Stopped flow kinetics of carbamylcholine binding to membrane bound acetylcholine receptor

Conformational changes upon binding of carbamylcholine to acetylcholine receptor-enriched membrane fragments have been observed by stopped-flow methods using the fluorescent probe ethidium bromide. A model consistent with both equilibrium and kinetic experiments is proposed in which the receptor binds two molecules of carbamylcholine with high affinity in a non-cooperative manner followed by binding of a third and possibly a fourth molecule with increasingly lower affinity. The receptor ligand precomplexes isomerize to different non-interconvertible complexes depending on the number of ligands bound. This kinetic model fits the data for carbamylcholine interactions with receptor prepared initially either in a low or high affinity form for ligands.     

Biochemical characterization of two nicotinic receptors from the optic lobe of the chick

We have studied putative nicotinic acetylcholine receptors in the optic lobe of the newborn chick, using 125I-labeled alpha-bungarotoxin, a specific blocker of acetylcholine receptors in the neuromuscular junction, and [3H]acetylcholine, a ligand which in the presence of atropine selectively labels binding sites of nicotinic character in rat brain cortex (Schwartz et al., 1982). [3H]Acetylcholine binds reversibly to a single class of high affinity binding sites (KD = 2.2 X 10(-8) M) which occur at a tissue concentration of 5.7 pmol/g. A large fraction (approximately 60%) of these binding sites is solubilized by Triton X-100, sodium cholate, or the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Solubilization increases the affinity for acetylcholine and several nicotinic drugs from 1.5- to 7-fold. The acetylcholine-binding macromolecule resembles the receptor for alpha-bungarotoxin present in the same tissue with respect to subcellular distribution, hydrodynamic properties, lectin binding, and agonist affinity rank order. It differs from the toxin receptor in affinity for nicotinic antagonists, sensitivity to thermal inactivation, and regional distribution. The solubilized [3H]acetylcholine binding activity is separated from the toxin receptor by incubation with agarose-linked acetylcholine, by affinity chromatography on immobilized Naja naja siamensis alpha-toxin, and by precipitation with a monoclonal antibody to chick optic lobe toxin receptor.     

The binding site of acetylcholine receptor as visualized in the X-Ray structure of a complex between alpha-bungarotoxin and a mimotope peptide.

We have determined the crystal structure at 1.8 A resolution of a complex of alpha-bungarotoxin with a high affinity 13-residue peptide that is homologous to the binding region of the alpha subunit of acetylcholine receptor. The peptide fits snugly to the toxin and adopts a beta hairpin conformation. The structures of the bound peptide and the homologous loop of acetylcholine binding protein, a soluble analog of the extracellular domain of acetylcholine receptor, are remarkably similar. Their superposition indicates that the toxin wraps around the receptor binding site loop, and in addition, binds tightly at the interface of two of the receptor subunits where it inserts a finger into the ligand binding site, thus blocking access to the acetylcholine binding site and explaining its strong antagonistic activity.     

A model of cooperative rearrangements of biological membranes without changes in the affinity of receptor binding centers

A mathematical model is proposed for cooperative rearrangements induced by specific ligand in certain biomembrane domains. They are considered as the N-valent receptors undergoing rearrangement when n less than or equal to N ligand-binding receptor sites are occupied. The model predicts distinct sigmoidal dependence for change of some structural parameter on ligand concentration when the receptor site-ligand affinity remains constant as binding rises (positive cooperativity is absent).     

Uses of fluorescent cholinergic analogues to study binding sites for cholinergic ligands in Torpedo californica acetylcholine receptor.

A series of synthetic 1,n-bis(3-aminopyridinio)-alkane fluorescent probes have been used to determine the ligand binding properties of the acetylcholine receptor purified from Torpedo californica electroplax. At equilibrium, the probes bound to a single class of sites. The binding affinity of the fluorescent decamethonium analogues increased progressively as the number of methylene groups (n) increased from 4 to 12 and decreased in the range of 16--18 such groups. The receptor bound 1,12-bis(3-aminopyridinio)dodecane and 1,14-bis(3-aminopyridinio)tetradecane with the highest affinity while related monofunctional probes such as 1-(3-amino-pyridinio)propane were bound with a substantially lower affinity. The data indicate that the receptor interacts strongly with both ends of a bifunctional probe such as 1,14-bis(3-aminopyridinio)tetradecane. Also, competition between bifunctional fluorescent probe binding and the binding of conventional cholinergic ligands, was investigated and led to the conclusion that the probes, which are antagonists, form ternary complexes in the presence of acetylcholine.     

Characterization of tachykinin NK2 receptor in the anterior pituitary gland

Tachykinins are a family of bioactive peptides that interact with three subtypes of receptors: NK1, NK2 and NK3. Substance P has greater affinity for NK1, and neurokinin A (NKA) for NK2 receptor subtype. Although only NK1 receptor has been characterized in the anterior pituitary gland, some evidence suggests the existence of NK2 receptors in this gland. Therefore, we investigated the presence of NK2 receptors in the anterior pituitary gland of male rats by radioligand binding studies using labeled SR48968, a non peptidic specific antagonist. [3H]SR48968 specific binding to cultured anterior pituitary cells was time-dependent and saturable, but with a lower affinity than previously reported values for cells expressing NK2 receptors. Unlabeled NKA inhibited only partially [(3)H]SR48968 specific binding to whole anterior pituitary cells. Since SR48968 is a non polar molecule, we performed experiments to discriminate surface from intracellular binding sites. SR48968 exhibited both surface and intracellular specific binding. Analysis of the surface-bound ligand indicated that [3H]SR48968 binds to one class of receptor with high affinity. Neurokinin A completely displaced [3H]SR48968 surface specific binding fitting to a two-site/two-state model with high and low affinity. Additionally, immunocytochemical studies showed that the NK2 receptor is expressed at least in a subset of lactotropes. These results demonstrate the presence of NK2 receptors in the anterior pituitary gland and suggest that NKA actions in this gland are mediated, at least in part, by the NK2 receptor subtype.     

Dissecting the conserved NPxxY motif of the M3 muscarinic acetylcholine receptor: critical role of Asp-7.49 for receptor signaling and multiprotein complex formation

Acetylcholine challenge produces M(3) muscarinic acetylcholine receptor activation and accessory/scaffold proteins recruitment into a signalsome complex. The dynamics of such a complex is not well understood but a conserved NPxxY motif located within transmembrane 7 and juxtamembrane helix 8 of the receptor was found to modulate G protein activation. Here by means of receptor mutagenesis we unravel the role of the conserved M(3) muscarinic acetylcholine receptor NPxxY motif on ligand binding, signaling and multiprotein complex formation. Interestingly, while a N7.49D receptor mutant showed normal ligand binding properties a N7.49A mutant had reduced antagonist binding and increased affinity for carbachol. Also, besides this last mutant was able to physically couple to Gα(q/11) after carbachol challenge it was neither capable to activate phospholipase C nor phospholipase D. On the other hand, we demonstrated that the Asn-7.49 is important for the interaction between M(3)R and ARF1 and also for the formation of the ARF/Rho/β γ signaling complex, a complex that might determine the rapid activation and desensitization of PLD. Overall, these results indicate that the NPxxY motif of the M(3) muscarinic acetylcholine receptor acts as key conformational switch for receptor signaling and multiprotein complex formation.     

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.     

The binding of [3H]R-PIA to A1 adenosine receptors produces a conversion of the high- to the low-affinity state

Kinetic evidence for negative cooperativity on the binding of [3H]R-PIA to A1 adenosine receptors was obtained from dissociation experiments at different ligand concentrations and from the equilibrium isotherm. The dissociation curves indicate that there is an apparent ligand-induced transformation of high- to low-affinity states of the receptor. At concentrations of 18.2 nM R-PIA or higher there was only found the low-affinity state of the receptor. In view of these results equilibrium binding data were analyzed by the usual two-state model (assuming that there is an interconversion between them) and by the negative cooperativity model employing the Hill equation.     

Profiling the interaction of 1‐phenylbenzimidazoles to cyclooxygenases

In the design of 1‐phenylbenzimidazoles as model cyclooxygenase (COX) inhibitors, docking to a series of crystallographic COX structures was performed to evaluate their potential for high‐affinity binding and to reproduce the interaction profile of well‐known COX inhibitors. The effect of ligand‐specific induced fit on the calculations was also studied. To quantitatively compare the pattern of interactions of model compounds to the profile of several cocrystallized COX inhibitors, a geometric parameter, denominated ligand‐receptor contact distance (LRCD), was developed. The interaction profile of several model complexes showed similarity to the profile of COX complexes with inhibitors such as iodosuprofen, iodoindomethacin, diclofenac, and flurbiprofen. Shaping of high‐affinity binding sites upon ligand‐specific induced fit mostly determined both the affinity and the binding mode of the ligands in the docking calculations. The results suggest potential of 1‐phenylbenzimidazole derivatives as COX inhibitors on the basis of their predicted affinity and interaction profile to COX enzymes. The analyses also provided insights into the role of induced fit in COX enzymes. While inhibitors produce different local structural changes at the COX ligand binding site, induced fit allows inhibitors in diverse chemical classes to share characteristic interaction patterns that ensure key contacts to be achieved. Different interaction patterns may also be associated with different inhibitory mechanisms.     

CC and CX3C chemokines differentially interact with the N terminus of the human cytomegalovirus-encoded US28 receptor

Human cytomegalovirus (HCMV) is the causative agent of life-threatening systemic diseases in immunocompromised patients as well as a risk factor for vascular pathologies, like atherosclerosis, in immunocompetent individuals. HCMV encodes a G-protein-coupled receptor (GPCR), referred to as US28, that displays homology to the human chemokine receptor CCR1 and binds several chemokines of the CC family as well as the CX3C chemokine fractalkine with high affinity. Most importantly, following HCMV infection, US28 activates several intracellular pathways, either constitutively or in a chemokine-dependent manner. In this study, our goal was to understand the molecular interactions between chemokines and the HCMV-encoded US28 receptor. To achieve this goal, a double approach has been used, consisting in the analysis of both receptor and ligand mutants. This approach has led us to identify several amino acids located in the N terminus of US28 that differentially contribute to the high affinity binding of CC versus CX3C chemokines. Additionally, our results highlight the importance of secondary modifications occurring at US28, such as sulfation, for ligand recognition. Finally, the effects of chemokine dimerization and interaction with glycosaminoglycans (GAGs) on chemokine binding and activation of US28 were investigated as well using CCL4 as model ligand. In line with the two-state model describing chemokine/receptor interaction, we show that an aromatic residue in the N-loop region of CCL4 promotes tight binding to US28, whereas receptor activation depends on the presence of the N terminus of CCL4, as shown previously for CCR5.     

Effects of carboxymethylation by a purified Torpedo californica methylase on the functional properties of the acetylcholine receptor in reconstituted membranes

The functional effects of carboxymethylation of Torpedo californica acetylcholine receptor by an endogenous Torpedo methylase were examined. Both the receptor and the methylase were purified to increase the level of methylation and the sensitivity of the functional assays. The methylase catalyzed the carboxymethylation of all four receptor subunits (alpha, beta, gamma, delta) with preferential labeling of the alpha and gamma subunits. For all the reactions, S-adenosylmethionine was used as the methyl donor. Functional effects of methylation were assessed by measuring ligand binding and ligand-activated ion permeability responses in reconstituted membranes containing purified acetylcholine receptors. Methylation of receptor to a level of 20 mol% had no significant effect on agonist or antagonist binding nor did methylation affect the transition from low-to-high affinity binding triggered by agonists. In contrast, 20% methylation led to a 20% reduction in the agonist-stimulated flux of cations across the receptor-containing membranes. The results suggest that methylation inhibits the ion permeability control properties of acetylcholine receptors.     

Structural states and transitions of carp hemoglobin.

The wide ligand affinity range previously observed for carp hemoglobin is bounded at both extremes by regions of constant affinity. Within these regions, pH, organic phosphates, and the extent of ligand binding have no effect on the measured affinity and the cooperativity of ligand binding is greatly reduced or absent. The rates of CO recombination to fully and partially unliganded carp hemoglobin, under various organic phosphate and pH conditions, are shown to reflect this behavior. Constant kinetic rates are seen to directly correspond to the regions of constant affinity. Therefore, these are taken to be single protein conformations, one of high and one of low ligand affinity. In the simplest view, these conformations represent the R and T states of a two-state model, and most of the properties of carp hemoglobin are explained quite well within this framework. Increases in either hydrogen or phosphate ion concentrations favor the stabilization of the low affinity structure of even fully liganded carp hemoglobin. We have studied the structural transition from high to low affinity by monitoring the absorption spectra of carp hemoglobins at constant pH as a function of organic phosphate concentration. We find that different spectra are induced in both carp methemoglobin and cyanomethemoglobin by inositol hexaphosphate addition. Furthermore, the dependence of the magnitude of the spectral changes on pH and organic phosphate concentration is the close agreement with that predicted from studies of the ligand binding properties of the molecule.     

Partial agonism in the glucagon receptor system is a consequence of the two-state rat hepatic receptor

We have previously demonstrated that the glucagon receptor binds hormone to form a low affinity complex which, by a time- and temperature-dependent mechanism, is converted to a high affinity complex (Horwitz, E.M., Jenkins, W.T., Hoosein, N.M., and Gurd, R.S. (1985) J. Biol. Chem. 260, 9307-9315). In this report we have investigated the effects of agonist concentration, potency, and intrinsic activity on the characteristics of the two, interconvertible states of the glucagon receptor. As the glucagon concentration is increased from 0.02 to 0.50 nM, the initial velocity of binding increases. The conversion of a low affinity to a high affinity complex is the rate-limiting step in the overall binding reaction and approaches its maximal velocity as the hormone concentration exceeds 0.20 nM. At equilibrium, 87-90% of the hormone-receptor complexes are in the high affinity state at all hormone concentrations examined. [S-methyl-Met27]glucagon, a full agonist with reduced potency, binds to the two-state system in a manner analogous to that of native glucagon. The binding of N alpha-biotinyl-N epsilon-acetimidoglucagon, a partial agonist with reduced potency, effects a two-state system where the high affinity state accounts for only 35% of the total hormone-receptor complexes at equilibrium. We conclude that the formation of the high affinity complex is the rate-limiting step involved in glucagon binding; reduction in binding potency with full agonism is due to a reduction in the affinity of the ligand for the unoccupied receptor and not to an alteration of the interconversion of the two states, and decreased intrinsic activity is due to a quantitative decrease in conversion of the low to high affinity state.     

Selective photoaffinity labeling of acetylcholine receptor using a cholinergic analogue

A bisazido derivative was synthesized from bis(3-aminopyridinium)-1,10-decane diiodide and it was shown that it was bound (KD congruent to 2.2 muM) specifically to purified acetylcholine receptor and fulfilled the requirements for a photoaffinity label. Like the parent compound the derivative could transform membrane-bound receptor from a low ligand affinity conformation(s) to a high ligand affinity form (s), a transition which is thought to resemble desensitization processes observed in vivo. Photolysis of 3H-labeled bisazido reagent was carried out in the presence of the receptor. After dodecyl sulfate-polyacrylamide gel electrophoresis of labeled purified receptor two of the four subunits (mol wt 40 000 and 60 000) contained 90% of the bound radioactivity while for membrane-bound receptor the subunits of mol wt 40 000 and 50 000 were labeled. The results favor the assumption that the specific ligand binding sites are located on mol wt 40 000 subunits and labeling of the other subunits reflects (a) their proximity to the ligand-binding site and (b) alterations in subunit topography between membrane-bound and solubilized states.     

Inhibition of ionotropic neurotransmitter receptors by antagonists: strategy to estimate the association and the dissociation rate constant of antagonists with very strong affinity to the receptors.

Since binding of an agonist to an ionotropic neurotransmitter receptor causes not only channel opening, but also desensitization of the receptor, inhibition of the receptor by the antagonist sometimes becomes very complicated. The transient state kinetics of ligand association and dissociation, and desensitization of the receptor were considered on the basis of the minimal model proposed by Hess' group, and the following possibilities were proposed. 1) When an agonist is simultaneously applied to the receptor with an antagonist whose affinity to the receptor is extremely strong and different from that of the agonist, it is usually impossible to estimate the real inhibition constant exactly from the responses because desensitization of the receptor proceeds before the equilibrium of the ligand binding. Simultaneous addition of the antagonist with strong affinity to the receptor may apparently accelerate inactivation (desensitization) of the receptor. The association rate constant of the antagonist can be estimated by analyses of the rate of the inactivation in the presence and the absence of the antagonist. 2) A preincubated antagonist with a slow dissociation rate constant, i.e., a very effective inhibitor, may cause apparent noncompetitive inhibition of the receptor, since the receptor is desensitized by an agonist as soon as the antagonist dissociates from the receptor and the dissociation of the antagonist from the receptor becomes the rate-determining step. A nicotinic acetylcholine receptor (nAChR) was expressed in Xenopus oocytes by injecting mRNA prepared from Electrophorus electricus electroplax and used for the experiments on inhibition by an antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)     

Squalene-derived flexible linkers for bioactive peptides

A regiochemical and stereochemical mixture of flexible linkers bearing terminal azide functionality was synthesized in two steps from squalene and was used to connect two high affinity NDP-alpha-MSH ligands or two low affinity MSH(4) ligands. The ligands were N-terminally acylated using N-hydroxysuccinimidoyl 5-hexynoate and were subsequently attached to the linker via copper-catalyzed 'click' 3+2 cyclization of the azide and alkyne moieties. In vitro biological evaluations showed that the binding affinity to the human melanocortin 4 receptor was not diminished for most linker-ligand combinations relative to the corresponding parental ligand. Statistical and cooperative binding effects were observed for dimeric constructs containing the low affinity ligand MSH(4), but not for dimeric NDP-alpha-MSH constructs, presumably due to slow off rates for this high affinity ligand.     

Molecular alteration of a muscarinic acetylcholine receptor system during synaptogenesis

Biochemical properties of the muscarinic acetylcholine receptor system of the avian retina were found to change during the period when synapses form in ovo. Comparison of ligand binding to membranes obtained before and after synaptogenesis showed a significant increase in the affinity, but not proportion, of the high affinity agonist-binding state. There was no change in receptor sensitivity to antagonists during this period. Pirenzepine binding, which can discriminate muscarinic receptor subtypes, showed the presence of a single population of low affinity sites (M2) before and after synaptogenesis. The change in agonist binding was not due to the late development of receptor function; tests for receptor-stimulated phosphatidylinositol turnover and for modulation of agonist binding by guanylylimidodiphosphate showed functional coupling to be present several days prior to the onset of synapse formation. However, detergent-solubilization of membranes eliminated differences in agonist binding between receptors from embryos and hatched chicks, suggesting a developmental change in interactions of the receptor with functionally related membrane components. A possible basis for altered interactions was obtained from isoelectric point data showing that the muscarinic receptor population underwent a transition from a predominantly low pI form (4.25) in 13 day embryos to a predominantly high pI form (4.50) in newly hatched chicks. The possibility that biochemical changes in the muscarinic receptor play a role in differentiation of the system by controlling receptor position on the surface of nerve cells is discussed.