Régis Grailhe

β2-Containing Nicotinic Receptors Contribute to the Organization of Sleep and Regulate Putative Micro-Arousals in Mice

The cholinergic system is involved in arousal and in rapid eye movement sleep (REMS). To evaluate the contribution of nicotinic acetylcholine receptors (nAChRs) to these functions, we studied with polygraphic recordings the regulation of sleep in mice lacking the β2 subunit gene of the nAChRs, a major component of high-affinity nicotine binding sites in the brain. Nicotine (1-2 mg/kg, i.p.) increased wakefulness in wild-type but not knock-out animals, indicating that β2-containing nAChRs mediate the arousing properties of nicotine. Under normal conditions, the β2-/- mice displayed the same amounts of waking, non-REM sleep (NREMS) and REMS as their wild-type counterparts. However, they exhibited longer REMS episodes and a reduced fragmentation of NREMS by events characterized notably by a transient drop in EEG power and frequently associated with EMG activation, tentatively referred to as micro-arousals. Respiration monitoring showed that these events were accompanied with, but not caused by, breathing irregularities. Sleep deprivation of β2-/- mice resulted in a normal increase in REMS episode duration and NREMS δ power but yielded a reduction of the number of micro-arousals in NREMS. In contrast, in β2-/- mice, a 1 hr immobilization stress failed to produce the normal rebound in REMS in the following 12 hr and, instead, was associated with increased NREMS fragmentation and sustained corticosterone levels. Our results show that the β2-containing nAChRs contribute to the organization of sleep by regulating the transient phasic activity in NREMS, the REMS onset and duration, and the REMS-promoting effect of stress.

β2 nicotinic acetylcholine receptor subunit modulates protective responses to stress: A receptor basis for sleep-disordered breathing after nicotine exposure

Nicotine exposure diminishes the protective breathing and arousal responses to stress (hypoxia). By exacerbating sleep-disordered breathing, this disturbance could underpin the well established association between smoking and the increased risk of sudden infant death syndrome. We show here that the protective responses to stress during sleep are partially regulated by particular nicotinic acetylcholine receptors (nAChRs). We compared responses of sleeping wild-type and mutant mice lacking the β2 subunit of the nAChR to episodic hypoxia. Arousal from sleep was diminished, and breathing drives accentuated in mutant mice indicating that these protective responses are partially regulated by β2-containing nAChRs. Brief exposure to nicotine significantly reduced breathing drives in sleeping wild-type mice, but had no effect in mutants. We propose that nicotine impairs breathing (and possibly arousal) responses to stress by disrupting functions normally regulated by β2-containing, high-affinity nAChRs.

Complex fluorescence of the cyan fluorescent protein: comparisons with the H148D variant and consequences for quantitative cell imaging.

We have studied the fluorescence decays of the purified enhanced cyan fluorescent protein (ECFP, with chromophore sequence Thr-Trp-Gly) and of its variant carrying the single H148D mutation characteristic of the brighter form Cerulean. Both proteins exhibit highly complex fluorescence decays showing strong temperature and pH dependences. At neutral pH, the H148D mutation leads (i) to a general increase in all fluorescence lifetimes and (ii) to the disappearance of a subpopulation, estimated to be more than 25% of the total ECFP molecules, characterized by a quenched and red-shifted fluorescence. The fluorescence lifetime distributions of ECFP and its H148D mutant remain otherwise very similar, indicating a high degree of structural and dynamic similarity of the two proteins in their major form. From thermodynamic analysis, we conclude that the multiexponential decay of ECFP cannot be simply ascribed, as is generally admitted, to the slow conformational exchange characterized by NMR and X-ray crystallographic studies [Seifert, M. H., et al. (2002) J. Am. Chem. Soc. 124, 7932-7942; Bae, J. H., et al. (2003) J. Mol. Biol. 328, 1071-1081]. Parallel measurements in living cells show that these fluorescence properties in neutral solution are very similar to those of cytosolic ECFP.

The role of nicotinic acetylcholine receptors in lymphocyte development.

The sizes of lymphocyte populations in lymphoid organs of nicotinic acetylcholine receptor knockout and chimera (knockout/wild-type) mice were studied by flow cytometry. The absence of beta2 subunit decreased, while nicotine treatment increased B lymphocyte numbers in the bone marrow. In chimera mice, either beta2 or alpha7 subunits influenced lymphocyte populations in primary lymphoid organs, while in the spleen, only alpha7 receptors were critical. More annexin V-positive B cells were found in the bone marrow of knockout than wild-type animals. We conclude that nicotinic receptors are involved in regulating lymphocyte development and control the B lymphocyte survival.

14-3-3 γ associates with muscle specific kinase and regulates synaptic gene transcription at vertebrate neuromuscular synapse

The muscle-specific receptor tyrosine kinase (MuSK) is part of a receptor complex, activated by neural agrin, that orchestrates the differentiation of the neuromuscular junction (NMJ). To gain insight into the function of the MuSK complex, we have developed a proteomic approach to identify new MuSK partners. MS analysis of MuSK crosslink products from postsynaptic membranes of the Torpedo electrocytes identified the adaptor protein 14-3-3 γ. The 14-3-3 γ protein was found localized at the adult rat NMJ. Cotransfection experiments in COS-7 cells showed that MuSK codistributed with the 14-3-3 γ protein at the plasma membrane. Furthermore, 14-3-3 γ was copurified by affinity chromatography with MuSK from transfected COS-7 cells and myotubes. The 14-3-3 γ protein did not colocalize with agrin-elicited acetylcholine receptor (AChR) aggregates in cultured myotubes, suggesting that it is not involved in AChR clustering. Expression of 14-3-3 γ specifically repressed the transcription of several synaptic reporter genes in cultured myotubes. This repression was potentiated by MuSK expression. Moreover, the expression of 14-3-3 γ in muscle fibers in vivo caused both the repression of synaptic genes transcription and morphological perturbations of the NMJ. Our data extend the notion that, apart from its well documented role in AChR clustering, the MuSK complex might also be involved in the regulation of synaptic gene expression at the NMJ.

Electron microscopic evidence for nucleation and growth of 3D acetylcholine receptor microcrystals in structured lipid–detergent matrices

Nicotinic acetylcholine receptors (AChRs) belong to a superfamily of oligomeric proteins that transduce electric signals across the cell membrane on binding of neurotransmitters. These receptors harbor a large extracellular ligand-binding domain directly linked to an ion-conducting channel-forming domain that spans the cell membrane 20 times and considerably extends into the cytoplasm. Thus far, none of these receptor channels has been crystallized in three dimensions. The crystallization of the AChR from Torpedo marmorata electric organs is challenged here in lipidic–detergent matrices. Detergent-soluble AChR complexed with α-bungarotoxin (αBTx), a polypeptidic competitive antagonist, was purified. The AChR–αBTx complex was reconstituted in a lipidic matrix composed of monoolein bilayers that are structured in three dimensions. The αBTx was conjugated to a photo-stable fluorophore, enabling us to monitor the physical behavior of the receptor–toxin complex in the lipidic matrix under light stereomicroscope, and to freeze fracture regions containing the receptor–toxin complex for visualization under a transmission electron microscope. Conditions were established for forming 2D receptor–toxin lattices that are stacked in the third dimension. 3D AChR nanocrystals were thereby grown inside the highly viscous lipidic 3D matrix. Slow emulsification of the lipidic matrix converted these nanocrystals into 3D elongated thin crystal plates of micrometer size. The latter are stable in detergent-containing aqueous solutions and can currently be used for seeding and epitaxial growth, en route to crystals of appropriate dimensions for x-ray diffraction studies.

Distinct subcellular targeting of fluorescent nicotinic α3β4 and serotoninergic 5-HT3A receptors in hippocampal neurons

The nicotinic acetylcholine receptors (nAChRs) and the 5‐HT3 serotonin receptor subtype belong to a superfamily of neurotransmitter‐gated ion channels involved in fast synaptic communication throughout the nervous system. Their trafficking to the neuron plasmalemma, as well as their targeting to specific subcellular compartments, is critical for understanding their physiological role. In order to investigate the cellular distribution of these receptors, we tagged the N‐termini of α3β4‐nAChR subunits and the 5‐HT3AR subunit with cyan and yellow fluorescent proteins (CFP, YFP). The fusion subunits were coexpressed in human embryonic kidney (HEK‐293) cells, where they assemble into functional receptor channels, as well as in primary cultures of hippocampal neurons. Fluorescence microscopy of living cells revealed that the heteropentameric α3CFP‐β4 and YFP‐α3β4 receptors are mainly distributed in the endoplasmic reticulum, while the homopentameric YFP‐5‐HT3A receptor was localized both to the plasma membrane and within intracellular compartments. Moreover, the YFP‐5‐HT3A receptor was found to be targeted to the micropodia in HEK‐293 cells and to the dendritic spines in hippocampal neurons, where it could be accessed by extracellularly applied specific fluorescent probes. The efficient targeting of the YFP‐5‐HT3A to the cytoplasmic membrane is in line with the large serotonin‐elicited currents (nA range) measured by whole‐cell voltage‐clamp recordings in transfected HEK‐293 cells. In contrast, α3β4‐nAChRs expressed in the same cells yielded weaker ACh‐evoked responses. Taken together, the fluorescent and electrophysiological studies presented here demonstrate the predominant intracellular location of α3β4‐nACh receptors and the predominant expression of the 5‐HT3AR in dendritic surface loci.

Mechanism of Cl- selection by a glutamate-gated chloride (GluCl) receptor revealed through mutations in the selectivity filter.

To learn about the mechanism of ion charge selectivity by invertebrate glutamate-gated chloride (GluCl) channels, we swapped segments between the GluClβ receptor of Caenorhabditis elegans and the vertebrate cationic α7-acetylcholine receptor and monitored anionic/cationic permeability ratios. Complete conversion of the ion charge selectivity in a set of receptor microchimeras indicates that the selectivity filter of the GluClβ receptor is created by a sequence connecting the first with the second transmembrane segments. A single substitution of a negatively charged residue within this sequence converted the selectivity of the GluClβ receptors pore from anionic to cationic. Unexpectedly, elimination of the charge of each basic residue of the selectivity filter, one at a time or concomitantly, moderately reduced the PCl/PNa ratios, but the GluClβ receptors mutants retained high capacity to select Cl- over Na+. These results indicate that, unlike the proposed case of anionic Gly- and γ-aminobutyric acid-gated ion channels, positively charged residues do not play the key role in the selection of ionic charge by the GluClβ receptor. Taken together with measurements of the effective open pore diameter and with structural modeling, the study presented here collectively indicates that in the most constricted part of the open GluClβ receptors channel, Cl- interacts with backbone amides, where it undergoes partial dehydration necessary for traversing the pore.

Regional changes in the cholinergic system in mice lacking monoamine oxidase A

Elevated brain monoamine concentrations resulting from monoamine oxidase A genetic ablation (MAOA knock-out mice) lead to changes in other neurotransmitter systems. To investigate the consequences of MAOA deficiency on the cholinergic system, we measured ligand binding to the high-affinity choline transporter (CHT1) and to muscarinic and nicotinic receptors in brain sections of MAOA knock-out (KO) and wild-type mice. A twofold increase in [(3)H]-hemicholinium-3 ([(3)H]-HC-3) binding to CHT1 was observed in the caudate putamen, nucleus accumbens, and motor cortex in MAOA KO mice as compared with wild-type (WT) mice. There was no difference in [(3)H]-HC-3 labeling in the hippocampus (dentate gyrus) between the two genotypes. Binding of [(125)I]-epibatidine ([(125)I]-Epi), [(125)I]-alpha-bungarotoxin ([(125)I]-BGT), [(3)H]-pirenzepine ([(3)H]-PZR), and [(3)H]-AFDX-384 ([(3)H]-AFX), which respectively label high- and low-affinity nicotinic receptors, M1 and M2 muscarinic cholinergic receptors, was not modified in the caudate putamen, nucleus accumbens, and motor cortex. A small but significant decrease of 19% in M1 binding densities was observed in the hippocampus (CA1 field) of KO mice. Next, we tested acetylcholinesterase activity and found that it was decreased by 25% in the striatum of KO mice as compared with WT mice. Our data suggest that genetic deficiency in MAOA enzyme is associated with changes in cholinergic activity, which may account for some of the behavioral alterations observed in mice and humans lacking MAOA.