Distance between the agonist and noncompetitive inhibitor sites on the nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor possesses an agonist binding site on each of the two alpha-subunits and an allosterically coupled noncompetitive inhibitor (NCI) site. The spatial relationships between these sites have been determined by fluorescence energy transfer employing lifetime and steady-state techniques with two donor-acceptor pairs. 6-(5-Dimethylaminonaphthalene-1-sulfonamido)hexanoic acid-beta-(N-trimethylammonium)ethyl ester (dansyl-C6-choline, an agonist) and bis(choline)-N-(4-nitrobenzo-2-oxa-1,3-diazol-7-yl)-iminodiprop rionate (BCNI, a competitive antagonist) were employed as energy donors bound to the agonist sites. Ethidium was employed as a specific probe of the NCI site and served as the energy acceptor for both donors. Under steady-state conditions, energy transfer was measured by monitoring BCNI fluorescence as a function of occupancy of ethidium. Changes in acceptor occupancy were achieved by titrating acetylcholine receptor-donor-acceptor complexes with phencyclidine, a nonfluorescent NCI ligand. Extrapolation of the data to 100% acceptor occupancy yielded a transfer efficiency of 38% for the BCNI-ethidium pair. In the second method, the transfer efficiency of the dansyl-C6-choline-ethidium pair was determined by analysis of the reduction of the donor-excited state fluorescence lifetime. The nanosecond decay rates for dansyl-C6-choline measured in the presence of phencyclidine are characterized by two lifetimes (tau 1 = 6.7; tau 2 = 17.1 ns) with an amplitude ratio, alpha 1/alpha 2 = 2.3. In the presence of ethidium, the two lifetimes were proportionally diminished while retaining a comparable ratio of amplitudes. Displacement of ethidium from the NCI site by phencyclidine restored the two lifetimes to their original values. These data indicate that the donors bound to the two agonist sites transferred energy with similar efficiencies to the acceptor. Thus, the lifetime data suggest that the NCI site is approximately equidistant from each of the agonist sites. The corrected efficiency of donor quenching by this method was 34%, a value in close accord with the steady-state measurements. The distance between the agonist sites and the NCI site was calculated to be between 21-35 A for the BCNI/ethidium pair and 22-40 A for the dansyl-C6-choline/ethidium pair. Consideration of these distances with respect to the molecular dimensions of the receptor and location of the agonist sites suggests a location for the NCI site near the ion channel at the extracellular surface of the membrane bilayer.     

Allosterically linked noncompetitive antagonist binding sites in the resting nicotinic acetylcholine receptor ion channel

Previous studies have established the presence of overlapping binding sites for the noncompetitive antagonists (NCAs) amobarbital, tetracaine, and 3-trifluoromethyl-3-(m-[(125)I]iodophenyl) diazirine ([(125)I]TID) within the ion channel of the Torpedo nicotinic acetylcholine receptor (AChR) in the resting state. These well-characterized NCAs and competitive radioligand binding and photolabeling experiments were employed to better characterize the interaction of the dissociative anesthetics ketamine and thienylcycloexylpiperidine (TCP) with the resting AChR. Our experiments yielded what appear to be conflicting results: (i) both ketamine and TCP potentiated [(125)I]TID photoincorporation into AChR subunits; and (ii) ketamine and TCP had very little effect on [(14)C]amobarbital binding. Nevertheless, (iii) both ketamine and TCP completely displaced [(3)H]tetracaine binding (K(i)s approximately 20.9 and 2.0 microM, respectively) by a mutually exclusive mechanism. To reconcile these results we propose that, in the resting ion channel, TCP and ketamine bind to a site that is spatially distinct from the TID and barbiturate locus, while tetracaine bridges both binding sites.     

Localization of agonist and competitive antagonist binding sites on nicotinic acetylcholine receptors

Identification of all residues involved in the recognition and binding of cholinergic ligands (e.g. agonists, competitive antagonists, and noncompetitive agonists) is a primary objective to understand which structural components are related to the physiological function of the nicotinic acetylcholine receptor (AChR). The picture for the localization of the agonist/competitive antagonist binding sites is now clearer in the light of newer and better experimental evidence. These sites are located mainly on both alpha subunits in a pocket approximately 30-35 A above the surface membrane. Since both alpha subunits are identical, the observed high and low affinity for different ligands on the receptor is conditioned by the interaction of the alpha subunit with other non-alpha subunits. This molecular interaction takes place at the interface formed by the different subunits. For example, the high-affinity acetylcholine (ACh) binding site of the muscle-type AChR is located on the alphadelta subunit interface, whereas the low-affinity ACh binding site is located on the alphagamma subunit interface. Regarding homomeric AChRs (e.g. alpha7, alpha8, and alpha9), up to five binding sites may be located on the alphaalpha subunit interfaces. From the point of view of subunit arrangement, the gamma subunit is in between both alpha subunits and the delta subunit follows the alpha aligned in a clockwise manner from the gamma. Although some competitive antagonists such as lophotoxin and alpha-bungarotoxin bind to the same high- and low-affinity sites as ACh, other cholinergic drugs may bind with opposite specificity. For instance, the location of the high- and the low-affinity binding site for curare-related drugs as well as for agonists such as the alkaloid nicotine and the potent analgesic epibatidine (only when the AChR is in the desensitized state) is determined by the alphagamma and the alphadelta subunit interface, respectively. The case of alpha-conotoxins (alpha-CoTxs) is unique since each alpha-CoTx from different species is recognized by a specific AChR type. In addition, the specificity of alpha-CoTxs for each subunit interface is species-dependent.In general terms we may state that both alpha subunits carry the principal component for the agonist/competitive antagonist binding sites, whereas the non-alpha subunits bear the complementary component. Concerning homomeric AChRs, both the principal and the complementary component exist on the alpha subunit. The principal component on the muscle-type AChR involves three loops-forming binding domains (loops A-C). Loop A (from mouse sequence) is mainly formed by residue Y(93), loop B is molded by amino acids W(149), Y(152), and probably G(153), while loop C is shaped by residues Y(190), C(192), C(193), and Y(198). The complementary component corresponding to each non-alpha subunit probably contributes with at least four loops. More specifically, the loops at the gamma subunit are: loop D which is formed by residue K(34), loop E that is designed by W(55) and E(57), loop F which is built by a stretch of amino acids comprising L(109), S(111), C(115), I(116), and Y(117), and finally loop G that is shaped by F(172) and by the negatively-charged amino acids D(174) and E(183). The complementary component on the delta subunit, which corresponds to the high-affinity ACh binding site, is formed by homologous loops. Regarding alpha-neurotoxins, several snake and alpha-CoTxs bear specific residues that are energetically coupled with their corresponding pairs on the AChR binding site. The principal component for snake alpha-neurotoxins is located on the residue sequence alpha1W(184)-D(200), which includes loop C. In addition, amino acid sequence 55-74 from the alpha1 subunit (which includes loop E), and residues gammaL(119) (close to loop F) and gammaE(176) (close to loop G) at the low-affinity binding site, or deltaL(121) (close to the homologous region of loop G) at the high-affinity binding site, are i     

A novel nicotinic acetylcholine receptor antagonist radioligand for PET studies

Using positron emission tomography (PET) with a specific and selective radioligand targeting nicotinic acetylcholine receptor (nAChR) would allow us to better understand various nAChR related CNS disorders. The use of radiolabeled nAChR antagonists would provide a much safer pharmacological profile, avoiding most peripheral side effects that might be generated from radiolabeled nAChR agonists even at the tracer level; thus, PET imaging with nAChR antagonists would facilitate clinical application. A potent and selective nAChR antagonist was labeled and characterized with PET in non-human primates. Its high brain uptake, high signal-to-noise ratio, and high specific binding strongly suggest a great potential to carry out imaging studies in humans. In addition, the use of a C-11 radiotracer would allow us to perform multiple PET studies in the same individual within a short time frame. The presence of an iodine atom in the molecule also allows the possibility to label with radioiodine for SPECT studies.     

Modeling noncompetitive antagonism of a nicotinic acetylcholine receptor

Models of closed and open channel pores of a muscle-type nicotinic acetylcholine receptor (nAChR) channel comprising M1 and M2 segments are presented. A model of the closed channel is proposed in which hydrophobic residues of the Equatorial Leucine ring screen the oxygen domain formed by the Serine ring, thereby preventing ion flux without completely occluding the pore. This model demonstrates a high similarity with the structure derived from a recent electron microscopy study. We propose that hydrophobic residues of the Equatorial Leucine ring are retracted when the pore is open. Our models provide a possible resolution of the nAChR gate controversy. We have also obtained explanations for the complex mechanisms underlying inhibition of nAChR by philanthotoxins (PhTXs). PhTX-343, containing a spermine moiety with a charge of +3, binds deep in the pore near the Serine ring where classical open channel blockers of nAChR bind. In contrast, PhTX-(12), which has a single charged amino group is unable to reach deeply located rings because of steric restrictions. Both philanthotoxins may bind to a hydrophobic site located close to the external entrance of the pore in a region that includes residues associated with the regulation of desensitization.     

A novel agonist effect on the nicotinic acetylcholine receptor exerted by the anticonvulsive drug Lamotrigine

The anticonvulsive drug Lamotrigine (LTG) is found to activate adult muscle nicotinic acetylcholine receptors (AChR). Single-channel patch-clamp recordings showed that LTG (0.05-400 μM) applied alone is able to open AChR channels. [¹²⁵I]α-bungarotoxin-binding studies further indicate that LTG does not bind to the canonical ACh-binding sites. Fluorescence experiments using the probe crystal violet demonstrate that LTG induces the transition from the resting state to the desensitized state of the AChR in the presence of excess α-bungarotoxin, that is, when the agonist site is blocked. Allosterically-potentiating ligands or the open-channel blocker QX-314 exhibited a behavior different from that of LTG. We conclude that LTG activates the AChR through a site that is different from those of full agonists/competitive antagonists and allosterically-potentiating ligands, respectively.     

Binding sites for alpha-bungarotoxin and the noncompetitive inhibitor phencyclidine on a synthetic peptide comprising residues 172-227 of the alpha-subunit of the nicotinic acetylcholine receptor.

The binding of the competitive antagonist alpha-bungarotoxin (alpha-Btx) and the noncompetitive inhibitor phencyclidine (PCP) to a synthetic peptide comprising residues 172-227 of the alpha-subunit of the Torpedo acetylcholine receptor has been characterized. 125I-alpha-Btx bound to the 172-227 peptide in a solid-phase assay and was competed by alpha-Btx (IC50 = 5.0 x 10(-8) M), d-tubocurarine (IC50 = 5.9 X 10(-5)M), and NaCl (IC50 = 7.9 x 10(-2)M). In the presence of 0.02% sodium dodecyl sulfate, 125I-alpha-Btx bound to the 56-residue peptide with a KD of 3.5 nM, as determined by equilibrium saturation binding studies. Because alpha-Btx binds to a peptide comprising residues 173-204 with the same affinity and does not bind to a peptide comprising residues 205-227, the competitive antagonist and hence agonist binding site lies between residues 173 and 204. After photoaffinity labeling, [3H]PCP was bound to the 172-227 peptide. [3H]PCP binding was inhibited by chlorpromazine (IC50 = 6.3 x 10(-5)M), tetracaine (IC50 = 4.2 x 10(-6)M), and dibucaine (IC50 = 2.7 x 10(-4)M). Equilibrium saturation binding studies in the presence of 0.02% sodium dodecyl sulfate showed that [3H]PCP bound at two sites, a major site of high affinity with an apparent KD of 0.4 microM and a minor low-affinity site with an apparent KD of 4.6 microM. High -affinity binding occurred at a single site on peptide 205-227 (KD = 0.27 microM) and was competed by chlorpromazine but not by alpha-Btx.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutations affecting agonist sensitivity of the nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor (AChR) is a pentameric transmembrane protein (alpha 2 beta gamma delta) that binds the neurotransmitter acetylcholine (ACh) and transduces this binding into the opening of a cation selective channel. The agonist, competitive antagonist, and snake toxin binding functions of the AChR are associated with the alpha subunit (Kao et al., 1984; Tzartos and Changeux, 1984; Wilson et al., 1985; Kao and Karlin, 1986; Pederson et al., 1986). We used site-directed mutagenesis and expression of AChR in Xenopus oocytes to identify amino acid residues critical for ligand binding and channel activation. Several mutations in the alpha subunit sequence were constructed based on information from sequence homology and from previous biochemical (Barkas et al., 1987; Dennis et al., 1988; Middleton and Cohen, 1990) and spectroscopic (Pearce and Hawrot, 1990; Pearce et al., 1990) studies. We have identified one mutation, Tyr190 to Phe (Y190F), that had a dramatic effect on ligand binding and channel activation. These mutant channels required more than 50-fold higher concentrations of ACh for channel activation than did wild type channels. This functional change is largely accounted for by a comparable shift in the agonist binding affinity, as assessed by the ability of ACh to compete with alpha-bungarotoxin binding. Other mutations at nearby conserved positions of the alpha subunit (H186F, P194S, Y198F) produce less dramatic changes in channel properties. Our results demonstrate that ligand binding and channel gating are separable properties of the receptor protein, and that Tyr190 appears to play a specific role in the receptor site for acetylcholine.     

Characterization and three-dimensional structure determination of psi-conotoxin Piiif,a novel noncompetitive antagonist of nicotinic acetylcholine receptors

A novel inhibitor of nicotinic acetylcholine receptors (nAChRs), psi-conotoxin Piiif, was isolated from the venom of Conus purpurascens and found to have the sequence GOOCCLYGSCROFOGCYNALCCRK-NH2. The sequence is highly homologous to that of psi-conotoxin Piiie, a previously identified noncompetitive inhibitor of Torpedo electroplax nAChR, also isolated from C. purpurascens. Both psi-conotoxins block Torpedo and mouse nicotinic acetylcholine receptors (nAChRs), but psi-Piiif is less potent by a factor of 10(1)-10(2). A high-resolution structure of psi-Piiif was determined by NMR and molecular modeling calculations. Psi-Piiif analogues containing [(13)C]-labeled cysteine at selected positions were synthesized to resolve spectral overlap of Cys side chain proton signals. The structures are well-converged, with backbone atom position RMSDs of 0.21 A for the main body of the peptide between residues 4 and 22 and 0.47 A for all residues. The overall backbone conformation is closely similar to psi-Piiie, the main difference being in the degree of conformational disorder at the two termini. Psi-Piiie and psi-Piiif have similar locations of positive charge density, although psi-Piiif has a lower overall charge. One disulfide bridge of psi-Piiif appears to undergo dynamic conformational fluctuations based on both the model and on experimental observation. Chimeras in which the three intercysteine loops were swapped between psi-Piiie and psi-Piiif were tested for inhibitory activity against Torpedo nAChRs. The third loop, which contains no charged residues in either peptide, is the prime determinant of potency in these psi-conotoxins.     

Affinity purification of a chimeric nicotinic acetylcholine receptor in the agonist and antagonist bound states

Nicotinic acetylcholine receptors (nAChRs) form ligand-gated ion channels that mediate fast signal transmission at synapses. These receptors are members of a large family of pentameric ion channels that are of active medical interest. An expression system utilizing a chimerical construct of the N-terminal extracellular ligand binding domain of alpha7 type nAChR and the C-terminal transmembrane portion of 5HT3 type receptor resulted high level of expressions. Two ligand affinity chromatography purification methods for this receptor have been developed. One method relies on the covalent immobilization of a high affinity small molecule alpha7 nAChR agonist, (R)-5-(4-aminophenyl)-N-(quinuclidin-3-yl) furan-2-carboxamide, and the other uses mono biotinylated alpha-bungarotoxin, an antagonist, that forms a quasi-irreversible complex with alpha7 nAChR. Detergent solubilized alpha7/5HT(3) chimeric receptors were selectively retained on the affinity resins and could be eluted with free ligand or biotin. The proteins purified by both methods were characterized by gel electrophoresis, mass spectra, amino acid composition analysis, and N-terminal sequence determination. These analyses confirmed the isolation of a mature alpha7/5HT(3) receptor with the signal peptide removed. These results suggest a scalable path forward to generate multi-milligram amounts of purified complexes for additional studies including protein crystallization.     

Channel permeant cations compete selectively with noncompetitive inhibitors of the nicotinic acetylcholine receptor.

Previous work suggests that noncompetitive inhibitor (NCI) ligands and channel permeant cations bind to sites within the nicotinic acetylcholine receptor ion channel. We have used ethidium as a fluorescent probe of the NCI site to investigate interactions between NCI ligands and channel permeant cations. We found that ethidium can be completely displaced from the receptor by a variety of inorganic monovalent and divalent cations. The rank order of monovalent cation affinities was found to be Tl+ greater than Rb+ greater than or equal to K+ greater than Cs+ greater than Na+ greater than Li+. The monovalent cation Kd values vary markedly over a 40-fold range, from 3 to 121 mM. The Kd values and rank order correspond to values determined previously from electrophysiological data. Hill plots of the back titrations yield slopes of 1.0 for all monovalent cations, indicating a single class of independent sites, as shown previously for NCI ligands. Scatchard analysis of ethidium binding in the presence of Tl+ reveals a reduction in affinity and no changes in the maximal number of sites. In the presence of agonist the kinetics of ethidium dissociation induced by the addition of phencyclidine or cations alone or the simultaneous addition of both are nearly identical. The ethidium dissociation rate induced by either phencyclidine or cations is regulated by the occupation of the agonist sites in a similar manner. These results indicate that the effect of cations on NCI ligand binding occurs by mutually exclusive competition. We suggest that NCIs can regulate cation binding at a physiological cation recognition site that is likely part of the cation permeation path through the receptor channel.     

Rapid laser flash photoaffinity labeling of binding sites for a noncompetitive inhibitor of the acetylcholine receptor

Photoaffinity labeling of the nicotinic acetylcholine receptor from Torpedo marmorata electric tissue was performed in the presence of cholinergic effectors in the millisecond to second time range by a combination of a stopped-flow apparatus and a high-energy pulse laser. The label applied was [3H]triphenylmethylphosphonium, a lipophilic cation previously shown to be a specific blocker of the acetylcholine receptor ion channel. With the receptor in the resting state most of the label was incorporated into the alpha polypeptide chains. In the presence of agonists and antagonists increasing incorporation into the delta- and (less pronounced) the beta-chain was observed. The time course of this increase had a half-life of about 0.4 s, being slower than receptor activation and channel opening. in the resting, active, and even rapidly desensitized state, the alpha polypeptide chains appear to be the primary targets of the photoaffinity reaction. The action spectrum of the photolabeling has a sharp maximum at lambda = 270 nm and a small-side maximum at lambda = 290 nm. It does not resemble the absorption spectrum of the label and may hint at amino acid side chains as the moieties activated by UV light causing the photolabeling. The effector specificity of the observed slow increase of label incorporation into the delta polypeptide chain was investigated. It does not prove that slow desensitization is the underlying event. The agonists acetylcholine and carbamoylcholine as well as treatment of receptor-rich membranes with phospholipase A2 (but not phospholipase D) triggered labeling of delta, but antagonists such as D-tubocurarine and most conspicuously flaxedil had a similar effect.     

Phosphorylation sites of the nicotinic acetylcholine receptor. A novel site detected in position delta S362.

The delta-subunit of the nicotinic acetylcholine receptor from Torpedo californica electric tissue isolated form receptor purified in the absence of protein phosphatase inhibitors contains a total of four phosphate groups. Three of these are shown to represent phosphoserine groups. The fourth possible represents phosphotyrosine. The phosphate groups are localized within the primary structure: We found phosphoserine in positions delta S361 and delta S377, the predicted sites phosphorylated by PKA and PKC, respectively. In addition, we found that position delta S362 is also phosphorylated. Phosphorylation experiments with the synthetic peptide delta L357-delta K368 show that phosphorylation of this novel site can be catalyzed by PKA and by PKC. It is concluded that the delat-subunit of the acetylcholine receptor is stably and not transiently phosphorylated. Implications for the physiological functions of receptor phosphorylation are discussed.     

Molecular mechanisms and binding site location for the noncompetitive antagonist crystal violet on nicotinic acetylcholine receptors

We investigated the molecular mechanisms and the binding site location for the fluorophor crystal violet (CrV), a noncompetitive antagonist of the nicotinic acetylcholine receptor (AChR). To this end, radiolabeled competition binding, fluorescence spectroscopy, Schild-type analysis, patch-clamp recordings, and molecular dynamics approaches were used. The results indicate that (i) CrV interacts with the desensitized Torpedo AChR with higher affinity than with the resting state at several temperatures (5-37 degrees C); (ii) CrV-induced inhibition of the phencyclidine (PCP) analogue [(3)H]thienylcyclohexylpiperidine binding to the desensitized or resting AChR is mediated by a steric mechanism; (iii) tetracaine inhibits CrV binding to the resting AChR, probably by a steric mechanism; (iv) barbiturates modulate CrV binding to the resting AChR by an allosteric mechanism; (v) CrV itself induces AChR desensitization; (vi) CrV decreases the peak of macroscopic currents by acting on the resting AChR but without affecting the desensitization rate from the open state; and (vii) two tertiary amino groups from CrV may bind to the alpha1-Glu(262) residues (located at position 20') in the resting state. We conclude that the CrV binding site overlaps the PCP locus in the resting and desensitized state. The noncompetitive action of CrV may be explained by an allosteric mechanism in which the binding of CrV to the extracellular mouth of the resting receptor leads to an inhibition of channel opening. Binding of CrV probably increases desensitization of the resting channel and stabilizes the desensitized state.     

Agonist-induced changes in the structure of the acetylcholine receptor M2 regions revealed by photoincorporation of an uncharged nicotinic noncompetitive antagonist.

To characterize structural changes induced in the nicotinic acetylcholine receptor (AChR) by agonists, we have mapped the sites of photoincorporation of the cholinergic noncompetitive antagonist 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine (]125I]TID) in the presence and absence of 50 microM carbamylcholine. [125I]TID binds to the AChR with similar affinity under both these conditions, but agonist inhibits photoincorporation into all subunits by greater than 75% (White, B. H., Howard, S., Cohen, S. G., and Cohen, J. B. (1991) J. Biol. Chem. 266, 21595-21607). [125I]TID-labeled sites on the beta- and delta-subunits were identified by amino-terminal sequencing of both cyanogen bromide (CNBr) and tryptic fragments purified by Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by reversed-phase high-performance liquid chromatography. In the absence of agonist, [125I]TID specifically labels homologous aliphatic residues (beta L-257, delta L-265, beta V-261, and delta V-269) in the M2 region of both subunits. In the presence of agonist, labeling of these residues is reduced approximately 90%, and the distribution of labeled residues is broadened to include a homologous set of serine residues at the amino terminus of M2. In the beta-subunit residues beta S-250, beta S-254, beta L-257, and beta V-261 are all labeled in the presence of carbamylcholine. This pattern of labeling supports an alpha-helical model for M2 with the labeled face forming the ion channel lumen. The observed redistribution of label in the resting and desensitized states provides the first direct evidence for an agonist-dependent rearrangement of the M2 helices. The efficient labeling of the resting state channel in a region capable of structural change also suggests a plausible model for AChR gating in which the aliphatic residues labeled by [125I]TID form a permeability barrier to the passage of ions. We also report increased labeling of the M1 region of the delta-subunit in the presence of agonist.     

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.     

A photoreactive probe that differentiates the binding sites of noncompetitive GABA receptor antagonists

γ-Aminobutyric acid (GABA) receptors are postsynaptic membrane protein complexes that are important not only in the regulation of the nervous system but also as targets of drugs and insecticides. We synthesized a photoreactive straight-chain noncompetitive antagonist (NCA), 2-nitro-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenyl 4-(4-methoxycarbonyl-1-butynyl)benzoate (NMB), to probe the NCA binding site. Our data show that this probe labels the NCA site and demonstrate that the NCA insecticide fipronil binds at a site distinct from that of other NCAs, such as picrotoxinin and 4′-ethynyl-4-n-propylbicycloorthobenzoate. The unique molecule NMB will be useful in identifying the cross-linking site of straight-chain NCAs in GABA receptors and mapping allosteric binding sites. Such studies should provide invaluable information in designing novel NCAs.     

Protein structural effects of agonist binding to the nicotinic acetylcholine receptor.

The effects on the protein structure produced by binding of cholinergic agonists to purified acetylcholine receptor (AcChR) reconstituted into lipid vesicles, has been studied by Fourier-transform infrared spectroscopy and differential scanning calorimetry. Spectral changes in the conformationally sensitive amide I infrared band indicates that the exposure of the AcChR to the agonist carbamylcholine, under conditions which drive the AcChR into the desensitized state, produces alterations in the protein secondary structure. Quantitative estimation of these agonist-induced alterations by band-fitting analysis of the amide I spectral band reveals no appreciable changes in the percent of alpha-helix, but a decrease in beta-sheet structure, concomitant with an increase in less ordered structures. Additionally, agonist binding results in a concentration-dependent increase in the protein thermal stability, as indicated by the temperature dependence of the protein infrared spectrum and by calorimetric analysis, which further suggest that AcChR desensitization induced by the cholinergic agonist implies significant rearrangements in the protein structure.