Positive allosteric action of eburnamonine on cardiac muscarinic acetylcholine receptors

It was discovered recently that alcuronium and strychnine (which is a precursor of alcuronium) allosterically increase the affinity of cardiac muscarinic receptors for the antagonist, N-methylscopolamine. We have now investigated the effects of l-eburnamonine and vincamine, which are both closely related to strychnine. In experiments on rat heart atria, l-eburnamonine was found to increase the binding of [3H]N-methylscopolamine with Ehlert's cooperativity coefficient alpha = 0.35, which indicates that the strength of its allosteric action is close to that of alcuronium and strychnine (alpha = 0.31 and 0.44, respectively). However, the affinity of l-eburnamonine for the cardiac muscarinic receptors is lower than the affinities of alcuronium and strychnine (KAR = 22.6 microM, 0.15 microM, and 3.4 microM, respectively). In spite of its extremely close similarity to l-eburnamonine, vincamine has a negative allosteric effect on the binding of [3H]N-methylscopolamine (alpha = 4.1; KAR = 22.8 microM). It is likely that a systematic investigation of the allosteric effects of the analogues of strychnine will not only yield new allosteric effectors on muscarinic receptors, but also clarify the structural features responsible for the direction (positive or negative) of their allosteric effect.     

Positive cooperativity of acetylcholine and other agonists with allosteric ligands on muscarinic acetylcholine receptors

At fertilization periodic Ca2+ oscillations release oocytes from meiotic arrest. The present study examined whether these oscillations have a long-term role in pre- and postimplantation development, independent of their immediate effect. Sr(2+)-containing medium was used to induce oscillations during exit from meiosis and first embryonic mitosis and Sr(2+)-activated parthenotes were compared to ethanol-activated parthenotes and embryos generated by in vitro fertilization. After embryo culture, blastocysts were differentially stained for the inner cell mass and trophectoderm. It was found that oscillations both during exit from meiosis and during mitosis acted to increase the number of inner cell mass cells. In contrast, the trophectoderm cell number was largest in ethanol-activated parthenotes and smallest in fertilized embryos. Postimplantation development was also modestly improved by extending the time of exposure to Sr(2+)-containing medium. Together these data suggest that Ca2+ oscillations have a role in long-term embryonic events and that they provide more than merely a stimulus for meiotic resumption.     

Search for lead structures to develop new allosteric modulators of muscarinic receptors

Various compounds are known to allosterically modulate the binding of ligands to muscarinic receptors. Most of these compounds have another predominant pharmacological action. Identification of the potent representatives should be useful for the development of allosteric modulators that are specific and highly active. For various reasons, a direct comparison of allosteric potencies on the basis of literature data is difficult. Therefore, a series of compounds was compared with regard to the allosteric delay of the dissociation of N-[3H]methylscopolamine from porcine heart M2 receptors under the following assay conditions: "Na,K,Pi buffer", 4 mM Na2HPO4, 1 mM KH2PO4, pH 7.4, 23 degrees C; "Mg,Tris,Cl,Pi buffer', 50 mM Tris-HCl, 3 mM MgHPO4,pH 7.3, 37 degrees C. Generally, the allosteric potency of the compounds was higher in the Na,K,Pi buffer, compared with the Mg,Tris,Cl,Pi buffer. However, the extent of the potency shift differed, ranging from approximately 2-fold for tacrine to approximately 100-fold for gallamine. The concentration retarding radioligand dissociation to half of the control rate (EC50) served as a measure of allosteric potency. Under both assay conditions, alcuronium was the most potent compound (EC50,Na,K,Pi = 4 nM and EC50,Mg,Tris,Cl,Pi = 55 nM), followed by alkane-bisammonium and bispyridinium compounds containing phthalimido moieties. Gallamine showed intermediate potency (EC50 values of 180 nM and 16,000 nM in Na,K,Pi buffer and Mg,Tris,Cl,Pi buffer, respectively). Obidoxime and hexamethonium, both known to antagonize allosteric actions, revealed submaximal efficacy and low potency (EC50,Na,K,Pi of approximately 100,000 nM). The relevance of these results, regarding the identification of lead structures for the development of new allosteric modulators, is discussed.     

Molecular mechanisms for the regulation of the expression and function of muscarinic acetylcholine receptors

The regulation of muscarinic acetylcholine receptor expression and function was investigated in cultured cells and in knockout mice. Muscarinic agonist exposure causes m2 receptor desensitization and sequestration and decreases the expression of cardiac potassium channels. The expression of m2 receptors in chick retina is regulated by a developmentally regulated secreted factor. Mice lacking the m1 receptor exhibit a loss of muscarinic regulation of M-current potassium channel activity and pilocarpine-induced seizures.     

On the unique binding and activating properties of xanomeline at the M1 muscarinic acetylcholine receptor

We investigated the molecular nature of the interaction between the functionally selective M1 muscarinic acetylcholine receptor (mAChR) agonist xanomeline and the human M1 mAChR expressed in Chinese hamster ovary (CHO) cells. In contrast to the non-subtype-selective agonist carbachol, xanomeline demonstrated M1 mAChR binding that was resistant to extensive washout, resulting in a significant reduction in apparent N-[3H]methylscopolamine saturation binding affinity in intact cells. Functional assays, using both M1 mAChR-mediated phosphoinositide hydrolysis and activation of neuronal nitric oxide synthase, confirmed that this persistent binding resulted in elevated basal levels of system activity. Furthermore, this phenomenon could be reversed by the addition of the antagonist atropine. However, pharmacological analysis of the inhibition by atropine of xanomeline-mediated functional responses indicated a possible element of noncompetitive behavior that was not evident in several kinetic and equilibrium binding experimental paradigms. Taken together, our findings indicate for the first time a novel mode of interaction between an mAChR agonist and the M1 mAChR, which may involve unusually avid binding of xanomeline to the receptor. This yields a fraction of added agonist that is retained at the level of the receptor compartment to persistently bind to and activate the receptor subsequent to washout. The results of the current study suggest that elucidation of the mechanism or mechanisms of interaction of xanomeline with the M1 mAChR is particularly important in relation to the potential therapeutic use of this agent in the treatment of Alzheimer's disease.     

Molecular rigidity and potency of bispyridinium type allosteric modulators at muscarinic M2-receptors

Several bispyridinium compounds have been shown to be potent allosteric modulators of ligand binding to muscarinic M2-receptors. ,,Uno compounds" are benzyl derivatives of the bispyridinium "TMB4" (trimethylene-bis-[4-hydroxy-iminomethyl-pyridinium]). To gain more insight into structure activity relationships, eleven derivatives with varying structure of the oxime-linked aromatic substituent were tested for their ability to inhibit the equilibrium-binding of [3H]N-methylscopolamine ([3H]NMS) in guinea pig cardiac membranes and to retard [3H]NMS-dissociation allosterically. At a concentration of 3 microM, all compounds reduced [3H]NMS-binding to about 40 % of the control level, indicating a similar potency to inhibit the association of [3H]NMS. Allosteric retardation of [3H]NMS-dissociation required higher concentrations. Comparing the effects of the compounds at 30 and 300 microM, respectively, revealed considerable differences in potency. Therefore, the concentration-dependency of the delay of [3H]NMS-dissociation was determined for selected compounds. The results indicate that introduction of a benzyl-moiety into TMB4 leads to a 20-fold increase in allosteric potency. A further increment by a factor of 10 is obtained with the 2,6-dichlorobenzyl-substitution and with the naphthyl-derivative. The other compounds were less potent. An inverse correlation was found between the rotational freedom of the aromatic substituent and the allosteric potency. In conclusion, the aromatic moiety of non-symmetric bispyridinium-type modulators does not seem to be part of the pharmacophore involved in the inhibitory effect on the association of [3H]NMS. In contrast, a rigid aromatic lateral moiety appears to be essential for the interaction with the recognition site mediating the allosteric delay of [3H]NMS dissociation from muscarinic M2-receptors.     

Volatile anaesthetics have differential effects on recombinant m1 and m3 muscarinic acetylcholine receptor function

Muscarinic acetylcholine signalling plays major roles in regulation of consciousness, cognitive functioning, pain perception and circulatory homeostasis. Halothane has been shown to inhibit m1 muscarinic signalling. However, no comparative data are available for desflurane, sevoflurane or isoflurane, nor have the anaesthetic effects on the m3 subtype (which is also prominent in the brain) been studied. Therefore, we have investigated the effects of these compounds on isolated m1 and m3 muscarinic receptor function. Defolliculated Xenopus oocytes expressing recombinant m1 or m3 muscarinic or (for comparison) AT1A angiotensin II receptors were voltage clamped, and Ca(2+)-activated Cl- currents (ICl(Ca)) induced by acetyl-beta-methylcholine (Mch) or angiotensin II were measured in the presence of clinically relevant concentrations of halothane, sevoflurane, desflurane or isoflurane. To determine the site of action of the volatile anaesthetics we compared anaesthetic effects on m1, m3 and AT1A receptor function and studied the effects of volatile anaesthetics on signalling induced by intracellular injection of the second messenger IP3. Desflurane had a biphasic effect on m1 signalling, enhancing at a concentration of 0.46 mmol litre-1 but depressing at 0.92 mmol litre-1. A similar, although not significant, trend was observed with m3 signalling. Isoflurane had no effect on m1 signalling, but profoundly inhibited m3 signalling. Sevoflurane depressed the function of m1 and m3 signalling in a dose-dependent manner. Halothane, similar to its known effect on m1 signalling, dose-dependently depressed m3 function. ICl(Ca) induced by intracellular injections of IP3 were unaffected by all four anaesthetics. Similarly, none of the anaesthetics tested affected AT1A signalling. Absence of interference with AT1A signalling and intracellular pathways suggest that the effects of anaesthetics on muscarinic signalling most likely result from interactions with the m1 or m3 receptor molecule. Multiple interaction sites with different affinities may explain the biphasic response to desflurane. Anaesthetic-specific effects on closely related receptor subtypes suggest defined sites of action for volatile anaesthetics on the receptor protein.     

Covalent labeling of muscarinic acetylcholine receptors by tritiated aryldiazonium photoprobes

Members of the Tc1/mariner superfamily of transposons isolated from fish appear to be transpositionally inactive due to the accumulation of mutations. Molecular phylogenetic data were used to construct a synthetic transposon, Sleeping Beauty, which could be identical or equivalent to an ancient element that dispersed in fish genomes in part by horizontal transmission between species. A consensus sequence of a transposase gene of the salmonid subfamily of elements was engineered by eliminating the inactivating mutations. Sleeping Beauty transposase binds to the inverted repeats of salmonid transposons in a substrate-specific manner, and it mediates precise cut-and-paste transposition in fish as well as in mouse and human cells. Sleeping Beauty is an active DNA-transposon system from vertebrates for genetic transformation and insertional mutagenesis.     

Two-step binding of green mamba toxin to muscarinic acetylcholine receptor

The mechanism of binding of toxin MT2 from venom of green mamba Dendroaspis angusticeps to muscarinic acetylcholine receptors from rat cerebral cortex was investigated by studying the kinetics of the toxin-receptor interaction. The muscarinic antagonist N-methyl-[3H]scopolamine was used as a 'reporter' ligand. Evidence for a mechanism of toxin-receptor interaction comprising at least two steps was obtained. Such a mechanism increases the potency of the toxin. The first step was fast with no competition between the toxin and the antagonist. The second step was slow with formation of a more stable toxin-receptor complex and inhibition of the antagonist binding. It is proposed that the snake toxin is a muscarinic agonist of slow action.     

Allosteric interactions of quaternary strychnine and brucine derivatives with muscarinic acetylcholine receptors

The affinity and allosteric properties of 22 quaternary derivatives of strychnine and brucine at the m1-m4 subtypes of muscarinic receptors have been analyzed and compared. The subtype selectivity, in terms of affinity, was in general m2 > m4 > m1 > m3. The highest affinities were found for N-benzyl, N-2-naphthylmethyl, and N-4-biphenylylmethyl strychnine (13, 14, and 18, respectively). All the strychnine and brucine derivatives were positively cooperative with the antagonist, N-methylscopolamine, at m2 receptors and, in the case of the strychnine analogues, were positively cooperative with N-methylscopolamine at least at one other subtype. The strychnine analogues were negatively cooperative with the neurotransmitter, acetylcholine, at all subtypes whereas brucine and five of the six derivatives examined were positively cooperative with acetylcholine at one or more subtypes (m1-m5) and exhibited different patterns of subtype selectivity. The ability to generate subtype-selective allosteric enhancers of acetylcholine binding and function may be of use in the development of drugs for the treatment of Alzheimer's disease.     

Endogenous acetylcholine in the dorsal hippocampus reduces anxiety through actions on nicotinic and muscarinic1 receptors

Dorsal hippocampal cholinergic modulation of behavior in different tests of anxiety was investigated by direct injection of the muscarinic M1 and M2 receptor antagonists, pirenzepine and gallamine, and the nicotinic receptor antagonist mecamylamine. In the social interaction test, the anxiogenic effect of pirenzepine (30-100 ng) provided evidence for a tonic cholinergic anxiolytic action mediated by postsynaptic M1 receptors. The anxiogenic action of mecamylamine (30 and 100 ng) was most likely mediated by its action of presynaptic nicotinic receptors to reduce acetylcholine release. Gallamine (10-1,000 ng) was without effect, suggesting that M2 receptors in this brain region do not play a significant role in this behavioral test. On Trial 1 in the elevated plus-maze, the receptor antagonists were without any effect, but in those with a previous 5-min experience of the plus-maze pirenzepine and mecamylamine had anxiogenic effects in the dose range of 30-300 ng; gallamine (100 and 300 ng) was without significant effect.     

Structure and function of glutamate and nicotinic acetylcholine receptors

The past year has seen remarkable progress in defining the structure of various ligand-gated ion channels. Images of opened and closed nicotinic acetylcholine receptors at 9 A resolution have now made it easier to identify the conformational changes underlying gating. In addition, recent studies on glutamate receptors have led to a radical revision of their postulated transmembrane topology: models for agonist-binding and allosteric domains now use sites previously thought to lie in cytoplasmic loops. Other areas that are being actively pursued include identification of the amino acids lining the ion channels, accurate measurements of Ca2+ fluxes, and tests of transmembrane topology in kainate receptor subunits.     

Endogenous acetylcholine in the dorsal hippocampus reduces anxiety through actions on nicotinic and muscarinic? receptors.

Dorsal hippocampal cholinergic modulation of behavior in different tests of anxiety was investigated by direct injection of the muscarinic M? and M? receptor antagonists, pirenzepine and gallamine, and the nicotinic receptor antagonist mecamylamine. In the social interaction test, the anxiogenic effect of pirenzepine (30–100 ng) provided evidence for a tonic cholinergic anxiolytic action mediated by postsynaptic M? receptors. The anxiogenic action of mecamylamine (30 and 100 ng) was most likely mediated by its action of presynaptic nicotinic receptors to reduce acetylcholine release. Gallamine (10–1,000 ng) was without effect, suggesting that M? receptors in this brain region do not play a significant role in this behavioral test. On Trial 1 in the elevated plus-maze, the receptor antagonists were without any effect, but in those with a previous 5-min experience of the plus-maze pirenzepine and mecamylamine had anxiogenic effects in the dose range of 30–300 ng; gallamine (100 and 300 ng) was without significant effect. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Colocalization of muscarinic acetylcholine receptors and protein kinase C gamma in rat parietal cortex

The present investigation analyzes the cellular distribution of muscarinic acetylcholine receptors (mAChRs) and the gamma isoform of protein kinase C (PKC) in the rat parietal cortex employing the monoclonal antibodies M35 and 36G9, respectively. Muscarinic cholinoceptive neurons were most present in layers 2, 3 and 5, whereas most PKC gamma-positive cells were found in layers 2, 5 and 6. Under normal, non-stimulated conditions, approximately 58% of all muscarinic cholinoceptive neurons were immunoreactive for PKC gamma. Conversely, nearly all PKC gamma-positive neurons were M35-immunoreactive. Although both pyramidal and nonpyramidal neurons express the two types of protein, the pyramidal cell type represents the vast majority. Of all cortical neurons, the large (15-25 microns in diameter) muscarinic cholinoceptive pyramidal neurons in layer 5 express the gamma isoform of PKC most abundantly and most frequently. Approximately 96% of these cells are immunoreactive for PKC gamma. Stimulation of mAChRs by the cholinergic agonist carbachol resulted in a pronounced increase in the intensity of 36G9 immunoreactivity, which may suggest that the mAChRs are functionally linked to the colocalized PKC gamma. No change was found in the number of 36G9-immunoreactive neurons. In contrast, the number of immunocytochemically detectable muscarinic cholinoceptive neurons increased by approximately 38% after carbachol stimulation. The high degree of codistribution in cortical neurons of both transduction proteins suggests a considerable cholinergic impact upon the regulation of PKC gamma, a candidate key enzyme in cortical learning and memory mechanisms.     

Interaction of positive allosteric modulators with human and Drosophila recombinant GABA receptors expressed in Xenopus laevis oocytes

1. A comparative study of the actions of structurally diverse allosteric modulators on mammalian (human alpha 3 beta 2 gamma 2L) or invertebrate (Drosophila melanogaster Rdl or a splice variant of Rdl) recombinant GABA receptors has been made using the Xenopus laevis oocyte expression system and the two electrode voltage-clamp technique. 2. Oocytes preinjected with the appropriate cRNAs responded to bath applied GABA with a concentration-dependent inward current. EC50 values of 102 +/- 18 microM; 152 +/- 10 microM and 9.8 +/- 1.7 microM were determined for human alpha 3, beta 1 gamma 2L, Rdl splice variant and the Rdl receptors respectively. 3. Pentobarbitone enhanced GABA-evoked currents mediated by either the mammalian or invertebrate receptors. Utilizing the appropriate GABA EC10, the EC50 for potentiation was estimated to be 45 +/- 1 microM, 312 +/- 8 microM and 837 +/- 25 microM for human alpha 3, beta 1 gamma 2L, Rdl splice variant and Rdl receptors respectively. Maximal enhancement (expressed relative to the current induced by the EC10 concentration of GABA where this latter response = 1) at the mammalian receptor (10.2 +/- 1 fold) was greater that at either the Rdl splice variant (5.5 +/- 1.3 fold) or Rdl (7.9 +/- 0.8 fold) receptors. 4. Pentobarbitone directly activated the human alpha 3 beta 1 gamma 2L receptor with an EC50 of 1.2 +/- 0.03 mM and had a maximal effect amounting to 3.3 +/- 0.4 fold of the response evoked by the EC10 concentration of GABA. Currents evoked by pentobarbitone were blocked by 10-30 microM picrotoxin and potentiated by 0.3 microM flunitrazepam. Pentobarbitone did not directly activate the invertebrate GABA receptors. 5. 5 alpha-Pregnan-3 alpha-ol-20-one potentiated GABA-evoked currents mediated by the human alpha 3 beta 1 gamma 2L receptor with an EC50 of 87 +/- 3 nM and a maximal enhancement of 6.7 +/- 0.8 fold of that produced by the GABA EC10 concentration. By contrast, relatively high concentrations (3-10 microM) of this steroid had only a modest effect on the Rdl receptor and its splice variant. 6. A small direct effect of 5 alpha-pregnan-3 alpha-ol-20-one (0.3-10 microM) was detected for the human alpha 3 beta 1 gamma 2L receptor (maximal effect only 0.08 +/- 0.01 times that of the GABA EC10). This response was antagonized by 30 microM picrotoxin and enhanced by flunitrazepam (0.3 microM). 5 alpha-Pregnan-3 alpha-ol-20-one did not directly activate the invertebrate GABA receptors. 7. Propofol enhanced GABA-evoked currents mediated by human alpha 3 beta 1 gamma 2L and Rdl splice variant receptors with EC50 values of 3.5 +/- 0.1 microM and 8 +/- 0.3 microM respectively. The maximal enhancement was similar at the two receptor types (human 11 +/- 1.8 fold; invertebrate 8.8 +/- 1.4 fold that of the GABA EC10). 8. Propofol directly activated the human alpha 3 beta 1 gamma 2L receptor with an EC50 of 129 +/- 10 microM, and at a maximally effective concentration, evoked a current amounting to 3.5 +/- 0.5 times that elicited by a concentration of GABA producing 10% of the maximal response. The response to propofol was blocked by 10-30 microM picrotoxin and enhanced by flunitrazepam (0.3 microM). Propofol did not directly activate the invertebrate Rdl splice variant receptor. 9. GABA-evoked currents mediated by the human alpha 3 beta 1 gamma 2L receptor were potentiated by etomidate (EC50 = 7.7 +/- 0.2 microM) and maximally enhanced to 8 +/- 0.8 fold of the response to an EC10 concentration of GABA. By contrast, the Rdl, or Rdl splice variant forms of the invertebrate GABA receptor were insensitive to the positive allosteric modulating actions of etomidate. Neither the mammalian nor the invertebrate receptors, were directly activated by etomidate. 10. delta-Hexachlorocyclohexane enhanced GABA-evoked currents with EC50 values of 3.4 +/- 0.1 microM and 3.0 +/- 0.1 microM for the human alpha 3 beta 1 gamma 2L receptor and the Rdl splice variant receptor respectively. The maximal enhancement was 4.5     

Opposite effects of lanthanum on different types of nicotinic acetylcholine receptors

The effects of lanthanum (La3+) were studied on muscle and neuronal nicotinic acetylcholine receptors (AChRs) expressed in Xenopus oocytes. La3+ exerts a dose-dependent positive modulation on alpha1 beta1 gamma8 muscle AChRs, whereas it modulates negatively either alpha2 beta2, alpha2 beta4 or alpha3 beta4 neuronal AChRs. Moreover, La3+ appears to accelerate the desensitization of neuronal receptors. In both muscle and neuronal AChRs, the respective potentiating or inhibiting effects of La3+ on the ACh-currents are voltage-independent, suggesting that La3+ is acting at a site located in the external domain of the receptor.     

Anticholinergic effects of strychnine in the cochlea do not involve muscarinic receptors

Central control of cochlear function is mediated by the cholinergic (medial) efferent system and both muscarinic and nicotinic acetylcholine receptors are thought to be present on outer hair cells. All the physiological effects of acetylcholine in the cochlea are blocked by strychnine and we therefore investigated whether strychnine interacts with muscarinic receptors in the cochlea. The effects of strychnine on both (3H)-quinuclidinyl benzylate binding and atropine sensitive carbachol-induced (3H)-inositol phosphate formation were examined. Strychnine (1 to 50 microM) has no effect on either quinuclidinyl benzylate binding or carbachol (1 mM)-induced inositol phosphate synthesis. Moreover, strychnine does not change basal inositol phosphate metabolism. These data indicate that muscarinic receptors are not sensitive to strychnine at concentrations which are known to block the effects of acetylcholine on outer hair cells.     

Single channel properties of synaptic acetylcholine receptors in dystrophic fibers

Neuromuscular transmission in dystrophic mice has been extensively studied through analysis of nerve-evoked endplate potentials. In the present study, single channel ACh-induced activity recorded from endplates of fast twitch muscle fibers from mdx and dy dystrophic mice, was compared with activity recorded from wild-type fiber endplates to ascertain whether expression of these phenotypes leads to changes in ACh receptor properties and synaptic transmission. An 89 pS class of ACh-induced events predominated in recordings from wild-type and both strains of dystrophic mice. The mean open times for this class of events was well described by two exponential components, one with a voltage-independent time constant of approximately 0.3 ms and the other with a time constant of 2-5 ms which increased e-fold with approximately 120 mV of hyperpolarization. The expression of the mdx or the dy phenotype was not associated with significant differences in the conductance, distribution of open durations, or the voltage-dependence of the mean open time for this class of ACh-induced events.     

Endothelin-1 increases the levels of mRNA and protein of muscarinic acetylcholine receptors and c-fos mRNA in cerebellar granule cells

Endothelin-1 (ET-1) induced a time- and dose-dependent increase in the levels of mRNA of m2- and m3-muscarinic acetylcholine receptors (mAChRs) in cultured cerebellar granule cells. The levels of immunoprecipitable m3-mAChR protein and total mAChR binding sites were also increased by ET-1 treatment. The up-regulation of m2- and m3-mAChR was blocked by phorbol ester pretreatment to inhibit ET-1-stimulated phosphoinositide hydrolysis and was preceded by an increase in c-fos mRNA levels. Treatments that prevented ET-1-induced c-fos mRNA increase also abolished the subsequent m2- and m3-mAChR mRNA up-regulation, suggesting that c-Fos protein is involved in the ET-1-induced mAChR expression.     

Critical elements determining diversity in agonist binding and desensitization of neuronal nicotinic acetylcholine receptors

To identify the molecular determinants underlying the pharmacological diversity of neuronal nicotinic acetylcholine receptors, we compared the alpha7 homo-oligomeric and alpha4beta2 hetero-oligomeric receptors. Sets of residues from the regions initially identified within the agonist binding site of the alpha4 subunit were introduced into the alpha7 agonist binding site, carried by the homo-oligomeric alpha7-V201-5HT3 chimera. Introduction of the alpha4 residues 183-191 into alpha7 subunit sequence (chimera C2) selectively increased the apparent affinities for equilibrium binding and for ion channel activation by acetylcholine, resulting in a receptor that no longer displays differences in the responses to acetylcholine and nicotine. Introduction of the alpha4 residues 151-155 (chimera B) produced a approximately 100-fold increase in the apparent affinity for both acetylcholine and nicotine in equilibrium binding measurements. In both cases electrophysiological recordings revealed a much smaller increase (three- to sevenfold) in the apparent affinity for activation, but the concentrations required to desensitize the mutant chimeras parallel the shifts in apparent binding affinity. The data were fitted by a two-state concerted model, and an alteration of the conformational isomerization constant leading to the desensitized state accounts for the chimera B phenotype, whereas alteration of the ligand binding site accounts for the chimera C2 phenotype. Point mutation analysis revealed that several residues in both fragments contribute to the phenotypes, with a critical effect of the G152K and T183N mutations. Transfer of alpha4 amino acids 151-155 and 183-191 into the alpha7-V201-5HT3 chimera thus confers physiological and pharmacological properties typical of the alpha4beta2 receptor.