Studies of reversible and irreversible interactions of an alkylating agonist with Torpedo californica acetylcholine receptor in membrane-bound and purified states.

The interaction of a cholinergic depolarizing agent, bromoacetylcholine, with acetylcholine receptor (AcChR) enriched membrane fragments and Triton-solubilized, purified AcChR from Torpedo californica has been studied. The reagent bound to membrane-bound AcChR reversibly with an apparent dissociation constant of 16 +/- 1 nM at equilibrium. This 600-fold higher affinity for the receptor than found from physiological studies [Kact congruent to 10 micrometers; Karlin, A. (1973) Fed. Proc. Fed. Am. Soc. Exp. Biol. 32, 1847--1853] can be attributed to a ligand-induced affinity change of the membrane-bound receptor upon preincubation with bromoacetylcholine. At equilibrium [3H]bromoacetylcholine, like acetylcholine, bound to half the number of alpha-bungarotoxin sites present in the preparation without apparent positive cooperativity, and this binding was competitively inhibited by acetylcholine. In the presence of dithiothreitol, [3H]bromoacetylcholine irreversibly alkylated both membrane-bound and solubilized, purified acetylcholine receptor, with a stoichiometry identical with that for reversible binding. NaDodSO4-polyacrylamide gel electrophoresis of the labeled acetylcholine receptor showed that only the 40 000-dalton subunit contained the label. From these results it is concluded that the 40 000-dalton subunit represents a major component of the agonist binding site of the receptor.     

Nonequivalence of alpha-bungarotoxin binding sites in the native nicotinic receptor molecule

In the native, membrane-bound form of the nicotinic acetylcholine receptor (M-AcChR) the two sites for the cholinergic antagonist alpha-bungarotoxin (alpha-BGT) have different binding properties. One site has high affinity, and the M-AcChR/alpha-BGT complexes thus formed dissociate very slowly, similar to the complexes formed with detergent-solubilized AcChR (S-AcChR). The second site has much lower affinity (KD approximately 59 +/- 35 nM) and forms quickly reversible complexes. The nondenaturing detergent Triton X-100 is known to solubilize the AcChR in a form unable, upon binding of cholinergic ligands, to open the ion channel and to become desensitized. Solubilization of the AcChR in Triton X-100 affects the binding properties of this second site and converts it to a high-affinity, slowly reversible site. Prolonged incubation of M-AcChR at 4 degrees C converts the low-affinity site to a high-affinity site similar to those observed in the presence of Triton X-100. Although the two sites have similar properties when the AcChR is solubilized in Triton X-100, their nonequivalence can be demonstrated by the effect on alpha-BGT binding of concanavalin A, which strongly reduces the association rate of one site only. The Bmax of alpha-BGT to either Triton-solubilized AcChR or M-AcChR is not affected by the presence of concanavalin A. Occupancy of the high-affinity, slowly reversible site in M-AcChR inhibits the Triton X-100 induced conversion to irreversibility of the second site. At difference with alpha-BGT, the long alpha-neurotoxin from Naja naja siamensis venom (alpha-NTX) binds with high affinity and in a very slowly reversible fashion to two sites in the M-AcChR (Conti-Tronconi & Raftery, 1986). We confirm here that Triton-solubilized AcChR or M-AcChR binds in a very slowly reversible fashion the same amount of alpha-NTX.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of thio-group modifications of Torpedo californica acetylcholine receptor on ion flux activation and inactivation kinetics

The effects of thio-group modifications on the ion permeability control and ligand binding properties of the acetylcholine receptor were measured in reconstituted membranes prepared from purified Torpedo californica acetylcholine receptor and soybean lipids (asolectin). A quench flow device was used to obtain subsecond time resolution for agonist-stimulated cation influx using carbamylcholine chloride (Carb) as the ligand and 86Rb+ as the cation. The effects of disulfide reduction with dithiothreitol (DTT), affinity alkylation with [4-(N-maleimido)benzyl]trimethylammonium ion and bromoacetylcholine, and nonspecific alkylation with N-ethylmaleimide and N-benzylmaleimide were examined. Activation, fast inactivation, and slow inactivation rates were measured on the chemically modified membranes. The flux results were compared with similar measurements on native membranes, and the role of vesicle size, heterogeneity, and influx time on ion flux results was analyzed. Major conclusions are that the binding sites that react with affinity labels are the same sites that mediate ligand-activated ion flux and that blockade of one of the two ligand binding sites is sufficient to block about 95% of the ion flux response. The main effect of DTT reduction is to shift the EC50 values for activation and slow inactivation to higher Carb concentrations, consistent with a decrease in binding affinity for Carb. The EC50 value for fast inactivation was not affected by DTT. However, the maximum rate of ion flux activation and the maximum rate of fast inactivation were decreased 2-fold after DTT treatment.     

Affinity-dependent cross-linking to neurotoxin sites of the acetylcholine receptor mediated by catechol oxidation

The choline homologue 3-[(trimethylammonio)methyl]catechol (TMC) has been synthesized, and the controllable features of its complex oxidation have been examined spectroscopically and correlated with its toxin binding inactivating reactions with the acetylcholine receptor (AcChR) from Torpedo californica electroplax. Affinity-dependent reactions of early intermediates in the oxidation of TMC are suggested to intercede covalently in this inactivation. At pH 7.4, where the oxidative polymerization of catechols proceeds spontaneously, pyrocatechol produced no effect on the toxin binding function of AcChR, whereas comparable concentrations of TMC led to inactivation of half of all available sites. Lower concentrations of TMC converted via oxidation with ceric salts to an in situ mixture of monohydroxylated catechols were shown to be effective in short-term incubations in inactivating approximately half of the toxin binding sites by covalent labeling of the receptor. Mixtures of dihydroxycatechol intermediates, hydroxy-p-quinones, and polymeric products led to nonspecific toxin binding site inactivation of AcChR in excess of half of all available sites. Collectively, the results suggest that both covalent labeling and oxygen reduction product inactivating mechanisms are operative in these model macromolecular site reactions and that catechol-containing affinity reagents may be useful in elucidating the molecular features of sites to which they are directed.     

Ligand-induced variations in the reactivity of thio groups of the alpha-subunit of the acetylcholine receptor from Torpedo californica

We have studied alkylation of the acetylcholine receptor by N-[3H]ethylmaleimide ([3H]NEM) under various conditions. The radiolabeled preparations were submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis to separate the receptor complex into subunits, and the incorporation of 3H into each type of chain was determined. We found the following: (i) When cysteines of native receptor in intact membranes were reacted with [3H]NEM, only the beta-subunit was labeled; the extent of alkylation did not change significantly if cholinergic effectors were present during this reaction. (ii) When the disulfide bonds of the receptor were reduced with dithiothreitol (DTT), the alpha- and beta-chains were labeled with [3H]NEM. The presence of receptor agonists and competitive antagonists during alkylation significantly altered the labeling patterns. Gallamine and hexamethonium markedly enhanced, while carbamylcholine and decamethonium markedly lessened, labeling of the alpha-subunit. Choline, d-tubocurarine, and alpha-neurotoxin induced small, but significant decreases in alkylation of the alpha-subunit, while procaine had no effect. (iii) When the same ligands were present during the reduction step, subsequent labeling with [3H]NEM produced patterns similar to those described in (ii). We also investigated the effects of gallamine and hexamethonium on reduction of the disulfide bond located near the acetylcholine binding site by using the affinity alkylating reagent (bromoacetyl)choline (BAC). Gallamine (0.1 mM) was able to increase the rate of reduction of this particular disulfide bond 3-fold in comparison to the control. In these experiments, alkylation by BAC blocked 50% of the toxin binding sites. Hexamethonium (1 mM) had a similar effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pyrenesulfonyl azide as a fluorescent label for the study of protein-lipid boundaries of acetylcholine receptors in membranes

Acetylcholine receptor (AcChR) enriched membrane fragments from Torpedo californica electroplax were labeled by in situ photogenerated nitrenes from a hydrophobic fluorescent probe, pyrene-1-sulfonyl azide. Preferential photolabeling of membrane proteins, mainly AcChR, has been achieved and there is a pronounced exposure of the 48,000 and 55,000 molecular weight subunits of AcChR to the lipid environment of the membrane core. Covalent attachment of the photogenerated fluorescence probe does not perturb the α-neurotoxins' binding properties of membrane-bound AcChR or the desensitization kinetics induced by prolonged exposures to cholinergic agonists. Non-covalent photoproducts can be conveniently removed from labeled membrane preparations by exchange into lipid vesicles prepared from electroplax membrane lipids. Fluorescence features of model pyrene sulfonyl amide derivatives, such as fine vibrational structure of emission spectra or fluorescence lifetimes, are highly sensitive to the solvent milieu. The covalently bound probe shows similar fluorescence properties in situ. PySA photoproducts have great potential to spectroscopically monitor neurotransmitter induced events on selected AcChR subunits exposed to the hydrophobic environment of membranes.     

Effects of thio-group modification and Ca2+ on agonist-specific state transitions of a central nicotinic acetylcholine receptor.

Agonist-binding affinities of central nervous system nicotinic acetylcholine receptors (nAcChR) are sensitive to the duration of exposure to agonist. These agonist-induced changes in receptor state may be mimicked by appropriate modification of receptor thio groups and/or by manipulation of solvent ionic composition. In the absence of Ca2+, the concentration of acetylcholine (AcCh) necessary to prevent half of specific 3H-labeled alpha-bungarotoxin binding is approximately 1 mM for nAcChR treated with dithiothreitol (DTT) or DTT-N-ethylmaleimide (low-affinity states) and approximately 40 microM for nAcChR treated with DTT-5,5'-dithiobis(2-nitrobenzoic acid) or for native nAcChR pretreated with AcCh (high-affinity states). Addition of Ca2+ results in an increase in the effectiveness of AcCh toward blocking toxin binding. None of these treatments alters toxin or antagonist binding nor are there observed differences in Hill numbers for agonist binding. Agonists competitively inhibit toxin binding to low-affinity states, but noncompetitive inhibition is observed for binding to high-affinity states. Values of AcCh dissociation constants estimated from these data fall within the range of values determined physiologically with nAcChR from other systems. The data indicate that the redox state of brain nAcChR thio groups and Ca2+ may mediate physiologically important changes in the receptor state during activation and desensitization.     

Binding of anti-acetylcholine receptor antibodies inhibits the acetylcholine receptor mediated cation flux

A fast kinetics, spectroscopic technique has been applied to the study of the transient cation flux associated to the binding of cholinergic agonist to native acetylcholine receptor (AcChR)-rich membrane vesicles in presence of anti-AcChR antibodies. The technique is based on the collisional quenching of an intravesicularly trapped fluorophore by externally added T1+ which substitutes for physiologically occurring cations. Presence of polyclonal Fab fragments from goat anti-AcChR antibodies bound to the membrane AcChR promotes a 80-90% inhibition on the observed rate constants of T1+ influx. The observed inhibition process appears to follow a non-competitive pattern between antibody and cholinergic ligand binding, suggesting that in the AcChR protein the antigenic sites responsible for ion translocation may be other than those involved in ligand binding.     

Monoclonal antibodies as probes of the alpha-bungarotoxin and cholinergic binding regions of the acetylcholine receptor

We have probed the acetylcholine receptor (AcChR) molecule with six anti-AcChR monoclonal antibodies (mAbs) whose binding to the AcChR is inhibited or blocked by alpha-bungarotoxin (alpha BgTx). mAbs bound with a maximum stoichiometry of either one mAb (387D, 247G) or two mAbs (383C, 572C, 370C, 249E) per AcChR monomer, and the extent to which they inhibited alpha BgTx binding directly correlated with their stoichiometry of binding. The effect of mAbs on the alpha BgTx and cholinergic ligand binding properties of the AcChR molecule defined three major categories of mAbs: those that block alpha BgTx and carbamylcholine (agonist) binding, but do not block d-tubocurarine (antagonist) binding (383C, 572C, 370C and 249E); mAb 387D, which blocks agonist binding and partially blocks alpha BgTx and d-tubocurarine binding; and mAb 247G, which does not affect agonist binding, blocks at most 50% of the alpha BgTx binding sites, and decreases the affinity of the high affinity component of d-tubocurarine binding (Mihovilovic, M., and Richman, D. P. (1984) J. Biol. Chem. 259, 15051-15059). Except for mAb 247G, these mAbs strongly competed with each other for binding to the AcChR. In contrast, mAb 247G blocks about 50% of the binding of all the other mAbs. The results demonstrate the ability of mAbs to stabilize different conformational states of the AcChR and to probe cholinergic epitopes of functional importance. They also indicate the nonequivalence of the two alpha-toxin binding regions of the AcChR molecule and suggest that it is possible to identify epitopes within the alpha BgTx binding region that when bound produce differential effects on the binding of the agonist (carbamylcholine) and the antagonist (d-tubocurarine).     

Covalent labeling of the acetylcholine receptor from Torpedo electric tissue with the channel blocker [3H]triphenylmethylphosphonium by ultraviolet irradiation

The lipophilic cation [3H]triphenylmethylphosphonium, frequently used as a voltage sensor in membrane systems, binds reversibly to a site different from the acetylcholine binding site. This is concluded from the different pH dependences of the binding of these two ligands. Furthermore [3H]triphenylmethylphosphonium, previously identified as a channel blocker, can be covalently incorporated into acetylcholine receptor-rich membranes from Torpedo electric tissue by UV irradiation of the receptor-ligand complex. In the absence of effector, predominantly the alpha-polypeptide chains (Mr 40000) of the receptor protein are labeled by the radioactive ligand. The agonist carbamoylcholine strongly stimulates the labeling, but it directs the label predominantly to the delta- and beta-polypeptide chains. The antagonist D-tubocurarine and the virtually irreversible competitive antagonist alpha-bungarotoxin have qualitatively the same effect as the agonist carbamoylcholine. Significant differences were obtained with receptor-rich membranes prepared from Torpedo marmorata and Torpedo californica: No agonist- or antagonist-stimulated reaction was observed with the latter. The results are interpreted as an indication of a rearrangement of the receptor's quaternary structure caused by cholinergic effector binding preceding discrimination between agonists and antagonists.     

Binding studies of Mn+2 to isolated acetylcholine receptor as a probe for cation-receptor interaction.

The paramagnetic cation Mn⁺² binds to $\text{Torpedo californica}$ acetylcholine receptor (AcChR) at sites with at least two different affinity constants. For each α-Bungarotoxin (α-Bgt) binding site AcChR has between 3 to 4 Mn⁺² sites with Kd values of 1.74 ± 1.0 × 10^?4 M. An additional 10–12 sites/α-Bgt site have a weaker affinity for Mn⁺² (Kd ? 1 mM). The α-Bgt does not displace bound Mn⁺², however Ca⁺² displaces all bound Mn⁺² in a competitive fashion with Kd of 0.90 × 10^?3 M and Mg⁺² is as effective as Ca⁺² in the displacement. Decamethonium, carbamylcholine and NaCl at high concentrations are also effective in displacing Mn⁺². A constant enhancement value (?_b) for the binary metal · AcChR complexes was obtained when simultaneous EPR measurements and the water proton relaxation rates were made. Similarity of the AcChR environment and/or coordination number for the Mn⁺² sites in AcChR is inferred. It appears that Mn⁺² binds to many AcChR sites, different from those responsible for binding cholinergic ligands. The Mn⁺² site seem to be the same as those responsible for binding the electrophysiologically significant Ca⁺².     

Phosphatidylcholine Biosynthesis in the Neuroblastoma-Glioma Hybrid Cell Line NG 108-15: Stimulation by Phorbol Esters

Studies were conducted on curaremimetic neurotoxin binding to the nicotinic acetylcholine receptor present on membrane fractions derived from the human medulloblastoma clonal line, TE671. High-affinity binding sites (KD = 2 nM for 1-h incubation at 20 degrees C) and low-affinity binding sites (KD = 40 nM) for 125I-labeled alpha-bungarotoxin are present in equal quantities (60 fmol/mg membrane protein). The kinetically determined dissociation constant for high-affinity binding of toxin is 0.56 nM (k1 = 6.3 X 10(-3) min-1 nM-1; k-1 = 3.5 X 10(-3) min-1) at 20 degrees C. Nicotine, d-tubocurarine, and acetylcholine are among the most effective inhibitors of high-affinity toxin binding. The quantity of toxin binding sites and their affinity for cholinergic agonists is sensitive to reduction, alkylation, and/or oxidation of membrane sulfhydryl residues. High-affinity toxin binding sites that have been subjected to reaction with the sulfhydryl reagent dithiothreitol are irreversibly blocked by the nicotinic receptor affinity reagent bromoacetylcholine. High-affinity toxin binding is inhibited in the presence of either of two polyclonal antisera or a monoclonal antibody raised against nicotinic acetylcholine receptors from fish electric tissue. Taken together, these results indicate that curaremimetic neurotoxin binding sites on membrane fractions of the TE671 cell line share some properties with nicotinic acetylcholine receptors of peripheral origin and with toxin binding sites on other neuronal tissues.     

Two binding sites in acetylcholine receptor from Torpedo marmorata electroplax

The sensitivity of acetylcholine receptor to eleven cholinergic drugs, phospholipase A, heat and pH provided evidence that the so-called high-affinity binding (K_d for acetylcholine 11 nm in 1% Triton) and low-affinity binding (K_d 562 nm) were related to two distinct binding sites. The low-affinity binding site was less sensitive to heat and several of the cholinergic drugs, but was a little more sensitive to bungarotoxin than the high-affinity site. Zinc (0.4 mm) and EDTA (10 mm) abolished acetylcholine binding to both sites; the EDTA inhibition was time-dependent.     

Equilibrium binding of [3H]tubocurarine and [3H]acetylcholine by Torpedo postsynaptic membranes: stoichiometry and ligand interactions

Studies are presented of the equilibrium binding of [3H]-d-tubocurarine (dTC) and [3H]acetylcholine (AcCh) to Torpedo postsynaptic membranes. The saturable binding of [3H]dTC is characterized by two affinities: Kd1 = 33 +/- 6 nM and Kd2 = 7.7 +/- 4.6 microM, with equal numbers of binding sites. Both components are completely inhibited by pretreatment with excess alpha-bungarotoxin or 100 microM nonradioactive dTC and competitively inhibited by carbamylcholine with a KI = 100 nM, but not affected by the local anesthetics dimethisoquin, proadifen, and meproadifen. The biphasic nature of [3H]dTC binding was unaltered in solutions of low ionic strength and by preparation of Torpedo membranes in the presence of N-ethylmaleimide, a treatment which yields dimeric AcCJ receptors. dTC competitively inhibits the binding of [3H]AcCH and decreases the fluorescence of 1-(5-dimethylaminonaphthalene-1-sulfonamido)ethane-2-trimethylammonium (Dns-Chol) in a manner quantitatively consistent with its directly measured binding properties. It decreases the initial rate of 3H-labeled Naja nigricollis alpha-toxin binding by 50% at 60 nM with an apparent Hill coefficient of 0.58. The stoichiometry of total dTC, AcCh, and alpha-neurotoxin binding sites in Torpedo membranes was determined by radiochemical techniques and by a novel fluorescence assay utilizing Dns-Chol as an indicator, yielding ratios of 0.9 +/- 0.1:0.9 +/- 0.2:1, respectively. The biphasic equilibrium binding function is not unique to dTC since other ligands inhibited [3h]acCh binding in a biphasic manner with apparent inhibition constants as follows: gallamine triethiodide (K11 = 2 microM, K12 = 1 mM); Me2dTC (K11 = 500 nM, K12 = 10 microM); decamethonium (K11 = 100 nM, K12 = 1.6 microM). Carbamylcholine, however, inhibited [3H]AcCh binding with a single KI = 100 nM. The observed competition between those ligands and [3H] AcCh cannot be completely accounted for by competitive interaction with two different affinities, and the deviations are discussed in terms of the positive cooperativity of the [3H] AcCh binding function itself. It is concluded that dTC binds only to the AcCh sites in Torpedo membranes and that those sites display two affinities for dTC but only one for AcCh.     

Synthesis and characterization of a heterobifunctional mercurial cross-linking agent: incorporation into cobratoxin and interaction with the nicotinic acetylcholine receptor

The heterobifunctional organomercurial reagents 3-(acetoxymercurio)- and 3-(chloromercurio)-5-nitrosalicylaldehyde were prepared, characterized in model studies, and used to probe the interaction between cobratoxin, purified from the venom of the Thailand cobra (Naja naja siamensis), and the affinity-purified nicotinic acetylcholine receptor (AcChR) from Torpedo california electroplax. These reagents may also be useful in introducing chemically well-defined heavy metal atoms into proteins containing no reactive thiols. Model reagent adducts were prepared in situ by reductive amination with N-butylamine and N alpha-acetyllysine-N-methylamide. The nitrophenolic pKaS of the amine adducts were similar to those of the aldehyde reagents through reduced by 1.3-1.5 units when compared with the hydroxylmethyl reduction product. Reaction of either mercuriosalicylaldehyde with cobratoxin led to a single major modification product incorporating 1 mol of the reagent into cobratoxin at Lys 23. The Lys 23 modified toxin had a reduced binding affinity for the AcChR over that of the native toxin (Kd 2.75 nM cf. 0.3 nM). Reduction of the purified AcChR with 1 mM dithiothreitol (DTT) followed by removal of excess thiol led to cross-linking reactions with the Lys 23 modified cobratoxin to both the alpha and beta subunits of the AcChR complex. Reaction of DTT-treated AcChR with N-ethylmaleimide (NEM) blocked cross-linking, while treatment of the initially cross-linked toxin-AcChR complex with mercaptoethanol leads to reversal of cross-linking. These observations strongly support cross-linking mediated by the formation of a mercury-sulfur bond and further lend support the identity of the respective interacting sites in AcChR and toxin.     

Thermal perturbation studies of membrane-bound acetylcholine receptor from Torpedo: effects of cholinergic ligands and membrane perturbants

Thermal perturbation techniques have been used to probe structural features of the nicotinic acetylcholine receptor (AcChR). The information obtained from differential scanning calorimetry (DSC) of AcChR membranes (M.C. Farach and M. Martinez-Carrion (1983) J. Biol. Chem. 258, 4176) in the absence and in the presence of cholinergic ligands and local anesthetics, is comparable to that obtained from a simpler technique of heat inactivation of the alpha-bungarotoxin (alpha-Bgt) binding sites on the AcChR protein in similar samples. When AcChR membranes are heated at approximately 1 degree C/min, heat inactivation of toxin binding sites has a characteristic T50 value (temperature at which 50% of the initial capacity to bind alpha-Bgt remains) of approximately 60 degrees C. When heated at a constant temperature during increasing periods of time, the rate at which heat inactivation occurs is also characteristic of the temperature chosen for the experiment. The above thermal parameters are also sensitive to perturbation of the AcChR membrane matrix by the presence of subsolubilizing concentrations of detergents. Moreover, elimination of detergents by dialysis allows us to evaluate the reversibility or irreversibility of AcChR thermal destabilization induced by detergents or other membrane perturbants. Under the experimental conditions used, structural destabilization induced by octylglucoside or cholate can be fully reversed by detergent dialysis, while that exerted by deoxycholate cannot. "Thermal gel" analysis of the aggregation of AcChR subunits induced by heat (G. Soler, J. R. Mattingly, and M. Martinez-Carrion (1984) Biochemistry 23, 4630) has also been used to assess the effects of detergent presence on the AcChR protein. When deoxycholate is used as the perturbing agent, there is a particularly effective sulfhydryl-mediated aggregation of the gamma-delta subunit group, which appears to correlate with the irreversible destabilization of alpha-Bgt binding sites induced by that detergent.     

14 beta-(Bromoacetamido)morphine irreversibly labels mu opioid receptors in rat brain membranes

The binding properties of 14 beta-(bromoacetamido)morphine (BAM) and the ability of BAM to irreversibly inhibit opioid binding to rat brain membranes were examined to characterize the affinity and selectivity of BAM as an irreversible affinity ligand for opioid receptors. BAM had the same receptor selectivity as morphine, with a 3-5-fold decrease in affinity for the different types of opioid receptors. When brain membranes were incubated with BAM, followed by extensive washing, opioid binding was restored to control levels. However, when membranes were incubated with dithiothreitol (DTT), followed by BAM, and subsequently washed, 90% of the 0.25 nM [3H] [D-Ala2,(Me)Phe4,Gly(ol)5]enkephalin (DAGO) binding was irreversibly inhibited as a result of the specific alkylation of a sulfhydryl group at the mu binding site. This inhibition was dependent on the concentrations of both DTT and BAM. The mu receptor specificity of BAM alkylation was demonstrated by the ability of BAM alkylated membranes to still bind the delta-selective peptide [3H] [D-penicillamine2,D-penicillamine5]enkephalin (DPDPE) and (-)-[3H]bremazocine in the presence of mu and delta blockers, selective for kappa binding sites. Under conditions where 90% of the 0.25 nM [3H]DAGO binding sites were blocked, 80% of the 0.8 nM [3H]naloxone binding and 50% of the 0.25 nM 125I-labeled beta h-endorphin binding were inhibited by BAM alkylation. Morphine and naloxone partially protected the binding site from alkylation with BAM, while ligands that did not bind to the mu site did not afford protection.2+hese studies have demonstrated that when a disulfide bond     

Photoaffinity labeling of the acetylcholine transporter.

The acetylcholine (AcCh) binding site in the AcCh transporter-vesamicol receptor (AcChT-VR) present in synaptic vesicles isolated from the electric organ of Torpedo was characterized. A high-affinity analogue of AcCh containing an aryl azido group, namely, cyclohexylmethyl cis-N-(4-azidophenacyl)-N-methylisonipecotate bromide (AzidoAcCh), was synthesized in nonradioactive and highly tritiated forms. AzidoAcCh was shown to be a competitive inhibitor of [3H]AcCh active transport and binding of [3H]-vesamicol to the allosteric site. The [3H]AzidoAcCh saturation curve was determined. In all cases the AcChT.AzidoAcCh complex exhibited an inhibition or dissociation constant of about 0.3 microM. Binding of [3H]AzidoAcCh was inhibited by vesamicol and AcCh. AzidoAcCh irreversibly blocked greater than 90% of the [3H]vesamicol binding sites after multiple rounds of photolysis and reequilibration with fresh ligand. Autofluorographs of synaptic vesicles photoaffinity-labeled with [3H]AzidoAcCh showed specific labeling of material exhibiting a continuous distribution from 50 to 250 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The result demonstrates that the AcChT has an unexpected structure highly suggestive of the synaptic vesicle proteoglycan.     

Ligand-induced changes in membrane-bound acetylcholine receptor observed by ethidium fluorescence. 1. Equilibrium studies.

The interactions between the fluorescent probe ethidium and acetylcholine receptor enriched membranes from Torpedo californica are described. One class of saturable ethidium sites was blocked by alpha-bungarotoxin and therefore reflects direct binding to the receptor (Kd approximately 3 micrometers; stoichiometry--one ethidium site per two alpha-bungarotoxin sites). The second class of sites was nonsaturable and unaffected by alpha-toxin and was therefore considered nonspecific in nature. The increase in fluorescence intensity observed upon addition of cholinergic agonists and antagonists accurately reflects the dissociation constant and stoichiometry of the high-affinity receptor sites for these ligands. The effects of local anaesthetics are complex in nature and depend on the structure of the ligand. For carbamylcholine, the increase in flourescence intensity was due to an increase in the quantum yield of the dye bound to the membrane rather than a dye uptake. In general, ethidium appears not to strongly alter the properties of the membrane-bound acetylcholine receptor and can therefore be profitably used as a spectroscopic probe.     

Alpha-conotoxins

alpha-Conotoxins (alpha-CgTxs) are a family of Cys-enriched peptides found in several marine snails from the genus Conus. These small peptides behave pharmacologically as competitive antagonists of the nicotinic acetylcholine receptor (AChR). The data indicate that (1) alpha-CgTxs are able to discriminate between muscle- and neuronal-type AChRs and even among distinct AChR subtypes; (2) the binding sites for alpha-CgTxs are located, like other cholinergic ligands, at the interface of alpha and non-alpha subunits (gamma, delta, and epsilon for the muscle-type AChR, and beta for several neuronal-type AChRs); (3) some alpha-CgTxs differentiate the high- from the low-affinity binding site found on either alpha/non-alpha subunit interface; and that (4) specific residues in the cholinergic binding site are energetically coupled with their corresponding pairs in the toxin stabilizing the alpha-CgTx-AChR complex. The alpha-CgTxs have proven to be excellent probes for studying the structure and function of the AChR family.