An intracellular redox sensor for reactive oxygen species at the M3‐M4 linker of GABAAρ1 receptors

Abstract

Reactive oxygen species (ROS) are normally involved in cell oxidative stress but also play a role as cellular messengers in redox signalling; for example, modulating the activity of neurotransmitter receptors and ion channels. However, the direct actions of ROS on GABAA receptors were not previously demonstrated. In the present work, we studied the effects of ROS on GABAA ρ1 receptor function. GABAA ρ1 receptors were expressed in oocytes and GABA-evoked responses electrophysiologically recorded in the presence or absence of ROS. Chemical protection of cysteines by selective sulfhydryl reagents and site-directed mutagenesis studies were used to identify protein residues involved in ROS actions. GABAA ρ1 receptor-mediated responses were significantly enhanced in a concentration-dependent and reversible manner by H₂O₂. Potentiating effects were attenuated by a free radical scavenger, lipoic acid or an inhibitor of the Fenton reaction, deferoxamine. Each ρ1 subunit contains only three cysteine residues, two extracellular at the Cys-loop (C¹⁷⁷ and C¹⁹¹) and one intracellular (C³⁶⁴) at the M3-M4 linker. Mutant GABAA ρ1 receptors in which C³⁶⁴ was exchanged by alanine were completely insensitive to modulation, implying that this site, rather than a cysteine in the Cys-loop, is essential for ROS modulation. Our results show that the function of GABAA ρ1 receptors is enhanced by ROS and that the intracellular C³⁶⁴ is the sensor for ROS actions.