Diazotization and Thiocyanate Differentiate Agonists from Antagonists for the High- and Low-Affinity Receptors of ?-Aminobutyric Acid

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Abstract
The differentiation of high- and low-affinitypostsynaptic y-aminobutyric acid (GABA) receptors wasexamined in a washed cortical membrane preparation of the rat. The selective elimination of the high- and low-affinity GABA sites by the chaotropic anion thiocyanateand diazotization by p-diazobenzenesulfonic acid (DSA), respectively, offered two model systems for the sepa-rate sites. The [3H]GABA displacing potencies of some GABA agonists [GABA, 4,5,6,7-tetrahydro-isoxa-zole[4,5c]pyridine-3-01 (THIP), and muscimol] and antagonists [bicuculline methiodide (BCM), 3-α-hydroxy-16-imino-5β;-l7-aza-androstan-ll-one (R-5 135), and d-tubo-curarine] and their slope factors were examined in thesemodel systems and in control membranes. The displacingpotency of the agonists was increased in the DSA-pre-treated membranes and decreased in the presence of thiocyanate. The displacing potency of the antagonists wasshifted in an opposite manner. The chaotropic effect ofthiocyanate was reversible and not additive with the inhibitory effect of diazotization on the specific binding of GABA. Inhibition of specific GABA binding by pyri-doxal-5-phosphate (PLP) could not be protected by GABA antagonists (BCM and R-5135) but only by agonists. The results can be interpreted in the frame work of a dual (agonist-antagonist) receptor model, postulating a hydrophobic accessory site at the low-affinity GABA re-ceptor. The effect of thiocyanate on the GABA receptor may result in the exposure of the hydrophobic accessory sites.