Synergistic inhibition of [3H]muscimol binding to calf-brain synaptic membranes in the presence of L-homocysteine and pyridoxal 5'-phosphate. A possible mechanism for homocysteine-induced seizures.

Abstract

L-Homocysteine and pyridoxal 5'-phosphate (PxyP) inhibited [3H]muscimol binding to freeze-thawed, Triton-treated calf brain membranes (containing high-affinity muscimol-binding sites: Kd 9.5 +/- 0.6 nM, Bmax 5.2 +/- 0.2 pmol/mg protein). The homocysteine--pyridoxal-5'-phosphate (Hcy-PxyP) thiazine complex had no effect. L-Homocysteine was found to be a partially competitive inhibitor, thus demonstrating an allosteric inhibition with Ki value of 1.96 mM for free receptor and Ki of 13 mM for receptor-muscimol complex. PxyP was shown to be a two-site pure competitive inhibitor of [3H]muscimol binding with cooperativity of PxyP binding such that Ki values for PxyP of 20 mM and 2.1 mM were found. L-Homocysteine and PxyP when added simultaneously to binding assays, caused a greater degree of inhibition than that observed at the same total specific concentration of either inhibitor alone. This synergistic inhibitory effect was shown to be due to a homocysteine-induced increase in the affinity of PxyP-binding sites. Three alternative models are suggested to explain the observed synergistic effects whereby it is proposed that PxyP and [3H]muscimol binding is mutually exclusive, while L-homocysteine with PxyP and L-homocysteine with muscimol, exhibit non-exclusivity. Non-linear regression analysis of binding data was undertaken in order to substantiate conclusions drawn from graphical procedures and in an attempt to ascertain which mathematical model best fitted the experimental data describing the synergistic inhibitory effects of L-homocysteine and PxyP. This synergistic inhibitory effect of PxyP and L-homocysteine on the post-synaptic gamma-aminobutyric acid receptor may provide a basis for explanation of the mechanism of homocysteine-induced seizures.