GABA binding and bicuculline in spinal cord and cortical membranes from adult rat and from mouse neurons in cell culture

Abstract

Sodium-independent [3H]GABA and [3H]muscimol binding was determined in adult rat cerebral cortical and spinal cord membranes and in membranes from fetal mouse cortical and spinal cord neurons in primary dissociated cell culture. In adult rat cerebral cortical membranes, [3H]GABA bound to two sites (Kd=8 nM, Bmax=0.62 pmol/mg protein; Kd=390 nM, Bmax=3.9 pmol/mg protein) whereas the GABA agonist, [3H]muscimol, bound only to a high affinity site (Kd=5.6 nM, Bmax=1.9 pmol/mg protein). In adult rat spinal cord, only a low affinity site was seen with [3H]GABA (Kd=340 nM, Bmax=9.8 pmol/mg protein) and only a high affinity site was seen with [3H]muscimol (Kd=5.6 nM, Bmax=0.25 pmol/mg protein). The inability to measure a high affinity [3H]GABA binding site in spinal cord probably reflects the high ratio of low to high affinity sites in spinal cord (39:1). In membranes from mouse neurons in cel; culture, [3H]GABA bound to two sites on cortical neurons (Kd=9 nM, Bmax=0.24 pmol/mg protein; Kd=510 nM, Bmax=1.3 pmol/mg protein) and spinal cord neurons (Kd=13 nM, Bmax=0.12 pmol/mg protein; Kd=640 nM, Bmax=3.2 pmol/mg protein). Again, the ratio of low to high affinity sites in cultured mouse spinal cord neurons was high (27:1).The effects of the potent GABA antagonist, (+)bicuculline, on both low and high affinity [3H]GABA binding was determined. Bicuculline appeared to inhibit binding to both sites competitively but the Ki for inhibiting the high affinity site was 5 [mu]M and for inhibiting the low affinity site was 115 [mu]M. Bicuculline inhibited [3H]muscimol binding in both brain and spinal cord competitively with Kis of 4[mu]M and 10 [mu]M respectively. Bicuculline inhibition of [3H]muscimol binding in cultured neuronal membranes was similar to that in adult rat membranes.The binding of the potent GABA agonist, muscimol, only to the high affinity site in both adult rat and cultured mouse neuronal membranes suggests that the high affinity site is the physiologically relevant postsynaptic GABA receptor. The fact that bicuculline inhibits the high affinity site (but not the low affinity site) in concentrations similar to those needed to block GABA-responses in physiological experiments28 supports this hypothesis.