Functional architecture of the acetylcholine nicotinic receptor as a model of ligandgated ion channel : investigations at the amino acid level

Información del proyecto

Identificador del acuerdo de subvención: BIO2930518

Proyecto cerrado

Fecha de inicio 1 Septiembre 1993

Fecha de finalización 31 Agosto 1996

Financiado con arreglo a

• Specific research and technological development programme (EEC) in the field of biotechnology, 1990-1994

Coste total

Sin datos

Aportación de la UE

€ 420 000,00

Coordinado por

INSTITUT PASTEUR
France

Objetivo

The primary objective of the concerted project was to identify, at the amino acid level, the structural features of the nicotinic receptor protein engaged in the transduction of the neurotransmitter signal into an ion channel opening and its regulation by allosteric effectors.
1) As preliminary to the experimental studies, theoretical models of the functional properties of the nAChR expressed in terms of allosteric mechanisms have been presented and discussed as structural models of the tertiary folding and quaternary organisation of the transmembrane organisation of the nAChR.
2) Recombinant chimeric subunits containing the N-terminal part of the nicotinic a7 subunit and the C terminal domain of the serotoninergic 5HT3 receptor subunit joined at conserved canonical residues (Y194 and V201) were found functional after expression in Xenopus oocyte. The V201 chimera was activated by nicotinic ligands with the same selectivity as a7, but the ionic channel activated by acetylcholine was blocked by Ca++, as in the serotoninergic channel. Moreover, external Ca++ ions which act as positive allosteric effectors of alpha7 receptor also potentiated the permeability response of the chimera to acetylcholine, thus demonstrating that the Ca++ regulatory site is located in the N-terminal synaptic domain.
3) The acetylcholine binding site overlaps the boundary between an alpha-subunit (principal component) and another subunit (complementary component) in the receptor oligomer. The complementary component was identified by mutating tryptophan 54 which is homologous, in the alpha7 subunit, to gamma Trp and delta Trp 57 of non-alpha-subunits of Torpedo receptor previously found, labeled by d-tubocurarine (Cohen et al., 1992, Abstract). Trp 54 was mutated into phenylalanine (F), alanine (A) and histidine (H): as a consequence, the dissociation constants for acetylcholine binding (Kp) (measured by following decreases in the initial rates of 125Ialpha-bungarotoxin binding increased progressively, from 83uM (WT) to 280 uM (W54F), to 840 (W54A) to 3500 (W54H), as well as the EC50 recorded electrophysiologically in the Xenopus oocytes. Decreases in binding affinity also took place for nicotine and dihydroberythroidine without significant modification in alpha-bungarotoxin binding. Interestingly, pharmacological differences were noticed among the various mutants.
4) The allosteric binding site for Ca++ ions, which regulates the potentiation of the neuronal nAChR have been identified in the aminoterminal domain within the canonical sequence [alpha7 161-172].
5) Analysis of the effect of the competitive antagonist methyllycaconitine (MLA) on the pleiotropic mutant alpha7 L 247T reveals that mutant molecules may exhibit spontaneous openings which are closed by methyllycaconitine or alpha-bungarotoxin.
6) The human nAChR alpha4b2 can be stably expressed in a functional state in HEK 293 cell lines.
7) A nAChR subunit from Caenorhabditis elegans has been cloned and identified; it forms functional homooligomers when expressed in Xenopus oocyte and displays striking sequence homologies with vertebrate alpha7 subunit; it thus appears the most primitive subunit within vertebrates. The pharmacological properties of two different receptors from Manduca sexta and from locust have been established.
MAJOR SCIENTIFIC BREAKTHROUGHS:
1) The finding that a chimaera can be constructed between two ligand-gated ion channels with different neurotransmitter specificities in a functional state supports the view that both receptors share a highly conserved architectural framework; the successful coupling of the neurotransmitter site domain and of the ion channel domain suggests a folding autonomy of these units.
2) Mutagenesis data on Trp 54 from alpha7 neuronal receptor provide evidence for a new `complementary component' for the nicotinic binding site distinct from its three-loop `principal component'; this component is homologous to the non-alpha component present on gamma and delta-subunits in Torpedo receptor.
3) A Ca++ binding site involved in the allosteric potentiation of neuronal nicotinic receptor has been identified within the large N terminal hydrophilic domain of the alpha7 receptor.
4) In a M2 mutant the ionic channel spontaneously opens and is closed by competitive antagonists.

Ámbito científico (EuroSciVoc)

• ciencias naturales › ciencias biológicas › bioquímica › biomoléculas › proteínas
• ciencias naturales › ciencias químicas › química orgánica › aminas

Programa(s)

• FP3-BIOTECH 1 - Specific research and technological development programme (EEC) in the field of biotechnology, 1990-1994

Tema(s)

• 1.1 - Protein structure and function

Convocatoria de propuestas

Datos no disponibles

Régimen de financiación

CSC - Cost-sharing contracts

Coordinador

INSTITUT PASTEUR

• Aportación de la UE: Sin datos
• Dirección: Rue du Docteur Roux 25 ; 75724 PARIS ; Francia
• Coste total: Sin datos

Participantes (2)

Centre Médical Universitaire

Suiza

• Aportación de la UE: Sin datos
• Dirección: 1,Michel Servet ; 1211 Genève 4
• Coste total: Sin datos

UNIVERSITY OF BATH

Reino Unido

• Aportación de la UE: Sin datos
• Dirección: Claverton Down ; BA2 7AY BATH
• Coste total: Sin datos