Pharmacological modulation of the diazepam-insensitive recombinant gamma-aminobutyric acidA receptors alpha 4 beta 2 gamma 2 and alpha 6 beta 2 gamma 2

We characterized modulation of the gamma-aminobutyric acid (GABA)-evoked responses of the diazepam-insensitive alpha 4 beta 2 gamma2 and alpha 6 beta 2 gamma 2 recombinant GABAA receptors. The partial agonist bretazenil potentiated the responses of both receptors with similar dose dependence but with a higher maximal enhancement at the alpha 4 beta 2 gamma 2 receptor. The bretazenil-induced potentiation was reduced by the benzodiazepine antagonist flumazenil. At a high concentration (10 microM), flumazenil was a weak potentiator of the GABA response. The partial agonist imidazenil was inactive. The imidazobenzodiazepine inverse agonist Ro 15-4513, which is known to bind with high affinity to the alpha 6 beta 2 gamma 2 receptor, potentiated the GABA responses of the alpha 4 beta 2 gamma 2 and alpha 6 beta 2 gamma 2 receptor subtypes with similar dose dependence over the concentration range of 0.1-10 microM. Methyl-6, 7-dimethoxy-4-ethyl-beta-carboline, a beta-carboline inverse agonist, had a similar potentiating effect when tested at a concentration of 10 microM. The alpha 4 beta 2 gamma 2 and alpha 6 beta 2 gamma 2 receptor-mediated currents had equal sensitivities to furosemide and Zn2+ ions, both of which reduced the GABA-evoked responses. The alpha 6 beta 2 gamma 2 receptor but not the alpha 4 beta 2 gamma 2 receptor exhibited a low level of spontaneous activity in the absence of GABA; this resting current could be directly potentiated by Ro 15-4513, methyl-6,7-dimethoxy-4-ethyl-beta-carboline, bretazenil and flumazenil and was blocked by picrotoxin. Thus, although the alpha 4 beta 2 gamma 2 receptors are insensitive to benzodiazepine binding site full agonists, such as diazepam, they can be modulated by certain ligands acting as partial and inverse agonists at diazepam-sensitive receptors and thereby contribute to the respective pharmacological profiles.     

(+)-Anatoxin-a is a potent agonist at neuronal nicotinic acetylcholine receptors

The effects of the nicotinic agonist (+)-anatoxin-a have been examined in four different preparations, representing at least two classes of neuronal nicotinic receptors. (+)-Anatoxin-a was most potent (EC50 = 48 nM) in stimulating 86Rb+ influx into M10 cells, which express the nicotinic receptor subtype comprising alpha 4 and beta 2 subunits. A presynaptic nicotinic receptor mediating acetylcholine release from hippocampal synaptosomes was similarly sensitive to (+)-anatoxin-a (EC50 = 140 nM). alpha-Bungarotoxin-sensitive neuronal nicotinic receptors, studied using patch-clamp recording techniques, required slightly higher concentrations of this alkaloid for activation: Nicotinic currents in hippocampal neurons were activated by (+)-anatoxin-a with an EC50 of 3.9 microM, whereas alpha 7 homooligomers reconstituted in Xenopus oocytes yielded an EC50 value of 0.58 microM for (+)-anatoxin-a. In these diverse preparations, (+)-anatoxin-a was between three and 50 times more potent than (-)-nicotine and approximately 20 times more potent than acetylcholine, making it the most efficacious nicotinic agonist thus far described.     

Rat alpha3/beta4 subtype of neuronal nicotinic acetylcholine receptor stably expressed in a transfected cell line: pharmacology of ligand binding and function

We stably transfected human kidney embryonic 293 cells with the rat neuronal nicotinic acetylcholine receptor (nAChR) alpha3 and beta4 subunit genes. This new cell line, KXalpha3 beta4R2, expresses a high level of the alpha3/beta4 receptor subtype, which binds (+/-)- [3H]epibatidine with a Kd value of 304+/-16 pM and a Bmax value of 8942 +/- 115 fmol/mg protein. Comparison of nicotinic drugs in competing for alpha3/beta4 receptor binding sites in this cell line and the binding sites in rat forebrain (predominantly alpha4/beta2 receptors) revealed marked differences in their Ki values, but similar rank orders of potency for agonists were observed, with the exception of anatoxin-A. The affinity of the competitive antagonist dihydro-beta-erythroidine is >7000 times higher at alpha4/beta2 receptors in rat forebrain than at the alpha3/beta4 receptors in these cells. The alpha3/beta4 nAChRs expressed in this cell line are functional, and in response to nicotinic agonists, 86Rb+ efflux was increased to levels 8-10 times the basal levels. Acetylcholine, (-)-nicotine, cytisine, carbachol, and (+/-)-epibatidine all stimulated 86Rb+ efflux, which was blocked by mecamylamine. The EC50 values for acetylcholine and (-)-nicotine to stimulate 86Rb+ effluxes were 114 +/- 24 and 28 +/- 4 microM, respectively. The rank order of potency of nicotinic antagonists in blocking the function of this alpha3/beta4 receptor was mecamylamine > d-tubocurarine > dihydro-beta-erythroidine > hexamethonium. Mecamylamine, d-tubocurarine, and hexamethonium blocked the function by a noncompetitive mechanism, whereas dihydro-beta-erythroidine blocked the function competitively. The KXalpha3 beta4R2 cell line should prove to be a very useful model for studying this subtype of nAChRs.     

Four pharmacologically distinct subtypes of alpha4beta2 nicotinic acetylcholine receptor expressed in Xenopus laevis oocytes

Nicotinic receptors generally are presumed to consist of two alpha and three non-alpha subunits. We varied the relative levels of expression of the neuronal nicotinic alpha4 and beta2 receptor subunits in Xenopus laevis oocytes by nuclear injection of cDNAs coding for these subunits in alpha:beta ratios of 9:1, 1:1, and 1:9. The sensitivities of the receptors to acetylcholine and d-tubocurarine were investigated in voltage-clamp experiments. For receptors expressed at the 9:1 and 1:1 alpha:beta ratios, the EC50 value of acetylcholine is approximately 60 microM. For the majority of the receptors expressed at the 1:9 alpha:beta ratio, the sensitivity to acetylcholine is enhanced 30-fold. No evidence for more than one type of acetylcholine binding site in a single receptor is obtained. The sensitivity to d-tubocurarine decreases with decreasing alpha:beta ratio. IC50 values of d-tubocurarine are 0.2, 0.5, and 2 microM for the 9:1, 1:1, and 1:9 alpha:beta ratios, respectively. At the 1:9 alpha:beta ratio, additional receptors with an IC50 value of 163 microM d-tubocurarine are expressed. At least two components with distinct sensitivities to d-tubocurarine are required to account for the shift in IC50. The combined agonist and antagonist effects reveal four distinct subtypes of alpha4beta2 nicotinic receptors. The results imply that the subunit stoichiometry of heteromeric alpha4beta2 acetylcholine receptors is not restricted to 2alpha:3beta.     

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HEK293 cells were stably transfected with rat neuronal nicotinic alpha4 and beta2 subunits. Binding of tritiated cytisine and nicotine to cell homogenates revealed the presence of a single class of high-affinity sites (dissociation constants 0.1 nM and 0.4 nM, respectively). Activation of nicotinic receptors was studied using whole-cell patch clamp methods, and acetylcholine, nicotine, dimethylphenylpiperazinium, and cytisine all produced a conductance increase. Responses desensitized to prolonged applications, at both positive and negative membrane potentials. The conductance was strongly rectifying, and outward currents were essentially absent. Responses were maximal at about 2 mM external calcium ion concentration and were reduced by about one-half at either nominally 0 or 10 mM external calcium. Di-hydro-beta-erythroidine blocked physiological responses to acetylcholine and nicotine (IC50, 2.5 nM), and reduced cytisine binding in a competitive manner (Ki 20 nM). Physostigmine enhanced the response to low concentrations of acetylcholine or nicotine. The anesthetic steroid (+)-3alpha-hydroxy-5alpha-androstane-17beta-carbonitrile blocked responses to acetylcholine (IC50, 1.3 microM), but had no effect on cytisine binding at a concentration of 30 microM. The binding properties of the receptors are those expected for rat neuronal nicotinic receptors composed of alpha4 and beta2 subunits. The pharmacological properties indicate that the responsiveness of the receptors may be allosterically enhanced or inhibited.     

Interactions between tachykinins and diverse, human nicotinic acetylcholine receptor subtypes

Nicotinic acetylcholine receptors (nAChR) are diverse members of the ligand-gated ion channel superfamily of neurotransmitter receptors and play critical roles in chemical signaling throughout the nervous system. Reports of effects of substance P (SP) on nAChR function prompted us to investigate interactions between several tachykinins and human nAChR subtypes using clonal cell lines as simple experimental models. Acute exposure to SP inhibits carbamylcholine- or nicotine-stimulated function measured using 86Rb+ efflux assays of human ganglionic (alpha 3 beta 4) nAChR expressed in SH-SY5Y neuroblastoma cells (IC50 approximately 2.3 microM) or of human muscle-type (alpha 1 beta 1 gamma delta) nAChR expressed in TE671/RD clonal cells (IC50 approximately 21 microM). SP also acutely blocks function of rat ganglionic nAChR expressed in PC12 pheochromocytoma cells (IC50 approximately 2.1 microM). Neurokinin A and eledoisin inhibit function (extrapolated IC50 values between 60 and 160 microM) of human muscle-type or ganglionic nAChR, but neurokinin B does not, and neither human nAChR is as sensitive as PC12 cell alpha 3 beta 4-nAChR to eledoisin or neurokinin A inhibition. At concentrations that produce blockade of nAChR function, SP fails to affect binding of [3H]acetylcholine to human muscle-type or ganglionic nAChR. SP-mediated blockade of rat or human ganglionic nAChR function is insurmountable by increasing agonist concentrations. Collectively, these results indicate that tachykinins act noncompetitively to inhibit human nAChR function with potencies that vary across tachykinins and nAChR subtypes. They also indicate that tachykinin actions at nAChR could further contribute to complex cross-talk between nicotinic cholinergic and tachykinin signals in regulation of nervous system activity.     

Anabaseine is a potent agonist on muscle and neuronal alpha-bungarotoxin-sensitive nicotinic receptors

We assessed the pharmacological activity of anabaseine, a toxin found in certain animal venoms, relative to nicotine and anabasine on a variety of vertebrate nicotinic receptors, using cultured cells, the Xenopus oocyte expression system, contractility assays with skeletal and smooth muscle strips containing nicotinic receptors and in vivo rat prostration assay involving direct injection into the lateral ventricle of the brain. Anabaseine stimulated every subtype of nicotinic receptor that was tested. It was the most potent frog skeletal muscle nicotinic receptor agonist. At higher concentrations it also blocked the BC3H1 (adult mouse) muscle type receptor ion channel. The affinities of the three nicotinoid compounds for rat brain membrane alpha-bungarotoxin binding sites and their potencies for stimulating Xenopus oocyte homomeric alpha7 receptors, expressed in terms of their active monocation concentrations, displayed the same rank order, anabaseine>anabasine> nicotine. Although the maximum currents generated by anabaseine and anabasine at alpha7 receptors were equivalent to that of acetylcholine, the maximum response to nicotine was only about 65% of the acetylcholine response. At alpha4-beta2 receptors the affinities and apparent efficacies of anabaseine and anabasine were much less than that of nicotine. Anabaseine, nicotine and anabasine were nearly equipotent on sympathetic (PC12) receptors, although parasympathetic (myenteric plexus) receptors were much more sensitive to anabaseine and nicotine but less sensitive to anabasine. These differences suggest that there may be different subunit combinations in these two autonomic nicotinic receptors. The preferential interactions of anabaseine, anabasine and nicotine with different receptor subtypes provides molecular clues that should be helpful in the design of selective nicotinic agonists.     

Adaptive resonance theory

We report that preapplication of ivermectin, in the micromolar range, strongly enhances the subsequent acetylcholine-evoked current of the neuronal chick or human alpha7 nicotinic acetylcholine receptors reconstituted in Xenopus laevis oocytes and K-28 cells. This potentiation does not result from nonspecific Cl- currents. The concomitant increase in apparent affinity and cooperativity of the dose-response curve suggest that ivermectin acts as a positive allosteric effector. This interpretation is supported by the observation of an increase in efficiency of a partial agonist associated with the potentiation and by the differential effect of ivermectin on mutants within the M2 channel domain. Ivermectin effects reveal a novel allosteric site for pharmacological agents on neuronal alpha7 nicotinic acetylcholine receptors.     

Benzodiazepine modulation of recombinant alpha1beta3gamma2 GABA(A) receptor function efficacy determination using the Cytosensor microphysiometer

The nonsteroidal antioestrogen tamoxifen has been shown to block a number of voltage-gated cation-selective channels but its effect on ligand-gated cation-selective channels has not been studied. We have investigated the action of tamoxifen and the related derivative toremifene on ligand-gated cationic nicotinic acetylcholine and 5-HT3 receptor channels. Tamoxifen and toremifene both inhibited cationic currents of adult-type human muscle nicotinic acetylcholine receptors expressed in Xenopus oocytes with similar IC50 values of 1.2 +/- 0.03 microM (nH = 0.84 +/- 0.02) and 1.2 +/- 0.1 microM (nH = 1.1 +/- 0.1), respectively. Tamoxifen could also block native 5-HT3 receptors in NG108-15 neuroblastoma/glioma hybrid cells with IC50 = 0.81 +/- 0.15 microM and nH of 1.3 +/- 0.3. The characteristics of block by tamoxifen at the 5-HT3 receptor were voltage- and use-independent. The inhibition of the 5-HT-evoked currents were not overcome by increasing concentrations of 5-HT consistent with a noncompetitive mechanism of block.     

Functional characterization of a mutated chicken alpha7 nicotinic acetylcholine receptor subunit with a leucine residue inserted in transmembrane domain 2

1. Site-directed mutagenesis was used to create an altered form of the chicken alpha7 nicotinic acetylcholine (ACh) receptor subunit (alpha7x61) in which a leucine residue was inserted between residues Leu9' and Ser10' in transmembrane domain 2. The properties of alpha7x61 receptors are distinct from those of the wild-type receptor. 2. Oocytes expressing wild-type alpha7 receptors responded to 10 microM nicotine with rapid inward currents that desensitized with a time-constant of 710+/-409 ms (mean+/-s.e.mean, n=5). However in alpha7x61 receptors 10 microM nicotine resulted in slower onset inward currents that desensitized with a time-constant of 5684+/-3403 ms (mean+/-s.e.mean, n = 4). No significant difference in the apparent affinity of nicotine or acetylcholine between mutant and wild-type receptors was observed. Dihydro-beta-erythroidine (DHbetaE) acted as an antagonist on both receptors. 3. Molecular modelling of the alpha7x61 receptor channel pore formed by a bundle of M2 alpha-helices suggested that three of the channel lining residues would be altered by the leucine insertion i.e.; Ser10 would be replaced by the leucine insertion, Val13' and Phe14' would be replaced, by Thr and Val, respectively. 4 When present in the LEV-1 nicotinic ACh receptor subunit from Caenorhabditis elegans the same alteration conferred resistance to levamisole anthelmintic drug. Levamisole blocked responses to nicotine of wild-type and alpha7x61 receptors. However, block was more dependent on membrane potential for the alpha7x61 receptors. 5. We conclude that the leucine insertion in transmembrane domain 2 has the unusual effect of slowing desensitization without altering apparent agonist affinity.     

Cloning and functional characterisation of two novel nicotinic acetylcholine receptor alpha subunits from the insect pest Myzus persicae

Nicotinic acetylcholine receptors play a major role in excitatory neurotransmission in insect CNSs and constitute an important target for insecticides. Here, we report the isolation and functional characterisation of two cDNAs encoding nicotinic acetylcholine receptor alpha subunits from a major insect pest, the peach-potato aphid Myzus persicae. These two subunits, termed Mp alpha1 and Mp alpha2, are respective structural homologues of the Drosophila D alpha2/Schistocerca gregaria alphaL1 alpha-subunit pair and the Drosophila ALS alpha subunit. Xenopus oocyte expression confirmed that each Myzus subunit can form functional acetylcholine- or nicotine-gated channels. However, some electrophysiological and pharmacological properties of the Myzus subunits were distinct from those encoded by the corresponding Drosophila subunits. Coexpression of the Myzus subunits with the chick beta2 subunit revealed other differences from the Drosophila system, as only very limited potentiation of agonist-induced currents was observed with Mp alpha2 and none with Mp alpha1. Available data therefore indicate that structurally homologous insect nicotinic acetylcholine receptor alpha subunits from different species can exhibit distinctive physiological and pharmacological characteristics.     

Developmental modulation of mouse hypoglossal nerve inspiratory output in vitro by noradrenergic receptor agonists

The ontogeny of the noradrenergic receptor subtypes modulating hypoglossal (XII) nerve inspiratory output was characterized. Noradrenergic agents were locally applied over the XII nucleus of rhythmically active medullary slice preparations isolated from mice between zero and 13 days of age (P0-P13) and the effects on XII inspiratory burst amplitude quantified. The alpha1 receptor agonist phenylephrine (PE, 0.1-10 microM) produced a dose-dependent, prazosin-sensitive (0.1-10 microM) increase in XII nerve inspiratory burst amplitude. The magnitude of this potentiation increased steadily from a maximum of 15+/-8% in P0 mice to 134+/-4% in P12-P13 mice. The beta receptor agonist isoproterenol (0.01-1.0 mM) produced a prazosin-insensitive, propranolol-sensitive potentiation of XII nerve burst amplitude. The isoproterenol-mediated potentiation increased with development from 27+/-5% in P0-P1 slices, to 37+/-3% in P3 slices and 45+/-4% in P9-P10 slices. The alpha2 receptor agonist clonidine (1 mM) reduced XII nerve inspiratory burst amplitude in P0-P3 slices by 29+/-5%, but had no effect on output from P12-P13 slices. An alpha2 receptor-mediated inhibition of inspiratory activity in neonates (P0-P3) was further supported by a 19+/-3% reduction in XII nerve burst amplitude when norepinephrine (NE, 100 microM) was applied in the presence of prazosin (10 microM) and propranolol (100 microM). Results indicate that developmental increases in potentiating alpha1 and, to a lesser extent, beta receptor mechanisms combine with a developmentally decreasing inhibitory mechanism, most likely mediated by alpha2 receptors, to determine the ontogenetic time course by which NE modulates XII MN inspiratory activity.     

Myosin light chain 2a and 2v identifies the embryonic outflow tract myocardium in the developing rodent heart

BACKGROUND: Volatile general anesthetics increase agonist-mediated ion flux through the gamma-aminobutyric acid(A), glycine, and 5-hydroxytryptamine3 (5-HT3) receptors. This action reflects an anesthetic-induced increase in the apparent agonist affinity of these receptors. In contrast, volatile anesthetics block ion flux through the nicotinic acetylcholine receptor (nAcChoR). The authors tested the hypothesis that in addition to blocking ion flux through the nAcChoR, isoflurane also increases the apparent affinity of the nAcChoR for agonist. METHODS: Nicotinic acetylcholine receptors were obtained from the electroplax organ of Torpedo nobiliana. The apparent agonist affinity of the nAcChoR was determined using a new stopped-flow fluorescence assay. This assay derives the apparent agonist affinity of the nAcChoR from the apparent rates with which agonists convert nAcChoRs from the resting state to the desensitized state. RESULTS: Isoflurane significantly increased the apparent affinity (decreased the apparent dissociation constant) of acetylcholine for the nAcChoR at clinically relevant concentrations. The apparent dissociation constant decreased exponentially with the isoflurane concentration from a control value of 44+/-4 microM to 1.0+/-0.1 microM in the presence of 1.5 mM isoflurane, the highest concentration studied. CONCLUSIONS: Isoflurane increases the apparent agonist affinity of the nAcChoR; however, this effect is poorly resolved in ion flux studies because isoflurane also causes channel blockade. The lack of saturation of isoflurane's effect on the apparent agonist affinity even at relatively high isoflurane concentrations argues against a single site of anesthetic action. However, it is consistent with isoflurane interactions with several receptor sites that exhibit a range of anesthetic affinities, sites within the membrane lipid, or both.     

Choline is a selective agonist of alpha7 nicotinic acetylcholine receptors in the rat brain neurons

In the present study, we demonstrate that choline, a precursor of acetylcholine (ACh) and a product of acetylcholine hydrolysis by acetylcholinesterase (AChE), acts as an efficient and relatively selective agonist of alpha7-containing nicotinic acetylcholine receptors (nAChR) in neurons cultured from the rat hippocampus, olfactory bulb and thalamus as well as in PC12 cells. Choline was able to activate postsynaptic and presynaptic alpha7 nAChRs, with the latter action resulting in the release of other neurotransmitters. Although choline was approximately one order of magnitude less potent than ACh (EC50 of 1.6 mM for choline and 0.13 mM for ACh), it acted as a full agonist at alpha7 nAChRs. In contrast, choline did not activate alpha4beta2 agonist-bearing nAChRs on hippocampal neurons, and acted as a partial agonist at alpha3beta4-containing nAChRs on PC12 cells. The ethyl alcohol moiety of choline is required for the selective action on alpha7 nAChR. Exposure of cultured hippocampal neurons for 10 min to choline (10-100 microM) resulted in desensitization of the native alpha7 nAChRs. Moreover, chronic exposure (10 days) of the cultured hippocampal neurons to a desensitizing concentration of choline (approximately 30 microM) decreased their responsiveness to ACh. The selective action of choline on native alpha7 nAChRs suggests that this naturally occurring compound may act in vivo as an endogenous ligand for these receptors. Putative physiological actions of choline include retrograde messenger activity during the development of the mammalian central nervous system and during periods of elevated synaptic activity that leads to long-term potentiation.     

Characterization of human recombinant neuronal nicotinic acetylcholine receptor subunit combinations alpha2beta4, alpha3beta4 and alpha4beta4 stably expressed in HEK293 cells

Human embryonic kidney (HEK293) cells were transfected with cDNA encoding the human beta4 neuronal nicotinic acetylcholine (ACh) receptor subunit in pairwise combination with human alpha2, alpha3 or alpha4 subunits. Cell lines A2B4, A3B4.2 and A4B4 were identified that stably express mRNA and protein corresponding to alpha2 and beta4, to alpha3 and beta4 and to alpha4 and beta4 subunits, respectively. Specific binding of [3H]epibatidine was detected in A2B4, A3B4.2 and A4B4 cells with Kd (mean +/- S.D. in pM) values of 42 +/- 10, 230 +/- 12 and 187 +/- 29 and with Bmax (fmol/mg protein) values of 1104 +/- 338, 2010 +/- 184 and 3683 +/- 1450, respectively. Whole-cell patch-clamp recordings in each cell line demonstrated that (-)nicotine (Nic), ACh, cytisine (Cyt) and 1, 1-dimethyl-4-phenylpiperazinium iodide (DMPP) elicit transient inward currents. The current-voltage (I-V) relation of these currents showed strong inward rectification. Pharmacological characterization of agonist-induced elevations of intracellular free Ca++ concentration revealed a distinct rank order of agonist potency for each subunit combination as follows: alpha2beta4, (+)epibatidine (Epi) > Cyt > suberyldicholine (Sub) = Nic = DMPP; alpha3beta4, Epi > DMPP = Cyt = Nic = Sub; alpha4beta4, Epi > Cyt = Sub > Nic > DMPP. The noncompetitive antagonists mecamylamine and d-tubocurarine did not display subtype selectivity. In contrast, the Kb value for the competitive antagonist dihydro-beta-erythroidine (DHbetaE) was highest at alpha3beta4 compared with alpha2beta4 or alpha4beta4 receptors. These data illustrate that the A2B4, A3B4.2 and A4B4 stable cell lines are powerful tools for examining the functional and pharmacological properties of human alpha2beta4, alpha3beta4 and alpha4beta4 neuronal nicotinic receptors.     

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.     

Human high affinity, Na(+)-dependent L-glutamate/L-aspartate transporter GLAST-1 (EAAT-1): gene structure and localization to chromosome 5p11-p12

Controversies remain over which ion channels are the most sensitive to ethanol. We have found that ethanol potently modulates the neuronal nicotinic acetylcholine receptor-channel at micromolar concentrations with an EC50 of 88.5 microM, which is significantly lower than most values previously reported for other ion channels. Prolonged application of ethanol accelerated the decay phase of acetylcholine-induced currents, caused single-channels to open in bursts, and shortened the mean open time, all of which reflect increased receptor desensitization. However, ethanol slowed the decay phase of the current induced by a brief application of acetylcholine, which may indicate that ethanol manifests its action by causing an increase in the affinity of the receptor for acetylcholine. These results suggest that neuronal nicotinic acetylcholine receptors may be important target sites of ethanol, particularly in the early stages of ethanol intoxication.     

Rapid increase in cytosolic calcium ion concentration mediated by acetylcholine receptors in cultured retinal neurons and Müller cells

PURPOSE: This study was conducted to detect the presence of muscarinic or nicotinic receptors in cultured retinal neurons and Müller cells. METHODS: Pure Müller cell cultures and cocultures of retinal neurons and Müller cells were used; the former, obtained from adult rabbit retinas, and the latter, retinal neurons from neonatal rats, were cocultured with Müller cells. Intracellular calcium ion concentration ([Ca2+]i) following the administration of acetylcholine, a cholinesterase inhibitor (trichlorfon), nicotine or muscarinic agonist with or without a receptor antagonist was monitored using the calcium ion indicator, fura-2. RESULTS: Acetylcholine and trichlorfon induced rapid increase in [Ca2+]i in half of either cell type. Trichlorfon induced positive response in coculture but not in the pure Müller cell cultures. This positive response was blocked only partially in the presence of atropine. Approximately 30-40% of neurons responded to nicotine at 5 microM, which was significantly blocked by alpha-bungarotoxin at 50 nM. No response to nicotine could be detected in Müller cells. Approximately 50% of neurons responded to muscarine at 50 microM, but 500 microM was required for the formation of calcium transients in 50% of Müller cells. The muscarine inducement of rapid increase in [Ca2+]i was blocked by atropine. The agonist of M1 (a muscarinic receptor subtype), McN-A-343, at 0.5 microM induced the most significant and rapid increase in [Ca2+]i both in neurons and Müller cells. McN-A-343 administration at 0.05 microM induced positive response in half the neurons, but only in approximately 10% of Müller cells. Such positive response was not observed following preincubation with the M1 antagonist, pirenzepine, at 50 microM. CONCLUSIONS: Cocultured retinal neurons enhance the release of acetylcholine following anticholinesterase administration, and approximately half the neurons were found to possess muscarinic and nicotinic receptors. However, Müller cells appeared to possess only the less sensitive muscarinic receptor. Muscarinic receptor subtypes on either type of cell contained at least M1.     

alpha-Conotoxin ImI exhibits subtype-specific nicotinic acetylcholine receptor blockade: preferential inhibition of homomeric alpha 7 and alpha 9 receptors

Through a study of cloned nicotinic receptors expressed in Xenopus oocytes, we provide evidence that alpha-conotoxin ImI, a peptide marine snail toxin that induces seizures in rodents, selectively blocks subtypes of nicotinic acetylcholine receptors. alpha-Conotoxin ImI blocks homomeric alpha 7 nicotinic receptors with the highest apparent affinity and homomeric alpha 9 receptors with 8-fold lower affinity. This toxin has no effect on receptors composed of alpha 2 beta 2, alpha 3 beta 2, alpha 4 beta 2, alpha 2 beta 4, alpha 3 beta 4, or alpha 4 beta 4 subunit combinations. In contrast to alpha-bungarotoxin, which has high affinity for alpha 7, alpha 9, and alpha 1 beta 1 gamma delta receptors, alpha-conotoxin ImI has low affinity for the muscle nAChR. Related Conus peptides, alpha-conotoxins MI and GI, exhibit a distinct specificity, strictly targeting the muscle subtype receptor but not alpha 7 or alpha 9 receptors. alpha-Conotoxins thus represent selective tools for the study of neuronal nicotinic acetylcholine receptors.     

Cyanotriphenylborate: subtype-specific blocker of glycine receptor chloride channels

The inhibitory glycine receptor is a ligand-gated ion-channel protein existing in different homo- and heterooligomeric isoforms. Here we show that the chloride channel of the recombinant alpha 1-subunit homooligomeric glycine receptor is efficiently blocked by cyanotriphenylborate (CTB) with a concentration effecting 50% inhibition (IC50) of 1.3 microM in the presence of 50 microM glycine. The antagonistic effect of CTB is noncompetitive, use dependent, and more pronounced at positive membrane potentials, suggesting open-channel block. In contrast to alpha 1-subunit receptors, alpha 2-subunit homooligomers are resistant to CTB (IC50 > 20 microM). By exchanging the channel-lining transmembrane segment M2 of the alpha 1 polypeptide by that of the alpha 2 polypeptide, we could transfer this resistance to alpha 1 channels, indicating that a single glycine residue at position 254 of the alpha 1 subunit is critical for CTB sensitivity. The blocker did not affect the cation-selective channel of the nicotinic acetylcholine receptor. Thus, CTB may prove useful as a tool to probe the subunit structure of native glycine receptors in mammalian neurons.