In year 2000, the Unit has demonstrated : the difference in physical properties between the two binding sites of Torpedo receptor ; the differential distribution of neuronal nicotinic receptor subunit mRNA in monkey's brain ; the differential mobilisation of Ca⁺⁺ ions by two distinct nicotinic receptors in dopaminergic neurons from substantia nigra ; the phosphorylation of Torpedo 43K-Rapsyn by a novel endogenous histidine kinase.

The activity of the Molecular Neurobiology Unit (Jean-Pierre Changeux) is centered on the study of the nicotinic acetylcholine receptor (nAChR), an "allosteric" membrane protein involved in the transduction of chemical neurotransmitter signal into an electrical response at the level of the postsynaptic membrane of the neuromuscular junction and of central nicotinic synapses.
The three main objectives are : (1) the identification, at the amino acid level, of the elementary structures engaged in the recognition of nicotinic ligands, in ionic selectivity and transport and in their diverse modes of "allosteric" coupling for activation and desensitization as well as for binding " up regulation " and physiological response potentiation following chronic incubation with nicotine ; (2) the regulatory mechanisms of gene expression (transcriptional and post-transcriptional) involved in the focal distribution of nicotinic receptors at the muscle endplate postsynaptic membrane (in the course of development and in the adult) ; and (3) the neural processes to which contribute neuronal nicotinic receptors at the level of the dendritic vs. axonal compartments of central neurons in learning and reward, nicotine addiction, nociception, anxiety, neuroprotection and various brain pathologies.
The methods used to achieve these goals include affinity labeling by specific ligands under rapid mixing conditions, peptide microsequencing, site-directed mutagenesis, stopped-flow with fluorescent ligands in wild type and mutant brain nicotinic receptors, crystallization of the purified protein (or specific fragments) and analysis of the three-dimensional structure by electron microscopy and X-ray diffraction. To achieve objectives 2 and 3, the most recent advances in recombinant DNA technologies, cloning and sequencing of cDNAs in situ hybridization, promoter analyses, in vivo and in vitro transfection experiments, cloning of transcription factors and conditional homologous recombination in vivo are being exploited, along with cell biology methods such as confocal microscopy.
Ongoing applications of the current work to medicine include : (1) the pharmacology of peripheral and central nicotinic synapses ; (2) the pathology of denervated skeletal muscle and myasthenia gravis ; and (3) brain defects resulting from (or associated with) alterations of nicotinic receptors : frontal lobe nocturnal epilepsy, Alzheimer's, Gilles de la Tourette, and Parkinson's diseases, analgesia, aging and nicotine addiction.
The original contributions of the Unit " Receptors and Cognition " in 2000, include: 1) the direct demonstration of structural differences in the two agonist binding sites carried by the receptor protein using time-resolved fluorescence spectroscopy (Martinez et al., 2000) (Collaboration with F. Mérola) ; 2) the anatomical distribution of the mRNAs coding for the diverse nAChR subunits in monkey's brain ; 3) the differential mobilization of Ca⁺⁺ ions by two distinct (a4b2 and a7) nAChRs in mouse substantia nigra, using knock-out mice ; 4) the specific histidine phosphorylation of Torpedo 43K-Raspsyn protein by a novel kinase involving thiamine triphosphate as the phosphate donor (Nghiêm et al., 2000) ; 5) the control of neural tube closure in the mouse by the nucleosome assembly protein -1- like 2 (Rogner et al., 2000) (Collaboration with P. Avner).

Jacqueline GEX jgex@pasteur.fr

Hoàng-Oanh NGHIEM, CR1, CNRS

Morten SUNESEN, P danois

Martine SOUDANT, Technicienne, Institut Pasteur