Functional modulation of homomeric rho1 GABAc receptors by ascorbic acid.

CI CALERO AND DJ CALVO

Chicago

Congreso; 39th Annual Meeting Society for Neuroscience; 2009

Society for Neuroscience

   The activity of many neurotransmitter receptors, including different members of the cys-loop ligand-gated receptor superfamily, can be regulated by redox mechanisms. In a previous work we demonstrated that, similarly to GABAA receptors, GABAC receptors (GABAr1R) can be pharmacologically modulated by reducing and oxidizing agents. Ascorbic acid (Asc) has a major role in cell physiology as antioxidant and was proposed to be a neuromodulator. Based on these data, we examined if GABAr1R function could be changed in the presence of this endogenous vitamin. Homomeric r1 GABAC receptors (GABAr1R) were expressed in Xenopus laevis oocytes and GABA-evoked responses (chloride currents) were recorded by two-electrode voltage clamp in the presence or absence of Asc.      Asc applications produced an enhancement of the GABAr1R responses. Asc actions were dose dependent, very fast and easily reversible. Potentiation induced by 3mM Asc on ionic currents elicited by low concentrations of GABA (0.3mM) was significant (+192.9 ± 87.5%; n=7) and a mild but still significant effect (+14.8 ± 3.4%; n=7) was observed at maximal saturating GABA concentrations (30mM). Asc produced a marked leftward shift in dose-response (D-R) curves for GABA (EC50control= 0.74 ± 0.01mM, Hill coefficient (nHill) 1.58 ± 0.04 and EC50Asc= 0.49 ± 0.01mM, nHill 1.88 ± 0.07 (n= 5-10; p<0.03) and a slight but significant increase in the current maximal values (13.xx ± X.xx %). L-dehydroascorbic acid and calcium ascorbate, two related molecules that lack the ability to donate protons, were unable to modulate GABAr1 responses. Meanwhile, D-isoascorbic acid, an stereoisomer of Asc which can also donate protons, was less active than Asc and significantly increased GABAr1 responses (GABA= 0.3mM) in a 33.5 ± 12.5%, (n=4; p< 0.2). N-ethylmaleimide (NEM) (30mM), a thiol alkylating agent that modified irreversibly sulfhydryl groups, precluded Asc effects at high GABA concentrations but did not prevent the shift induced by Asc in the D-R curve.      Our results indicate that Asc induces at least two types of reversible redox modifications in GABAr1R. Good candidates for targeting Asc actions are the two cysteine residues (C207 and C221) that form the characteristic cys-loop in these receptors. In addition, redox sensitive/NEM insensitive aminoacidic residues are probably involved in the functional modulation of GABAr1R by Asc. Supported by CONICET and FONCyT.