Location of ligand-binding sites on the nicotinic acetylcholine receptor alpha-subunit

The portions of the Torpedo californica nicotinic acetylcholine receptor (AChR) alpha-subunit that contribute to the allosteric antagonist-binding site and to the agonist-binding site have been localized by affinity labeling and proteolytic mapping. [3H]Meproadifen mustard was employed as an affinity label for the allosteric antagonist-binding site and [3H]tubocurare as a photoaffinity label for the agonist-binding site. Both labels were found in a 20-kDa proteolytic fragment generated from the AChR alpha-subunit by Staphylococcus aureus V8 protease. This 20-kDa peptide also contains the 3H-labeled 4-(N-maleimido)-alpha-benzyltrimethylammonium iodide-reactive site and binds 125I-alpha-bungarotoxin. N-terminal sequencing established that the 20-kDa fragment began at Ser-173 of the alpha-subunit. Fluorescein isothiocyanate-conjugated concanavalin A could be bound to the second of the two major V8 cleavage products, an 18-kDa peptide. This peptide was also sensitive to treatment with endo-beta-N-acetyl-glucosaminidase H, consistent with the presence of N-linked carbohydrate on this fragment. The N terminus of this peptide was found to be Val-46 of the alpha-subunit sequence. Experiments designed to map disulfide bonds within the AChR alpha-subunit indicate that no bonds exist between the 18-kDa fragment (containing Cys-128 and Cys-142) and the 20-kDa fragment (containing Cys-192, Cys-193, and Cys-222). These results establish that the 20-kDa fragment contributes to both the acetylcholine and the allosteric antagonist-binding sites, whereas there is no evidence that the 18-kDa fragment is part of either site.     

Localization of a highly immunogenic region on the acetylcholine receptor alpha-subunit

Antibodies to synthetic peptides were employed in order to map domains on the alpha-subunit of the acetylcholine receptor to which several monoclonal antibodies are directed. Five peptides corresponding to residues 1-20, 126-143, 169-181, 330-340 and 351-368 of the receptor alpha-subunit were synthesized and antibodies against them were elicited. The anti-peptide antibodies were employed along with the monoclonal antibodies to identify fragments of S. aureus V8 protease digested- alpha-subunit in immunoblotting experiments. Our results demonstrate that a highly immunogenic region of the alpha-subunit is located on a carboxy-terminal 14 kDa portion of the alpha-subunit. This region also seems to undergo antigenic changes during muscle development. A monoclonal antibody directed against the cholinergic binding site of the acetylcholine receptor reacted with an 18 kDa segment of the alpha-subunit which bound alpha-bungarotoxin as well as antibodies directed against peptide 169-181.     

Human nicotinic acetylcholine receptor alpha-subunit isoforms: origins and expression.

A majority of the autoantibodies in the disease myasthenia gravis (MG) are directed against the alpha-subunit of the muscle nicotinic acetylcholine receptor (AChR). Unlike AChR alpha-subunits previously characterised from other species, the human alpha-subunit exists as two isoforms. The isoforms are generated by alternate splicing of an additional exon located between exons P3 and P4, termed P3A. The 25 amino acids encoded by the P3A exon are incorporated into the extracellular region of the alpha-subunit, and so may be relevant to the pathogenesis of MG. Genomic sequences from rhesus monkey, and from dog and cat, which are susceptible to MG, were characterised between AChR alpha-subunit exons P3 and P4. Although regions homologous to the P3A exon were identified for each of these species, analysis by RT-PCR showed that they are not expressed. At variance with a previous report, constitutive expression of mRNA encoding the human P3A+ alpha-subunit isoform was not detected in heart, kidney, liver, lung or brain. Differential expression of the two alpha-subunit isoforms was not seen during fetal muscle development or in muscle from MG patients. In all cases where mRNAs encoding the two alpha-subunit isoforms have been detected, they are present at an approximate 1:1 ratio.     

Low chemical specificity of the nicotinic acetylcholine receptor sterol activation site

The nicotinic acetylcholine receptor (nAcChoR) has an absolute requirement for cholesterol if agonist-stimulated channel opening is to occur [Biochemistry 25 (1986) 830]. Certain non-polar analogs could replace cholesterol in vectorial vesicle permeability assays. Using a stopped-flow fluorescence assay to avoid the limitations of permeability assays imposed by vesicle morphology, it was shown that polar conjugates of cholesterol could also satisfy the sterol requirement [Biochim. Biophys. Acta 1370 (1998) 299]. Here this assay is used to explore the chemical specificity of sterols. Affinity-purified nAcChoRs from Torpedo were reconstituted into bilayers at mole ratios of 58:12:30 [1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dioleoyl-sn-glycero-3-phosphate (DOPA)/steroid]. When the enantiomer of cholesterol was used, or when the stereochemistry at the 3-hydroxy group was changed from beta to alpha by substituting epicholesterol for cholesterol, activation was still supported. The importance of cholesterol's planar ring structure was tested by comparing planar cholestanol (5alpha-cholestan-3beta-ol) with nonplanar coprostanol (5beta-cholestan-3beta-ol). Both supported activation. Thus, these steroids support activation independent of structural features known to be important for modulation of lipid bilayer properties. This provides indirect support for a steroid binding site possessing very lax structural requirements.     

Low chemical specificity of the nicotinic acetylcholine receptor sterol activation site

The nicotinic acetylcholine receptor (nAcChoR) has an absolute requirement for cholesterol if agonist-stimulated channel opening is to occur [Biochemistry 25 (1986) 830]. Certain non-polar analogs could replace cholesterol in vectorial vesicle permeability assays. Using a stopped-flow fluorescence assay to avoid the limitations of permeability assays imposed by vesicle morphology, it was shown that polar conjugates of cholesterol could also satisfy the sterol requirement [Biochim. Biophys. Acta 1370 (1998) 299]. Here this assay is used to explore the chemical specificity of sterols. Affinity-purified nAcChoRs from Torpedo were reconstituted into bilayers at mole ratios of 58:12:30 [1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dioleoyl-sn-glycero-3-phosphate (DOPA)/steroid]. When the enantiomer of cholesterol was used, or when the stereochemistry at the 3-hydroxy group was changed from β to α by substituting epicholesterol for cholesterol, activation was still supported. The importance of cholesterol's planar ring structure was tested by comparing planar cholestanol (5α-cholestan-3β-ol) with nonplanar coprostanol (5β-cholestan-3β-ol). Both supported activation. Thus, these steroids support activation independent of structural features known to be important for modulation of lipid bilayer properties. This provides indirect support for a steroid binding site possessing very lax structural requirements.     

Identification of a cytoplasmic region of the Torpedo nicotinic acetylcholine receptor alpha-subunit by epitope mapping.

Analysis of the binding of monoclonal antibodies (mAbs) by Torpedo nicotinic acetylcholine receptor (AChR) has demonstrated that a region of the alpha-subunit between alpha-156 and alpha-179 is exposed on the cytoplasmic surface of the nicotinic post-synaptic membrane. A panel of mAbs was produced that recognized sodium dodecyl sulfate-denatured subunits of the Torpedo AChR. Antibodies recognizing alpha-subunit were distinguished in terms of their ability to bind alpha-subunit fragments generated by Staphylococcus aureus V8 protease: an 18-kDa fragment beginning at Val-46, a 20-kDa fragment beginning at Ser-173/Ser-162, and a 10 kDa fragment beginning at Asn-339. Three mAbs, selected for binding to each of the V8-protease alpha-subunit fragments, respectively, were characterized in detail. The location of epitopes recognized by both anti-V8-18 and anti-V8-20 mAbs was determined to be within alpha-156 to alpha-179 by isolation of small immunoreactive peptides from proteolytic digests of the alpha-subunit, while the mAb reactive to V8-10 was bound to an epitope within alpha-339 to alpha-386. Quantitative evaluation of binding of the anti-V8-18 and anti-V8-20 mAbs to overlapping synthetic peptides corresponding to alpha-147 to alpha-179 localized the epitopes to distinct portions of this region. Further screening of the panel of mAbs using these synthetic peptides revealed three additional mAbs that bind in this region. The mAbs that bound the three distinct V8-protease alpha-subunit fragments were shown to bind to native AChR by indirect immunofluorescence on frozen sections of Torpedo electric organ. Binding to the native AChR was to the cytoplasmic surface of the AChR since the mAbs could bind to AChR in native vesicles, in which the AChR is oriented right-side-out, only after permeabilization of the vesicles by alkaline treatment or after scrambling of the orientation of the AChR by solubilization and reconstitution into liposomes. The location of the mAb-binding sites at the cytoplasmic surface of the AChR was visualized directly by freeze-etch immunoelectron microscopy. The identification of alpha-156 and alpha-179 as containing a cytoplasmic exposed sequence implies the existence of two non-hydrophobic transmembrane sequences between the site of N-glycosylation (Asn-141) and Cys-192, a site alkylated by the cholinergic affinity labels.     

Localization of the insulin-binding site to the cysteine-rich region of the insulin receptor alpha-subunit

Affinity-purified insulin receptor was photoaffinity labeled with a cleavable radioactive insulin photoprobe. Exhaustive digestion of the labeled alpha-subunit with endoproteinase Glu-C produced a major radioactive fragment of 23 kDa as a part of the putative insulin-binding domain. This fragment could contain either residues 205-316 or 518-633 of the alpha-subunit. Rat hepatoma cells and Chinese hamster ovary cells were transfected with cDNA encoding a human insulin receptor mutant with a deletion of the cysteine-rich region spanning amino acid residues 124-319. Insulin binding by these cells was not increased in spite of high numbers of the mutant insulin receptors being expressed. A panel of monoclonal antibodies which was specific for the receptor alpha-subunit and inhibited insulin binding immunoprecipitated the photolabeled 23-kDa receptor fragment but not the receptor mutant. A synthetic peptide containing residues 243-251 was specifically bound by agarose-insulin beads. We therefore suggest that the 23-kDa fragment contains residues 205-316, and that insulin binding occurs, in part, in the cysteine-rich region of the alpha-subunit.     

Mapping the alpha-subunit site photolabeled by the noncompetitive inhibitor [3H]quinacrine azide in the active state of the nicotinic acetylcholine receptor

We have characterized the time-resolved labeling of a site on the Torpedo californica electrocyte acetylcholine receptor (ACHR) by the photoreactive noncompetitive inhibitor derivative quinacrine azide (QA). The dependence of [3H]QA labeling on acetylcholine (ACH) concentration and on time is consistent with the preferential labeling by [3H]QA of ACHR in the open state. The ACH-dependent [3H]QA labeling, which was associated predominantly with the alpha-subunit, was blocked by other noncompetitive inhibitors including quinacrine, chlorpromazine, proadifen, histrionicotoxin, and bupivacaine. alpha-Subunit from ACHR labeled with [3H]QA 20 ms after the addition of ACH was cleaved with CNBr, and the fragments were separated by high pressure liquid chromatography. A peptide containing a major site of specific labeling was purified on two different reverse-phase columns. By N-terminal sequencing, amino acid composition, binding to mercurial-agarose, and apparent molecular weight, this [3H]QA-labeled peptide was identified as alpha-208-243, a CNBr fragment containing the putative membrane-spanning helix M1.     

The role of tyrosine at the ligand-binding site of the nicotinic acetylcholine receptor

Identification of the critical residues in a receptor's ligand-binding site provides valuable structural information important for understanding the basis for ligand recognition. The design of specific ligands targeted for receptor action will depend to a great extent on detailed structural knowledge of this kind. Although the nicotinic acetylcholine receptor (nAChR) is perhaps the best characterized of all receptors, the detailed configuration of the ligand-binding site remains unknown. Structural comparisons of nicotinic agonists and antagonists have long predicted a negative subsite on the receptor to interact with the positively charged alkyl-ammonium moiety common to nearly all nicotinic agents. We have used intrinsic fluorescence spectroscopic analyses together with binding studies of selectively modified peptide fragments of the nAChR to suggest that one or two invariant tyrosine residues at positions 190 and 198 on the alpha-subunit provide the critical negative subsite required for ligand binding. Tyrosines may similarly be part of the negative subsite of muscarinic receptors and other neurotransmitter receptors that bind cationic ligands.     

An analog of lophotoxin reacts covalently with Tyr190 in the alpha-subunit of the nicotinic acetylcholine receptor

Lophotoxin and lophotoxin analog-1 are natural diterpenes from coral that inhibit nicotinic acetylcholine receptors by covalent reaction with the acetylcholine recognition sites on the alpha-subunits. Although both toxins contain potentially reactive epoxides and alpha,beta-unsaturated aldehydes, the mechanism of their covalent reaction with the receptor is not known. The role of the alpha,beta-unsaturated aldehyde in analog-1 was investigated by reduction of the aldehyde to an alcohol with [3H]NaBH4. The reduced [3H]analog-1 bound selectively and covalently to the alpha-subunit of the receptor. Covalent binding was inhibited by agonists and antagonists, but not by noncompetitive allosteric inhibitors. The apparent dissociation constant of the reduced [3H]analog-1 was approximately 1.5 x 10(-6) M. These results demonstrate that the alpha,beta-unsaturated aldehyde in analog-1 is not an absolute requirement for covalent reaction with the receptor. Receptors were treated with the reduced-[3H]analog-1, and the labeled alpha-subunits were isolated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and digested with staphylococcal V8 protease. A labeled 20-kDa V8 protease fragment was purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse-phase high performance liquid chromatography and subjected to sequence analysis. A peptide beginning at Ser173 was identified, and the label appeared in the 18th step corresponding to Tyr190. This assignment was confirmed by digestion of the labeled 20-kDa V8 protease fragment with cyanogen bromide, followed by purification of the labeled cyanogen bromide peptide on reverse-phase high performance liquid chromatography. A peptide beginning at Lys179 was identified, and the label appeared in the 12th step, again corresponding to Tyr190. Tyr190 may react with the coral toxin by nucleophilic addition at one of the carbons associated with an epoxide, and may form part of the alkylammonium-binding subsite of the acetylcholine recognition site.     

Molecular investigations on the nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor (nAChR) is a well-understood member of the ligand-gated ion channels superfamily. The members of this signaling proteins group, including 5HT₃, GABA_A, glycine, and ionotropic glutamate receptors, are thought to share common secondary, tertiary, and quaternary structures on the basis of a very high degree of sequence similarity. Despite the absence of X-ray crystallographic data, considerable progress on structural analysis of nAChR was achieved from biochemical, mutational, and electron microscopy data allowing the emergence of a three-dimensional image. Photoaffinity labeling and site-directed mutagenesis gave information on the tertiary structure with respect to the agonist/antagonist binding sites, the ion channel, and its selectivity filter. nAChR is an allosterical protein that undergoes interconversion among several conformational states. Time-resolved photolabeling was used in an attempt to elucidate the structural changes that occur in nAChR on neurotransmitter activation. Tertiary and quaternary rearrangements were found in the cholinergic binding pocket and in the channel lumen, but the structural determinant and the functional link between the binding of agonist and the channel gating remain unknown. Time-resolved photolabeling of the functional activated A state using photosensitive agonists might help in understanding the dynamic process leading to the interconversion of the different states.     

Molecular environment of the phencyclidine binding site in the nicotinic acetylcholine receptor membrane

Summary Phencyclidine is a highly specific noncompetitive inhibitor of the nicotinic acetylcholine receptor. In a novel approach to study this site, a spin-labeled analogue of phencyclindine. 4-phenyl-4-(1-piperidinyl)-2.2.6.6.-tetramethylpiperidinoxyl (PPT) was synthesized. The binding of PPT inhibits⁸⁶Rb flux (IC₅₀=6.6m), and [³H] phencyclidine binding to both resting and desensitized acetylcholine receptor (IC₅₀=17 m and 0.22 m, respectively). From an indirect Hill plot of the inhibition of [³H]phencyclidine binding by PPT. a Hill coefficient of approximately one was obtained in the presence of carbamylcholine and 0.8 in -bungarotoxin-treated preparations. Taken together, these results indicate that PPt mimics phencyclidine in its ability to bind to the noncompetitive inhibitor site and is functionally active in blocking ion flux across the acetylcholine receptor channel. Analysis of the electron spin resonance signal of the bound PPT suggests that the environment surrounding the probe within the ion channel is hydrophobic, with a hydrophobicity parameter of 1.09. A dielectric constant for the binding site was estimated to be in the range of 2–3 units.     

Expression and characterization of the chick nicotinic acetylcholine receptor alpha-subunit in insect cells using a baculovirus vector

A baculovirus transfer vector was constructed containing an entire cDNA copy of the chick nicotinic acetylcholine receptor (nAChR) alpha-subunit under control of the Autographa californica nuclear polyhedrosis virus (AcNPV) polyhedrin gene promoter. Recombinant baculovirus was obtained by co-transfection of Spodoptera frugiperda cells with infectious, wild-type AcNPV DNA and the transfer vector. Polyhedrin-negative, recombinant viruses were identified which expressed the nAChR alpha-subunit. The insect cell-expressed alpha-subunit protein had a molecular mass of 42 kDa and was shown to be targeted to the plasma membrane by fluorescence microscopy and toxin-binding assays. The levels of expression were low, approximately 1-2% of cell proteins, when compared with the levels of natural polyhedrin protein. The expressed receptor alpha-subunit was recognised by polyclonal antisera raised against purified Torpedo nAChR alpha-subunit and carried the binding site for the snake venom toxin, alpha-bungarotoxin. Bound alpha-bungarotoxin was displaced in competition binding assays by alpha-cobra toxin, carbamylcholine and d-tubocurarine, and thus had a similar pharmacological profile to that obtained with authentic receptors in muscle cells and receptors expressed in other systems i.e. Xenopus oocytes and mammalian cells. We have also shown that when the chick nAChR alpha-subunit is expressed in the absence of other receptor subunits, unexpectedly high concentrations of nicotine (10 mM) were required to displace bound alpha-bungarotoxin.     

Aryldiazonium salts as photoaffinity labels of the nicotinic acetylcholine receptor PCP binding site

Several aryldiazonium salts are described as irreversible blockers of the phencyclidine binding site of the nicotinic cholinergic receptor. A partial hydrophobic character increases the affinity of these salts for the phencyclidine binding site. Photoaffinity labelling with a tritiated diazonium salt in the presence of either carbamylcholine or alpha-bungarotoxin leads to incorporation of radioactivity into the 4 subunits of the receptor. Among these diazonium salts, an imidazole derivative is unique in that the photoinduced irreversible blocking in only effective when the receptor is in a desensitised state.     

Three possible disulfides in the acetylcholine receptor alpha-subunit

The cysteinyl residues of the acetylcholine receptor alpha-subunit of Torpedo californica were analyzed. All seven cysteines could be accounted for. Three possible disulfide bridges and one unpaired cysteine were indicated. The disulfide linkages were as follows: Cys128 to Cys142; Cys192 to Cys193; Cys412 to Cys418 (Cys222 is unpaired). The identification of cysteinyl residues was accomplished by a modified protein blot procedure. Cysteinyl residues of intact nicotinic acetylcholine receptor were selectively biotinylated with 3-(N-maleimidopropionyl)biocytin and subsequently detected by the 125I-labeled avidin overlay of blotted Staphylococcus aureus V8 proteolyzed alpha-subunits. Two pairs of cysteines (Cys128/Cys142 and Cys412/Cys418) could be demonstrated only after Na(BH4) reduction of the acetylcholine receptor. Cysteine residues 192 and 193 are particularly sensitive to reduction; 0.1 mM dithiothreitol is sufficient.