Structurally similar allosteric modulators of α7 nicotinic acetylcholine receptors exhibit five distinct pharmacological effects.

Activation of nicotinic acetylcholine receptors (nAChRs) is associated with the binding of agonists such as acetylcholine to an extracellular site that is located at the interface between two adjacent receptor subunits. More recently, there has been considerable interest in compounds, such as positive and negative allosteric modulators (PAMs and NAMs), that are able to modulate nAChR function by binding to distinct allosteric sites. Here we examined a series of compounds differing only in methyl substitution of a single aromatic ring. This series of compounds includes a previously described α7-selective allosteric agonist, cis-cis-4-p-tolyl-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide (4MP-TQS), together with all other possible combinations of methyl substitution at a phenyl ring (18 additional compounds). Studies conducted with this series of compounds have revealed five distinct pharmacological effects on α7 nAChRs. These five effects can be summarized as: 1) nondesensitizing activation (allosteric agonists), 2) potentiation associated with minimal effects on receptor desensitization (type I PAMs), 3) potentiation associated with reduced desensitization (type II PAMs), 4) noncompetitive antagonism (NAMs), and 5) compounds that have no effect on orthosteric agonist responses but block allosteric modulation (silent allosteric modulators (SAMs)). Several lines of experimental evidence are consistent with all of these compounds acting at a common, transmembrane allosteric site. Notably, all of these chemically similar compounds that have been classified as nondesensitizing allosteric agonists or as nondesensitizing (type II) PAMs are cis-cis-diastereoisomers, whereas all of the NAMs, SAMs, and type I PAMs are cis-trans-diastereoisomers. Our data illustrate the remarkable pharmacological diversity of allosteric modulators acting on nAChRs

Distribution of α7 Nicotinic Acetylcholine Receptor Subunit mRNA in the Developing Mouse.

Homomeric α7 nicotinic acetylcholine receptors (nAChRs) are abundantly expressed in the central and peripheral nervous system (CNS and PNS, respectively), and spinal cord. In addition, expression and functional responses have been reported in non-neuronal tissue. In the nervous system, α7 nAChR subunit expression appears early during embryonic development and is often transiently upregulated, but little is known about their prenatal expression outside of the nervous system. For understanding potential short-term and long-term effects of gestational nicotine exposure, it is important to know the temporal and spatial expression of α7 nAChRs throughout the body. To that end, we studied the expression of α7 nAChR subunit mRNA using highly sensitive isotopic in situ hybridization in embryonic and neonatal whole-body mouse sections starting at gestational day 13. The results revealed expression of α7 mRNA as early as embryonic day 13 in the PNS, including dorsal root ganglia, parasympathetic and sympathetic ganglia, with the strongest expression in the superior cervical ganglion, and low to moderate levels were detected in brain and spinal cord, respectively, which rapidly increased in intensity with embryonic age. In addition, robust α7 mRNA expression was detected in the adrenal medulla, and low to moderate expression in selected peripheral tissues during embryonic development, potentially related to cells derived from the neural crest. Little or no mRNA expression was detected in thymus or spleen, sites of immune cell maturation. The results suggest that prenatal nicotine exposure could potentially affect the nervous system with limited effects in non-neural tissues

Neurotransmitter-gated ion channels at fast chemical synapses: from structure to pathology

Fil: Bouzat, Cecilia Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentin

An in vivo biosensor for neurotransmitter release and in situ receptor activity.

Tools from molecular biology, combined with in vivo optical imaging techniques, provide new mechanisms for noninvasively observing brain processes. Current approaches primarily probe cell-based variables, such as cytosolic calcium or membrane potential, but not cell-to-cell signaling. We devised cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) to address this challenge and monitor in situ neurotransmitter receptor activation. CNiFERs are cultured cells that are engineered to express a chosen metabotropic receptor, use the G(q) protein-coupled receptor cascade to transform receptor activity into a rise in cytosolic [Ca(2+)] and report [Ca(2+)] with a genetically encoded fluorescent Ca(2+) sensor. The initial realization of CNiFERs detected acetylcholine release via activation of M1 muscarinic receptors. We used chronic implantation of M1-CNiFERs in frontal cortex of the adult rat to elucidate the muscarinic action of the atypical neuroleptics clozapine and olanzapine. We found that these drugs potently inhibited in situ muscarinic receptor activity

The distribution of charged amino acid residues and the Ca(2+) permeability of nicotinic acetylcholine receptors: a predictive model

Nicotinic acetylcholine receptors (nAChRs) are cation-selective ligand-gated ion channels exhibiting variable Ca(2+) permeability depending on their subunit composition. The Ca(2+) permeability is a crucial functional parameter to understand the physiological role of nAChRs, in particular considering their ability to modulate Ca(2+)-dependent processes such as neurotransmitter release. The rings of extracellular and intracellular charged amino acid residues adjacent to the pore-lining TM2 transmembrane segment have been shown to play a key role in the cation selectivity of these receptor channels, but to date a quantitative relationship between these structural determinants and the Ca(2+) permeability of nAChRs is lacking. In the last years the Ca(2+) permeability of several nAChR subtypes has been experimentally evaluated, in terms of fractional Ca(2+) current (Pf, i.e., the percentage of the total current carried by Ca(2+) ions). In the present study, the available Pf-values of nAChRs are used to build a simplified modular model describing the contribution of the charged residues in defined regions flanking TM2 to the selectivity filter controlling Ca(2+) influx. This model allows to predict the currently unknown Pf-values of existing nAChRs, as well as the hypothetical Ca(2+) permeability of subunit combinations not able to assemble into functional receptors. In particular, basing on the amino acid sequences, a Pf > 50% would be associated with homomeric nAChRs composed by different α subunits, excluding α7, α9, and α10. Furthermore, according to the model, human α7β2 receptors should have Pf-values ranging from 3.6% (4:1 ratio) to 0.1% (1:4 ratio), much lower than the 11.4% of homomeric α7 nAChR. These results help to understand the evolution and the function of the large diversity of the nicotinic receptor family

Ion channels as insecticide targets.

Published onlineJournal ArticleIon channels remain the primary target of most of the small molecule insecticides. This review examines how the subunit composition of heterologously expressed receptors determines their insecticide-specific pharmacology and how the pharmacology of expressed receptors differs from those found in the insect nervous system. We find that the insecticide-specific pharmacology of some receptors, like that containing subunits of the Rdl encoded GABA receptor, can be reconstituted with very few of the naturally occurring subunits expressed. In contrast, workers have struggled even to express functional insect nicotinic acetylcholine receptors (nAChRs), and work has therefore often relied upon the expression of vertebrate receptor subunits in their place. We also examine the extent to which insecticide-resistance-associated mutations, such as those in the para encoded voltage-gated sodium channel, can reveal details of insecticide-binding sites and mode of action. In particular, we examine whether mutations are present in the insecticide-binding site and/or at sites that allosterically affect the drug preferred conformation of the receptor. We also discuss the ryanodine receptor as a target for the recently developed diamides. Finally, we examine the lethality of the genes encoding these receptor subunits and discuss how this might determine the degree of conservation of the resistance-associated mutations found

Mutations in M2 alter the selectivity of the mouse nicotinic acetylcholine receptor for organic and alkali metal cations

We measured the permeability ratios (PX/PNa) of 3 wild-type, 1 hybrid, 2 subunit-deficient, and 22 mutant nicotinic receptors expressed in Xenopus oocytes for alkali metal and organic cations using shifts in the bi-ionic reversal potential of the macroscopic current. Mutations at three positions (2', 6', 10') in M2 affected ion selectivity. Mutations at position 2' (alpha Thr244, beta Gly255, gamma Thr253, delta Ser258) near the intracellular end of M2 changed the organic cation permeability ratios as much as twofold and reduced PCs/PNa and PK/PNa by 16-18%. Mutations at positions 6' and 10' increased the glycine ethyl ester/Na+ and glycine methyl ester/Na+ permeability ratios. Two subunit alterations also affected selectivity: omission of the delta subunit reduced PCs/PNa by 16%, and substitution of Xenopus delta for mouse delta increased Pguanidinium/PNa more than twofold and reduced PCs/PNa by 34% and PLi/PNa by 20%. The wild-type mouse receptor displayed a surprising interaction with the primary ammonium cations; relative permeability peaked at a chain length equal to four carbons. Analysis of the organic permeability ratios for the wild-type mouse receptor shows that (a) the diameter of the narrowest part of the pore is 8.4 A; (b) the mouse receptor departs significantly from size selectivity for monovalent organic cations; and (c) lowering the temperature reduces Pguanidinium/PNa by 38% and Pbutylammonium/PNa more than twofold. The results reinforce present views that positions -1' and 2' are the narrowest part of the pore and suggest that positions 6' and 10' align some permeant organic cations in the pore in an interaction similar to that with channel blocker, QX-222

High-throughput screening in larval zebrafish identifies novel potent sedative-hypnotics

BACKGROUND: Many general anesthetics were discovered empirically, but primary screens to find new sedative-hypnotics in drug libraries have not used animals, limiting the types of drugs discovered. The authors hypothesized that a sedative-hypnotic screening approach using zebrafish larvae responses to sensory stimuli would perform comparably to standard assays, and efficiently identify new active compounds. METHODS: The authors developed a binary outcome photomotor response assay for zebrafish larvae using a computerized system that tracked individual motions of up to 96 animals simultaneously. The assay was validated against tadpole loss of righting reflexes, using sedative-hypnotics of widely varying potencies that affect various molecular targets. A total of 374 representative compounds from a larger library were screened in zebrafish larvae for hypnotic activity at 10 µM. Molecular mechanisms of hits were explored in anesthetic-sensitive ion channels using electrophysiology, or in zebrafish using a specific reversal agent. RESULTS: Zebrafish larvae assays required far less drug, time, and effort than tadpoles. In validation experiments, zebrafish and tadpole screening for hypnotic activity agreed 100% (n = 11; P = 0.002), and potencies were very similar (Pearson correlation, r > 0.999). Two reversible and potent sedative-hypnotics were discovered in the library subset. CMLD003237 (EC50, ~11 µM) weakly modulated γ-aminobutyric acid type A receptors and inhibited neuronal nicotinic receptors. CMLD006025 (EC50, ~13 µM) inhibited both N-methyl-D-aspartate and neuronal nicotinic receptors. CONCLUSIONS: Photomotor response assays in zebrafish larvae are a mechanism-independent platform for high-throughput screening to identify novel sedative-hypnotics. The variety of chemotypes producing hypnosis is likely much larger than currently known.This work was supported by grants from Shanghai Jiaotong University School of Medicine, Shanghai, China, and the Chinese Medical Association, Beijing, China (both to Dr. Yang). The Department of Anesthesia, Critical Care and Pain Medicine of Massachusetts General Hospital, Boston, Massachusetts, supported this work through a Research Scholars Award and an Innovation Grant (both to Dr. Forman). Contributions to this research from the Boston University Center for Molecular Discovery, Boston, Massachusetts (to Drs. Porco, Brown, Schaus, and Xu, and to Mr. Trilles), were supported by a grant from the National Institutes of Health, Bethesda, Maryland (grant No. R24 GM111625). (Shanghai Jiaotong University School of Medicine, Shanghai, China; Chinese Medical Association, Beijing, China; Department of Anesthesia, Critical Care and Pain Medicine of Massachusetts General Hospital, Boston, Massachusetts; R24 GM111625 - National Institutes of Health, Bethesda, Maryland)Accepted manuscript2019-09-0