some components of nAChR-mediated signalling in the arterial wall are highly specific for individual arteries.