Reconstitution of the purified porcine atrial muscarinic acetylcholine receptor with purified porcine atrial inhibitory guanine nucleotide binding protein

Purified porcine atrial muscarinic receptor (mAcChR) was reconstituted with purified porcine atrial inhibitory guanine nucleotide binding protein (Gi) in a lipid mixture consisting of phosphatidylcholine, phosphatidylserine, and cholesterol (1:1:0.1 w/w). 5'-Guanylyl imidodiphosphate (0.1 mM) had no effect on the binding of the muscarinic antagonist L-quinuclidinyl benzilate but converted high-affinity carbachol binding sites (Kd equal to 1 microM) in the reconstituted preparation to the low-affinity state (Kd equal to about 100 microM). Steady-state kinetic measurements of GTPase activity showed that the turnover number was increased from 0.19 min-1 in the presence of the muscarinic antagonist L-hyoscyamine to 2.11 min-1 for the agonist carbachol. The affinity of Gi for GDP was reduced by about 50-fold upon interaction with the carbachol-mAcChR complex, and the observed rate constant for GDP dissociation was increased by 38-fold from 0.12 to 4.5 min-1. Thus, the increase in steady-state GTPase activity observed for muscarinic agonists is largely, if not exclusively, due to the increase in GDP dissociation from Gi--probably the rate-limiting step in the steady-state mechanism. Carbachol-stimulated GTPase was sensitive to ADP-ribosylation of the reconstituted Gi by pertussis toxin, but the high-affinity agonist binding was uncoupled only when the reconstituted preparation was treated with pertussis toxin in the presence of GTP and the agonist acetylcholine. These results suggest that association with the mAcChR protects Gi from ADP-ribosylation by pertussis toxin.     

Partial functional reconstitution of the cardiac muscarinic cholinergic receptor

Digitonin-solubilized cardiac muscarinic receptors were reconstituted by dialysis into human erythrocyte acceptor membranes which lack high-affinity muscarinic receptors. The number of receptors reconstituted was proportional to the quantity of soluble receptors added to the reconstitution system. Specific [3H](-)-quinuclidinyl benzilate binding to the reconstituted receptor was found to be saturable with a Kd (dissociation constant) equal to 48 +/- 4 pM and a Bmax (maximal density of binding sites) equal to 50 +/- 5 fmol/mg of protein. Competitive binding studies indicated that the reconstituted receptors showed stereoselectivity and drug specificity consistent with a high-affinity muscarinic receptor. Agonist binding to the reconstituted receptor was decreased by the addition of guanyl-5'-yl imidodiphosphate. Sixty per cent of the reconstituted receptors were found to be integral membrane proteins. The molecular weight of the reconstituted receptor as determined by sodium dodecyl sulfate-gel electrophoresis was 76,000 +/- 2,000 and was identical to the molecular weight of the muscarinic receptor in the original cardiac membranes. The data indicate that a partially functional, intact muscarinic receptor was reconstituted into human erythrocyte acceptor membranes and that membrane constituents may be required to stabilize the receptor in a high-affinity state for antagonists.     

Photoaffinity labeling of the solubilized, partially purified muscarinic acetylcholine receptor from porcine atria by p-azidoatropine methyl iodide

The synthesis of a tritiated photoaffinity analogue of the muscarinic antagonist atropine, [3H]-p-azidoatropine methyl iodide is described. The compound appeared to bind to a single class of sites in membrane-bound, solubilized, and partially purified preparations of muscarinic receptor from porcine atria with a dissociation constant (determined by competition vs. [3H]-L-quinuclidinyl benzilate) of about 1.0 X 10(-7) M. This value was in agreement with the apparent dissociation constant (8.5 X 10(-8)M) determined by measuring the concentration dependence of covalent incorporation into a partially purified receptor preparation. Competition experiments indicated that the specific covalent labeling could be blocked by the muscarinic agonist carbamylcholine and the antagonists L-quinuclidinyl benzilate and atropine. An apparent molecular weight of 75 000 +/- 5000 was found for specifically labeled peptide(s) in a solubilized, partially purified receptor preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Engineering of three-finger fold toxins creates ligands with original pharmacological profiles for muscarinic and adrenergic receptors

Protein engineering approaches are often a combination of rational design and directed evolution using display technologies. Here, we test "loop grafting," a rational design method, on three-finger fold proteins. These small reticulated proteins have exceptional affinity and specificity for their diverse molecular targets, display protease-resistance, and are highly stable and poorly immunogenic. The wealth of structural knowledge makes them good candidates for protein engineering of new functionality. Our goal is to enhance the efficacy of these mini-proteins by modifying their pharmacological properties in order to extend their use in imaging, diagnostics and therapeutic applications. Using the interaction of three-finger fold toxins with muscarinic and adrenergic receptors as a model, chimeric toxins have been engineered by substituting loops on toxin MT7 by those from toxin MT1. The pharmacological impact of these grafts was examined using binding experiments on muscarinic receptors M1 and M4 and on the α(1A)-adrenoceptor. Some of the designed chimeric proteins have impressive gain of function on certain receptor subtypes achieving an original selectivity profile with high affinity for muscarinic receptor M1 and α(1A)-adrenoceptor. Structure-function analysis supported by crystallographic data for MT1 and two chimeras permits a molecular based interpretation of these gains and details the merits of this protein engineering technique. The results obtained shed light on how loop permutation can be used to design new three-finger proteins with original pharmacological profiles.     

Quinuclidinyl Benzilate Binding in House Fly Heads and Rat Brain

Abstract: House fly heads contain a binding site for 3-quinuclidinyl benzilate (QNB) that is quite similar in pharmacology to the muscarinic acetylcholine receptor of vertebrate tissues. The house fly site binds [³H]QNB reversibly with a K _d of 260 PM and B_max of 1 pmol/g of heads from direct binding measurements. The K_d calculated from the ratio of the dissociation rate constant (2 × 10⁻⁴ sec⁻¹) to the association rate constant (2.5 × 10⁶ M⁻¹ Sec⁻¹) was 80 pM. The house fly site binds (-)quinuclidinyl benzilate preferentially, as do classic muscarinic receptors. The binding is also sensitive to other muscarinic antagonists and agonists. Nicotinic and other drugs are no more effective on the house fly site than they are on the rat brain muscarinic receptor itself. These binding studies suggest that the house fly QNB binding site is a muscarinic receptor.     

MNT inhibits the migration of human hepatocellular carcinoma SMMC7721 cells

Muscarinic toxins (MTs) are snake venom peptides found to selectively target specific subtypes of G-protein-coupled receptors. In here, we have attached a glycosylphosphatidylinositol (GPI) tail to three different toxin molecules and evaluated their receptor-blocking effects in a heterologous expression system. MT7-GPI remained anchored to the cell surface and selectively inhibited M(1) muscarinic receptor signaling expressed in the same cell. To further demonstrate the utility of the GPI tail, we generated MT3- and MTα-like gene sequences and fused these to the signal sequence for GPI attachment. Functional assessment of these membrane-anchored toxins on coexpressed target receptors indicated a prominent antagonistic effect. In ligand binding experiments the GPI-anchored toxins were found to exhibit similar selection profiles among receptor subtypes as the soluble toxins. The results indicate that GPI attachment of MTs and related receptor toxins could be used to assess the role of receptor subtypes in specific organs or even cells in vivo by transgenic approaches.     

Evidence for Multiple Muscarinic Receptor Subtypes in Human Brain

Pirenzepine, a compound with selective antimuscarinic activity, was used to distinguish muscarinic acetylcholine receptor subtypes in normal human brain. Hill coefficients and IC50 values derived from the inhibition of specific [3H]L-quinuclidinyl benzilate receptor binding suggest the presence of two muscarinic binding sites, differing both in affinity for pirenzepine and in tissue distribution.     

Snake toxins with high selectivity for subtypes of muscarinic acetylcholine receptors

There are five subtypes of muscarinic acetylcholine receptors (M(1) to M(5)) which control a large number of physiological processes, such as the function of heart and smooth muscles, glandular secretion, release of neurotransmitters, gene expression and cognitive functions as learning and memory. A selective ligand is very useful for studying the function of a subtype in presence of other subtypes, which is the most common situation, since a cell or an organ usually has several subtypes. There are many non-selective muscarinic ligands, but only few selective ones. Mambas, African snakes of genus Dendroaspis have toxins, muscarinic toxins, that are selective for M(1), M(2) and M(4) receptors. They consist of 63-66 amino acids and four disulfides which form four loops. They are members of a large group of snake toxins, three-finger toxins; three loops are extended like the middle fingers of a hand and the disulfides and the shortest loop are in the palm of the hand. Some of the toxins target the allosteric site which is located in a cleft of the receptor molecule close to its extracellular part. A possible explanation to the good selectivity is that the toxins bind to the allosteric site, but because of their size they probably also bind to extracellular parts of the receptors which are rather different in the various subtypes. Some other allosteric ligands also have good selectivity, the alkaloid brucine and derivatives are selective for M(1), M(3) and M(4) receptors. Muscarinic toxins have been used in several types of experiments. For instance radioactively labeled M(1) and M(4) selective toxins were used in autoradiography of hippocampus from Alzheimer patients. One significant change in the receptor content was detected in one region of the hippocampus, dentate gyrus, where M(4) receptors were reduced by 50% in patients as compared to age-matched controls. Hippocampus is essential for memory consolidation. M(4) receptors in dentate gyrus may play a role, since they decreased in Alzheimers disease which destroys the memory. Another indication of the role of M(4) receptors for memory is that injection of the M(4) selective antagonist muscarinic toxin 3 (M(4)-toxin 1) into rat hippocampus produced amnesia.     

Regulation of the solubilized bovine cerebral cortex muscarinic receptor by GTP and Na+

The muscarinic acetylcholine receptor was solubilized, in a sensitive form for GTP and Na+, from bovine cerebral cortex using a zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate. The solubilized muscarinic receptor displayed characteristics as follows: (1) high affinity to nanomolar concentration of Z-[3H]quinuclidinyl benzilate; (2) muscarinic agonists and antagonists had similar inhibitory potencies as on the membrane-bound receptor; (3) without Na+, GTP did not significantly alter the binding affinity of muscarinic agonists and antagonists; (4) GTP in the presence of Na+, selectively decreased the affinity of muscarinic agonists, carbamylcholine and oxotremoline, but not the antagonist binding affinity; (5) Na+ in the absence or presence of GTP, reduced both muscarinic agonist and antagonist affinities.     

Antagonism by 8-hydroxy-2(di-n-propylamino)tetraline and other serotonin agonists of muscarinic M1-type receptors coupled to inositol phospholipid breakdown in human IMR-32 and SK-N-MC neuroblastoma cells.

IMR-32 and SK-N-MC cells were found to contain [3H]quinuclidinyl benzilate specific binding sites inhibited by pirenzepine in a manner suggesting the presence of both M1-type and M2-type muscarinic receptor recognition sites. Neither cell had detectable [3H]8-OH-DPAT binding sites. Carbachol stimulated the rate of inositol phospholipid breakdown in IMR-32 and SK-N-MC human neuroblastoma cells with an EC50 value of about 50 microM in both cases. Pirenzepine inhibited the carbachol (100 microM)-stimulated inositol phospholipid breakdown in both cells with Hill slopes of unity and IC50 values of 15 nM (IMR-32) and 12 nM (SK-N-MC). The 5-HT1A receptor agonist 8-OH-DPAT competitively inhibited carbachol-stimulated inositol phospholipid breakdown with pA2 values of 5.78 (IMR-32) and 5.61 (SK-N-MC). These values are consistent with the inhibitory potency of 8-OH-DPAT towards [3H]quinuclidinyl benzilate binding in these cells. The 5-HT agonists 5-MeODMT and buspirone at micromolar concentrations inhibited carbachol-stimulated breakdown in IMR-32 cells. The inhibition by 8-OH-DPAT and 5-MeODMT was not affected by preincubation with (-)alprenolol. 5-HT (10-100 microM) was without effect on either basal or carbachol-stimulated breakdown. It is concluded that IMR-32 and SK-N-MC neuroblastoma cells express muscarinic M1-type but not serotoninergic receptors coupled to phosphoinositide-specific phospholipase C. 8-OH-DPAT acts as a weak antagonist at these muscarinic receptors.     

Endogenous Inhibitor of Ligand Binding to the Muscarinic Acetylcholine Receptor

An endogenous inhibitor of L-[3H]quinuclinidinyl benzilate binding to the brain muscarinic acetylcholine receptor was identified. [3H]Quinuclinidinyl benzilate binding to rat brain synaptosomes was measured using a filtration assay. The inhibitor was prepared from several calf tissues and was found in highest specific activity in thymus. The loss of binding activity was slow, requiring a 30-40 min preincubation of the synaptosomes with the inhibitor, and reversed by removing the inhibitor by washing the membranes. Scatchard analysis of the binding data showed that the inhibition was noncompetitive resulting from both a decrease in affinity and a decrease in the number of binding sites. Zn2+ was required in low concentrations for this effect. Muscarinic acetylcholine receptor in synaptic membranes and in membranes free of most peripheral membrane proteins was still sensitive to inhibition. Preliminary characterization of the inhibitory molecule showed that it is of low molecular weight, moderately heat-stable, and acidic. The inhibitor was inactivated by reagents that are nonspecific for nucleophiles, but not by reagents specific for primary amine or thiol groups.     

Binding of N-[propionyl-3H]propionylated alpha-bungarotoxin and L-[benzilic-4,4'-3H] quinuclidinyl benzilate to CNS extracts of the cockroach Periplaneta americana

The nerve cord of the cockroach (Periplaneta americana) contains distinct saturable components of specific binding for the ligands N-[propionyl-3H]propionylated alpha-bungarotoxin and L-[benzilic-4,4'-3H]quinuclidinyl benzilate. N-[Propionyl-3H]propionylated alpha-bungarotoxin bound reversibly to homogenates with a Kd of 4.8 nM and Bmax of 910 fmol mg-1. The association rate constant (1.9 X 10(5) M-1 s-1) and dissociation rate constant (1.2 X 10(-4) s-1) yielded a Kd of 0.6 nM. Nicotinic ligands were found to displace toxin binding most effectively. The binding sites characterized in this way showed many similarities with the properties of the vertebrate neuronal alpha-bungarotoxin binding site. For a range of cholinergic ligands, inhibition constants calculated from toxin binding studies closely corresponded to their effectiveness in blocking the depolarizing response to acetylcholine recorded by electrophysiological methods from an identified cockroach motoneurone. The N-[propionyl-3H]propionylated alpha-bungarotoxin binding component therefore appears to be a constituent of a functional CNS acetylcholine receptor. Binding of L-[benzilic-4,4'-3H]quinuclidinyl benzilate was reversible with a Kd of 8 nM and Bmax of 138 fmol mg-1, determined from equilibrium binding experiments. The Kd calculated from the association rate constant (2.4 X 10(5) M-1 s-1) and dissociation rate constant (1.3 X 10(-4) s-1) was 1.9 nM. Muscarinic ligands were the most potent inhibitors of quinuclidinyl benzilate binding. The characteristics of this binding site resembled those of vertebrate CNS muscarinic cholinergic receptors. In contrast with vertebrate CNS, the nerve cord of Periplaneta americana contains more (approximately X 7) alpha-bungarotoxin binding sites than quinuclidinyl benzilate binding sites.     

Muscarinic toxin-like proteins from cobra venom.

Three new polypeptides were isolated from the venom of the Thailand cobra Naja kaouthia and their amino-acid sequences determined. They consist of 65-amino-acid residues and have four disulfide bridges. A comparison of the amino-acid sequences of the new polypeptides with those of snake toxins shows that two of them (MTLP-1 and MTLP-2) share a high degree of similarity (55-74% sequence identity) with muscarinic toxins from the mamba. The third polypeptide (MTLP-3) is similar to muscarinic toxins with respect to the position of cysteine residues and the size of the disulfide-confined loops, but shows less similarity to these toxins (30-34% sequence identity). It is almost identical with a neurotoxin-like protein from Bungarus multicinctus (TrEMBL accession number Q9W727), the sequence of which has been deduced from cloned cDNA only. The binding affinities of the isolated muscarinic toxin-like proteins towards the different muscarinic acetylcholine receptor (mAChR) subtypes (m1-m5) was determined in competition experiments with N-[3H]methylscopolamine using membrane preparations from CHO-K1 cells, which express these receptors. We found that MTLP-1 competed weakly with radioactive ligand for binding to all mAChR subtypes. The most pronounced effect was observed for the m3 subtype; here an IC50 value of about 3 microM was determined. MTLP-2 had no effect on ligand binding to any of the mAChR subtypes at concentrations up to 1 microM. MTLP-1 showed no inhibitory effect on alpha-cobratoxin binding to the nicotinic acetylcholine receptor from Torpedo californica at concentrations up to 20 microM.     

A system for the directed evolution of the insecticidal protein from Bacillus thuringiensis

Theoretically, the activity of AB-type toxin molecules such as the insecticidal toxin (Cry toxin) from B. thuringiensis, which have one active site and two binding site, is improved in parallel with the binding affinity to its receptor. In this experiment, we tried to devise a method for the directed evolution of Cry toxins to increase the binding affinity to the insect receptor. Using a commercial T7 phage-display system, we expressed Cry1Aa toxin on the phage surface as fusions with the capsid protein 10B. These recombinant phages bound to a cadherin-like protein that is one of the Cry1Aa toxin receptors in the model target insect Bombyx mori. The apparent affinity of Cry1Aa-expressing phage for the receptor was higher than that of Cry1Ab-expressing phage. Phages expressing Cry1Aa were isolated from a mixed suspension of phages expressing Cry1Ab and concentrated by up to 130,000-fold. Finally, random mutations were made in amino acid residues 369–375 in domain 2 of Cry1Aa toxin, the mutant toxins were expressed on phages, and the resulting phage library was screened with cadherin-like protein-coated beads. As a result, phages expressing abnormal or low-affinity mutant toxins were excluded, and phages with high-affinity mutant toxins were selected. These results indicate that a method combining T7 phage display with selection using cadherin-like protein-coated magnetic beads can be used to increase the activity of easily obtained, low-activity Cry toxins from bacteria.     

Kinetic aspects of l-quinuclidinyl benzilate interaction with muscarinic receptor

l-Quinuclidinyl benzilate is undoubtedly the most widely used radioactive reporter ligand for studies on muscarinic receptor. In the present Commentary the kinetic aspects of the interaction of this ligand with muscarinic receptor are summarized. On the basis of these results a kinetic mechanism has been proposed involving consequential isomerization of the receptor-ligand complex and cooperative regulation of this process by the excess of the ligand. In addition, the data give evidence of at least two different types of binding sites on the receptor. Owing to the solubilization of the receptor protein there occur remarkable changes in its kinetic properties. The kinetic analysis points to inadequacy of the simple one-step equilibrium binding scheme for l-quinuclidinyl benzilate interaction with muscarinic receptor, which may explain the apparently contradictory data in the literature, such as the large scattering of the K_d values and the different regularities described in the case of the receptor-ligand complex dissociation reaction. That points to the conclusion that l-quinuclidinyl benzilate is an “inconvenient” ligand for receptor studies, which call for true equilibrium conditions of the system.     

α2-Adrenergic and Muscarinic Cholinergic Receptors Have Oposing Actions on Cyclic AMP Levels in SK-N-SH Human Neuroblastoma Cells

Forskolin and vasoactive intestinal polypeptide (VIP) were shown to increase cyclic AMP accumulation in a human neuroblastoma cell line, SK-N-SH cells. The alpha 2-adrenergic agonist UK 14304 decreased forskolin-stimulated cyclic AMP levels by 40 +/- 2%, with an EC50 of 83 +/- 20 nM. This response was blocked by pretreatment with pertussis toxin (PT) (EC50 = 1 ng/ml) or by the alpha 2-antagonists yohimbine, idazoxan, and phentolamine. Antagonist IC50 values were 0.3 +/- 0.1, 2.2 +/- 0.3, and 1.4 +/- 0.1 microM, respectively. This finding suggests the presence of normal inhibitory coupling of SK-N-SH cell alpha 2-adrenergic receptors to adenylate cyclase via the inhibitory GTP-binding protein species, Gi. Muscarinic receptors in many target cell types are coupled to inhibition of adenylate cyclase. However, in SK-N-SH cells, muscarinic agonists synergistically increased (67-95%) the level of cyclic AMP accumulation elicited by forskolin or VIP. EC50 values for carbamylcholine (CCh) and oxotremorine facilitation of the forskolin response were 1.2 +/- 0.2 and 0.3 +/- 0.1 microM, respectively. Pharmacological studies using the muscarinic receptor subtype-preferring antagonists 4-diphenylacetoxy-N-methylpiperidine, pirenzepine, and AF-DX 116 indicated mediation of this response by the M3 subtype. IC50 values were 14 +/- 1, 16,857 +/- 757, and 148,043 +/- 16,209 nM, respectively. CCh-elicited responses were unaffected by PT pretreatment. Muscarinic agonist binding affinity was indirectly measured by the ability of CCh to compete for [3H]quinuclidinyl benzilate binding sites on SK-N-SH cell membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation, sequence, and functional expression of the mouse M1 muscarinic acetylcholine receptor gene

A genomic clone encoding the gene for the mouse M1 muscarinic acetylcholine receptor has been isolated, placed under the control of the zinc-inducible mouse metallothionein promoter, and transfected into mouse Y1 adrenal cells. The receptor concentration was about 300 fmol/mg membrane protein in the absence of zinc and could be increased to 4000 fmol/mg membrane protein in the presence of increasing concentrations of zinc. The receptor expressed in zinc-induced cells exhibits the high affinity binding for quinuclidinyl benzilate, atropine, and pirenzepine expected of the M1 muscarinic receptor. The M1 receptor when expressed in Y1 or L cells is physiologically active, as measured by agonist-dependent stimulation of phosphatidylinositol metabolism, but does not inhibit forskolin stimulation of cAMP accumulation. In contrast, a cloned M2 muscarinic receptor when expressed in Y1 cells is able to inhibit forskolin stimulation of cAMP accumulation, but is unable to stimulate phosphatidylinositol metabolism. The stimulation of phosphatidylinositol metabolism mediated by the M1 receptor was not altered by prior treatment of Y1 cells with concentrations of islet-activating protein sufficient to eliminate M2 receptor-mediated inhibition of adenylate cyclase. The cloned M1 receptor gene thus exhibits both the pharmacological and physiological properties expected of the M1 muscarinic acetylcholine receptor. In addition, these results indicate that different subtypes of the muscarinic receptor are coupled to different physiological responses.     

Localization of the voltage-sensor toxin receptor on KvAP

A variety of venomous animals produce small protein toxins that impair the function of voltage-dependent cation channels by affecting the motions of the voltage-sensor domains and altering the energetics of the opening of the channel. In this study, we investigate the location of the receptor for tarantula venom voltage-sensor toxins on the voltage-dependent K+ channel from Aeropyrum pernix (KvAP), an archeabacterial channel that is functionally inhibited by members of this toxin family. We show that it is possible to purify the same set of toxins from venom of the tarantula Grammostola spatulata using either the purified KvAP voltage-sensor domain or the full-length KvAP channel. The equivalence of toxin retention profiles for the two channel proteins implies that the tarantula voltage-sensor toxin receptor resides exclusively on the voltage-sensor domain and that the pore is not required for the toxin-channel interaction. We have identified and characterized the functional properties of a subset of the tarantula toxins that bind to the KvAP voltage-sensor domain. Some of these toxins, VSTX1 and GSMTX4, have been previously isolated, while others, VSTX2 and VSTX3, are new members of the tarantula voltage-sensor toxin family. Some but not all toxins that bind to the voltage-sensor domain affect voltage-dependent gating of KvAP channels in lipid membranes.     

Kinetic Effects of Terbium Ions on Muscarinic Acetylcholine Receptors of Murine Neuroblastoma Cells

Preincubation of murine neuroblastoma cells (clone N1E-115) with terbium chloride resulted in a significant potentiation of carbachol-mediated increase in cyclic GMP formation. This effect was accompanied by a shift of the peak response from 30 s to 120 s and a 6-fold decrease in carbachol concentration producing half-maximal responses, in addition to a significant increase in the Hill coefficient. Terbium ions also caused a significant decrease in the affinity and an increase in the maximum binding of [3H]quinuclidinyl benzilate to muscarinic receptors, the change in affinity being mainly due to a decrease in the association rate. Preincubation of cells with 1 mM carbachol for 4 h (the desensitized state of the muscarinic receptor) resulted in a decrease in the ability of terbium to alter [3H]quinuclidinyl benzilate binding. The effects of terbium reported here might be due to its affecting muscarinic receptor-effector coupling, which is considered to be lost upon receptor desensitization.     

An alkylating derivative of oxotremorine interacts irreversibly with the muscarinic receptor

A 2-chloroethylamine derivative of oxotremorine was studied in pharmacological experiments and muscarinic receptor binding assays. The compound, N-[4-(2-chloroethylmethylamino)-2-butynyl]-2-pyrrolidone (BM 123), forms an aziridinium ion in aqueous solution at neutral pH that stimulates contractions of the guinea pig ileum with a potency similar to that of oxotremorine. Following the initial stimulation, there is a long lasting period of lack of sensitivity of the guinea pig ileum to muscarinic agonists. BM 123 also produces muscarinic effects in vivo. When homogenates of the rat cerebral cortex were incubated with BM 123 and assayed subsequently in muscarinic receptor binding assays, a loss of binding capacity for the muscarinic antagonist, [3H]N-methylscopolamine ( [3H]NMS), was noted without a change in affinity. Similar observations were made in [3H]1-3-quinuclidinyl benzilate ( [3H]1-QNB) binding assays on the forebrains of mice that had been injected with BM 123 24 hr earlier. The loss in receptor capacity for both [3H]NMS and [3H]1-QNB was prevented by atropine treatment. Kinetic studies of the interaction of BM 123 with homogenates of the rat cerebral cortex in vitro showed that the half-time for the loss of [3H]1-QNB binding sites increased from 10 to 45 min as the concentration of BM 123 decreased from 10 to 1 microM. In contrast to the aziridinium ion, the parent 2-chloroethylamine compound and the alcoholic hydrolysis product were largely devoid of pharmacological and binding activity.