Mutaciones en el sitio de union de acetilcolina de reptores alfa9 alfa10

SAVINO J; PLAZAS PV; KATZ E; ELGOYHEN AB

Congreso; XXXIX Reunion Anual de la Sociedad Argentina de Farmacologia Experimental; 2007

A conserved feature of all nicotinic receptors is the presence of a disulfide bond between Cys-192 and Cys-193, adjacent to the acetylcholine (ACh) binding site. In order to characterize the relative importance of these residues in the α9α10 receptor we performed site directed mutagenesis of the Cys to Ser. ACh-gated currents were recorded in two-electrode voltage-clamped X. laevis oocytes injected with the receptor subunits. Mutant receptors showed an increase in the EC50 (α9α10: 13.8±1.7μM, n=6; α9α10*: 53.52±2.1μM, n=5; α9*α10: 146.7±5.6μM, n=5). A decrease in blocking potency for nicotine, atropine and strychnine was observed. The desensitization rate was similar to wild type receptors for the single mutant α9*α10 (I20sec/Ipeak=39.7±3.9%, n=2) but was lower in α9α10* (I20sec/Ipeak=90.6±5.5%, n=3). Treatment of α9*α10injected oocytes with the Ca2+ chelator BAPTA-AM resulted in a 94.2 ±1.1% (n=4) decrease in ACh-evoked currents, showing that mutant receptors retained the high Ca2+ permeability previously described for the wild type α9α10 receptor. The macroscopic channel properties of the receptors were not altered by the mutations as shown by the current-voltage relationships. The mutations C192S/C193S in α9α10 appear to alter the affinity of the ligand binding site or, alternatively, the coupling of ligand-binding to channel opening. Our results demonstrate that both α9 as well as α10 can form the principal component of the ligand binding site of the receptor.