Identifying of important sites of the conducting pore of nicotinic acetycholine receptor through two-electrode voltage-clamp

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 T