Sonia Bertrand

A neuronal nicotinic acetylcholine receptor subunit (α7) is developmentally regulated and forms a homo-oligomeric channel blocked by α-BTX

cDNA and genomic clones encoding alpha 7, a novel neuronal nicotinic acetylcholine receptor (nAChR) alpha subunit, were isolated and sequenced. The mature alpha 7 protein (479 residues) has moderate homology with all other alpha and non-alpha nAChR subunits and probably assumes the same transmembrane topology. alpha 7 transcripts transiently accumulate in the developing optic tectum between E5 and E16. They are present in both the deep and the superficial layers of E12 tectum. In Xenopus oocytes, the alpha 7 protein assembles into a homo-oligomeric channel responding to acetylcholine and nicotine. The alpha 7 channel desensitizes very rapidly, rectifies strongly above -20 mV, and is blocked by alpha-bungarotoxin. A bacterial fusion protein encompassing residues 124-239 of alpha 7 binds labeled alpha-bungarotoxin. We conclude that alpha-bungarotoxin binding proteins in the vertebrate nervous system can function as nAChRs.

A Novel Positive Allosteric Modulator of the α7 Neuronal Nicotinic Acetylcholine Receptor: In Vitro and In Vivo Characterization

Several lines of evidence suggest a link between the α7 neuronal nicotinic acetylcholine receptor (nAChR) and brain disorders including schizophrenia, Alzheimers disease, and traumatic brain injury. The present work describes a novel molecule, 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea (PNU-120596), which acts as a powerful positive allosteric modulator of the α7 nAChR. Discovered in a high-throughput screen, PNU-120596 increased agonist-evoked calcium flux mediated by an engineered variant of the human α7 nAChR. Electrophysiology studies confirmed that PNU-120596 increased peak agonist-evoked currents mediated by wild-type receptors and also demonstrated a pronounced prolongation of the evoked response in the continued presence of agonist. In contrast, PNU-120596 produced no detectable change in currents mediated by α4β2, α3β4, and α9α10 nAChRs. PNU-120596 increased the channel mean open time of α7 nAChRs but had no effect on ion selectivity and relatively little, if any, effect on unitary conductance. When applied to acute hippocampal slices, PNU-120596 increased the frequency of ACh-evoked GABAergic postsynaptic currents measured in pyramidal neurons; this effect was suppressed by TTX, suggesting that PNU-120596 modulated the function of α7 nAChRs located on the somatodendritic membrane of hippocampal interneurons. Accordingly, PNU-120596 greatly enhanced the ACh-evoked inward currents in these interneurons. Systemic administration of PNU-120596 to rats improved the auditory gating deficit caused by amphetamine, a model proposed to reflect a circuit level disturbance associated with schizophrenia. Together, these results suggest that PNU-120596 represents a new class of molecule that enhances α7 nAChR function and thus has the potential to treat psychiatric and neurological disorders.

Neuronal nicotinic alpha 7 receptor expressed in Xenopus oocytes presents five putative binding sites for methyllycaconitine.

1. The recently isolated compound methyllycaconitine (MLA) is a plant toxin which is a competitive inhibitor of nicotinic acetylcholine receptors (nAChRs). We found that homomeric alpha 7 receptors display a very high sensitivity to MLA with an IC50 in the picomolar range. 2. The competitive nature of the alpha 7 MLA blockade was reinforced by the observation that this compound has no action on wild‐type serotoninergic receptors (5‐HT3), whereas it is a powerful antagonist of chimaeric receptors alpha 7‐5‐HT3. 3. The time course of MLA inhibition of the wild‐type (WT) alpha 7 follows a monotonic exponential decay whose time constant is proportional to the MLA concentration and could be described by a bimolecular mechanism with a forward rate constant (k+) of 2.7 x 10(7) S‐1 M‐1. In contrast, recovery from MLA inhibition displays an S‐shaped time course that is incompatible with a simple bimolecular reaction. 4. Given the pentameric nature of the neuronal nicotinic receptors, a linear chain model, including five putative MLA binding sites corresponding to the homomeric nature of alpha 7, is proposed. 5. Both onset and recovery data obtained on the alpha 7 wild‐type receptor are adequately described by this model assuming that a single MLA molecule is sufficient to block receptor function. 6. Analysis of MLA blockade and recovery of reconstituted heteromeric alpha 4 beta 2 receptors reveals, as expected, a time course compatible with only two binding sites for the toxin and, thus, further supports the validity of our model.

Functional significance of aromatic amino acids from three peptide loops of the α7 neuronal nicotinic receptor site investigated by site-directed mutagenesis

Three aromatic amino acids, Tyr92, Trp148 and Tyr187 belonging to three separate domains of the α7‐subunit of neuronal nicotinic acetylcholine receptor were mutated to phenylalanine, and the electrophysiological response of the resulting mutant receptors analyzed in the Xenopus oocyte expression system. All mutations significantly decreased the apparent affinities for acetylcholine and nicotine, and to a lesser extent, those for the competitive antagonists dihydro‐β‐erythroidine and α‐bungarotoxin. Other properties investigated, such as the voltage dependency of the ion response as well as its sensitivity to the open channel blocker QX222, were not significantly changed, indicating that the mutations affected selectively the recognition of cholinergic ligands by the receptor protein. The maximal rates for the rapid desensitization process were slightly modified, suggesting that the contribution of Tyr92, Trp148 and Tyr187 to the binding area might differ in the various conformations of the nicotinic receptor. Other mutations at nearby positions (S94N, W153F, G151D and G82E) did not affect the properties of the electrophysiological response. These data point to the functional significance of Tyr92, Trp148 and Tyr187 in the binding of cholinergic ligands and ion channel activation of the nicotinic receptor, thus supporting a multiple loop model [(1990) J. Biol. Chem. 265, 10430–10437] for the ligand binding area.

Properties of neuronal nicotinic acetylcholine receptor mutants from humans suffering from autosomal dominant nocturnal frontal lobe epilepsy

1 Physiological and pharmacological properties of the human neuronal α4β2 nicotinic AChR and mutants found in patients suffering from autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) were studied. 2 Investigations of nicotinic AChRs reconstituted in Xenopus oocytes with the control or mutated α4 subunits revealed that both mutation S248F as well as the Leucine insertion (776ins3) result in major but different changes in the physiological and pharmacological properties of the receptors. 3 Mutation S248F causes a decrease in apparent affinity to ACh of about 7 fold. In addition, this receptor already desensitizes during exposure to agonist concentration 3000 times lower than the control. 4 776ins3 provokes a 10 fold increase of apparent ACh affinity, an increase in the IC50 caused by prolonged ACh exposures and a slowing down of the response decay. 5 At saturating ACh concentration cells expressing the S248F mutant display average currents that are about five times smaller than control. 6 When measured at very low concentration, agonist sensitivities of the control and mutated receptors to ACh, nicotine and epibatidine exhibit differences that match those observed for higher agonist concentrations. 7 Mutation 776ins3 increases the apparent efficacy to cytisine. 8 Data presented herein suggest that mutation S248F mainly affects the desensitization properties of the receptor while the leucine insertion (776ins3) increases the probability of transition to the active state. Although these mutations differentially affect the receptor properties they both result in reduced permeability to calcium and enhanced desensitization sensitivity that might account for the ADNFLE phenotype.

The long internal loop of the alpha 3 subunit targets nAChRs to subdomains within individual synapses on neurons in vivo.

Different types of neurotransmitter receptors coexist within single neurons and must be targeted to discrete synaptic regions for proper function. In chick ciliary ganglion neurons, nicotinic acetylcholine receptors (nAChRs) containing α3 and α5 subunits are concentrated in the postsynaptic membrane, whereas α-bungarotoxin receptors composed of α7 subunits are localized perisynaptically and excluded from the synapse. Using retroviral vector-mediated gene transfer in vivo, we show that the long cytoplasmic loop of α3 targets chimeric α7 subunits to the synapse and reduces endogenous nAChR surface levels, whereas the α5 loop does neither. These results show that a particular domain of one subunit targets specific receptor subtypes to the interneuronal synapse in vivo. Moreover, our findings suggest a difference in the mechanisms that govern assembly of interneuronal synapses as compared to the neuromuscular junction in vertebrates.

Mutated nicotinic receptors responsible for autosomal dominant nocturnal frontal lobe epilepsy are more sensitive to carbamazepine.

Summary: Purpose: The recent linkage between a genetically transmissible form of epilepsy (ADNFLE) and mutations within the α4 subunit, one component of the major brain neuronal nicotinic acetylcholine receptor (nAChR), raises the question of the role of this receptor in epileptogenesis. Although acting by different mechanisms, the two genetic alterations so far identified both render the nAChR less efficient. In view of the high sensitivity of ADNFLE to carbamazepine (CBZ), we studied the effects of this drug and of valproate (VPA) on the human α4β2 nAChR and its mutations.

Pentameric ligand-gated ion channel ELIC is activated by GABA and modulated by benzodiazepines

GABAA receptors are pentameric ligand-gated ion channels involved in fast inhibitory neurotransmission and are allosterically modulated by the anxiolytic, anticonvulsant, and sedative-hypnotic benzodiazepines. Here we show that the prokaryotic homolog ELIC also is activated by GABA and is modulated by benzodiazepines with effects comparable to those at GABAA receptors. Crystal structures reveal important features of GABA recognition and indicate that benzodiazepines, depending on their concentration, occupy two possible sites in ELIC. An intrasubunit site is adjacent to the GABA-recognition site but faces the channel vestibule. A second intersubunit site partially overlaps with the GABA site and likely corresponds to a low-affinity benzodiazepine-binding site in GABAA receptors that mediates inhibitory effects of the benzodiazepine flurazepam. Our study offers a structural view how GABA and benzodiazepines are recognized at a GABA-activated ion channel.

Positive Allosteric Modulation of the α7 Nicotinic Acetylcholine Receptor : Ligand Interactions with Distinct Binding Sites and Evidence for a Prominent Role of the M2-M3 Segment

The α7 nicotinic acetylcholine receptor (nAChR), a homopentameric, rapidly activating and desensitizing ligand-gated ion channel with relatively high degree of calcium permeability, is expressed in the mammalian central nervous system, including regions associated with cognitive processing. Selective agonists targeting the α7 nAChR have shown efficacy in animal models of cognitive dysfunction. Use of positive allosteric modulators selective for the α7 receptor is another strategy that is envisaged in the design of active compounds aiming at improving attention and cognitive dysfunction. The recent discovery of novel positive allosteric modulators such as 1-(5-chloro-2-hydroxyphenyl)-3-(2-chloro-5-trifluoromethylphenyl)urea (NS-1738) and 1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)urea (PNU-120596) that are selective for the α7 nAChRs but display significant phenotypic differences in their profile of allosteric modulation, suggests that these molecules may act at different sites on the receptor. Taking advantage of the possibility to obtain functional receptors by the fusion of proteins domains from the α7 and the 5-HT3 receptor, we examined the structural determinants required for positive allosteric modulation. This strategy revealed that the extracellular N-terminal domain of α7 plays a critical role in allosteric modulation by NS-1738. In addition, α7-5HT3 chimeras harboring the M2-M3 segment showed that spontaneous activity in response to NS-1738, which confirmed the critical contribution of this small extracellular segment in the receptor gating. In contrast to NS-1738, positive allosteric modulation by PNU-120596 could not be restored in the α7-5HT3 chimeras but was selectively observed in the reverse 5HT3-α7 chimera. All together, these data illustrate the existence of distinct allosteric binding sites with specificity of different profiles of allosteric modulators and open new possibilities to investigate the α7 receptor function.

Multisite Binding of a General Anesthetic to the Prokaryotic Pentameric Erwinia chrysanthemi Ligand-gated Ion Channel (ELIC)

Background: Pentameric ligand-gated ion channels are modulated by general anesthetics. Results: The crystal structure of ELIC in complex with bromoform reveals anesthetic binding in the channel pore and in novel sites in the transmembrane and extracellular domain. Conclusion: General anesthetics allosterically modulate channel function via multisite binding. Significance: Our data reveal detailed insight into multisite recognition of general anesthetics at the structural level. Pentameric ligand-gated ion channels (pLGICs), such as nicotinic acetylcholine, glycine, γ-aminobutyric acid GABAA/C receptors, and the Gloeobacter violaceus ligand-gated ion channel (GLIC), are receptors that contain multiple allosteric binding sites for a variety of therapeutics, including general anesthetics. Here, we report the x-ray crystal structure of the Erwinia chrysanthemi ligand-gated ion channel (ELIC) in complex with a derivative of chloroform, which reveals important features of anesthetic recognition, involving multiple binding at three different sites. One site is located in the channel pore and equates with a noncompetitive inhibitor site found in many pLGICs. A second transmembrane site is novel and is located in the lower part of the transmembrane domain, at an interface formed between adjacent subunits. A third site is also novel and is located in the extracellular domain in a hydrophobic pocket between the β7–β10 strands. Together, these results extend our understanding of pLGIC modulation and reveal several specific binding interactions that may contribute to modulator recognition, further substantiating a multisite model of allosteric modulation in this family of ion channels.