Naturally occurring mutations at the acetylcholine receptor binding site independently alter ACh binding and channel gating

By defining functional defects in a congenital myasthenic syndrome (CMS), we show that two mutant residues, located in a binding site region of the acetylcholine receptor (AChR) epsilon subunit, exert opposite effects on ACh binding and suppress channel gating. Single channel kinetic analysis reveals that the first mutation, epsilon N182Y, increases ACh affinity for receptors in the resting closed state, which promotes sequential occupancy of the binding sites and discloses rate constants for ACh occupancy of the nonmutant alphadelta site. Studies of the analogous mutation in the delta subunit, deltaN187Y, disclose rate constants for ACh occupancy of the nonmutant alpha epsilon site. The second CMS mutation, epsilon D175N, reduces ACh affinity for receptors in the resting closed state; occupancy of the mutant site still promotes gating because a large difference in affinity is maintained between closed and open states. epsilon D175N impairs overall gating, however, through an effect independent of ACh occupancy. When mapped on a structural model of the AChR binding site, epsilon N182Y localizes to the interface with the alpha subunit, and epsilon D175 to the entrance of the ACh binding cavity. Both epsilon N182Y and epsilon D175 show state specificity in affecting closed relative to desensitized state affinities, suggesting that the protein chain harboring epsilon N182 and epsilon D175 rearranges in the course of receptor desensitization. The overall results show that key residues at the ACh binding site differentially stabilize the agonist bound to closed, open and desensitized states, and provide a set point for gating of the channel.     

Mutation in the M1 Domain of the Acetylcholine Receptor α Subunit Decreases the Rate of Agonist Dissociation

We describe the kinetic consequences of the mutation N217K in the M1 domain of the acetylcholine receptor (AChR) α subunit that causes a slow channel congenital myasthenic syndrome (SCCMS). We previously showed that receptors containing αN217K expressed in 293 HEK cells open in prolonged activation episodes strikingly similar to those observed at the SCCMS end plates. Here we use single channel kinetic analysis to show that the prolonged activation episodes result primarily from slowing of the rate of acetylcholine (ACh) dissociation from the binding site. Rate constants for channel opening and closing are also slowed but to much smaller extents. The rate constants derived from kinetic analysis also describe the concentration dependence of receptor activation, revealing a 20-fold shift in the EC₅₀ to lower agonist concentrations for αN217K. The apparent affinity of ACh binding, measured by competition against the rate of ¹²⁵I-α-bungarotoxin binding, is also enhanced 20-fold by αN217K. Both the slowing of ACh dissociation and enhanced apparent affinity are specific to the lysine substitution, as the glutamine and glutamate substitutions have no effect. Substituting lysine for the equivalent asparagine in the β, ε, or δ subunits does not affect the kinetics of receptor activation or apparent agonist affinity. The results show that a mutation in the amino-terminal portion of the M1 domain produces a localized perturbation that stabilizes agonist bound to the resting state of the AChR.     

Acetylcholine and epibatidine binding to muscle acetylcholine receptors distinguish between concerted and uncoupled models.

The muscle acetylcholine receptor (AChR) has served as a prototype for understanding allosteric mechanisms of neurotransmitter-gated ion channels. The phenomenon of cooperative agonist binding is described by the model of Monod et al. (Monod, J., Wyman, J., and Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118; MWC model), which requires concerted switching of the two binding sites between low and high affinity states. The present study examines binding of acetylcholine (ACh) and epibatidine, agonists with opposite selectivity for the two binding sites of mouse muscle AChRs. We expressed either fetal or adult AChRs in 293 HEK cells and measured agonist binding by competition against the initial rate of 125I-alpha-bungarotoxin binding. We fit predictions of the MWC model to epibatidine and ACh binding data simultaneously, taking as constants previously determined parameters for agonist binding and channel gating steps, and varying the agonist-independent parameters. We find that the MWC model describes the apparent dissociation constants for both agonists but predicts Hill coefficients that are far too steep. An Uncoupled model, which relaxes the requirement of concerted state transitions, accurately describes binding of both ACh and epibatidine and provides parameters for agonist-independent steps consistent with known aspects of AChR function.     

Design and synthesis of peptides that bind α-bungarotoxin with high affinity and mimic the three-dimensional structure of the binding-site of acetylcholine receptor

α-Bungarotoxin (α-BTX) is a highly toxic snake neurotoxin that binds to acetylcholine receptor (AChR) at the neuromuscular junction, and is a potent inhibitor of this receptor. In the following we review multi-phase research of the design, synthesis and structure analysis of peptides that bind α-BTX and inhibit its binding to AChR. Structure-based design concomitant with biological information of the α-BTX/AChR system yielded 13-mer peptides that bind to α-BTX with high affinity and are potent inhibitors of α-BTX binding to AChR (IC₅₀ of 2 nM). X-Ray and NMR spectroscopy reveal that the high-affinity peptides fold into an anti-parallel β-hairpin structure when bound to α-BTX. The structures of the bound peptides and the homologous loop of acetylcholine binding protein, a soluble analog of AChR, are remarkably similar. Their superposition indicates that the toxin wraps around the binding-site loop, and in addition, binds tightly at the interface of two of the receptor subunits and blocks access of acetylcholine to its binding site. The procedure described in this article may serve as a paradigm for obtaining high-affinity peptides in biochemical systems that contain a ligand and a receptor molecule.     

The hydrophobic photoreagent 3-(trifluoromethyl)-3-m-([125I] iodophenyl) diazirine is a novel noncompetitive antagonist of the nicotinic acetylcholine receptor

We have shown previously that the lipophilic photoreagent 3-(trifluoromethyl)3-m-([125I]iodophenyl)-diazirine ([125I]TID) photolabels all four subunits of the Torpedo nicotinic acetylcholine receptor (AChR) and that greater than 70% of this photoincorporation is inhibited by cholinergic agonists and some noncompetitive antagonists, including histrionicotoxin (HTX), but not phencyclidine (PCP; White, B.H., and Cohen, J.B. (1988) Biochemistry 27, 8741-8751). We have now examined the effects of nonradioactive TID on (a) AChR photoincorporation of [125I]TID, (b) AChR-mediated ion transport, and (c) AChR binding of several cholinergic ligands. We find that TID inhibits [125I]TID photoincorporation into the AChR to the same extent as carbamylcholine. The saturable component of [125I]TID photolabeling is half-maximal at 4 microM [125I]TID with 0.5 mol specifically incorporated per mol of AChR after 30 min photolysis with 60 microM [125I]TID. Repeated labeling of membranes at a fixed [125I]TID concentration gave results consistent with a maximal incorporation of one [125I]TID molecule per AChR. Nonradioactive TID also noncompetitively inhibits agonist-stimulated 22Na+ efflux from Torpedo vesicles with an IC50 of 1 microM. Furthermore, TID inhibits allosterically the binding of [3H]HTX, decreasing its affinity for the AChR 5-fold both in the presence and absence of agonist. In contrast, TID has little effect on [3H]PCP binding in the absence of agonist but completely inhibits it in the presence of agonist. TID enhances the cooperativity of [3H]nicotine binding. [125I]TID is thus a photoaffinity label for a novel noncompetitive antagonist binding site on the AChR that is linked allosterically to the binding sites of both agonists and other noncompetitive antagonists. The [125I]TID site is presumably located within the central pore of the AChR.     

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.     

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     

Acetylcholine Receptor Channels Activated by a Single Agonist Molecule

The neuromuscular acetylcholine receptor (AChR) is an allosteric protein that alternatively adopts inactive versus active conformations (R↔R^∗). The R^∗ shape has a higher agonist affinity and ionic conductance than R. To understand how agonists trigger this gating isomerization, we examined single-channel currents from adult mouse muscle AChRs that isomerize normally without agonists but have only a single site able to use agonist binding energy to motivate gating. We estimated the monoliganded gating equilibrium constant E₁ and the energy change associated with the R versus R^∗ change in affinity for agonists. AChRs with only one operational binding site gave rise to a single population of currents, indicating that the two transmitter binding sites have approximately the same affinity for the transmitter ACh. The results indicated that E₁ ≈ 4.3 × 10⁻³ with ACh, and ≈1.7 × 10⁻⁴ with the partial-agonist choline. From these values and the diliganded gating equilibrium constants, we estimate that the unliganded AChR gating constant is E₀ ≈ 6.5 × 10⁻⁷. Gating changes the stability of the ligand-protein complex by ∼5.2 kcal/mol for ACh and ∼3.3 kcal/mol for choline.     

Site-specific charge interactions of alpha-conotoxin MI with the nicotinic acetylcholine receptor

We have tested the importance of charge interactions for alpha-conotoxin MI binding to the nicotinic acetylcholine receptor (AChR). Ionic residues on alpha-conotoxin MI were altered by site-directed mutagenesis or by chemical modification. In physiological buffer, removal of charges at the N terminus, His-5, and Lys-10 had small (2-4-fold) effects on binding affinity to the mouse muscle AChR and the Torpedo AChR. It was also demonstrated that conotoxin had no effect on the conformational equilibrium of either receptor, as assessed by the effects of the noncompetitive antagonist proadifen on conotoxin binding and, conversely, the effect of conotoxin on the affinity of phencyclidine, proadifen, and ethidium. Conotoxin displayed higher binding affinity in low ionic strength buffer; neutralization of Lys-10 and the N terminus by acetylation blocked this affinity shift at the alphadelta site but not at the alphagamma site. It is concluded that Ctx residues Lys-10 and the N terminal interact with oppositely charged receptor residues only at the alphadelta site, and the two sites have distinct arrangements of charged residues. Ethidium fluorescence experiments demonstrated that conotoxin is formally competitive with a small cholinergic ligand, tetramethylammonium. Thus, alpha-conotoxin MI appears to interact with the portion of the binding site responsible for stabilizing agonist cations but does not do so with a cationic residue and is, consequently, incapable of inducing a conformational change.     

Biochemical investigations of ionic channels in excitable membranes

Summary Ion channels of excitable membranes are composed of a gating device and a selectivity filter. Two strategies are discussed in this review for the biochemical isolation and characterization of these two functional subunits of channels: Membrane molecules involved in ion translocation can be identified in vitro by their pharmacological properties, i.e. by binding assays with radioactive drugs known to selectively affect a special channel in vivo. More desirable is an assay of their true biological function, i.e. translocation of ions through a membrane. Ion flux measurements with natural and reconstituted membrane systems in vitro are recently available.This article summarizes our present knowledge of electrically excitable sodium and potassium channels of nerve membranes and of the chemically excitable sodium/potassium channel of cholinergic synapses, the acetylcholine receptor complex (AChR). Because of the availability of a great variety of drugs binding with high affinity to axonal sodium channels its investigation is more advanced than that of the axonal potassium channel. The lack of high affinity labels for the latter can be possibly overcome by photoaffinity labels which label components of the channel in situ. Initial success is reported with a photoafinity label derived from the potassium channel blocker TEA.Most advanced is the biochemical investigation of the acetylcholine receptor (AChR) which has been purified in milligram quantities. It represents a protein complex composed of different polypeptide chains with different functions regulating the sodium/potassium permeability of cholinergic postsynaptic membranes. Experiments are described to elucidate the quaternary structure, the site of binding of cholinergic ligands and neurotoxins and to prove dynamic conformation changes of the protein which may be the cause for permeability changes of the membrane. The gating device and the ion translocation system (selectivity filter, ionophor) appear to be present in the receptor complex though located possible in different subunits. This is evidenced by reconstitution of excitable membranes from purified AChR and exogenous lipids by a novel and reproducible method.An invited review article.     

Monoclonal antibodies to Torpedo acetylcholine receptor. Characterisation of antigenic determinants within the cholinergic binding site

Thirteen monoclonal antibodies (mAb) to the acetylcholine receptor (AChR) from Torpedo marmorata showed high avidity for the receptor but none exhibited binding to muscle AChR solubilised from seven other animal species. Five mAb and Fab monomer fragments prepared from two of them, inhibited alpha-bungarotoxin (alpha BuTx) binding to receptor by a maximum of 50%. In the presence of excess mAb the 125I-alpha BuTx bound could be precipitated by anti-IgG indicating that the mAb bound to only one of the two alpha BuTx binding sites on each AChR monomer. This site appeared to have a lower affinity for d-tubocurarine and decamethonium than the non-mAb site. Binding of five anti-site mAb was mutually competitive and four of them (AS2-AS5) were inhibited by other cholinergic ligands and influenced by four non-toxin binding site antibodies. One (AS1) bound within the toxin binding site yet outside the main neurotransmitter binding region. It is concluded that these five mAb distinguish between the two alpha BuTx binding sites on the Torpedo AChR, and bind only to the site which displays lower affinity for d-tubocurarine and other competitive ligands.     

An Accessory Agonist Binding Site Promotes Activation of α4β2* Nicotinic Acetylcholine Receptors

Neuronal nicotinic acetylcholine receptors containing α4, β2, and sometimes other subunits (α4β2* nAChRs) regulate addictive and other behavioral effects of nicotine. These nAChRs exist in several stoichiometries, typically with two high affinity acetylcholine (ACh) binding sites at the interface of α4 and β2 subunits and a fifth accessory subunit. A third low affinity ACh binding site is formed when this accessory subunit is α4 but not if it is β2. Agonists selective for the accessory ACh site, such as 3-[3-(3-pyridyl)-1,2,4-oxadiazol-5-yl]benzonitrile (NS9283), cannot alone activate a nAChR but can facilitate more efficient activation in combination with agonists at the canonical α4β2 sites. We therefore suggest categorizing agonists according to their site selectivity. NS9283 binds to the accessory ACh binding site; thus it is termed an accessory site-selective agonist. We expressed (α4β2)₂ concatamers in Xenopus oocytes with free accessory subunits to obtain defined nAChR stoichiometries and α4/accessory subunit interfaces. We show that α2, α3, α4, and α6 accessory subunits can form binding sites for ACh and NS9283 at interfaces with α4 subunits, but β2 and β4 accessory subunits cannot. To permit selective blockage of the accessory site, α4 threonine 126 located on the minus side of α4 that contributes to the accessory site, but not the α4β2 sites, was mutated to cysteine. Alkylation of this cysteine with a thioreactive reagent blocked activity of ACh and NS9283 at the accessory site. Accessory agonist binding sites are promising drug targets.     

Energy for Wild-Type Acetylcholine Receptor Channel Gating from Different Choline Derivatives

Agonists, including the neurotransmitter acetylcholine (ACh), bind at two sites in the neuromuscular ACh receptor channel (AChR) to promote a reversible, global change in protein conformation that regulates the flow of ions across the muscle cell membrane. In the synaptic cleft, ACh is hydrolyzed to acetate and choline. Replacement of the transmitter’s ester acetyl group with a hydroxyl (ACh→choline) results in a +1.8 kcal/mol reduction in the energy for gating generated by each agonist molecule from a low- to high-affinity change of the transmitter binding site (ΔG_B). To understand the distinct actions of structurally related agonist molecules, we measured ΔG_B for 10 related choline derivatives. Replacing the hydroxyl group of choline with different substituents, such as hydrogen, chloride, methyl, or amine, increased the energy for gating (i.e., it made ΔG_B more negative relative to choline). Extending the ethyl hydroxide tail of choline to propyl and butyl hydroxide also increased this energy. Our findings reveal the amount of energy that is available for the AChR conformational change provided by different, structurally related agonists. We speculate that a hydrogen bond between the choline hydroxyl and the backbone carbonyl of αW149 positions this agonist’s quaternary ammonium group so as to reduce the cation-π interaction between this moiety and the aromatic groups at the binding site.     

Modal affinities of endplate acetylcholine receptors caused by loop C mutations

The time course of the endplate current is determined by the rate and equilibrium constants for acetylcholine receptor (AChR) activation. We measured these constants in single-channel currents from AChRs with mutations at the neurotransmitter-binding sites, in loop C. The main findings are: (a) Almost all perturbations of loop C generate heterogeneity in the channel open probability (“modes”). (b) Modes are generated by different affinities for ACh that can be either higher or lower than in the wild-type receptors. (c) The modes are stable, in so far as each receptor maintains its affinity for at least several minutes. (d) Different agonists show different degrees of modal activity. With the loop C mutation αP197A, there are four modes with ACh but only two with partial agonists. (e) The affinity variations arise exclusively from the αδ-binding site. (f) Substituting four γ-subunit residues into the δ subunit (three in loop E and one in the β5–β5′ linker) reduces modal activity. (g) At each neurotransmitter-binding site, affinity is determined by a core of five aromatic residues. Modes are eliminated by an alanine mutation at δW57 but not at the other aromatics. (h) Modes are eliminated by a phenylalanine substitution at all core aromatics except αY93. The results suggest that, at the αδ agonist site, loop C and the complementary subunit surface can each adopt alternative conformations and interact with each other to influence the position of δW57 with respect to the aromatic core and, hence, affinity.     

Agonists block currents through acetylcholine receptor channels

We have examined the effects of high concentrations of cholinergic agonists on currents through single acetylcholine receptor (AChR) channels on clonal BC3H1 cells. We find that raised concentrations of acetylcholine (ACh; above 300 microM) or carbamylcholine (Carb; above 1,000 microM) produce a voltage- and concentration-dependent reduction in the mean single-channel current. Raised concentrations of suberyldicholine (Sub; above 3 microM) produce a voltage- and concentration-dependent increase in the number of brief duration low-conductance interruptions of open-channel currents. These observations can be quantitatively described by a model in which agonist molecules enter and transiently occlude the ion-channel of the AChR.     

An ER‐resident membrane protein complex regulates nicotinic acetylcholine receptor subunit composition at the synapse

Nicotinic acetylcholine receptors (nAChRs) are homo‐ or heteropentameric ligand‐gated ion channels mediating excitatory neurotransmission and muscle activation. Regulation of nAChR subunit assembly and transfer of correctly assembled pentamers to the cell surface is only partially understood. Here, we characterize an ER transmembrane (TM) protein complex that influences nAChR cell‐surface expression and functional properties in Caenorhabditis elegans muscle. Loss of either type I TM protein, NRA‐2 or NRA‐4 (n icotinic r eceptor a ssociated), affects two different types of muscle nAChRs and causes in vivo resistance to cholinergic agonists. Sensitivity to subtype‐specific agonists of these nAChRs is altered differently, as demonstrated by whole‐cell voltage‐clamp of dissected adult muscle, when applying exogenous agonists or after photo‐evoked, channelrhodopsin‐2 (ChR2) mediated acetylcholine (ACh) release, as well as in single‐channel recordings in cultured embryonic muscle. These data suggest that nAChRs desensitize faster in nra‐2 mutants. Cell‐surface expression of different subunits of the ‘levamisole‐sensitive’ nAChR (L‐AChR) is differentially affected in the absence of NRA‐2 or NRA‐4, suggesting that they control nAChR subunit composition or allow only certain receptor assemblies to leave the ER.     

Loop C and the mechanism of acetylcholine receptor–channel gating

Agonist molecules at the two neuromuscular acetylcholine (ACh) receptor (AChR) transmitter-binding sites increase the probability of channel opening. In one hypothesis for AChR activation (“priming”), the capping of loop C at each binding site transfers energy independently to the distant gate over a discrete structural pathway. We used single-channel analyses to examine the experimental support for this proposal with regard to brief unliganded openings, the effects of loop-C modifications, the effects of mutations to residues either on or off the putative pathway, and state models for describing currents at low [ACh]. The results show that (a) diliganded and brief unliganded openings are generated by the same essential, global transition; (b) the radical manipulation of loop C does not prevent channel opening but impairs agonist binding; (c) both on- and off-pathway mutations alter gating by changing the relative stability of the open-channel conformation by local interactions rather than by perturbing a specific site–gate communication link; and (d) it is possible to estimate directly the rate constants for agonist dissociation from and association to both the low and high affinity forms of the AChR-binding site by using a cyclic kinetic model. We conclude that the mechanism of energy transfer between the binding sites and the gate remains an open question.     

Binding of phencyclidine to the detergent solubilized acetylcholine receptor from Torpedo marmorata

The binding of phencyclidine to the acetylcholine receptor from Torpedo marmorata electroplaque was measured following solubilization of the receptor in sodium cholate followed by the exchange of cholate for Tween 80. In both the membrane-bound and solubilized AChR, the addition of cholinergic agonists simultaneously with the addition of PCP results in a 100 to 1000 fold increase in the PCP association rate and a 5 to 10 fold increase in the dissociation rate as compared to the unliganded AChR or AChR equilibrated with agonist prior to PCP addition. In addition, the number of binding sites and the pharmacological properties of the binding are not markedly changed in the soluble receptor. These results suggest that the acetylcholine receptor can undergo similar conformational transitions in the membrane-bound and the Tween 80 solubilized form and that phencyclidine can monitor these transitions in both cases.     

Action of nicotine and analogs on acetylcholine receptors having mutations of transmitter-binding site residue αG153

A primary target for nicotine is the acetylcholine receptor channel (AChR). Some of the ability of nicotine to activate differentially AChR subtypes has been traced to a transmitter-binding site amino acid that is glycine in lower affinity and lysine in higher affinity AChRs. We studied the effects of mutations of this residue (αG153) in neuromuscular AChRs activated by nicotine and eight other agonists including nornicotine and anabasine. All of the mutations increased the unliganded gating equilibrium constant. The affinity of the resting receptor (K_d) and the net binding energy from the agonist for gating (ΔG_B) were estimated by cross-concentration fitting of single-channel currents. In all but one of the agonist/mutant combinations there was a moderate decrease in K_d and essentially no change in ΔG_B. The exceptional case was nicotine plus lysine, which showed a large, >8,000-fold decrease in K_d but no change in ΔG_B. The extraordinary specificity of this combination leads us to speculate that AChRs with a lysine at position αG153 may be exposed to a nicotine-like compound in vivo.     

Conformation of acetylcholine receptor in the presence of agonists and antagonists

The conformations of acetylcholine receptor fromTorpedo californica in the absence and presence of agonists, antagonists, and local anesthetics were studied by circular dichroism (CD). Without ligands, the receptor had about 40% helix, 20% -sheets, and 10% -turns as analyzed from its far-UV CD spectrum. Its near-UV CD spectrum resembled that of acetylcholinesterase from the same source. None of the ligands studied altered the far-UV spectrum of the receptor. However, in the near-UV region, carbamylcholine and acetylcholine shifted the Phe and Tyr bands of AChR to less negative, whereas hexamethonium changed the Tyr bands to more negative, indicating that the site of binding of agonists and antagonists and their effect on the conformation of the receptor may be different. Decamethonium, procaine, and lidocaine had no effect on both the far- and near-UV CD spectra of acetylcholine receptor.