Anne Devillers-Thiéry

Functional architecture of the nicotinic acetylcholine receptor: A prototype of ligand-gated ion channels

The nicotinic acetylcholine receptor (nAChR) ~ is involved in chemo-electrical transduction at the neuromuscular junction and at cholinergic synapses of the central nervous system. At the motor endplate, invasion of the motor nerve ending by an action potential causes the release in the synaptic cleft of a brief pulse of acetylcholine (ACh), whose local concentration reaches 0. I to 1 mM (Katz & Miledi, 1977) for less than 1 msec (see also Clements et al., 1992 in the case of GluR). ACh diffuses through the cleft and binds to the nAChR present in the postsynaptic membrane, where it triggers the all-ornone opening of cation-selective ion channels through which Na+/K + ions flow passively. When depolarization reaches a threshold, muscle contraction occurs. In the cleft, ACh concentration rapidly declines to background levels (10 .9 M) as a consequence of diffusion and degradation by ACh esterase (Kuffler & Yoshikami, 1975; Katz & Miledi, 1977).

Functional significance of aromatic amino acids from three peptide loops of the α7 neuronal nicotinic receptor site investigated by site-directed mutagenesis

Three aromatic amino acids, Tyr92, Trp148 and Tyr187 belonging to three separate domains of the α7‐subunit of neuronal nicotinic acetylcholine receptor were mutated to phenylalanine, and the electrophysiological response of the resulting mutant receptors analyzed in the Xenopus oocyte expression system. All mutations significantly decreased the apparent affinities for acetylcholine and nicotine, and to a lesser extent, those for the competitive antagonists dihydro‐β‐erythroidine and α‐bungarotoxin. Other properties investigated, such as the voltage dependency of the ion response as well as its sensitivity to the open channel blocker QX222, were not significantly changed, indicating that the mutations affected selectively the recognition of cholinergic ligands by the receptor protein. The maximal rates for the rapid desensitization process were slightly modified, suggesting that the contribution of Tyr92, Trp148 and Tyr187 to the binding area might differ in the various conformations of the nicotinic receptor. Other mutations at nearby positions (S94N, W153F, G151D and G82E) did not affect the properties of the electrophysiological response. These data point to the functional significance of Tyr92, Trp148 and Tyr187 in the binding of cholinergic ligands and ion channel activation of the nicotinic receptor, thus supporting a multiple loop model [(1990) J. Biol. Chem. 265, 10430–10437] for the ligand binding area.

Mutational Analysis of the Charge Selectivity Filter of the α7 Nicotinic Acetylcholine Receptor

In the alpha7 nicotinic acetylcholine receptors, we analyze the contribution of mutations E237A and V251T, together with the proline insertion P236, in the conversion of the charge selectivity from cationic to anionic. We show that the triple mutant exhibits spontaneous openings displaying anionic selectivity. Furthermore, at position 251, hydrophilic or even negatively charged residues are compatible with an anionic channel. In contrast, the additional proline yields an anionic channel only when inserted between positions 234 and 237; insertion before 234 yields a cationic channel and after 238 alters the receptor surface expression. The coiled 234-238 loop thus directly contributes to the charge selectivity filter of the alpha7 channel.

Identification of the Amino Acid Subsets Accounting for the Ligand Binding Specificity of a Glutamate Receptor

In a situation so far unique among neurotransmitter receptors, glutamate receptors share amino acid sequence similarities with the bacterial periplasmic binding proteins (PBPs). On the basis of the primary structure similarity of two bacterial periplasmic proteins (lysine/arginine/ornithine- and phosphate-binding proteins) with the chick cerebellar kainate-binding protein (KBP), a member of the ionotropic glutamate receptor family, we have generated a three-dimensional model structure of the KBP extracellular domain. By an interplay between homology modeling and site-directed mutagenesis, we have investigated the kainate binding properties of 55 different mutants (corresponding to 43 positions) and studied the interactions of some of these mutants with various glutamatergic ligands. As a result, we present here the subsets of amino acids accounting for the binding free energies and specificities of KBP for kainate, glutamate, and CNQX and propose a three-dimensional model, at the microarchitectural level, of the glutamatergic binding domain.

The amino-terminal sequence of the 40 000 molecular weight subunit of the acetylcholine receptor protein from Torpedo marmorata

The membrane receptor for the neurotransmitter acetylcholine (ACh) is now a well characterized protein that has been isolated and purified in milligram quantities from fish electric organ and vertebrate skeletal muscle, in a state that binds cholinergic (nicotinic) ligands (reviewed [ 11). Although its exact quatemary structure is still a matter of controversy, general agreement exists on two major issues: G>