Modulation of neuronal and recombinant GABA(A) receptors by redox reagents

1. The functional role played by the postulated disulphide bridge in gamma- aminobutyric acid type A (GABA(A)) receptors and its susceptibility to oxid ation and reduction were studied using recombinant (murine receptor subunit s expressed in human embryonic kidney cells) and rat neuronal GABA(A) recep tors in conjunction with whole-cell and single channel patch-clamp techniqu es. 2. The reducing agent dithiothreitol (DTT) reversibly potentiated GABA-acti vated responses (I-GABA) of alpha 1 beta 1 or alpha 1 beta 2 receptors whil e the oxidizing reagent 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) caused inhibition. Redox modulation of I-GABA was independent of GABA concentrati on, membrane potential and the receptor agonist and did not affect the GABA EC50 or Hill coefficient. The endogenous antioxidant reduced glutathione ( GSH) also potentiated I-GABA in alpha 1 beta 2 receptors, while both the ox idized form of DTT and glutathione (GSSG) caused small inhibitory effects. 3. Recombinant receptors composed of alpha 1 beta 1 gamma 2S or alpha 1 bet a 2 gamma 2S were considerably less sensitive to DTT and DTNB. 4. For neuronal GABA(A) receptors, I-GABA was enhanced flurazepam and relat ively unaffected by redox reagents. However, in cultured sympathetic neuron es, nicotinic acetylcholine-activated responses were inhibited by DTT whils t in cerebellar granule neurones, NMDA-activated currents were potentiated by DTT and inhibited by: DTNB. 5. Single GABA-activated ion channel currents exhibited a conductance of 16 pS for alpha 1 beta 1 constructs. DTT did not affect the conductance or in dividual open time constants determined from dwell time histograms, but inc reased the mean open time by affecting the channel open probability without increasing the number of cell surface receptors. 6. A kinetic model of the effects of DTT and DTNB suggested that the recept or existed in equilibrium between oxidized and reduced forms. DTT increased the rate of entry into reduced receptor forms and also into desensitized s tates. DTNB reversed these kinetic effects. 7. Our results. indicate that GABA(A) receptors formed by alpha and beta su bunits are susceptible to regulation by redox agents. Inclusion of the gamm a 2 subunit in the receptor, or recording from some neuronal GABA, receptor s, resulted in reduced sensitivity to DTT and DTNB. Given the suggested exi stence of alpha beta subunit complexes in some areas of the central nervous system together with the generation and release of endogenous redox compou nds, native GABA(A) receptors may be subject to regulation by redox mechani sms.