Functional Study of the Mechanism Underling Redox Modulation of Homomeric rho1 GABAC Receptor

CALERO CI; CALVO DJ

Córdoba, Argentina

Congreso; I IRCN. Primera Reunion Conjunta de Neurociencias de la Sociedad Argentina de Neurociencias y el Taller Argentino de Neurociencias; 2009

Redox mechanisms can regulate the activity of many neurotransmitter receptors including the members of the cys-loop receptor family. In a previous work we demonstrated that, similarly to GABAA receptors, GABAC receptors can be redox modulated. Two extracellular sulfhydryl groups (-SH) form the characteristic cys-loop in GABAC receptors, they are located in cys 207 and 221 and are promising targets for redox modulation. To test this hypothesis we studied if chemical manipulation of these aminoacidic residues can affect GABAC receptor modulation by redox agents.   Homomeric GABAr1 receptors (GABAr1R) were expressed in X. laevis oocytes and GABA-evoked Cl- currents (GABAr1 responses) recorded by two-electrode voltage clamp. The application of N-ethylmaleimide (NEM), an irreversible thiol alkylating agent that forms covalent bonds with free -SH and prevents further chemical reactions with these sites, modulated GABAr1 responses both, shifting to the left D-R curves and significantly increasing maximal response values. Meanwhile, as previously shown, the -SH reducing agent DTT also potentiated GABAr1R, but in a reversely way (Calero, et al., 2008). As GABAr1R do not have additional –SH in their extracellular domain, and in order to study if NEM effects were indeed due to the chemical modification of aminoacids forming the cys-loop, we examined DTT actions on GABAr1 responses before and after the application of NEM. Consistently with the participation of the cys 207 and 221, NEM treatment prevented DTT-induced potentiation of GABAr1 responses. Similar experiments were performed using other endogenous redox agents who are known to modulate GABAr1R, that is ascorbic acid (Asc) and GSH. In contrast to that observed for DTT, redox modulation of GABAr1 responses induced by Asc and GSH was only partially prevented by NEM.      Our results indicate that redox agents can induce at least two types of reversible modifications at the GABAr1R. The effects of redox agents can be explained in part by modification of the -SH that form the cys-loop (e.g: DTT), but also by the reaction with redox-sensitive/NEM-insensitive residues (e.g: Asc and GSH) which are probably involved in the functional modulation of GABAr1R. Supported by CONICET and FONCyT.