NMR structures of the transmembrane domains of the α4β2 nAChR

The α4β2 nicotinic acetylcholine receptor (nAChR) is the predominant heteromeric subtype of nAChRs in the brain, which has been implicated in numerous neurological conditions. The structural information specifically for the α4β2 and other neuronal nAChRs is presently limited. In this study, we determined structures of the transmembrane (TM) domains of the α4 and β2 subunits in lauryldimethylamine-oxide (LDAO) micelles using solution NMR spectroscopy. NMR experiments and size exclusion chromatography-multi-angle light scattering (SEC-MALS) analysis demonstrated that the TM domains of α4 and β2 interacted with each other and spontaneously formed pentameric assemblies in the LDAO micelles. The Na(+) flux assay revealed that α4β2 formed Na(+) permeable channels in lipid vesicles. Efflux of Na(+) through the α4β2 channels reduced intra-vesicle Sodium Green? fluorescence in a time-dependent manner that was not observed in vesicles without incorporating α4β2. The study provides structural insight into the TM domains of the α4β2 nAChR. It offers a valuable structural framework for rationalizing extensive biochemical data collected previously on the α4β2 nAChR and for designing new therapeutic modulators.     

The crystal structures of the α-subunit of the α2β2 tetrameric Glycyl-tRNA synthetase

Aminoacyl-tRNA synthetases (AARSs) are ligases (EC.6.1.1.-) that catalyze the acylation of amino acids to their cognate tRNAs in the process of translating genetic information from mRNA to protein. Their amino acid and tRNA specificity are crucial for correctly translating the genetic code. Glycine is the smallest amino acid and the glycyl-tRNA synthetase (GlyRS) belongs to Class II AARSs. The enzyme is unusual because it can assume different quaternary structures. In eukaryotes, archaebacteria and some bacteria, it forms an ??₂ homodimer. In some bacteria, GlyRS is an ??₂??₂ heterotetramer and shows a distant similarity to ??₂ GlyRSs. The human pathogen eubacterium Campylobacter jejuni GlyRS (CjGlyRS) is an ??₂??₂ heterotetramer and is similar to Escherichia coli GlyRS; both are members of Class IIc AARSs. The two-step aminoacylation reaction of tetrameric GlyRSs requires the involvement of both ??- and ??-subunits. At present, the structure of the GlyRS ??₂??₂ class and the details of the enzymatic mechanism of this enzyme remain unknown. Here we report the crystal structures of the catalytic ??-subunit of CjGlyRS and its complexes with ATP, and ATP and glycine. These structures provide detailed information on substrate binding and show evidence for a proposed mechanism for amino acid activation and the formation of the glycyl-adenylate intermediate for Class II AARSs.     

Anesthetic effects on the structure and dynamics of the second transmembrane domains of nAChR α4β2

Channel functions of the neuronal α4β2 nicotinic acetylcholine receptor (nAChR), one of the most widely expressed subtypes in the brain, can be inhibited by volatile anesthetics. Our Na⁺ flux experiments confirmed that the second transmembrane domains (TM2) of α4 and β2 in 2:3 stoichiometry, (α4)₂(β2)₃, could form pentameric channels, whereas the α4 TM2 alone could not. The structure, topology, and dynamics of the α4 TM2 and (α4)₂(β2)₃ TM2 in magnetically aligned phospholipid bicelles were investigated using solid-state NMR spectroscopy in the absence and presence of halothane and isoflurane, two clinically used volatile anesthetics. ²H NMR demonstrated that anesthetics increased lipid conformational heterogeneity. Such anesthetic effects on lipids became more profound in the presence of transmembrane proteins. PISEMA experiments on the selectively ¹⁵N-labeled α4 TM2 showed that the TM2 formed transmembrane helices with tilt angles of 12° ± 1° and 16° ± 1° relative to the bicelle normal for the α4 and (α4)₂(β2)₃ samples, respectively. Anesthetics changed the tilt angle of the α4 TM2 from 12° ± 1° to 14° ± 1°, but had only a subtle effect on the tilt angle of the (α4)₂(β2)₃ TM2. A small degree of wobbling motion of the helix axis occurred in the (α4)₂(β2)₃ TM2. In addition, a subset of the (α4)₂(β2)₃ TM2 exhibited counterclockwise rotational motion around the helix axis on a time scale slower than 10^- 4 s in the presence of anesthetics. Both helical tilting and rotational motions have been identified computationally as critical elements for ion channel functions. This study suggested that anesthetics could alter these motions to modulate channel functions.     

Synthesis and initial evaluation of novel,non-peptidic antagonists of the αv-integrins αvβ3 and αvβ5

The discovery, synthesis and preliminary SAR of a novel class of non-peptidic antagonists of the α_v-integrins α_vβ₃ and α_vβ₅ is described. High-throughput screening of an extensive series of ECLiPS? compound libraries led to the identification of compound 1 as a dual inhibitor of the α_v-integrins α_vβ₃ and α_vβ₅. Optimization of compound 1 involving, in part, introduction of two novel constraints led to the discovery of compounds 15a and 15b with reduced PSA and much improved potency for both the α_vβ₃ and α_vβ₅ integrins. Compounds 15a and 15b were shown to have promising activity in functional cellular assays and compound 15a also exhibited a promising Caco-2 permeability profile.     

NMR resolved multiple anesthetic binding sites in the TM domains of the α4β2 nAChR

The α4β2 nicotinic acetylcholine receptor (nAChR) has significant roles in nervous system function and disease. It is also a molecular target of general anesthetics. Anesthetics inhibit the α4β2 nAChR at clinically relevant concentrations, but their binding sites in α4β2 remain unclear. The recently determined NMR structures of the α4β2 nAChR transmembrane (TM) domains provide valuable frameworks for identifying the binding sites. In this study, we performed solution NMR experiments on the α4β2 TM domains in the absence and presence of halothane and ketamine. Both anesthetics were found in an intra-subunit cavity near the extracellular end of the β2 transmembrane helices, homologous to a common anesthetic binding site observed in X-ray structures of anesthetic-bound GLIC (Nury et al., [32]). Halothane, but not ketamine, was also found in cavities adjacent to the common anesthetic site at the interface of α4 and β2. In addition, both anesthetics bound to cavities near the ion selectivity filter at the intracellular end of the TM domains. Anesthetic binding induced profound changes in protein conformational exchanges. A number of residues, close to or remote from the binding sites, showed resonance signal splitting from single to double peaks, signifying that anesthetics decreased conformation exchange rates. It was also evident that anesthetics shifted population of two conformations. Altogether, the study comprehensively resolved anesthetic binding sites in the α4β2 nAChR. Furthermore, the study provided compelling experimental evidence of anesthetic-induced changes in protein dynamics, especially near regions of the hydrophobic gate and ion selectivity filter that directly regulate channel functions.     

Polymorphisms in the α4 integrin of neotropical primates: insights for binding of natural ligands and HIV-1 gp120 to the human α4β7

The α4 integrin subunit associates with β7 and β1 and plays important roles in immune function and cell trafficking. The gut-homing receptor α4β7 has been recently described as a new receptor for HIV. Here, we describe polymorphisms of ITGA4 gene in New World primates (NWP), and tested their impact on the binding to monoclonal antibodies, natural ligands (MAdCAM and VCAM), and several gp120 HIV-1 envelope proteins. Genomic DNA of NWP specimens comprising all genera of the group had their exons 5 and 6 (encoding the region of binding to the ligands studied) analyzed. The polymorphisms found were introduced into an ITGA4 cDNA clone encoding the human α4 subunit. Mutant α4 proteins were co-expressed with β7 and were tested for binding of mAbs, MAdCAM, VCAM and gp120 of HIV-1, which was compared to the wild-type (human) α4. Mutant α4 proteins harboring the K201E/I/N substitution had reduced binding of all ligands tested, including HIV-1 gp120 envelopes. The mAbs found with reduced biding included one from which a clinically-approved drug for the treatment of neurological disorders has been derived. α4 polymorphisms in other primate species may influence outcomes in the development and treatment of infectious and autoimmune diseases in humans and in non-human primates.     

Computational neural network analysis of the affinity of N-n-alkylnicotinium salts for the α4β2* nicotinic acetylcholine receptor

Based on an 85 molecule database, linear regression with different size datasets and an artificial neural network approach have been used to build mathematical relationships to fit experimentally obtained affinity values (K_i) of a series of mono- and bis-quaternary ammonium salts from [³H]nicotine binding assays using rat striatal membrane preparations. The fitted results were then used to analyze the pattern among the experimental K_i values of a set of N-n-alkylnicotinium analogs with increasing n-alkyl chain length from 1 to 20 carbons. The affinity of these N-n-alkylnicotinium compounds was shown to parabolically vary with increasing numbers of carbon atoms in the n-alkyl chain, with a local minimum for the C₄ (n-butyl) analogue. A decrease in K_i value between C₁₂ and C₁₃ was also observed. The statistical results for the best neural network fit of the 85 experimental K_i values are r² = 0.84, rmsd = 0.39; r_cv² = 0.68, and loormsd = 0.56. The generated neural network model with the 85 molecule training set may also be of value for future predictions of K_i values for new virtual compounds, which can then be identified, subsequently synthesized, and tested experimentally.     

SHP2 Mediates the Localized Activation of Fyn Downstream of the α6β4 Integrin To Promote Carcinoma Invasion

Src family kinase (SFK) activity is elevated in many cancers, and this activity correlates with aggressive tumor behavior. The α6β4 integrin, which is also associated with a poor prognosis in many tumor types, can stimulate SFK activation; however, the mechanism by which it does so is not known. In the current study, we provide novel mechanistic insight into how the α6β4 integrin selectively activates the Src family member Fyn in response to receptor engagement. Both catalytic and noncatalytic functions of SHP2 are required for Fyn activation by α6β4. Specifically, the tyrosine phosphatase SHP2 is recruited to α6β4 and its catalytic activity is stimulated through a specific interaction of its N-terminal SH2 domain with pY1494 in the β4 subunit. Fyn is recruited to the α6β4/SHP2 complex through an interaction with phospho-Y580 in the C terminus of SHP2. In addition to activating Fyn, this interaction with Y580-SHP2 localizes Fyn to sites of receptor engagement, which is required for α6β4-dependent invasion. Of significance for tumor progression, phosphorylation of Y580-SHP2 and SFK activation are increased in orthotopic human breast tumors that express α6β4 and activation of this pathway is dependent upon Y1494.Expression of the α6β4 integrin, a laminin receptor, is associated with poor patient prognosis and reduced survival in many human cancers (32). For this reason, there is considerable interest in understanding how this integrin is regulated and how it functions to promote tumor progression. In normal tissues, the α6β4 integrin plays a major role in maintaining the integrity of epithelia by binding to laminins in the basement membrane and regulating the assembly of hemidesmosomes on the basal epithelial cell surface (7, 17). In pathophysiological conditions such as wound healing and cancer, the stable adhesive interactions of the α6β4 receptor are disrupted by phosphorylation of the β4 cytoplasmic domain, converting α6β4 to a signaling-competent receptor that promotes dynamic adhesion and invasion (18). Phosphorylation of the β4 subunit cytoplasmic domain on serine residues contributes to the dynamic adhesive functions of the receptor by disrupting interactions with hemidesmosomal proteins that regulate stable adhesion (33, 37). Phosphorylation of the β4 cytoplasmic domain on tyrosine residues may also contribute to the regulation of hemidesmosomes, but it is likely that the major contribution of tyrosyl phosphorylation is to mediate interactions that stimulate downstream signaling from the receptor (22).In transformed cells, engagement of the α6β4 integrin stimulates the activation of several signaling molecules, including phosphatidylinositol-3 kinase (PI3K), mitogen-activated protein kinases (MAPK), NFκB, and Src family kinases (SFKs) (10, 12, 21, 40). In previous studies, we identified Y1494 in the β4 subunit cytoplasmic domain as an important mediator of α6β4-dependent signaling by demonstrating that mutation of Y1494 inhibits the ability of α6β4 to stimulate PI3K, MAPK, and SFK activation (10, 39). Restoration of both PI3K and SFK signaling, but not MAPK signaling, rescues invasion in tumor cells expressing Y1494F-β4, indicating that PI3K and SFK signaling pathways cooperate downstream of Y1494 to promote α6β4-dependent invasion (10). Y1494 is localized within an immunoreceptor tyrosine-based inhibition motif (ITIM), a canonical binding site for Src-homology-2 (SH2) domain-containing protein-tyrosine phosphatase 1 (SHP1) and SHP2 (44). Examination of a chimeric receptor containing the extracellular domain of TrkB and the transmembrane and cytoplasmic domains of the β4 subunit demonstrated that SHP2 binds to and is activated by sequences in the β4 cytoplasmic domain in response to dimerization (23). Moreover, Y1494 is one of three tyrosine residues, along with Y1257 and Y1440, that mediate the interaction of SHP2 with the β4 subunit cytoplasmic domain in response to c-Met signaling (6). Importantly, SHP2 is essential for the activation of SFKs both by the chimeric TrkB/β4 receptor and when the β4 subunit functions as a signaling adaptor for c-Met (6, 23). However, the mechanism by which SHP2 activates SFKs in response to α6β4 engagement has not been established.Elevated SFK activity correlates strongly with breast cancer invasion and metastasis, and these kinases are frequently activated in human cancers (15). Given the parallels between α6β4 expression and SFK activation in cancer, investigation of how α6β4 contributes to the activation of this invasion-promoting pathway is warranted. In the current study, we sought to elucidate the mechanism by which engagement of α6β4 activates SFKs and the significance of the β4/SHP2/SFK signaling axis for tumor progression. Our results reveal a novel mechanism for SHP2-dependent activation of the SFK family member Fyn which involves Y580 in the C terminus of SHP2.     

β-Sheet-breaking peptides inhibit the fibrillation of human α-synuclein

α-Synuclein is the major components of the intracellular protein-aggregates, found in the dopaminergic neurons of Parkinson’s disease patients. Previously, we screened for α-synuclein substitution mutants that prevent fibril formation of both wild-type and Parkinson’s disease-linked α-synuclein variants. In the present study, we show that short synthetic peptides derived from these mutant sequences not only prevented α-synuclein fibrillation but also dissolved preformed α-synuclein aggregates in vitro. The hexapeptide PGVTAV, which was the shortest peptide that retained the ability to block α-synuclein fibrillation, may serve as a lead compound for the development of therapeutics for Parkinson’s disease.     

Short form of α9 promotes α9β1 integrin-dependent cell adhesion by modulating the function of the full-length α9 subunit

The α9β1 integrin is a multifunctional receptor that interacts with a variety of ligands including vascular cell adhesion molecule 1, tenascin-C, and osteopontin. A 2.3-kb truncated form of α9 integrin subunit cDNA was identified by searching the Medline database. This splice variant, which we called the short form of α9 integrin (SFα9), encodes a 632-aa isoform lacking transmembrane and cytoplasmic domains, and its authentic expression was verified by PCR and Western blotting. SFα9 is expressed on the cell surface but cannot bind ligand in the absence of the full-length α9 subunit. Over-expression of SFα9 in cells expressing full-length α9 promotes α9-dependent cell adhesion. This promoting effect of SFα9 requires the authentic cytoplasmic domain of the co-expressed full-length α9 subunit. Thus, SFα9 is a novel functional modulator of α9β1 integrin by inside-out signaling.     

Structural Basis of the Differential Binding of Engineered Knottins to Integrins αVβ3 and α5β1

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Modeling the molecular basis for α4β1 integrin antagonism

We report a 3D QSAR study of almost 300 structurally diverse small molecule antagonists of the integrin α4β1 whose biological activity spans six orders of magnitude. The alignment of the molecules was based on the conformation of a structurally related ligand bound to the αIIBβ3 and αvβ3 integrins in X-ray crystallographic studies. The molecular field method, CoMSIA, was used to generate the 3D QSAR models. The resulting models showed that the lipophilic properties were the most important, with hydrogen bond donor and steric properties less relevant. The models were highly significant (r(2)=0.89, q2(LOO)=0.67, r(2) (test set)=0.76), and could make robust predictions of the data (SEE=0.46, SEP=0.78, SEP (test set)=0.66). We predicted the antagonist activities of a further ten compounds with useful accuracy. The model appears capable of predicting α4β1 integrin antagonist activity to within a factor of five for compounds within its domain of applicability. The implications for design of improved integrin antagonists will be discussed.     

Cryptic Determinant of α4β7 Binding in the V2 Loop of HIV-1 gp120

The peptide segment of the second variable loop of HIV-1 spanning positions 166–181 harbors two functionally important sites. The first, spanning positions 179–181, engages the human α4β7 integrin receptor which is involved in T-cell gut-homing and may play a role in human immunodeficiency virus (HIV)-host cell interactions. The second, at positions 166–178, is a major target of anti-V2 antibodies elicited by the ALVAC/AIDSVAX vaccine used in the RV144 clinical trial. Notably, these two sites are directly adjacent, but do not overlap. Here, we report the identity of a second determinant of α4β7 binding located at positions 170–172 of the V2 loop. This segment – tripeptide QRV^170–172– is located within the second site, yet functionally affects the first site. The absence of this segment abrogates α4β7 binding in peptides bearing the same sequence from position 173–185 as the V2 loops of the RV144 vaccines. However, peptides exhibiting V2 loop sequences from heterologous HIV-1 strains that include this QRV^170–172 motif bind the α4β7 receptor on cells. Therefore, the peptide segment at positions 166–178 of the V2 loop of HIV-1 viruses appears to harbor a cryptic determinant of α4β7 binding. Prior studies show that the anti-V2 antibody response elicited by the RV144 vaccine, along with immune pressure inferred from a sieve analysis, is directed to this same region of the V2 loop. Accordingly, the anti-V2 antibodies that apparently reduced the risk of infection in the RV144 trial may have functioned by blocking α4β7-mediated HIV-host cell interactions via this cryptic determinant.     

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.     

Elucidation of Molecular Impediments in the α6 Subunit for in Vitro Expression of Functional α6β4* Nicotinic Acetylcholine Receptors

Explorations into the α6-containing nicotinic acetylcholine receptors (α6* nAChRs) as putative drug targets have been severely hampered by the inefficient functional expression of the receptors in heterologous expression systems. In this study, the molecular basis for the problem was investigated through the construction of chimeric α6/α3 and mutant α3 and α6 subunits and functional characterization of these co-expressed with β4 or β4β3 subunits in tsA201 cells in a fluorescence-based assay and in Xenopus oocytes using two-electrode voltage clamp electrophysiology. Substitution of a small C-terminal segment in the second intracellular loop or the Phe²²³ residue in transmembrane helix 1 of α6 with the corresponding α3 segment or residue was found to enhance α6β4 functionality in tsA201 cells significantly, in part due to increased cell surface expression of the receptors. The gain-of-function effects of these substitutions appeared to be additive since incorporation of both α3 elements into α6 resulted in assembly of α6β4* receptors exhibiting robust functional responses to acetylcholine. The pharmacological properties exhibited by α6β4β3 receptors comprising one of these novel α6/α3 chimeras in oocytes were found to be in good agreement with those from previous studies of α6* nAChRs formed from other surrogate α6 subunits or concatenated subunits and studies of other heteromeric nAChRs. In contrast, co-expression of this α6/α3 chimera with β2 or β2β3 subunits in oocytes did not result in efficient formation of functional receptors, indicating that the identified molecular elements in α6 could be specific impediments for the expression of functional α6β4* nAChRs.     

Contribution of Position α4S336 on Functional Expression and Up-regulation of α4β2 Neuronal Nicotinic Receptors

Phosphorylation of the nicotinic acetylcholine receptor (nAChR) is believed to play a critical role in its nicotine-induced desensitization and up-regulation. We examined the contribution of a consensus PKC site in the α4 M3/M4 intracellular loop (α4S336) on the desensitization and up-regulation of α4β2 nAChRs expressed in oocytes. Position α4S336 was replaced with either alanine to abolish potential phosphorylation at this site or with aspartic acid to mimic phosphorylation at this same site. Mutations α4S336A and α4S336D displayed a threefold increase in the ACh-induced response and an increase in ACh EC₅₀. Epibatidine binding revealed a three and sevenfold increase in surface expression for the α4S336A and α4S336D mutations, respectively, relative to wild-type, therefore, both mutations enhanced expression of the α4β2 nAChR. Interestingly, the EC₅₀’s and peak currents for nicotine activation remained unaffected in both mutants. Both mutations abolished the nicotine-induced up-regulation that is normally observed in the wild-type. The present data suggest that adding or removing a negative charge at this phosphorylation site cannot be explained by a simple straightforward on-and-off mechanism; rather a more complex mechanism(s) may govern the functional expression of the α4β2 nAChR. Along the same line, our data support the idea that phosphorylation at multiple consensus sites in the α4 subunit could play a remarkable role on the regulation of the functional expression of the α4β2 nAChR.     

Modification at the 5-Position of 4-Deoxypyridoxol and α4-Norpyridoxol

In an attempt to relate structure to anticoccidial activity, a number of 5-modified analogs of 4-deoxypyridoxol (4-DOP) and α⁴-norpyridoxol have been synthesized and their biological activities examined. The compounds prepared include the 5-(3-hydroxypropyl), 5-(2-hydroxyethyl), 5-(1-hydroxyethyl), formyl and acetyl analogs of 4-DOP, and 5-(3-hydroxypropyl), formyl, ethoxycarbonyl, carbamoyl and hydroxyl analogs of α⁴-norpyridoxol. Among these compounds, 4-deoxyisopyridoxal and α⁴-norisopyridoxal were found to exhibit anticoccidal activity.     

PAMAM G4 dendrimers affect the aggregation of α-synuclein

α-Synuclein (ASN) aggregation plays a key role in neurodegenerative disorders including Parkinson's disease, and inhibition of fibril formation is a potential therapeutic strategy for these conditions. The aim of the present study was to investigate polyamidoamine (PAMAM) dendrimers (generations 4 and 3.5) as inhibitors of fibril formation in vitro by examining their interaction with ASN intrinsic tyrosine fluorescence. Furthermore, the effect of dendrimers on ASN aggregation was studied using circular dichroism (CD) spectroscopy and CD studies were complemented by a fluorescence assays using the dye thioflavin T (ThT). The PAMAM G4 dendrimer caused an increase in tyrosine residue fluorescence, and inhibited fibrillation of ASN; inhibited fibrillation was not observed with PAMAM G3.5 dendrimers.     

Homology modeling and dynamics of the extracellular domain of rat and human neuronal nicotinic acetylcholine receptor subtypes α4β2 and α7

In recent years, it has become clear that the neuronal nicotinic acetylcholine receptor (nAChR) is a valid target in the treatment of a variety of diseases, including Alzheimer’s disease, anxiety, and nicotine addiction. As with most membrane proteins, information on the three-dimensional (3D) structure of nAChR is limited to data from electron microscopy, at a resolution that makes the application of structure-based design approaches to develop specific ligands difficult. Based on a high-resolution crystal structure of AChBP, homology models of the extracellular domain of the neuronal rat and human nAChR subtypes α4β2 and α7 (the subtypes most abundant in brain) were built, and their stability assessed with molecular dynamics (MD). All models built showed conformational stability over time, confirming the quality of the starting 3D model. Lipophilicity and electrostatic potential studies performed on the rat and human α4β2 and α7 nicotinic models were compared to AChBP, revealing the importance of the hydrophobic aromatic pocket and the critical role of the α-subunit Trp—the homolog of AChBP-Trp 143—for ligand binding. The models presented provide a valuable framework for the structure-based design of specific α4β2 nAChR subtype ligands aimed at improving therapeutic and diagnostic applications. Figure Electrostatic surface potential of the binding site cavity of the neuronal nicotinic acetylcholine receptor (nAChR). Nicotinic models performed with the MOLCAD program: a rat α7, b rat α4β2, c human α7, d human α4β2. All residues labeled are part of the α7 (a,c) or α4 (b,d) subunit with the exception of Phe 117, which belongs to subunit β2 (d). Violet Very negative, blue negative, yellow neutral, red very positive