Abstract: Objective To detect the main amino acid sites in the ion-conducting pore of the human α7 nicotinic acetycholine receptor(α7nAChR) by two-electrode voltage-clamp. Methods The amino acids in the transmembrane region M2 of α7 nAChR subunit (from Leu248 to Ala258) were mutated to hydrophilic serine by site-directed mutagenesis. cRNA was synthesized in vitro and injected into Xenopus oocytes. After 1～3 days, the function of wild type α7 nAChR and its mutants were detected by two-electrode voltage-clamp. Results After the hydrophilic mutation on the second transmembrane domain, it was indicated that the mutations of the hydrophobic residues (L248,V252,L255 and L256) to hydrophilic ones significantly decrease the EC₅₀ values of the receptor to agonist acetylcholine. The hydrophilic mutations of these four M2 residues resulted in a dramatic leftward shift of the dose-response curve and decrease desensitization. Moreover, the desensitization rate was positively correlated to the EC₅₀ values in log scale. Conclusions The residues L248, V252, L256 and L255 in the transmembrane M2 of α7 nAChR are the main sites to construct the ion-conducting pore.

Key words: α7 nicotine receptor, two-electrode voltage-clamp, ion-conducting pore