The extracelluar cysteines are critical for the potentiation of homomeric rho1 GABAC receptors by nitric oxide.

GASULLA J; BELTRÁN GONZALEZ A; CALVO DJ

New Orleans

Congreso; 42th meeting of the Society for Neuroscience; 2012

SFN

The extracelluar cysteines are critical for the potentiation of homomeric rho1 GABAC receptors by nitric oxide Gasulla J, Beltran Gonzalez AN, Calvo DJ. Nitric Oxide (NO) is a gas messenger produced in neurons by the neural nitric oxide synthase (nNOS) that can modulate the activity of neurotransmitter receptors (Garthwaite, 2008; Steinert et al., 2011). Previous experiments from our lab showed that the application of the NO donors 1,1-diethyl-2-hydroxy-2-nitroso-hydrazine sodium (DEA) and S-nitrosoglutathion (GSNO) potentiate homomeric ρ1 GABAC receptor (GABAρ1R) function in a dose-dependent, fast and reversible manner. The specific NO scavenger 2-(4-carboxyphenyl)-4,4,5,5 tetramethylimidazoline-1-oxyl-3-oxide potassium salt (CPTIO) prevents DEA effects indicating that modulation is exerted by NO itself and not by other reaction intermediates. In the present work, we examined the mechanisms underlying NO potentiation of GABAρ1R. Earlier studies in NMDA receptors shown that specific cysteine residues, critical for channel function, can be S-nitrosylated by NO (Ahern et al., 1999; Choi et al., 2000; Eu et al., 2000). Thus, we examined if GABAρ1R can undergo analogous modifications. Human GABAρ1R were expressed in Xenopus laevis oocytes and GABA-evoked responses electrophysiologically recorded by two-electrode voltage clamp. In order to test the involvement of GABAρ1R cysteine residues in NO effects, we examined the potentiation of GABAρ1R receptors induced by NO in the presence of reagents that modify cysteine thiol groups. Pre-incubation of 2.5 mM (2-aminoethyl) methanethiosulfonate (MTSEA) reduced DEA potentiation from 38.7 ± 4.1 % to 4.6 ± 1.6 % (n = 8-10; p < 0.0001). In addition, the irreversible alkylating reagent N-ethyl maleimide (NEM) prevented DEA effects on GABAρ1R in a dose dependent manner (% Pcontrol = 36.9 ± 5.0 %; % PNEM 300 μM = 0.2 ± 4.5 %; n = 4; p < 0.01, % PNEM 60 μM = 13.4 ± 1.5 %; n = 5; p < 0.05; % PNEM 30 μM = 37.6 ± 4.2 %; n = 4; n.s.). Each GABAρ1R subunit contains three cysteine residues, namely: two extracellular at the cys-loop (C177 and C191) and one intracellular (C364). Site directed mutagenesis of the C177 and C191 renders non-functional receptors, but C364 can be safely exchanged by alanine. Wild type receptors and mutant GABAρ1C364AR were similarly potentiated by DEA (% P GABAρ1C364A = 46.1 ± 9.3 vs. % P GABAρ1wt = 37.6 ± 3.9; n = 6; n.s.) suggesting that the intracellular C364 is not involved in NO effects. Taken together, these results indicate that extracellular cysteines C177 and C191 forming the characteristic cys-loop are crucial for NO action and that these amino acidic residues can be chemically modify by NO to reversibly potentiate GABAρ1R function.