[3H]1-aminocyclopropanecarboxylic acid, a novel probe for strychnine-insensitive glycine receptors

Abstract

[³H]1-Aminocyclopropanecarboxylic acid (ACPC) exhibits high affinity, specific binding to strychnine-insensitive glycine receptors. In extensively washed rat forebrain membranes, the specific binding of [³H]ACPC was optimal at 25°C in the presence of 10 mM MgCl₂. Comparable levels of specific [³H]ACPC binding were obtained using centrifugation and filtration for separation of bound from free radioligand. [³H]ACPC labels two sites with K_d1 and B_max1 values of 129±34 nM and 2.30±0.37 pmol/mg protein and K_d2 and B_max2 values of 7.26±1.69 μM and 20.6±2.2 pmol/mg protein for the high and low affinity sites, respectively. The K_d of [³H]ACPC (66 nM) estimated under non-equilibrium conditions (k_off=8.91±0.78×10⁻³ s⁻¹; k_on=1.35×10⁻⁴ nM⁻¹ s⁻¹) was similar to the value obtained for the high affinity site obtained by equilibrium binding. The K_d1 of [³H]ACPC is in good agreement with the previously reported K_i values of ACPC to inhibit the binding of other glycinergic ligands including [³H]glycine, [³H]5,7-dichlorokynurenic acid (5,7-DCKA) and [³H]L-689,560 ((±)-4-(trans)-2-carboxy-5,7-dichloro-4-phenylaminocarbonylamino-1,2,3,4-tetrahydroquinoline). Moreover, the potencies of a series of glycine site ligands, including glycine, ACPC, 1-aminocyclobutanecarboxylic acid (ACBC), 5,7-DCKA, 7-chlorokynurenic acid (7-CKA), R(+)-3-amino-1-hydroxy-2-pyrrolidine (HA-966) and D-serine, to inhibit [³H]ACPC binding were highly correlated with their potencies to inhibit [³H]glycine and [³H]5,7-DCKA binding (r²=0.98−0.51). These results demonstrate that [³H]ACPC is a useful tool for examining the neurochemical and pharmacological properties of strychnine-insensitive glycine receptors.