INVESTIGADORES
BOUZAT Cecilia Beatriz
congresos y reuniones científicas
Título:
Activation and Desensitization of Homomeric Cys-loop Receptors: Functional Relationships Between Agonist Binding Sites and Coupling Regions
Autor/es:
BOUZAT, C.
Reunión:
Conferencia; Nicotinic Receptors 2011. Wellcome Trust Conference Centre; 2011
Resumen:
Activation and Desensitization of Homomeric Cys-loop Receptors: Functional Relationships Between Agonist Binding Sites and Coupling Regions Homomeric receptors are the structural simplest class of receptors of the Cys-loop superfamily and are therefore invaluable models for probing fundamental relationships between structure and function. To determine the number and locations of agonist binding sites required for maximal open channel stability, we applied an electrical fingerprinting strategy in the homomeric receptor model, a7-5HT3A. To vary the number of agonist binding sites, we installed mutations that prevent agonist binding, and to report the presence of the mutant subunit, we installed mutations that alter single-channel conductance. We find that agonist occupancy of only one binding site allows channel opening although open channel duration is brief; occupancy of three consecutive or two non-consecutive sites produces openings with intermediate duration; and occupancy of three binding sites at non-consecutive sites exhibits maximal open channel lifetime. The conformational changes initiated at the binding site are propagated to the gate through the extracellular-transmembrane interface, named as coupling region. We show that structural differences in the coupling region of homomeric a7 and 5-HT3A receptors account for the large differences in open channel lifetime and time of desensitization onset between these contrasting members of the superfamily. The chimeric a7-5HT3A receptor exhibits functional properties intermediate between those of the parent receptors, but the functional signatures of each parent are reconstituted after substituting the major loops at the coupling region from the corresponding parent receptor. By applying the electrical fingerprinting strategy, we find that each coupling region in the pentamer contributes an equal increment to the stability of the open channel. We also determine minimal requirements for channel opening regardless of stability, and find that channel opening can be detected in a receptor with one active coupling region flanked by functional binding sites, or with one active binding site flanked by functional coupling regions. The overall findings show that whereas the agonist binding sites contribute inter-dependently and asymmetrically to open channel stability, the coupling regions contribute independently and symmetrically.