Interaction of La 3+ with GABA A receptors in rat cerebrocortical membranes as detected with [ 35S]t-butylbicyclophosphorothionate binding

Abstract

Lanthanides at micromolar concentrations stimulated binding of [ 35S]t-butylbicyclophosphorothionate (TBPS), a highly sensitive marker for allosteric modulation of the chloride channel of GABA A receptors, to purified rat cerebrocortical synaptosomal membranes in the absence of GABA. This trivalent cation effect appeared to reflect a specific and direct interaction with GABA A receptors, and could not be mimicked by divalent metal ions including Ca 2+, Cd 2+, Co 2+, Sr 2+, Mg 2+ and Mn 2+. On the other hand, Zn 2+, a known allosteric regulator of GABA A receptors, inhibited TBPS binding, but its presence could not prevent stimulation of TBPS binding by La 3+. We further examined the lanthanide binding site by monitoring La 3+-induced changes in TBPS binding. La 3+ reduced the K d for TBPS without affecting B max. Stimulation of TBPS by La 3+ was not additive with the increased TBPS binding induced by other agonists, including GABA (at nanomolar concentrations), neurosteroids or benzodiazepines (in the absence of GABA). GABA at micromolar concentrations inhibited TBPS binding and its inhibitory effect was potentiated by neurosteroids and benzodiazepines, but not by La 3+. Thus, GABA, in the presence of La 3+, inhibited TBPS binding in a monophasic manner with a Hill coefficient of approximately 2 and an IC 50 of 6 μM. In the absence of La 3+, the dose-response profile for GABA became biphasic with an apperance of a stimulatory phase at concentrations of GABA lower than 1 μM. Analysis of the initial stimulation yielded a Hill coefficient of 1 and an EC 50 of 88 nM. These data are consistent with the stimulation of TBPS binding by GABA resulting from the occupation of its high affinity site and its inhibition of TBPS from binding occupation of a low affinity site. It appears that GABA binding at the high affinity site induced the conformational change(s) at the TBPS binding site (which is presumably located near the mouth of the chloride channel as the picrotoxin binding site), which is qualitatively similar to the conformational change induced by La 3+, neurosteroids or benzodiazepines (in the absence of GABA). These similar and nonadditive effects on TBS binding may reflect changes on the multimeric GABA A receptors prior to opening of the chloride channels. Finally, the lanthanide site on GABA A receptors examined here with La 3+ appears to be different from the Zn 2+ site, and from the previously reported monovalent and divalent cation recognition sites (Squires and Saederup, 1982, Mol. Pharmacol. 22, 327).