Characterization of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) binding to GABAA receptors in postmortem human brain

Abstract

Background and purpose: The aim of the present study was to determine whether binding of [(35)S]t-butylbicyclophosphorothionate ([(35)S]TBPS) to the convulsant binding site of GABA(A) receptors in human postmortem brain samples can be used as an in vitro index of the functional activation of these receptors.

Experimental approach: Postmortem stability of [(35)S]TBPS binding was assessed in rat brain samples harvested at various times after death and the binding properties of [(35)S]TBPS binding (K(D) and B(max)) were determined in human postmortem brain using radioligand binding studies. In addition, the ability of human brain [(35)S]TBPS binding to be allosterically modulated by compounds that bind at recognition sites distinct from the convulsant binding site was measured.

Key results: Whereas binding of [(3)H]Ro 15-1788 to the benzodiazepine binding site and [(3)H]muscimol to the agonist (GABA) binding site were retained over a 20 h postmortem interval, there was a significant decrease in the affinity and number of [(35)S]TBPS binding sites. Nevertheless, [(35)S]TBPS binding in human brain could be inhibited by TBPS, picrotoxin, loreclezole and pentobarbital and modulated by GABA with potencies comparable to those observed in rats. In addition, the GABA-induced reduction in human brain [(35)S]TBPS binding could be modulated by benzodiazepine site ligands in a manner that reflected their intrinsic efficacies.

Conclusions and implications: These results suggest that allosteric coupling between the [(35)S]TBPS, GABA and benzodiazepine binding sites is preserved in postmortem human brain and that [(35)S]TBPS binding in this tissue may be used to study functional characteristics of native human GABA(A) receptors.

Figure 1

Saturation isotherms of (a) [³⁵S]TBPS; (b) [³H]Ro15-1788; and (c) [³H]muscimol binding to P₂ membranes prepared from whole-rat brains obtained either immediately (t=0) or 5, 10 or 20 h after sacrificing. Values shown are mean±s.e.m. of 3–4 separate animals.

Figure 2

Saturation isotherms for [³⁵S]TBPS binding to rat and human cortex (left) and cerebellum (right). Values shown are mean±s.e.m. (n=3–4).The K_D and B_max values derived from these data are shown in Table 2.

Figure 3

Inhibition of [³⁵S]TBPS binding by TBPS, picrotoxin, GABA, loreclezole and pentobarbital in P₂ membranes derived from (a) human cortex; (b) human cerebellum; (c) rat cortex; and (d) rat cerebellum. Values shown are mean±s.e.m. (n=3–4).

Figure 4

The kinetics of (a) association and (b) dissociation of specific [³⁵S]TBPS binding in P₂ membrane of human cerebral cortex observed in the absence and presence of 0.3, 0.6 and 1 μM GABA. The association and dissociation data were transformed into Ln(B_e/B_e−B) and Ln(B_t/B₀) functions, respectively (insets) and the kinetic constants determined by linear regression, where B_e = binding at equilibrium; B = binding at any time point in the association phase; B_t = binding at any time point during dissociation and B₀ = binding at t=0.

Figure 5

In (a), the association (K_ob) and in (b), the dissociation constants (K_off) for [³⁵S]TBPS binding to human and rat cortex (Cx) and cerebellum (Cb) are plotted as a function of GABA concentration. Values shown are mean±s.e.m. (n=3–4).

Figure 6

Modulation of the GABA-mediated inhibition of [³⁵S]TBPS binding to membranes of (a) human cortex (Cx) and (b) human cerebellum (Cb) by benzodiazepine site ligands with differing intrinsic efficacies, as measured under equilibrium binding conditions. The concentration–effect curve was essentially unaltered by the benzodiazepine site antagonist Ro15-1788 (100 nM) but shifted leftward by the agonist diazepam (1 μM) and shifted rightward by the inverse agonist DMCM (10 μM). Values shown are mean±s.e.m. (n=3–4).