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

Abstract

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.

Cytoplasmic regions adjacent to the M3 and M4 transmembrane segments influence expression and function of α7 nicotinic acetylcholine receptors. A study with single amino acid mutants

“…In a previously published model (Le Novère et al . 1999), this region appeared with α‐helical structure, the so‐called helix F. Deletion or substitution of this helix in the V201 α7–5HT 3A chimera induced lower levels of receptor expression (Devillers‐Thiéry et al . 2003).…”

A review of experimental techniques used for the heterologous expression of nicotinic acetylcholine receptors

“…and trafficking [184][185][186][187]. Other studies involving the use of subunit chimeras have helped to investigate receptor properties such as agonist sensitivity [188][189][190][191][192][193][194][195], antagonist sensitivity [190,192,[196][197][198], modulation by allosteric modulators [199,200], desensitization [188], inactivation [201] and channel gating [202][203][204].…”

Nicotinic cholinergic intercellular communication: Implications for the developing auditory system

“…NAChRs are heterooligomers consisting of Wve subunits, each with four transmembrane domains (Devillers-Thiery et al, 2003). All nAChRs possess a cys loop, which places them in a "cys loop" superfamily of the ligand-gated channels (Le Novere and Changeux, 1995) along with GABA A , glycine, 5HT 3 , and several invertebrate receptors (Reeves and Lummis, 2002).…”

“…A sequence in the cytoplasmic loop located between the third and fourth transmembrane regions targets the receptor to a microregion of the cell (Devillers-Thiery et al, 2003). For example, a myctagged chimeric 7/ 3 nAChR retargets the normally perisynaptic 7 nAChR to speciWc interneuronal postsynaptic synapses (Temburni et al, 2000).…”