Domain Of Nicotinic Acetylcholine Receptor Research Articles

Drysdalin, a snake neurotoxin with higher affinity for soluble acetylcholine binding protein from Aplysia californica than from Lymnaea stagnalis

Acetylcholine binding proteins (AChBPs), structural and functional surrogates of the extracellular binding domain of nicotinic acetylcholine receptor (nAChRs), in complex with various antagonists and agonists have provided detailed insights into the neurotransmitter binding site of nAChRs. The classical long-chain α-neurotoxins bungarotoxin (44-fold) and cobratoxin (7-fold) bind to Lymnaea stagnalis (Ls)-AChBP with higher affinity compared to Aplysia californica (Ac)-AChBP. In this study, we describe a novel long chain α-neurotoxin Drysdalin, which has higher binding affinity (7-fold) to Ac-AChBP when compared to Ls-AChBP. This suggests an involvement of different regions or modes of interaction of drysdalin, when compared to the bungarotoxin and cobratoxin. We also found that the C-terminal 24-amino acid residues of drysdalin are critical for the binding to Ac-AChBP and its removal caused ~90-fold reduction in affinity. Further to understand the interaction of drysdalin with Ac-AChBP, we studied the role of three non-conserved amino acid residues of drysdalin, namely Arg30, Leu34 and Ala37. Substitution of Arg30 with the conserved Phe residue caused a ~100-fold reduction, Leu34 with conserved Arg caused a ~6-fold reduction, whereas substitution of Ala37 with conserved Arg enhanced the binding by 3-fold. The dramatic influence of this carboxyl terminal sequence enriched in arginine and proline residues suggests that the toxin binding pose is influenced primarily by this extended sequence.

Spider acetylcholine binding proteins: An alternative model to study the interaction between insect nAChRs and neonicotinoids

Acetylcholine binding proteins (AChBPs) are homologs of extracellular domains of nicotinic acetylcholine receptors (nAChRs) and serve as models for studies on nAChRs. Particularly, studies on invertebrate nAChRs that are limited due to difficulties in their heterologous expression have benefitted from the discovery of AChBPs. Thus far, AChBPs have been characterized only in aquatic mollusks, which have shown low sensitivity to neonicotinoids, the insecticides targeting insect nAChRs. However, AChBPs were also found in spiders based on the sequence and tissue expression analysis. Here, we report five AChBP subunits in Pardosa pseudoannulata, a predator enemy against rice insect pests. Spider AChBP subunits shared higher sequence similarities with nAChR subunits of both insects and mammals compared with mollusk AChBP subunits. The AChBP1 subunit of P. pseudoannulata (Pp-AChBP) was then expressed in Sf9 cells. The Ls-AChBP from Lymnaea stagnalis was also expressed for comparison. In both AChBPs, one ligand site per subunit was present at each interface between two adjacent subunits. Neonicotinoids had higher affinities (7.9–18.4 times based on Kd or Ki values) for Pp-AChBP than for Ls-AChBP, although epibatidine and α-bungarotoxin showed higher affinities for Ls-AChBP. These results indicate that spider AChBP could be used as an alternative model to study the interaction between insect nAChRs and neonicotinoids.

From crystal structure of α-conotoxin GIC in complex with Ac-AChBP to molecular determinants of its high selectivity for α3β2 nAChR.

Acetylcholine binding proteins (AChBPs) are unique spatial homologs of the ligand-binding domains of nicotinic acetylcholine receptors (nAChRs), and they reproduce some pharmacological properties of nAChRs. X-ray crystal structures of AСhBP in complex with α-conotoxins provide important insights into the interactions of α-conotoxins with distinct nAChR subtypes. Although considerable efforts have been made to understand why α-conotoxin GIC is strongly selective for α3β2 nAChR, this question has not yet been solved. Here we present the structure of α-conotoxin GIC in complex with Aplysia californica AChBP (Ac-AChBP) at a resolution of 2.1 Å. Based on this co-crystal structure complemented with molecular docking data, we suggest the key residues of GIC in determining its high affinity and selectivity for human α3β2 vs α3β4 nAChRs. These suggestions were checked by radioligand and electrophysiology experiments, which confirmed the functional role of detected contacts for GIC interactions with Ac-AChBP and α3β2 nAChR subtypes. While GIC elements responsible for its high affinity binding with Ac-AChBP and α3β2 nAChR were identified, our study also showed the limitations of computer modelling in extending the data from the X-ray structures of the AChBP complexes to all nAChR subtypes.

Marine Natural Products Acting on the Acetylcholine-Binding Protein and Nicotinic Receptors: From Computer Modeling to Binding Studies and Electrophysiology

For a small library of natural products from marine sponges and ascidians, in silico docking to the Lymnaea stagnalis acetylcholine-binding protein (AChBP), a model for the ligand-binding domains of nicotinic acetylcholine receptors (nAChRs), was carried out and the possibility of complex formation was revealed. It was further experimentally confirmed via competition with radioiodinated α-bungarotoxin ([125I]-αBgt) for binding to AChBP of the majority of analyzed compounds. Alkaloids pibocin, varacin and makaluvamines С and G had relatively high affinities (Ki 0.5–1.3 μM). With the muscle-type nAChR from Torpedo californica ray and human neuronal α7 nAChR, heterologously expressed in the GH4C1 cell line, no competition with [125I]-αBgt was detected in four compounds, while the rest showed an inhibition. Makaluvamines (Ki ~ 1.5 μM) were the most active compounds, but only makaluvamine G and crambescidine 359 revealed a weak selectivity towards muscle-type nAChR. Rhizochalin, aglycone of rhizochalin, pibocin, makaluvamine G, monanchocidin, crambescidine 359 and aaptamine showed inhibitory activities in electrophysiology experiments on the mouse muscle and human α7 nAChRs, expressed in Xenopus laevis oocytes. Thus, our results confirm the utility of the modeling studies on AChBPs in a search for natural compounds with cholinergic activity and demonstrate the presence of the latter in the analyzed marine biological sources.

Analysis of rare variations reveals roles of amino acid residues in the N-terminal extracellular domain of nicotinic acetylcholine receptor (nAChR) alpha6 subunit in the functional expression of human alpha6*-nAChRs.

BackgroundFunctional heterologous expression of naturally-expressed and apparently functional mammalian α6*-nicotinic acetylcholine receptors (nAChRs; where ‘*’ indicates presence of additional subunits) has been difficult. Here we wanted to investigate the role of N-terminal domain (NTD) residues of human (h) nAChR α6 subunit in the functional expression of hα6*-nAChRs. To this end, instead of adopting random mutagenesis as a tool, we used 15 NTD rare variations (i.e., Ser43Pro, Asn46Lys, Asp57Asn, Arg87Cys, Asp92Glu, Arg96His, Glu101Lys, Ala112Val, Ser156Arg, Asn171Lys, Ala184Asp, Asp199Tyr, Asn203Thr, Ile226Thr and Ser233Cys) in nAChR hα6 subunit to probe for their effect on the functional expression of hα6*-nAChRs.ResultsN-terminal α-helix (Asp57); complementary face/inner β-fold (Arg87 or Asp92) and principal face/outer β-fold (Ser156 or Asn171) residues in the hα6 subunit are crucial for functional expression of the hα6*-nAChRs as variations in these residues reduce or abrogate the function of hα6hβ2*-, hα6hβ4- and hα6hβ4hβ3-nAChRs. While variations at residues Ser43 or Asn46 (both in N-terminal α-helix) in hα6 subunit reduce hα6hβ2*-nAChRs function those at residues Arg96 (β2-β3 loop), Asp199 (loop F) or Ser233 (β10-strand) increase hα6hβ2*-nAChR function. Similarly substitution of NTD α-helix (Asn46), loop F (Asp199), loop A (Ala112), loop B (Ala184), or loop C (Ile226) residues in hα6 subunit increase the function of hα6hβ4-nAChRs. All other variations in hα6 subunit do not affect the function of hα6hβ2*- and hα6hβ4*-nAChRs. Incorporation of nAChR hβ3 subunits always increase the function of wild-type or variant hα6hβ4-nAChRs except for those of hα6(D57N, S156R, R87C or N171K)hβ4-nAChRs. It appears Asp57Lys, Ser156Arg or Asn171Lys variations in hα6 subunit drive the hα6hβ4hβ3-nAChRs into a nonfunctional state as at spontaneously open hα6(D57N, S156R or N171K)hβ4hβ3V9’S-nAChRs (V9’S; transmembrane II 9’ valine-to-serine mutation) agonists act as antagonists. Agonist sensitivity of hα6hβ4- and/or hα6hβ4hβ3-nAChRs is nominally increased due to Arg96His, Ala184Asp, Asp199Tyr or Ser233Cys variation in hα6 subunit.ConclusionsHence investigating functional consequences of natural variations in nAChR hα6 subunit we have discovered additional bases for cell surface functional expression of various subtypes of hα6*-nAChRs. Variations (Asp57Asn, Arg87Cys, Asp92Glu, Ser156Arg or Asn171Lys) in hα6 subunit that compromise hα6*-nAChR function are expected to contribute to individual differences in responses to smoked nicotine.

Synthesis of Selective Agonists for the α7 Nicotinic Acetylcholine Receptor with In Situ Click-Chemistry on Acetylcholine-Binding Protein Templates

The acetylcholine-binding proteins (AChBPs), which serve as structural surrogates for the extracellular domain of nicotinic acetylcholine receptors (nAChRs), were used as reaction templates for in situ click-chemistry reactions to generate a congeneric series of triazoles from azide and alkyne building blocks. The catalysis of in situ azide-alkyne cycloaddition reactions at a dynamic subunit interface facilitated the synthesis of potentially selective compounds for nAChRs. We investigated compound sets generated in situ with soluble AChBP templates through pharmacological characterization with α7 and α4β2 nAChRs and 5-hydroxytryptamine type 3A receptors. Analysis of activity differences between the triazole 1,5-syn- and 1,4-anti-isomers showed a preference for the 1,4-anti-triazole regioisomers among nAChRs. To improve nAChR subtype selectivity, the highest-potency building block for α7 nAChRs, i.e., 3α-azido-N-methylammonium tropane, was used for additional in situ reactions with a mutated Aplysia californica AChBP that was made to resemble the ligand-binding domain of the α7 nAChR. Fourteen of 50 possible triazole products were identified, and their corresponding tertiary analogs were synthesized. Pharmacological assays revealed that the mutated binding protein template provided enhanced selectivity of ligands through in situ reactions. Discrete trends in pharmacological profiles were evident, with most compounds emerging as α7 nAChR agonists and α4β2 nAChR antagonists. Triazoles bearing quaternary tropanes and aromatic groups were most potent for α7 nAChRs. Pharmacological characterization of the in situ reaction products established that click-chemistry synthesis with surrogate receptor templates offered novel extensions of fragment-based drug design that were applicable to multisubunit ion channels.

Tryptophan scanning mutagenesis reveals distortions in the helical structure of the δM4 transmembrane domain of the Torpedo californica nicotinic acetylcholine receptor

The lipid-protein interface is an important domain of the nicotinic acetylcholine receptor (nAChR) that has recently garnered increased relevance. Several studies have made significant advances toward determining the structure and dynamics of the lipid-exposed domains of the nAChR. However, there is still a need to gain insight into the mechanism by which lipid-protein interactions regulate the function and conformational transitions of the nAChR. In this study, we extended the tryptophan scanning mutagenesis (TrpScanM) approach to dissect secondary structure and monitor the conformational changes experienced by the δM4 transmembrane domain (TMD) of the Torpedo californica nAChR, and to identify which positions on this domain are potentially linked to the regulation of ion channel kinetics. The difference in oscillation patterns between the closed- and open-channel states suggests a substantial conformational change along this domain as a consequence of channel activation. Furthermore, TrpScanM revealed distortions along the helical structure of this TMD that are not present on current models of the nAChR. Our results show that a Thr-Pro motif at positions 462–463 markedly bends the helical structure of the TMD, consistent with the recent crystallographic structure of the GluCl Cys-loop receptor which reveals a highly bent TMD4 in each subunit. This Thr-Pro motif acts as a molecular hinge that delineates two gating blocks in the δM4 TMD. These results suggest a model in which a hinge-bending motion that tilts the helical structure is combined with a spring-like motion during transition between the closed- and open-channel states of the δM4 TMD.

NMR structure and dynamics of a designed water-soluble transmembrane domain of nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor (nAChR) is an important therapeutic target for a wide range of pathophysiological conditions, for which rational drug designs often require receptor structures at atomic resolution. Recent proof-of-concept studies demonstrated a water-solubilization approach to structure determination of membrane proteins by NMR (Slovic et al., PNAS, 101: 1828–1833, 2004; Ma et al., PNAS, 105: 16537–42, 2008). We report here the computational design and experimental characterization of WSA, a water-soluble protein with ~83% sequence identity to the transmembrane (TM) domain of the nAChR α1 subunit. Although the design was based on a low-resolution structural template, the resulting high-resolution NMR structure agrees remarkably well with the recent crystal structure of the TM domains of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC), demonstrating the robustness and general applicability of the approach. NMR T2 dispersion measurements showed that the TM2 domain of the designed protein was dynamic, undergoing conformational exchange on the NMR timescale. Photoaffinity labeling with isoflurane and propofol photolabels identified a common binding site in the immediate proximity of the anesthetic binding site found in the crystal structure of the anesthetic-GLIC complex. Our results illustrate the usefulness of high-resolution NMR analyses of water-solubilized channel proteins for the discovery of potential drug binding sites.

Discrimination of agonists versus antagonists of nicotinic ligands based on docking onto AChBP structures

Numerous high-resolution crystallographic structures of the acetylcholine binding protein (AChBP), a molluscan cholinergic protein, homologous to the extracellular domain of nicotinic acetylcholine receptors, are available. This offers opportunities to model the interaction between various ligands and the acetylcholine binding site. Herein we present a study of the interplay between ligand binding and motions of the C-loop capping the binding site. Nicotinic agonists and antagonists were docked on AChBP X-ray structures. It is shown that the studied agonists and antagonists can be discriminated according to their higher affinities for structures respectively obtained in the presence of agonists or antagonists, highlighting the fact that AChBP structures retain a pharmacological footprint of the compound used in crystallography experiments. A detailed analysis of the binding site cavities suggests that this property is mainly related to the shape of the cavities.

Structural determinants for interaction of partial agonists with acetylcholine binding protein and neuronal α7 nicotinic acetylcholine receptor

The pentameric acetylcholine-binding protein (AChBP) is a soluble surrogate of the ligand binding domain of nicotinic acetylcholine receptors. Agonists bind within a nest of aromatic side chains contributed by loops C and F on opposing faces of each subunit interface. Crystal structures of Aplysia AChBP bound with the agonist anabaseine, two partial agonists selectively activating the alpha7 receptor, 3-(2,4-dimethoxybenzylidene)-anabaseine and its 4-hydroxy metabolite, and an indole-containing partial agonist, tropisetron, were solved at 2.7-1.75 A resolution. All structures identify the Trp 147 carbonyl oxygen as the hydrogen bond acceptor for the agonist-protonated nitrogen. In the partial agonist complexes, the benzylidene and indole substituent positions, dictated by tight interactions with loop F, preclude loop C from adopting the closed conformation seen for full agonists. Fluctuation in loop C position and duality in ligand binding orientations suggest molecular bases for partial agonism at full-length receptors. This study, while pointing to loop F as a major determinant of receptor subtype selectivity, also identifies a new template region for designing alpha7-selective partial agonists to treat cognitive deficits in mental and neurodegenerative disorders.

Influence of the M3–M4 intracellular domain upon nicotinic acetylcholine receptor assembly, targeting and function

The aim of this study was to investigate the influence of the intracellular domain of nicotinic acetylcholine receptor (nAChR) subunits upon receptor assembly, targeting and functional properties. Because most nAChR subunits form functional receptors only as heteromeric complexes, it can be difficult to examine the influence of individual subunits or subunit domains in isolation. A series of subunit chimaeras was constructed which contain the intracellular loop region (located between the M3 and M4 transmembrane domains) from nAChR subunits alpha1-alpha10 or beta1-beta4. All of these chimaeras contain common extracellular and transmembrane domains (from the nAChR alpha7 subunit and the 5-hydroxytryptamine receptor 5-HT(3A) subunit, respectively), thereby facilitating both homomeric receptor assembly and detection with radiolabelled or fluorescent alpha-bungarotoxin. The nAChR M3-M4 intracellular loop domain had no significant effect upon levels of total subunit protein detected in transfected cells but had a significant influence upon levels of both cell surface and intracellular assembled receptors. Comparisons of functional properties revealed a significant influence of the intracellular loop domain upon both single-channel conductance and receptor desensitization. In addition, studies conducted in polarized epithelial cells demonstrate that the nAChR loop can influence receptor targeting, resulting in either polarized (apical) or non-polarized distribution. Evidence has been obtained which demonstrates that the large intracellular loop domain of nAChR subunits can exert a profound influence upon receptor assembly, targeting and ion channel properties.

Extracellular Domain Nicotinic Acetylcholine Receptors Formed by α4 and β2 Subunits

Models of the extracellular ligand-binding domain of nicotinic acetylcholine receptors (nAChRs), which are pentameric integral membrane proteins, are attractive for structural studies because they potentially are water-soluble and better candidates for x-ray crystallography and because their smaller size is more amenable for NMR spectroscopy. The complete N-terminal extracellular domain is a promising foundation for such models, based on previous studies of alpha7 and muscle-type subunits. Specific design requirements leading to high structural fidelity between extracellular domain nAChRs and full-length nAChRs, however, are not well understood. To study these requirements in heteromeric nAChRs, the extracellular domains of alpha4 and beta2 subunits with or without the first transmembrane domain (M1) were expressed in Xenopus oocytes and compared with alpha4beta2 nAChRs based on ligand binding and subunit assembly properties. Ligand affinities of detergent-solubilized, extracellular domain alpha4beta2 nAChRs formed from subunits with M1 were nearly identical to affinities of alpha4beta2 nAChRs when measured with [3H]epibatidine, cytisine, nicotine, and acetylcholine. Velocity sedimentation suggested that these extracellular domain nAChRs predominantly formed pentamers. The yield of these extracellular domain nAChRs was about half the yield of alpha4beta2 nAChRs. In contrast, [3H]epibatidine binding was not detected from the extracellular domain alpha4 and beta2 subunits without M1, implying no detectable expression of extracellular domain nAChRs from these subunits. These results suggest that M1 domains on both alpha4 and beta2 play an important role for efficient expression of extracellular domain alpha4beta2 nAChRs that are high fidelity structural models of full-length alpha4beta2 nAChRs.

Intracellular domain of nicotinic acetylcholine receptor: the importance of being unfolded

Bioinformatics methods with subsequent verification by experimental data were applied to the structural investigation of the intracellular loop of the delta-subunit of the nicotinic acetylcholine receptor (nAChR). Three complementary methods were used: prediction of secondary structure elements, prediction of ordered/disordered protein regions and prediction of short functional binding motifs. The output of five different algorithms was used for the secondary structure construction. Most of the intracellular domain is predicted to be unfolded. The predictions correlate well with the experimental data of limited proteolysis and NMR performed on the mostly monomeric fraction of heterologously expressed Torpedo intracellular domain protein. Twelve functional binding motifs within the disordered regions of the nAChR intracellular domain are predicted. Identification of proteins that interact with the intracellular domain will provide a better understanding of protein-protein interactions involved in nAChR assembly, trafficking and clustering.

Curariform Antagonists Bind in Different Orientations to Acetylcholine-binding Protein

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 approximately 170 degrees rotation and approximately 30 degrees 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.

Structural Evaluation of Distant Homology. A 3-D Model of the Ligand Binding Domain of the Nicotinic Acetylcholine Receptor Based on Acetylcholinesterase: Consistency with Experimental Data

Acetylcholine is a ligand for both acetylcholinesterases and nicotinic acetylcholine receptors. Hence, at least some local sequence and structural similarities between the acetylcholinesterases and the receptors which recognize acetylcholine (ACh) might be expected. Peterson [2] produced an alignment of the ACh binding region between these two types of ACh–binding molecules, featuring a number of well conserved residues. The extent of this region of sequence similarity suggests the possible existence of a common ancenstral ACh binding module. To attempt to further validate Peterson′s sequence alignment we have built a homology model of the ACh binding domain of the human neuromuscular nicotinic acetylcholine receptor based on the structure of acetylcholinesterase from Torpedo californica. Using this 3–D model we have examined the residues which were previously shown to interact with the endogenous ligand by various methods (mapping, site–directed mutagenesis). The consistency of such data with the model provides further support for a structural similarity and possibly a divergent evolutionary relationship between the ACh–binding domains of these two classes of proteins. Results suggest that this model may be able to contribute to an understanding of the structure and function of the ACh receptor. Using this case as an example, we propose that 3–dimensional computer modeling can be used as a tool to evaluate distant homologies when adequate experimental data (e.g., site–directed mutagenesis) is available.