Interaction of t‐Butylbicyclophosphorothionate with γ‐Aminobutyric Acid‐Gated Chloride Channels in Cultured Cerebral Neurons

Abstract

The role of t-butylbicyclophosphorothionate (TBPS) as an antagonist of .gamma.-aminobutyric acid (GABA) was studied with primary cultures of neurons from the chick embryo cerebrum. The addition of GABA stimulated the uptake of 36Cl- by neurons and the dose dependence of this effect followed hyperbolic kinetics with a K0.5 = 1.3 .mu.M for GABA. TBPS proved to be a potent inhibitor of GABA-dependent Cl- uptake (IC50 = 0.30 .mu.M). Analysis of the kinetics of this process revealed that TBPS is a noncompetitive inhibitor (Ki = 0.15 .mu.M) with respect to GABA. Scatchard analysis of direct binding of [35S]TBPS to membranes isolated from neuronal cultures gave curvilinear plots. These could be resolved by nonlinear regression methods into two components with KD values of 3.1 nM and 270 nM. The TBPS binding constant for this lower affinity site agreed well with the IC50 and Ki values for inhibition of Cl- flux, suggesting that this site is physiologically relevant to GABA antagonism. GABA was a noncompetitive displacer of [35S]TBPS binding to the lower affinity site. The Ki value for this displacement by GABA (1.7 .mu.M) was comparable to the value for GABA enhancement of Cl- flux. The binding of [35S]TBPS to its low-affinity site on neuronal membranes was ninefold higher in the presence of Cl- than with gluconate, an impermeant anion. The rank order for anion stimulation of [35S]TBPS binding was Br- .gtoreq. SCN- > Cl- .gtoreq. NO3- > I- > F- > gluconate. The EC50 value for Cl- enhancement of [35S] TBPS binding (160 mM) agreed well with the Km for Cl- influx via GABA-gated channels (140 mM). These results indicate that TBPS acts as a GABA antagonist via direct blockade of neuronal Cl- channels. A minimum density of 6.5 .times. 104 chloride channels per neuron was obtained from TBPS binding at saturation.