Muscarinic Ligands Research Articles

Constitutive muscarinic receptors are involved in the growth and differentiation of friend erythroleukemia cells.

Binding experiments with the specific muscarinic ligand [3H]N-methylscopolamine (3H-NMS) have shown the presence of constitutive muscarinic acetylcholine receptors (mAChR) on Friend murine erythroleukemia cells (MELC). Competition experiments with a panel of specific antagonists indicated that the mAChR were predominantly of the M3 subtype. This was confirmed by the rt-PCR analysis of mRNA levels for M1-M5 AChR. Uninduced MELC expressed approximately 2,100 and 1,200 binding sites per cell of growing and resting populations, respectively. The dissociation constant (K(D)) for 3H-NMS was in the picomolar range. The modulation of mAChR upon induction suggested that MELC growth and maturation might be under control of a cholinergic system since mAChR were markedly decreased or virtually absent in MELC induced to terminal division by dimethyl sulfoxide (DMSO) or hexamethylene bisacetamide (HMBA), respectively. In turn, the number of mAChR on MELC committed to polyploidization by colcemid was either increased over or maintained at the control levels when receptor densities were expressed per cell or surface unit (square micrometers), respectively. Moreover, the muscarinic agonist carbachol was found to inhibit MELC differentiation by decreasing by approximately 35% the amount of benzidine-positive (B+) cells in HMBA-induced cultures and, to a lesser degree, also AChE levels. The carbachol effect on erythroid differentiation was reverted by atropine that was found to restore the original amount of B+ cells, while it reduced acetylcholinesterase (AChE) to levels of approximately 66% of control. Such a selective atropine-mediated inhibition of AChE expression was observed also in HMBA-induced MELC supplemented with the antagonist. These results have suggested that mAChR on MELC are functional and might play a role in modulating the expression of either the erythroid or megakaryocytic traits of these cells.

A quantitation of stereoselectivity by the Free-Wilson method

The stereoselectivity of muscarinic receptors was quantified by the Free-Wilson method. The analysis was performed with the affinity data, published by Gualtieri for certain muscarinic ligands (1,3-oxathiolanes) in three different tissues. The 2 and 3 positions in the 1,3-oxathiolane ring were important to differentiate between the receptors in those tissues. The results also showed that the three chiral centers in the 1,3-oxathiolanes displayed different stereoselective discrimination. The results of the Free-Wilson analysis were compared with those of a previous study on the same compounds obtained by epimeric eudismic analysis. The agreements and differences between the results obtained by these two methods are discussed in depth. Chirality 11:139–143, 1999. © 1999 Wiley-Liss, Inc.

Muscarinic agonists exhibit functional dopamine antagonism in unilaterally 6-OHDA lesioned rats.

(5R,6R) 6-(3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[3.2.1]oc tane (PTAC) is a selective muscarinic ligand with high affinity for central muscarinic receptors, agonist mode of action at the muscarinic M2 and M4 receptor subtypes and substantially less or no affinity for central dopamine receptors. In the present study PTAC, as well as the muscarinic agonists oxotremorine, RS86 and pilocarpine, inhibited dopamine D1 and D2 receptor agonist induced contralateral rotation in unilaterally 6-OHDA lesioned rats. The dose of SKF 38393 used to induce contralateral rotation also caused an intense Fos protein immunoreactivity in the rat dorsolateral striatum on the lesioned site which was inhibited by PTAC indicating that the inhibition of rotation by PTAC was not due to non-specific peripheral side effects.

Universal template approach to drug design: polyamines as selective muscarinic receptor antagonists.

The concept that polyamines may represent a universal template in the receptor recognition process is embodied in the design of new selective muscarinic ligands. Tetraamines 4-7 and 16-20 and diamine diamides 8-15 were synthesized, and their pharmacological profiles at muscarinic receptor subtypes were assessed by functional experiments in isolated guinea pig left atrium (M2) and ileum (M3) and by binding assays in rat cortex (M1), heart (M2), submaxillary gland (M3), and NG 108-15 cells (M4). It has been confirmed that appropriate substituents on the terminal nitrogens of a tetraamine template can tune both affinity and selectivity for muscarinic receptors. The novel tetraamine C-tripitramine (17) was able to discriminate significantly M1 and M2 receptors versus the other subtypes, and in addition it was 100-fold more lipophilic than the lead compound tripitramine. Compound 14 (tripinamide), in which the tetraamine backbone was transformed into a diamine diamide one, retained high affinity for muscarinic subtypes, displaying a binding affinity profile (M2 > M1 > M4 > M3) qualitatively similar to that of tripitramine. Both these ligands, owing to their improved lipophilicity relative to tripitramine and methoctramine, could serve as tools in investigating cholinergic functions in the central nervous system. Furthermore, notwithstanding the fact that the highest affinity was always associated with muscarinic M2 receptors, for the first time polyamines were shown to display high pA2 values also toward muscarinic M3 receptors.

Antagonist binding profile of the split chimeric muscarinic m2-trunc/m3-tail receptor

Recent evidence suggests that G-protein-coupled receptors can behave as multiple subunit receptors, and can be split into parts, maintaining their binding ability. Transfection of a truncated muscarinic m2 receptor (containing transmembrane domains I–V, named m2-trunc) with a gene fragment coding for the carboxyl-terminal receptor portion of the muscarinic m3 receptor (containing transmembrane domains VI and VII, named m3-tail) results in the formation of a binding site with a high affinity for the muscarinic ligand N-[ 3 H ]methylscopolamine. In this paper we analyse the antagonist binding profile of this chimeric m2-trunc/m3-tail receptor in comparison with the wild-type muscarinic m2 and m3 receptors. While many of the substances tested had an intermediate affinity for the chimeric m2-trunc/m3-tail receptor compared with m2 and m3, some compounds were able to distinguish between the chimeric m2-trunc/m3-tail receptor on the one hand and the m2 or the m3 receptor on the other. Among them, tripitramine (a high-affinity M 2 receptor antagonist) bound to the m2-trunc/m3-tail receptor with the same affinity as m2, but it bound to the m3 receptor with a 103-fold lower affinity; pirenzepine (a selective muscarinic M 1 receptor antagonist) bound to the chimeric receptor with an affinity that was 12- and 3-fold higher than that of m2 and m3, respectively. The results of this study demonstrate that the chimeric m2-trunc/m3-tail receptor has a pharmacological profile distinct from that of the originating muscarinic m2 and m3 receptors.

The relationship between agonist intrinsic activity and the rate of endocytosis of muscarinic receptors in a human neuroblastoma cell line.

The molecular mechanisms underlying the internalization of G protein-coupled receptors are still poorly understood. Normally agonists but not antagonists cause internalization (defined here as a reduction in the number of receptors at the cell surface), suggesting a functional relationship between agonist activity and internalization. In this study we investigated the effects of eight muscarinic ligands on the rate constants for endocytosis and recycling of m3 muscarinic acetylcholine receptors in human SH-SY5Y neuroblastoma cells. We found that there was a linear correlation between the intrinsic activity of the ligand and its ability to increase the rate constant for endocytosis, suggesting that the same active conformation of the receptor is responsible for stimulating both second messenger generation and receptor endocytosis. In contrast, the rate constant for recycling did not depend on which agonist had triggered receptor endocytosis, suggesting that recycling is a purely constitutive process. Because receptor internalization depends on the rate constants for both endocytosis and recycling, the relationship between internalization and intrinsic activity is nonlinear. In particular, mathematical modeling of receptor trafficking revealed that under certain conditions very small (3% or less) increases in the rate constant for endocytosis are sufficient to cause substantial receptor internalization. An important implication of this analysis is that extremely weak partial agonists (which may in practice be indistinguishable from antagonists) may produce significant receptor internalization.

Identification and localization of muscarinic acetylcholine receptors in the ocular tissues of the chick

The purpose of this study was to characterize the distribution of muscarinic acetylcholine receptors (mAChRs) in the ocular tissues of hatched chicks. In the chick, different isoforms of these receptors have been detected in the brain, heart, and retina, and mAChRs in ocular tissues have been implicated in the pathogenesis of form-deprivation myopia. However, the precise anatomical distribution of mAChRs within the retina, retinal pigment epithelium, choroid, ciliary body, and ciliary ganglion remains unknown. We used affinity-purified, type-specific antibodies directed to three different chick mAChR subtypes (cm2, cm3, and cm4) to detect receptor immunoreactivity in sections and extracts of these ocular tissues. We found cm2, cm3, and cm4 in the retina, retinal pigment epithelium, choroid, and ciliary body. Within the retina, cm2 was expressed in numerous amacrine and ganglion cells; cm3 was expressed in many bipolar cells and small subsets of amacrine cells; and cm4 was found in most, if not all, amacrine and ganglion cells. Each mAChR was localized to distinct strata within the inner plexiform layer that cumulatively form three broad bands that closely match previously described localizations of subtype-nonspecific muscarinic ligand binding. Only cm3 was detected in the outer plexiform layer, and only cm4 was detected in the ciliary ganglion. We propose that each mAChR subtype has unique functions in each ocular tissue.

Effects of nicotinic and muscarinic ligands on embryonic neurones of Periplaneta americana in primary culture: a whole cell clamp study

The pharmacological properties of acetylcholine (ACh) receptors of cultured neurones from embryonic cockroach brains were studied using the whole-cell configuration of the patch-clamp technique. More than 90% of the studied neurones responded to ACh by a monophasic inward current, the intensity of which varied from cell to cell. The sequence of potency of the five tested agonists was ACh > nicotine=carbamylcholine > suberyldicholine=oxotremorine. The dose–response relationship was complex, suggesting the existence of two populations of receptors: high-affinity receptors (extrapolated K d around 10 −7 M) and low-affinity receptors (extrapolated K d around 5×10 −5 M). The current–voltage relationship of the induced current was linear between −80 and −40 mV and the extrapolated reversal potential was not significantly different from 0 mV. The sequence of decreasing potency of the antagonists of the ACh response was: methyllycaconitine > α-bungarotoxin > mecamylamine > curare > strychnine > bicuculline > atropine > picrotoxin. These results show: (1) that, in embryonic brain neurones, the response to ACh corresponds to the opening of non-selective cationic channels; and (2) that the pharmacology of the ACh receptors is mainly but not solely nicotinic. The nature of the single events which underlie this response, as well as the structure of the channels (homo or hetero-oligomeric) remain to be investigated.

Use of the Supermolecule Approach to Derive Molecular Similarity Descriptors for QSAR Analysis

A relevant aspect in quantitative structure-activity (QSAR) and structure-selectivity (QSSR) relationships studies is the choice of the most appropriate molecular descriptors both with respect to the molecular series considered and the known or hypothetical mechanism of drug action. We have recently shown that ad hoc derived size and shape descriptors have been successful to derive QSAR and QSSR models for α1-adrenergic antagonists, 5-HT1A serotoninergic receptor ligands and M1 muscarinic ligands, especially when dealing with non congeneric series of molecules. These descriptors describe the size-shape similarity with respect to a reference supermolecule which is obtained by superposition of the most active (selective) and structural different compounds, better if rigids. Molecular similarity indices based on molecular electrostatic potential (MEP) have found, as well, widespread use in the QSAR area.

Imidacloprid actions on insect neuronal acetylcholine receptors

The neonicotinoid insecticide Imidacloprid acts at three pharmacologically distinct acetylcholine receptor (AChR) subtypes in the cockroach (Periplaneta americana) nervous system, but is ineffective on muscarinic receptors. Imidacloprid (3-100µmoll-1) induced dose-dependent depolarizations at cockroach cercal afferent/giant interneurone synapses. These responses were insensitive to 20µmoll-1 atropine but were completely blocked by the nicotinic antagonist mecamylamine (50µmoll-1). Similarly, Imidacloprid-induced depolarizations of cultured cockroach dorsal unpaired median (DUM) neurones dissociated from the same (terminal abdominal) ganglion were also completely blocked by 100µmoll-1 mecamylamine. However, two components of the response could be distinguished on the basis of their differential sensitivities to 0.1µmoll-1-bungarotoxin (-BTX), which selectively blocks AChRs with 'mixed' nicotinic/muscarinic pharmacology in this preparation. This indicates that Imidacloprid affects both AChRs sensitive to -BTX and -BTX-insensitive nicotinic acetylcholine receptors (nAChRs). Thus, in the cockroach, Imidacloprid activates -BTX-sensitive synaptic nAChRs in giant interneurones, -BTX-insensitive extrasynaptic nAChRs in DUM neurones, and a recently characterized DUM neurone 'mixed' AChR that is sensitive to both nicotinic and muscarinic ligands. Imidacloprid does not act on muscarinic acetylcholine receptors (mAChRs) present on DUM neurone cell bodies and at the cercal afferent/giant interneurone synapses. This study shows that Imidacloprid can act on pharmacologically diverse nAChR subtypes.

Methylene blue is a muscarinic antagonist in cardiac myocytes.

We studied the mechanism of action of methylene blue (Mblue), a putative guanylyl cyclase inhibitor, on the L-type calcium current (ICa) and the muscarinic activated K+ current (IK,ACh) in rat ventricular and atrial myocytes, respectively, and on the binding of [3H]quinuclidinyl benzylate in rat ventricular membranes. Superfusion, but not internal dialysis, with 30 microM Mblue antagonized the inhibitory effect of acetylcholine (ACh, 1 microM) on beta-adrenergic stimulation of ICa with isoprenaline (Iso, 10 nM or 1 microM). However, Mblue had no effect on the basal ICa or on the stimulation of ICa by Iso in the absence of ACh. The activation of IK,ACh by 3 microM ACh was also antagonized by Mblue in a dose-dependent manner. In contrast, Mblue had no effect on the activation of IK,ACh by either guanosine-5'-O-(3-thio)triphosphate or guanosine-5'-(beta,gamma-imido)triphosphate. Chlorpromazine (CPZ), a piperazine derivative like Mblue, also inhibited the muscarinic activation of IK,ACh in a dose-dependent manner. The specific binding of [3H]QNB, a muscarinic ligand, to rat ventricular membranes was displaced in a dose-dependent manner by Mblue and CPZ. The piperazine derivatives behaved like competitive antagonists of [3H]QNB binding, exhibiting equilibrium dissociation constant (Ki) values of 187 nM for Mblue and 366 nM for CPZ. In conclusion, Mblue exerts antimuscarinic effects on ICa and IK,ACh in rat cardiac myocytes that are best explained by the binding of Mblue to the M2 subtype of muscarinic receptors. This property probably contributes to the antimuscarinic effect of the putative guanylyl cyclase inhibitor reported in previous studies.

Cerebrovascular hypoperfusion: a risk factor for Alzheimer's disease? Animal model and postmortem human studies.

Although cognitive impairment during aging is usually associated with neuronal alterations, the cerebrovascular system undergoes prominent alterations in aging as well. Using electron microscopy we previously showed a progressive deterioration of the capillary wall in the cerebral cortex of aged rats. In aged rats the capillary basement membrane (BM) is thickened, massive bundles of collagen fibrils are deposited within the BM, and pericytes are degenerating. A compromized cerebral circulation (e.g., in rats with chronic hypertension) is characterized by an increased number of capillary alterations. In autopsy material (gray matter, gyrus cinguli) of carefully diagnosed patient groups (controls, AD, Lewy body disease, MID and demented Lewy body disease patients) we observed significantly more morphological changes in the capillary bed of demented versus non-demented patients. In both animal and human material morphological evidence points to a relation between energy-dependent nutrient transport across the blood-brain barrier and the ultrastructural deviations. In the AD cases we did not find a correlation between the stage of the disease (Braak I-VI) and the incidence of capillary aberrations, which indicates that the capillary alterations are not a consequence of AD pathology. Simultaneously, we are conducting animal model studies to determine the effects of cerebral hypoperfusion in the rat. Permanent bilateral occlusion of the carotid arteries shifts the behavioral profile of the rats (Morris maze, open field) towards that of aged rats, while the sensitivity for muscarinic ligand agents is altered.

ChemInform Abstract: Synthesis and Binding Affinity of New Muscarinic Ligands Structurally Related to Oxotremorine.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Synthesis and binding affinity of new muscarinic ligands structurally related to oxotremorine

The synthesis and radioligand binding assays of a group of muscarinic ligands related to oxotremorine are reported. The new compounds displayed binding affinities comparable to those of the parent molecule with the exception of the trimethylammonium salt 6, which behaved as a full muscarinic agonist and showed a pronounced selectivity for M 2 versus M 1 muscarinic receptor subtypes.

Covalent labeling of muscarinic acetylcholine receptors by tritiated aryldiazonium photoprobes

p-dimethylamino (A) and p-dibutylamino (B) benzenediazonium salts, previously characterized as efficient labels of membrane-bound and solubilized muscarinic receptor sites, are endowed with overall interesting photochemical and alkylating properties that allow their use as structural probes of the muscarinic ligand binding domain to be considered. Under reversible binding conditions, these antagonists display no binding selectivity towards the 5 muscarinic acetylcholine receptor (mAChR) subtypes. They were used here, in a tritiated form, as photoaffinity labels of purified muscarinic receptors from porcine striatum, and their irreversible binding was assessed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. When irradiated under energy transfer conditions, [ 3H]A and [ 3H]B were both found to covalently label purified muscarinic receptor sites in a light-dependent and atropine-protectable manner. The electrophoretic migration properties of the alkylated sites were similar to those of [ 3H]propylbenzilylcholine mustard (PrBCM)-labeled mAChRs. Specific radioactive incorporation showed a clear dependency on probe concentration. Labeling efficiency was rather high, with up to 30% and even 60% of the receptor population being photolabeled by [ 3H]A and [ 3H]B, respectively. These two photoactivatable ligands have proven to be powerful tools for the structural analysis of other cholinergic targets (acetylcholinesterase and the nicotinic acetylcholine receptor) by allowing the characterization of a number of different residues belonging to their acetylcholine-binding domain. Altogether, these results reinforce the interest of our site-directed labeling approach because [ 3H]A- and [ 3H]B-alkylated mAChRs may now be considered as suitable materials to investigate the muscarinic receptorbinding pocket through peptide mapping, sequence analyses, and identification of radiolabeled amino acid residues.

Expression of human muscarinic cholinergic receptors in tobacco.

We expressed human m1, m2 and chimeric muscarinic cholinergic receptors (MAChR) in tobacco plants and in cultured BY2 tobacco cells using Agrobacterium-mediated transformation. The membranes of most transgenic plants and calli bound muscarinic ligands with appropriate affinities, kinetics and pharmacologic specificity, as determined by direct and competitive binding measurements using the muscarinic ligand [3H]quinuclidinyl benzylate (QNB). Membranes of untransformed plants and calli or those transformed with vector alone did not bind [3H]QNB. Preliminary experiments did not suggest regulation of endogenous plant G protein signalling pathways by the recombinant receptors. Membranes from one callus clone expressed m1 MAChR at the level of 2.0-2.5 pmol [3H]QNB bound per mg membrane protein, more than the number of m1 MAChR in mammalian brain and comparable to that expressed in Sf9 insect cells using baculovirus vectors. This work demonstrates high level expression of active G protein-coupled receptors in plants, such that signaling might be genetically reconstituted by co-expression of appropriate G proteins and effectors.

Synthesis and structural determination of stereoisomers of muscarinic ligands of the (3‐propylthio‐1,2,5‐thiadiazol‐4‐yl)‐1‐azabicycloalkane type

Methods for the synthesis of each of the four stereoisomers of 6-(3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[3.2.1]octane (10, 11, 12, and 13) and 3-(3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[2.2.1]heptane (18, 19, 20, and 21), and the two stereoisomers of 3-(3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[2.2.2]octane (27 and 28) were developed. The relative configuration of the compounds was determined on the basis of previously described 1H NOE experiments, and the absolute configuration of 6-(3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[3.2.1]octanes (10, 11, 12, and 13) and 3-(3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[2.2.2]octane (27 and 28) was determined by single crystal X-ray crystallography. Optical purity was determined by capillary electrophoresis (CE) using chiral selectors as trimethyl-β-cyclodextrin and heparin dissolved in the running buffer. All the 3-(3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicycles had low nanomolar affinity for muscarinic receptors as determined by displacement of radiolabelled oxotremorine-M (3H-Oxo-M) and pirenzepine (3H-Pz) from cortical rat brain homogenates. The binding assay discriminated between diastereomers, but only a minor degree of enantioselectivity was observed in the binding assays. Chirality 9:739–749, 1997. © 1997 Wiley-Liss, Inc.

Synthesis and structural determination of stereoisomers of muscarinic ligands of the (3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicycloalkane type.

Methods for the synthesis of each of the four stereoisomers of 6-(3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[3.2.1]oc tane (10, 11, 12, and 13) and 3-(3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[2.2.1]he ptane (18, 19, 20, and 21), and the two stereoisomers of 3-(3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[2.2.2]oc tane (27 and 28) were developed. The relative configuration of the compounds was determined on the basis of previously described 1H NOE experiments, and the absolute configuration of 6-(3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[3.2.1]oc tanes (10, 11, 12, and 13) and 3-(3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[2.2.2]oc tane (27 and 28) was determined by single crystal X-ray crystallography. Optical purity was determined by capillary electrophoresis (CE) using chiral selectors as trimethyl-beta-cyclodextrin and heparin dissolved in the running buffer. All the 3-(3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicycles had low nanomolar affinity for muscarinic receptors as determined by displacement of radiolabelled oxotremorine-M (3H-Oxo-M) and pirenzepine (3H-Pz) from cortical rat brain homogenates. The binding assay discriminated between diastereomers, but only a minor degree of enantioselectivity was observed in the binding assays.

Functional Role of the Third Cytoplasmic Loop in Muscarinic Receptor Dimerization

By means of the expression of two chimeric receptors, alpha2/m3 and m3/alpha2, in which the carboxyl-terminal receptor portions, containing transmembrane (TM) domains VI and VII, were exchanged between the alpha2C adrenergic and the m3 muscarinic receptor, Maggio et al. (Maggio, R., Vogel, Z., and Wess, J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 3103-31073) demonstrated that G protein-linked receptors are able to interact functionally with each other at the molecular level to form (hetero)dimers. In the present study we tested the hypothesis that interaction between receptors might depend on the presence of a long third intracellular (i3) loop and that shortening this loop could impair the capability of receptors to form dimers. To address this question, we initially created short chimeric alpha2 adrenergic/m3 muscarinic receptors in which 196 amino acids were deleted from the i3 loop (alpha2/m3-short and m3/alpha2-short). Although co-transfection of alpha2/m3 and m3/alpha2 resulted in the appearance of specific binding, the co-expression of the two short constructs (alpha2/m3-short and m3/alpha2-short), either together or in combination, respectively, with m3/alpha2 and alpha2/m3 did not result in any detectable binding activity. In another set of experiments, a mutant m3 receptor, m3/m2(16aa), containing 16 amino acids of the m2 receptor sequence at the amino terminus of the third cytoplasmic loop, which was capable of binding muscarinic ligands but was virtually unable to stimulate phosphatidylinositol hydrolysis, was also mutated in the i3 loop, resulting in the m3/m2(16aa)-short receptor. Although co-transfection of m3/m2(16aa) with a truncated form of the m3 receptor (m3-trunc, containing an in frame stop codon after amino acid codon 272 of the rat m3 sequence) resulted in a considerable carbachol-stimulated phosphatidylinositol breakdown, the co-transfection of m3/m2(16aa)-short with the truncated form of the m3 receptor did not result in any recovery of the functional activity. Thus, these data suggest that intermolecular interaction between muscarinic receptors, involving the exchange of amino-terminal (containing TM domains I-V) and carboxyl-terminal (containing TM domains VI and VII) receptor fragments depends on the presence of a long i3 loop. One may speculate that when alternative forms of receptors with a different length of the i3 loop exist, they could have a different propensity to dimerize.

Characterization of muscarinic binding sites in the central nervous system of larval Manduca sexta

Putative muscarinic receptors (mAChRs) were characterized in the CNS of larval Manduca sexta through the binding of 3H-quinuclidyl benzilate ([ 3H]-QNB). Specific binding isotherms revealed a high affinity binding site in both crude homogenates (K a=3.22±0.62 nM 1 Kd 0.311 nM), B max=65.4±9.8 fmoles/mg protein) and in purified membrane preparations (K a=7.61±1.78 nM 1(K d 0.130 nM), B max=22.8±2.15 fmoles/mg protein). In purified membrane preparations the binding was complex, consisting of at least two sites (Hill coefficient =0.514±0.041). Because of the high proportion of non-specific binding at QNB concentrations greater than 10 nM, the binding parameters for the additional low affinity site could not be determined accurately but the Kd was estimated to be greater than 8 nM. Complex binding was also exhibited in the kinetics of [ 3H]-QNB association and dissociation in purified membranes. Analysis resolved two high affinity sites (dissociation rate constants =80.4 and 1.2 min, Kds=0.272 and 0.909 nM, consisting of 65.9±3.7 and 26±4.9% of the sites respectively). Presumably, the high affinity site identified in saturation studies consists of these two components. The primary high affinity site was isolated using a dissociative method and its pharmacology determined in competition studies. [ 3H]-QNB binding at this site was displaced by pirenzepine and methoctramine (Kis=248 and 707 nM) closely matching the pharmacology of the cloned Drosophila mAChR. Competition data for 4-DAMP were better fitted by a two site model (K/s=37 and 547 nM). These results demonstrate unexpected complexity in muscarinic ligand binding and are consistent with mAChR heterogeneity in the CNS of Manduca.