Intracellular Cys-364 is essential for ROS-induced potentiation of the GABAρ1 receptor function.

BELTRÁN GONZÁLEZ AN, GASULLA J, CALVO DJ.

Buenos Aires

Congreso; 5th Special Conference of the International Society of Neurochemistry, ?Synapses and dendritic spines in health and disease?.; 2012

the International Society of Neurochemistry

Reactive oxygen species (ROS), including hydrogen peroxide (H2O2), superoxide anion (O2-.) and hydroxyl radical (OH.), are generated as by-products of the cellular oxidative metabolism and secondary to NMDA and AMPA receptors activation. In brain cells ROS have been implicated as intracellular regulators of neuronal activity, as diffusible messengers for neuron-glia signaling and interneuronal communication, including the regulation of synaptic transmission and neural plasticity. Increased intracellular production of ROS results in oxidative stress and damage to the central nervous system, as seen in normal ageing and neurodegenerative disorders such as Parkinsons´s disease, Alzheimer´s disease, amyotrophic lateral sclerosis and ischemia-reperfusion injury. Diverse neurotransmission systems are modulated by ROS, including the adrenergic, dopaminergic, serotonergic and GABAergic. For example, H2O2 was shown to increase GABA release in spinal cord neurons and inhibit TBPS binding to GABAA receptors. However, the modulation of GABAC receptors by these species had not been determined yet. Previous experiments from our lab showed that H2O2 significantly potentiates homomeric ρ1 GABAC receptor (GABAρ1R) function. In the present study we characterized the mechanism underlying this modulation. Homomeric GABAρ1R were expressed in Xenopus laevis oocytes and GABA-evoked chloride currents recorded by two-electrode voltage-clamp in the presence or absence of H2O2. Potentiation of GABAρ1R by H2O2 was dose-dependent, reversible, voltage independent and strongly depended on GABA concentration. The co-application of lipoic acid (a scavenger of free radicals) and deferoxamine (an iron chelator that inhibits the formation of OH. through the Fenton reaction) decreased the potentiation induced by H2O2, suggesting that oxygen free radicals mediate the potentiation of the GABAρ1R function by H2O2. GABAρ1R receptors subunits contain three cysteine residues: two extracellular at the cys-loop (C177 and C191) and one intracellular (C364). Chemical protection of cysteine residues by N-ethyl maleimide (a membrane-permeable sulfhydryl alkylating reagent) prevented H2O2 potentiation. Furthermore, site directed mutagenesis of the intracellular cysteine by alanine (C364A) and serine (C364S) rendered receptors insensitive to H2O2, suggesting a single modulatory site.