Redox modulation of recombinant human GABA(A) receptors

We previously reported that GABA-evoked currents of rat retinal ganglion ce lls were modulated by redox agents. In this study, vie further characterize d the effects of redox modulation on GABA receptors using recombinant human subunits in the Xenopus oocyte expression system with two-electrode Voltag e-clamp recording. GABA receptors composed of subunits alpha(1-3). beta(1-3 ), gamma(1), gamma(2S). and rho(1) were expressed. The sulfhydryl reducing agent dithiothreitol reversibly potentiated the responses of various combin ations of functional recombinant GABA(A) subunits, whether expressed as tri plets (alpha(1)beta(1-3)gamma(1,2S)) pairs (alpha(1-3)beta(1-3); beta(1-3)g amma(1,2S)), or singly (beta(2)) These effects of dithiothreitol were rapid ly reversible, and the oxidizing agent 5-5'-dithiobis-2-nitrobenzoic acid e xerted the opposite effect. In contrast to these effects on GABA(A) recepto rs, dithiothreitol had no effect on the responses of homomeric GABA rho(1) (GABA(C)) receptors. The degree of dithiothreitol potentiation of GABA(A) r eceptor responses depended on subunit composition. Go-expression of gamma(2 S) With alpha(1)beta(1-3) subunits resulted in markedly less dithiothreitol potentiation of GABA-evoked currents than that observed for alpha(1-3)beta (1-3) subunits in the absence of gamma(2S). None the less, the magnitude of dithiothreitol potentiation could be restored by using a combination of lo wer GABA concentrations (5-10 mu M) and higher dithiothreitol concentration s (5-20 mM). N,N,N',N'-tetrakis(2-pyridyl-methyl)ethylenediamine a high-aff inity Zn2+ chelator, also potentiated GABA(A) receptor currents. However, t he potentiation produced by 10 mM dithiothreitol was larger than that produ ced by saturating concentrations of hi,N,N',N'-tetrakis(2-pyridyl-methyl)et hylenediamine (100 mu M), implying that at least part of the effect of dith iothreitol was due to redox modulation rather than Zn2+ chelation. Dithioth reitol also potentiated the spontaneous current of homomeric GABA(A) recept ors composed of beta subunits. Mutation of a single cysteine residue in the M3 domain, yielding homomeric beta(3)(C313A) receptors, abrogated dithioth reitol potentiation of the spontaneous current. In summary, this study further characterizes the modulatory effects of redo x agents on recombinant GABA(A) receptors. The degree of redox modulation o f GABA(A) receptors depended on subunit composition. In contrast to their e ffect on GABA(A) receptors, redox agents were not found to modulate GABA(C) receptors composed of homomeric rho(1) subunits. Using site-directed mutag enesis, a cysteine residue was located in the beta(3) subunit which may com prise one of the redox-active sites that underlies the modulation of hetero meric GABA(A) receptors by reducing and oxidizing agents. (C) 2000 IBRO. Pu blished by Elsevier Science Ltd. AU rights reserved.