INGEBI   02650
INSTITUTO DE INVESTIGACIONES EN INGENIERIA GENETICA Y BIOLOGIA MOLECULAR "DR. HECTOR N TORRES"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Mutational analysis of the ligand binding site of the á9á10 nicotinic cholinergic receptor.
Autor/es:
SAVINO, J; PLAZAS, PV; KATZ, E; ELGOYHEN, AB
Lugar:
Buenos Aires
Reunión:
Congreso; XXXIX Asociación Argentina de Farmacología; 2007
Resumen:
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 á9á10 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 (á9á10: 13.8±1.7ìM, n=6; á9á10*: 53.52±2.1ìM, n=5; á9*á10: 146.7±5.6ìM, 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 á9*á10 (I20sec/Ipeak=39.7±3.9%, n=2) but was lower in á9á10* (I20sec/Ipeak=90.6±5.5%, n=3). Treatment of á9*á10 injected 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 á9á10 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 á9á10 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 á9 as well as á10 can form the principal component of the ligand binding site of the receptor.