Exploring the structural and mechanistic basis of activation and drug modulation of Cys-loop receptors at the singlechannel level

CECILIA BOUZAT

Montpellier

Simposio; 12 Congreso Anual de la Sociedad Francesa de Neurociencias; 2015

Sociedad Francesa de Neurociencias

Cys-loop receptors are pentameric ligand-gated ion channels that include serotonin (5-HT3), nicotinic (AChR), GABA and glycine receptors. They are involved in a large number of physiological functions and neurological disorders, and they are modulated by endogenous and exogenous compounds. We have combined site-directed mutagenesis and cell expression with single-channel kinetic analysis to delineate molecular mechanisms and structures underlying the activation process and drug modulation in two homomeric members of this family: 7 AChR and 5-HT3A receptors. We have developed a strategy, called electrical fingerprinting, that allows the determination of the subunit stoichiometry of individual receptors from single-channel recordings. We found that 7 receptors can be fully activated with only one intact agonist binding site. Replacing the membrane-embedded domain of 7 by that of the related 5-HT3A receptor increases the number of sites that need to be occupied to achieve maximal open-channel lifetime, thus revealing a novel interdependence between the detector and actuator domains of these receptors. The unique ability to elicit a full biological response with a single occupied binding site adapts 7 to volume transmission, a prevalent mechanism of ACh-mediated signaling in the nervous system and non-neuronal cells. In contrast, the five coupling regions of 7, which are located at the interface between binding and pore domains, contribute additively to open-channel lifetime. By single-channel kinetic analysis we delineated mechanisms and sites of action of positive allosteric modulators of 7, which represent promising therapeutic tools for the treatment of several neurological disorders, and we generated models that describe activation by partial agonists of 5-HT3A receptors, which led to a novel concept of partial agonism. Deciphering the molecular basis underlying responses of Cys-loop receptors has implications for the design of novel and more specific therapeutic compounds.