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Mutations of a Conserved Lysine Residue in the N-Terminal Domain of 7 Nicotinic Receptors Affect Gating and Binding of Nicotinic Agonists

Instituto de Neurociencias de Alicante, Universidad Miguel Hernández-Consejo Superior de Investigaciones Cientificas, Alicante, Spain

Activation of nicotinic acetylcholine receptors is initiated by binding of agonists, and as a consequence, specific domains transmit the chemical signal to the channel gate through a sequence of conformational changes. Recent high-resolution structural data from a snail acetylcholine binding protein have shown that the side chain of a lysine residue, located in the -strand 7 and strictly conserved in subunits of nicotinic receptors, systematically moves upon agonist binding, suggesting that it might be involved in both binding and gating. To test this hypothesis in neuronal nicotinic receptors, Lys145 was substituted by other amino acids in the 7 nicotinic receptor, and expression levels and electrophysiological responses for several nicotinic agonists and antagonists were determined. Substitutions of Lys145 showed a variety of functional effects: 1) strong reductions in the functional responses to acetylcholine, nicotine, and dimethylphenylpiperazinium, the latter becoming an antagonist; 2) increases in the agonist EC₅₀ values (up to 80-fold with acetylcholine); 3) heterogeneous behavior of the different agonists, with epibatidine and cytisine being less affected by the substitutions; 4) decreases of agonist affinities for the desensitized receptors; and 5) small changes in the affinity of nicotinic antagonists. It is concluded that the presence of a polar or positively charged side chain at this position improves the gating function with acetylcholine and nicotine, although the lysine side chain seems to be necessary for retaining the binding properties of acetylcholine. The results are compatible with the involvement of Lys145 in the early steps of channel activation by acetylcholine.

Address correspondence to: Dr. Francisco Sala, Instituto de Neurociencias de Alicante, Universidad Miguel Hernández-CSIC, Apartado 18, 03550-Sant Joan d'Alacant, Alicante, Spain. E-mail: fsala{at}umh.es

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