Critical role of the C-terminal segment in the maturation and export to the cell surface of the homopentameric alpha 7-5HT3A receptor.

International audienceMany neurological pathologies are related to misfolded proteins. During folding and assembly in the endoplasmic reticulum, the nicotinic acetylcholine receptor (nAChR) subunits undergo several conformational changes to acquire the ability to bind ligands. After folding and maturation, by mechanisms largely unknown, receptors are exported to the cell surface. We investigated the maturational role of the extracellular C-terminal segment located at the boundary between the extracellular and the transmembrane domains. In the functional chimeric alpha7-5HT3A receptor used as a model system, amino acids from the C-terminal segment were successively deleted or mutated. Upon progressive shortening of the peptide we observed less and less alpha-bungarotoxin binding sites until no sites could be detected when the entire peptide had been deleted (chimera Del 5). Protein synthesis and pentameric assembly were not altered. In Del 5 transfected cells, pentameric receptors present in the endoplasmic reticulum were not detected on the cell surface where Del 5 proteins appeared as patches. With the Del 5 chimera, export of proteins to the cell surface diminished to about half that of wild-type. We propose that the C-terminal segment plays a double role: (i) through an interaction between the penultimate tyrosine residue of the C-terminal segment and the Cys loop of the N-terminal domain, it locks the receptor in a mature alpha-bungarotoxin binding conformation; (ii) this mature conformation, in turn, masks a retention signal present in the first transmembrane segment allowing properly assembled and matured receptors to escape to the cell surface

Chicken neuronal acetylcholine receptor alpha 2-subunit gene exhibits neuron-specific expression in the brain and spinal cord of transgenic mice.

Transgenic mice carrying the complete structural gene of the alpha 2 subunit of the chicken neuronal nicotinic acetylcholine receptor (nAChR) and 7 kilobase pairs (kbp) of 5' upstream and 3 kbp of 3' downstream sequences have been generated. The transgene was stably integrated in transgenic lines and transmitted to their progeny. Avian transgene expression was predominant in the central nervous system as detected by specific alpha 2-subunit cDNA amplification. Moreover, in at least two independent mouse lines, its expression appeared to be neuron-specific and reproducibly restricted to subregions in the brain and spinal cord, as revealed by in situ hybridization histochemistry. Most cranial motor nuclei were positive, and several of the alpha 2-subunit transgene-expressing structures corresponded to cholinergic areas in rodents. This study reveals that regulatory mechanisms giving rise to neuronal-specific gene expression have been conserved at least in part between birds and mammals

An in vitro study of the sub-cellular distribution of nicotinic receptors

Nicotinic and serotoninergic 5HT3 receptors share important sequence identities except for their cytoplasmic loop. Both ends of this loop display conserved 3D helical structures with distinct primary sequences. We decided to check whether these two helices named F and G play a role in the sub-cellular distribution of different nicotinic receptors. We systematically exchanged each helix with the equivalent sequence of neuronal nicotinic and a4, b2 and a7 subunits in the functional chimeric a7–5HT3 receptor used as a model system. The new chimeras were expressed in vitro in polarized epithelial cells from pig kidney. We quantified synthesis and export of the receptors to the cell surface by measuring a–bungarotoxin binding sites. Immunogold labelling was used, at the electron microscope level, to determine the amount of each chimera present at either domain, apical and/or basolateral, of these cells. We noticed that in epithelial cells the majority of a–bungarotoxin binding sites remained sequestered in the cytoplasm as already observed in neurons in vivo. The majority of the pentamers present at the cell surface were located at the apical domain. Our results suggest that helix F and G differently regulate assembly and export to the cell surface of a–bungarotoxin binding receptors. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved