The pore domain of the nicotinic acetylcholine receptor: molecular modeling, pore dimensions, and electrostatics.

The pore domain of the nicotinic acetylcholine receptor has been modeled as a bundle of five kinked M2 helices. Models were generated via molecular dynamics simulations incorporating restraints derived from 9-A resolution cryoelectron microscopy data (Unwin, 1993; 1995), and from mutagenesis data that identify channel-lining side chains. Thus, these models conform to current experimental data but will require revision as higher resolution data become available. Models of the open and closed states of a homopentameric alpha 7 pore are compared. The minimum radius of the closed-state model is less than 2 A; the minimum radius of the open-state models is approximately 6 A. It is suggested that the presence of "bound" water molecules within the pore may reduce the effective minimum radii below these values by up to approximately 3 A. Poisson-Boltzmann calculations are used to obtain a first approximation to the potential energy of a monovalent cation as it moves along the pore axis. The differences in electrostatic potential energy profiles between the open-state models of alpha 7 and of a mutant of alpha 7 are consistent with the experimentally observed change in ion selectivity from cationic to anionic. Models of the open state of the heteropentameric Torpedo nicotinic acetylcholine receptor pore domain are also described. Relatively small differences in pore radius and electrostatic potential energy profiles are seen when the Torpedo and alpha 7 models are compared.     

A structural model of the acetylcholine receptor channel based on partition energy and helix packing calculations.

A structural model of the transmembrane portion of the acetylcholine receptor was developed from sequences of all its subunits by using transfer energy calculations to locate transmembrane alpha-helices and to calculate which helical side chains should be in contact with water inside the channel, with portions of other transmembrane helices, or with lipid hydrocarbon chains. "Knobs-into-holes" side chain packing calculations were used with other factors to stack the transmembrane alpha-helices together. In the model each subunit has the following structures in order along the sequence from the NH2 terminus: a large extracellular domain of undetermined structure, a short apolar alpha-helix that lies on the extracellular lipid surface of the membrane; three apolar transmembrane alpha-helices (I, II, and III), a cytoplasmic domain of undetermined structure, an amphipathic transmembrane alpha-helix (L) that forms the channel lining, a short extracellular alpha-helix, another apolar transmembrane alpha-helix (IV), and a small cytoplasmic domain formed by the COOH-terminal end of the chain. Three concentric layers form the pore. A bundle of five amphipathic L helices forms the channel lining. This bundle is surrounded by a bundle of 10 alternating II and III helices. Helices I and IV cover portions of the outer surface of the bundle formed by helices II and III. Positions of disulfide bridges are predicted and a mechanism for opening and closing conformational changes is proposed that requires tilting transmembrane helices and possibly a thiol-disulfide interchange reaction.     

Homology model of the GABAA receptor examined using Brownian dynamics

We have developed a homology model of the GABA(A) receptor, using the subunit combination of alpha1beta2gamma2, the most prevalent type in the mammalian brain. The model is produced in two parts: the membrane-embedded channel domain and the extracellular N-terminal domain. The pentameric transmembrane domain model is built by modeling each subunit by homology with the equivalent subunit of the heteropentameric acetylcholine receptor transmembrane domain. This segment is then joined with the extracellular domain built by homology with the acetylcholine binding protein. The all-atom model forms a wide extracellular vestibule that is connected to an oval chamber near the external surface of the membrane. A narrow, cylindrical transmembrane channel links the outer segment of the pore to a shallow intracellular vestibule. The physiological properties of the model so constructed are examined using electrostatic calculations and Brownian dynamics simulations. A deep energy well of approximately 80 kT accommodates three Cl(-) ions in the narrow transmembrane channel and seven Cl(-) ions in the external vestibule. Inward permeation takes place when one of the ions queued in the external vestibule enters the narrow segment and ejects the innermost ion. The model, when incorporated into Brownian dynamics, reproduces key experimental features, such as the single-channel current-voltage-concentration profiles. Finally, we simulate the gamma2 K289M epilepsy inducing mutation and examine Cl(-) ion permeation through the mutant receptor.     

Extracellular domain nicotinic acetylcholine receptors formed by alpha4 and beta2 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.     

The beta subunit determines the ion selectivity of the GABAA receptor

The gamma-aminobutyric acid, type A (GABA(A)) receptor is a chloride-conducting receptor composed of alpha, beta, and gamma subunits assembled in a pentameric structure forming a central pore. Each subunit has a large extracellular agonist binding domain and four transmembrane domains (M1-M4), with the second transmembrane (M2) domain lining the pore. Mutation of five amino acids in the M1-M2 loop of the beta(3) subunit to the corresponding amino acids of the alpha(7) nicotinic acetylcholine subunit rendered the GABA(A) receptor cation-selective upon co-expression with wild type alpha(2) and gamma(2) subunits. Similar mutations in the alpha(2) or gamma(2) subunits did not lead to such a change in ion selectivity. This suggests a unique role for the beta(3) subunit in determining the ion selectivity of the GABA(A) receptor. The pharmacology of the mutated GABA(A) receptor is similar to that of the wild type receptor, with respect to muscimol binding, Zn(2+) and bicuculline sensitivity, flumazenil binding, and potentiation of GABA-evoked currents by diazepam. There was, however, an increase in GABA sensitivity (EC(50) = 1.3 microm) compared with the wild type receptor (EC(50) = 6.4 microm) and a loss of desensitization to GABA of the mutant receptor.     

Nanosecond-timescale conformational dynamics of the human alpha7 nicotinic acetylcholine receptor

We explore the conformational dynamics of a homology model of the human alpha7 nicotinic acetylcholine receptor using molecular dynamics simulation and analyses of root mean-square fluctuations, block partitioning of segmental motion, and principal component analysis. The results reveal flexible regions and concerted global motions of the subunits encompassing extracellular and transmembrane domains of the subunits. The most relevant motions comprise a bending, hinged at the beta10-M1 region, accompanied by concerted tilting of the M2 helices that widens the intracellular end of the channel. Despite the nanosecond timescale, the observations suggest that tilting of the M2 helices may initiate opening of the pore. The results also reveal direct coupling between a twisting motion of the extracellular domain and dynamic changes of M2. Covariance analysis of interresidue motions shows that this coupling arises through a network of residues within the Cys and M2-M3 loops where Phe135 is stabilized within a hydrophobic pocket formed by Leu270 and Ile271. The resulting concerted motion causes a downward shift of the M2 helices that disrupts a hydrophobic girdle formed by 9' and 13' residues.     

Expression of fusion proteins of the nicotinic acetylcholine receptor from mammalian muscle identifies the membrane-spanning regions in the alpha and delta subunits

We have investigated the topology of the alpha and delta subunits of the nicotinic acetylcholine receptor (AChR) from mammalian muscle synthesized in an in vitro translation system supplemented with dog pancreatic microsomes. Fusion proteins were expressed in which a carboxy-terminal fragment of bovine prolactin was attached downstream of each of the major putative transmembrane domains, M1-M4 and MA, in the AChR subunits. The orientation of the prolactin domain relative to the microsomal membrane was then determined for each protein by a proteolysis protection assay. Since the prolactin domain contains no information which either directs or prevents its translocation, its transmembrane orientation depends solely on sequences within the AChR subunit portion of the fusion protein. When subunit-prolactin fusion proteins with the prolactin domain fused after either M2 or M4 were tested, prolactin-immunoreactive peptides that were larger than the prolactin domain itself were recovered. No prolactin-immunoreactive peptides were recovered after proteolysis of fusion proteins containing prolactin fused after M1, M3, or MA. These results support a model of AChR subunit topology in which M1-M4, but not MA, are transmembrane domains and the carboxy terminus is extracellular.     

Transmembrane topography of nicotinic acetylcholine receptor: immunochemical tests contradict theoretical predictions based on hydrophobicity profiles

In our preceding paper [Ratnam, M., Sargent, P. B., Sarin, V., Fox, J. L., Le Nguyen, D., Rivier, J., Criado, M., & Lindstrom, J. (1986) Biochemistry (preceding paper in this issue)], we presented results from peptide mapping studies of purified subunits of the Torpedo acetylcholine receptor which suggested that the sequence beta 429-441 is on the cytoplasmic surface of the receptor. Since this finding contradicts earlier theoretical models of the transmembrane structure of the receptor, which placed this sequence of the beta subunit on the extracellular surface, we investigated the location of the corresponding sequence (389-408) and adjacent sequences of the alpha subunit by a more direct approach. We synthesized peptides including the sequences alpha 330-346, alpha 349-364, alpha 360-378, alpha 379-385, and alpha 389-408 and shorter parts of these peptides. These peptides corresponded to a highly immunogenic region, and by using 125I-labeled peptides as antigens, we were able to detect in our library of monoclonal antibodies to alpha subunits between two and six which bound specifically to each of these peptides, except alpha 389-408. We obtained antibodies specific for alpha 389-408 both from antisera against the denatured alpha subunit and from antisera made against the peptide. These antibodies were specific to alpha 389-396. In binding assays, antibodies specific for all of these five peptides bound to receptor-rich membrane vesicles only after permeabilization of the vesicles to permit access of the antibodies to the cytoplasmic surface of the receptors, suggesting that the receptor sequences which bound these antibodies were located on the intracellular side of the membrane. Electron microscopy using colloidal gold to visualize the bound antibodies was used to conclusively demonstrate that all of these sequences are exposed on the cytoplasmic surface of the receptor. These results, along with our previous demonstration that the C-terminal 10 amino acids of each subunit are exposed on the cytoplasmic surface, show that the hydrophobic domain M4 (alpha 409-426), previously predicted from hydropathy profiles to be transmembranous, does not, in fact, cross the membrane. Further, these results show that the putative amphipathic transmembrane domain M5 (alpha 364-399) also does not cross the membrane. Our results thus indicate that the transmembrane topology of a membrane protein cannot be deduced strictly from the hydropathy profile of its primary amino acid sequence. We present a model for the transmembrane orientation of receptor subunit polypeptide chains which is consistent with current data.     

Normal mode analysis suggests a quaternary twist model for the nicotinic receptor gating mechanism

We present a three-dimensional model of the homopentameric alpha7 nicotinic acetylcholine receptor (nAChR), that includes the extracellular and membrane domains, developed by comparative modeling on the basis of: 1), the x-ray crystal structure of the snail acetylcholine binding protein, an homolog of the extracellular domain of nAChRs; and 2), cryo-electron microscopy data of the membrane domain collected on Torpedo marmorata nAChRs. We performed normal mode analysis on the complete three-dimensional model to explore protein flexibility. Among the first 10 lowest frequency modes, only the first mode produces a structural reorganization compatible with channel gating: a wide opening of the channel pore caused by a concerted symmetrical quaternary twist motion of the protein with opposing rotations of the upper (extracellular) and lower (transmembrane) domains. Still, significant reorganizations are observed within each subunit, that involve their bending at the domain interface, an increase of angle between the two beta-sheets composing the extracellular domain, the internal beta-sheet being significantly correlated to the movement of the M2 alpha-helical segment. This global symmetrical twist motion of the pentameric protein complex, which resembles the opening transition of other multimeric ion channels, reasonably accounts for the available experimental data and thus likely describes the nAChR gating process.     

Aromaticity at the water-hydrocarbon core interface of the membrane: consequences on the nicotinic acetylcholine receptor

Almost all lipid-exposed transmembrane domains of integral proteins contain aromatic residues flanking the hydrophobic segment of the domains. These residues generally reside close to the carbonyl region of the membrane, and several structural and functional roles have been associated to these residues. Although the roles and physicochemical reasons for aromatic preference have been extensively studied using model systems, few studies have been done in a native membrane system. To gain insight about the mechanistic implication for this aromatic preference, we selected position alpha F426 of the muscle-type nicotinic acetylcholine receptor (nAChR). alpha F426 is a lipid-exposed residue at the extracellular segment of the alpha M4 transmembrane domain and is highly conserved among different nAChR subunits and species. We used site-directed mutagenesis, alpha-Bungarotoxin-binding assay, and two-electrodes voltage clamp in Xenopus laevis oocytes to characterize mutations at position alpha F426, which impart different physicochemical properties like volume, polarity, hydrogen bonds, aromaticity and net electrical charge. All mutations except the aromatic residues resulted in a significant reduction of the nAChR cell-surface levels and the macroscopic currents to acetylcholine. These results suggest that position alpha F426 contributes to structural stability and open-close transitions of the nAChR. Finally, the present study also provides information about how intermolecular interactions at position alpha 426 modulate open-close transitions of the nAChR.     

Membrane tethering enables an extracellular domain of the acetylcholine receptor alpha subunit to form a heterodimeric ligand-binding site

The first step of assembly of the nicotinic acetylcholine receptor (AChR) of adult skeletal muscle is the specific association of the alpha subunit with either delta or epsilon subunits to form a heterodimer with a ligand-binding site. Previous experiments have suggested that het erodimer formation in the ER arises from interaction between the luminal, NH2-terminal domains of the subunits. When expressed in COS cells with the delta subunit, however, the truncated NH2-terminal domain of the subunit folded correctly but did not form a heterodimer. Association with the delta subunit occurred only when the NH2-terminal domain was retained in the ER and was tethered to the membrane by its own M1 transmembrane domain, by the transmembrane domain of another protein, or by a glycolipid link. In each case, the ligand-binding sites of the resulting heterodimers were indistinguishable from that formed when the full-length alpha subunit was used. Attachment to the membrane may promote interaction by concentrating or orienting the subunit; alternatively, a membrane-bound factor may facilitate subunit association.     

Structural organization of nicotinic acetylcholine receptors

Nicotinic acetylcholine receptor of the electric ray Torpedo is the most comprehensively characterized neurotransmitter receptor. It consists of five subunits (alpha2beta gammadelta) amino acid sequences of which were determined by cDNA cloning and sequencing. The shape and size of the receptor were determined by electron cryomicroscopy. It has two agonist/competitive antagonist binding sites which are located between subunits near the membrane surface. The receptor ion channel is formed by five transmembrane helices (M2) of all five subunits. The position of the binding site for noncompetitive ion channel blockers was found by photoaffinity labelling and site-directed mutagenesis. The intrinsic feature of the receptor structure is the position of the agonist/competitive antagonist binding sites in close vicinity to the ion channel spanning the bilayer membrane. This peculiarity may substantially enhance allosteric transitions transforming the ligand binding into the channel opening and physiological response. Muscle nicotinic acetylcholine receptors from birds and mammals are also pentaoligomers consisting of four different subunits (alpha2beta gammadelta or alpha2beta epsilondelta) with high homology to the Torpedo receptor. Apparently, the pentaoligomeric structure is the main feature of all nicotinic, both muscle and neuronal, receptors. However, the neuronal receptors are formed only by two subunit types (alpha and beta) or are even pentahomomers (alpha7 neuronal receptors). All nicotinic receptors are ligand-gated ion channel, the properties of the channels being essentially determined by amino acid residues forming M2 transmembrane fragments.     

Role of a key cysteine residue in the gating of the acetylcholine receptor

We have examined changes in single-channel behavior that result from conservative amino acid substitutions at the Cys230 residue in the putative first transmembrane region (M1) of the murine nicotinic acetylcholine receptor. Mutations made in the gamma subunit altered the energy barrier for a single closing rate constant in proportion to the size of the substituted side chain. One of these substitutions, when made in the alpha subunits, had no effect on gating. No mutations altered permeation. We conclude that the region surrounding the M1 Cys is involved in the gating of the nicotinic acetylcholine receptor and that the gamma subunit contributes significantly to the control of channel closure.     

Cell-free expression and functional reconstitution of homo-oligomeric alpha7 nicotinic acetylcholine receptors into planar lipid bilayers.

The alpha7 nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel that modulates neurotransmitter release in the central nervous system. We show here that functional, homo-oligomeric alpha7 nAChRs can be synthesized in vitro with a rabbit reticulocyte lysate translation system supplemented with endoplasmic reticulum microsomes, reconstituted into planar lipid bilayers, and evaluated using single-channel recording techniques. Because wild-type alpha7 nAChRs desensitize rapidly, we used a nondesensitizing form of the alpha7 receptor with mutations in the second transmembrane domain (S2'T and L9'T) to record channel activity in the continuous presence of agonist. Endoglycosidase H treatment of microsomes containing nascent alpha7 S2'T/L9'T nAChRs indicated that the receptors were glycosylated. A proteinase K protection assay revealed a 36-kDa fragment in the ER lumen, consistent with a large extracellular domain predicted by most topological models, indicating that the protein was folded integrally through the ER membrane. alpha7 S2'T/L9'T receptors reconstituted into planar lipid bilayers had a unitary conductance of approximately 50 pS, were highly selective for monovalent cations over Cl(-), were nonselective between K(+) and Na(+), and were blocked by alpha-bungarotoxin. This is the first demonstration that a functional ligand-gated ion channel can be synthesized using an in vitro expression system.     

Tryptophan scanning of the acetylcholine receptor's βM4 transmembrane domain: Decoding allosteric linkage at the lipid-protein interface with ion-channel gating

The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel protein that mediates fast excitatory synaptic transmission in the peripheral and central nervous systems. Changes in the structure and function of the AChR can lead to serious impairment of physiological processes. In this study, we combined site-directed mutagenesis, radioligand binding assays, electrophysiological recordings, and Fourier analyses to characterize the functional role and structural aspects of the βM4 transmembrane domain of the Torpedo AChR. We performed tryptophan replacements, from residues L438 through F455, along the βM4 transmembrane domain. Expression levels of mutants F439W-G450W and F452W-I454W produced peak currents similar to or lower than those in wild-type (WT). Tryptophan substitutions at positions L438 and T451 led to a deficiency in either subunit expression or receptor assembly. Mutations L440W, V442W, C447W, and S453W produced a gain-of-function response. Mutation F455W produced a loss of ion channel function. The periodicity profile of the normalized expression level (closed state) and EC50 (open state) revealed a minor conformational change of 0.4 residues/turn of the βM4 domain. These findings suggest that a minor movement of the βM4 domain occurs during channel activation.     

Dual role of the RIC-3 protein in trafficking of serotonin and nicotinic acetylcholine receptors

The ric-3 gene is required for maturation of nicotinic acetylcholine receptors in Caenorhabditis elegans. The human homolog of RIC-3, hRIC-3, enhances expression of alpha7 nicotinic receptors in Xenopus laevis oocytes, whereas it totally abolishes expression of alpha4beta2 nicotinic and 5-HT3 serotonergic receptors. Both the N-terminal region of hRIC-3, which contains two transmembrane segments, and the C-terminal region are needed for these differential effects. hRIC-3 inhibits receptor expression by hindering export of mature receptors to the cell membrane. By using chimeric proteins made of alpha7 and 5-HT3 receptors, we have shown that the presence of an extracellular isoleucine close to the first transmembrane receptor fragment is responsible for the transport arrest induced by hRIC-3. Enhancement of alpha7 receptor expression occurs, at least, at two levels: by increasing the number of mature receptors and facilitating its transport to the membrane. Certain amino acids of a putative amphipathic helix present at the large cytoplasmic region of the alpha7 subunit are required for these actions. Therefore, hRIC-3 can act as a specific regulator of receptor expression at different levels.     

Subunit-selective role of the M3 transmembrane domain of the nicotinic acetylcholine receptor in channel gating

The nicotinic acetylcholine receptor (AChR) can be either hetero-pentameric, composed of alpha and non-alpha subunits, or homo-pentameric, composed of alpha7 subunits. To explore the subunit-selective contributions of transmembrane domains to channel gating we analyzed single-channel activity of chimeric muscle AChRs. We exchanged M3 between alpha1 and epsilon or alpha7 subunits. The replacement of M3 in alpha1 by epsilonM3 significantly alters activation properties. Channel activity appears as bursts of openings whose durations are 20-fold longer than those of wild-type AChRs. In contrast, 7-fold briefer openings are observed in AChRs containing the reverse epsilon chimeric subunit. The duration of the open state decreases with the increase in the number of alpha1M3 segments, indicating additive contributions of M3 of all subunits to channel closing. Each alpha1M3 segment decreases the energy barrier of the closing process by approximately 0.8 kcal/mol. Partial chimeric subunits show that small stretches of the M3 segment contribute additively to the open duration. The replacement of alpha1 sequence by alpha7 in M3 leads to 3-fold briefer openings whereas in M1 it leads to 10-fold prolonged openings, revealing that the subunit-selective role is unique to each transmembrane segment.     

Decrypting the sequence of structural events during the gating transition of pentameric ligand-gated ion channels based on an interpolated elastic network model

Despite many experimental and computational studies of the gating transition of pentameric ligand-gated ion channels (pLGICs), the structural basis of how ligand binding couples to channel gating remains unknown. By using a newly developed interpolated elastic network model (iENM), we have attempted to compute a likely transition pathway from the closed- to the open-channel conformation of pLGICs as captured by the crystal structures of two prokaryotic pLGICs. The iENM pathway predicts a sequence of structural events that begins at the ligand-binding loops and is followed by the displacements of two key loops (loop 2 and loop 7) at the interface between the extracellular and transmembrane domain, the tilting/bending of the pore-lining M2 helix, and subsequent movements of M4, M3 and M1 helices in the transmembrane domain. The predicted order of structural events is in broad agreement with the Φ-value analysis of α subunit of nicotinic acetylcholine receptor mutants, which supports a conserved core mechanism for ligand-gated channel opening in pLGICs. Further perturbation analysis has supported the critical role of certain intra-subunit and inter-subunit interactions in dictating the above sequence of events.     

NMR structural analysis of alpha-bungarotoxin and its complex with the principal alpha-neurotoxin-binding sequence on the alpha 7 subunit of a neuronal nicotinic acetylcholine receptor.

We report a new, higher resolution NMR structure of alpha-bungarotoxin that defines the structure-determining disulfide core and beta-sheet regions. We further report the NMR structure of the stoichiometric complex formed between alpha-bungarotoxin and a recombinantly expressed 19-mer peptide ((178)IPGKRTESFYECCKEPYPD(196)) derived from the alpha7 subunit of the chick neuronal nicotinic acetylcholine receptor. A comparison of these two structures reveals binding-induced stabilization of the flexible tip of finger II in alpha-bungarotoxin. The conformational rearrangements in the toxin create an extensive binding surface involving both sides of the alpha7 19-mer hairpin-like structure. At the contact zone, Ala(7), Ser(9), and Ile(11) in finger I and Arg(36), Lys(38), Val(39), and Val(40) in finger II of alpha-bungarotoxin interface with Phe(186), Tyr(187), Glu(188), and Tyr(194) in the alpha7 19-mer underscoring the importance of receptor aromatic residues as critical neurotoxin-binding determinants. Superimposing the structure of the complex onto that of the acetylcholine-binding protein (1I9B), a soluble homologue of the extracellular domain of the alpha7 receptor, places alpha-bungarotoxin at the peripheral surface of the inter-subunit interface occluding the agonist-binding site. The disulfide-rich core of alpha-bungarotoxin is suggested to be tilted in the direction of the membrane surface with finger II extending into the proposed ligand-binding cavity.     

Creating an α7 nicotinic acetylcholine recognition domain from the acetylcholine-binding protein: crystallographic and ligand selectivity analyses

Determining the structure of the ligand-binding domain of the nicotinic acetylcholine receptor (nAChR) has been a long standing goal in the design of selective drugs useful in implicated diseases for this prevalent receptor family. Acetylcholine-binding proteins have proven to be valuable surrogates with structural similarity and sequence identity to the extracellular domain of the nicotinic receptor, yet these soluble proteins have their unique features and do not serve as exact replicates of the nAChRs of interest. Here we systematically modify the sequence of these proteins toward the homomeric human α7 nAChR. These chimeric proteins exhibit a shift in affinities to reflect α7 binding characteristics yet maintain expression levels and stability conducive for crystallization. We also present a pentameric humanoid nAChR extracellular domain with the structural determination of the α7 nAChR glycosylation site.