Identification of a New Component of the Agonist Binding Site of the Nicotinic α7 Homooligomeric Receptor

Abstract

Tryptophan 54 of the α7 neuronal nicotinic homooligomeric receptor is homologous to γ-Trp-55 and δ-Trp-57 of non-α subunits of Torpedo receptor labeled by d-tubocurarine. This residue was mutated on the α7-V201-5-hydroxytryptamine (5HT)3 homooligomeric chimera, which displays α7 nicotinic pharmacology, and for which both equilibrium binding studies and electrophysiological recordings could be carried out in parallel. Replacement of Trp-54 by a Phe, Ala, or His causes a progressive decrease both in binding affinity and in responses (EC50 or IC50) for acetylcholine, nicotine, and dihydro-β-erythroidine, without significant modification in α-Bgtx binding. Except for Gln-56, comparatively small effects are observed when the other residues of the 52-58 region are mutated into alanine. These data support the participation of Trp-54 to ligand binding, and provide evidence for a new “complementary component” of the α7 nicotinic binding site, distinct from its three-loop “principal component,” and homologous to the “non-α component” present on γ and δ subunits. Tryptophan 54 of the α7 neuronal nicotinic homooligomeric receptor is homologous to γ-Trp-55 and δ-Trp-57 of non-α subunits of Torpedo receptor labeled by d-tubocurarine. This residue was mutated on the α7-V201-5-hydroxytryptamine (5HT)3 homooligomeric chimera, which displays α7 nicotinic pharmacology, and for which both equilibrium binding studies and electrophysiological recordings could be carried out in parallel. Replacement of Trp-54 by a Phe, Ala, or His causes a progressive decrease both in binding affinity and in responses (EC50 or IC50) for acetylcholine, nicotine, and dihydro-β-erythroidine, without significant modification in α-Bgtx binding. Except for Gln-56, comparatively small effects are observed when the other residues of the 52-58 region are mutated into alanine. These data support the participation of Trp-54 to ligand binding, and provide evidence for a new “complementary component” of the α7 nicotinic binding site, distinct from its three-loop “principal component,” and homologous to the “non-α component” present on γ and δ subunits.