CHARACTERIZATION OF BICUCULLINE BACLOFEN-INSENSITIVE GAMMA-AMINOBUTYRIC-ACID RECEPTORS EXPRESSED IN XENOPUS OOCYTES .1. EFFECTS OF CL- CHANNEL INHIBITORS

Poly(A)+ RNA from bovine retina expressed gamma-aminobutyric acid (GABA)-activated membrane current responses in Xenopus oocytes, consisting of two pharmacologically distinct components. One component (I(G-Aret)) was mediated by GABA(A) receptors, and the other component (K(G-BR)) by atypical GABA receptors that were resistant to inhibtion by bicuculline and insensitive to activation by baclofen. To further characterize the bicuculline/baclofen-insensitive GABA receptors, electrical recordings were made measuring the sensitivity of I(G-BR) to two Cl- channel inhibitors, t-butylbicyclophosphorothionate (TBPS) and picrotoxin. For purposes of comparsion, effects of TBPS and picrotoxin were also assayed on currents mediated by GABA(A) receptors expressed in oocytes by rat cerebral cortex RNA (I(G-Actx)). The main finding of this study was that TBPS was a surprisingly weak inhibitor of I(G-BR), whereas I(G-Actx) was potently suppressed. Assays on maximum responses indicated that I(G-Actx) was at least 500 times more sensitive to TBPS than was I(G-BR) (IC50 values of approximately 0.2-mu-M and > 50-mu-M, respectively). Morevoer, inhibition of I(G-Actx) by micromolar concentrations of TBPS was largely insurmountable, whereas the weak inhibitory effect on I(G-BR) showed strong dependence on agonist concentration. For For example, 10-mu-M TBPS reduced maximum I(G-Actx) by > 90%, an effect that was not significantly reversed by 10-fold increases in the concentration of agonist. In contrast, the same concentration of TBPS caused a 2-fold increase in the EC50 for I(G-BR) but had only marginal (< 5%) inhibitory effects on maximum responses. Picrotoxin inhibited both types of current maximum responses. Picrotoxin inhibited both types of current, but assays on maximum responses indicated that I(G-Actx) was approximately 30 times more sensitive than I(G-BR) (IC50 values of approximately 1 and 30-mu-M, respectively). Inhibitory effects of picrotoxin on I(G-BR) again showed strong dependence on agonist concentration, but in this case there was also a clear insurmountable component. Comparisons between I(G-Actx) and I(G-Aret) suggested that GABA(A) receptors expressed by either brain or retina RNA showed approximately the same sensitivity to TBPS and picrotoxin. Our experiments indicate that the bicuculline/baclofen-insensitive GABA receptors expressed by retina RNA differ markedly from GABA(A) receptors in their sensitivity to TBPS and picrotoxin. Defining the structural features responsible for these differences at the molecular level will provide a further means of investigating the complex mechanisms underlying interactions between inhibitors and GABA-activated Cl- channels.