Identification of extrasynaptic binding sites for [3H]GABA in peripheral nerve.

The binding of radiolabelled gamma-aminobutyric acid ( [3H]GABA) to extrasynaptic sites in peripheral nerve was examined in order to characterize the receptor species mediating the depolarizing action of GABA on myelinated axons. Binding of [3H]GABA was carried out under Na+-free conditions with fresh homogenates of amphibian sciatic nerve. The kinetics of association of the radioligand suggested the presence of a rapidly associating, reversible binding site, and a slowly associating, apparently irreversible one. Scatchard plots of the reversible component of binding were linear and yielded a mean affinity (Kd) of 22 nM. This stands in contrast with the ED50 of 90 microM for GABA-evoked depolarization of frog nerve; and therefore the high affinity binding sites are unlikely to be involved in such depolarization. The rank order of potency of agonists that competed with [3H]GABA was muscimol greater than GABA much greater than delta-aminovaleric acid greater than beta-alanine. However, the GABAA analogues 3-aminopropanesulfonic acid, beta-guanidinopropionic acid, and guanidoacetic acid as well as the GABAB agonist baclofen all failed to displace the ligand. Bicuculline methiodide, picrotoxin, and nipecotic acid also did not compete. The bicuculline-insensitive, baclofen-insensitive high affinity binding sites identified here appear to be unique, as they are distinctly different from the classical GABAA and GABAB receptors.