The cys-loop is essential for nitric oxide potentiation of the homomeric rho1 GABA receptors

J GASULLA; A BELTRÁN GONZÁLEZ; DJ CALVO

New Orleans

Congreso; Neuroscience 2012; 2012

Society for Neuroscience

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 rho1 GABAC receptor (GABArho1R) 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 GABArho1R. 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 GABArho1R can undergo analogous modifications. Human GABArho1R were expressed in Xenopus laevis oocytes and GABA-evoked responses electrophysiologically recorded by two-electrode voltage clamp. In order to test the involvement of GABArho1R cysteine residues in NO effects, we examined the potentiation of GABArho1R 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 GABArho1R 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 GABArho1R 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 GABArho1C364AR were similarly potentiated by DEA (% P GABArho1C364A = 46.1 ± 9.3 vs. % P GABArho1wt = 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 GABArho1R function.