Binding of [3H] (2S,1′S,2′S)-2-(9-xanthylmethyl)-2-(2′-carboxycycloprophyl) glycine ([3H]LY341495) to cell membranes expressing recombinant human group III metabotropic glutamate receptor subtypes

LY341495 is a highly potent and selective antagonist for group II mGlu receptors (mGlu2 and mGglu3). High affinity binding of [H-3]LY341495 to recombinant human group II mGlu receptors (mGlu2 and mGlu3), and in rat brain homogenates (K-d similar to 1 nM), has been previously described. Although LY341495 is a very selective nM-potent antagonist for group II mGlu receptors, it is also a relatively potent antagonist for group III mGlu receptors at high nanomolar to low micromolar concentrations. In this study we examined and characterized the binding of [H-3]LY341495 to membranes of cells expressing recombinant human group III mGlu receptors. Using up to 100 nM of [H-3]LY341495, the level of specific binding in human mGlu4a receptor-expressing cell membranes was not appreciable and binding to this site was not examined further. In contrast, we demonstrated sufficient specific binding of [H-3]LY341495 to human mGlu6, mGlu7a and mGlu8a receptor-expressing cell membranes to allow for further characterizations. [H-3]LY341495 binding was saturable and rapidly reversible. [H-3]LY341495 bound to a single site in each cell line, with K-d and B-max values of 31.6+/-6.8 nM and 3.3+/-0.7 pmol/mg protein (mGlu6), 72.7+/-22.0 nM and 3.7+/-0.4 pmol/mg protein (mGlu8a), and 14.0+/-1.1 nM and 3.0+/-0.2 pmol/mg protein (mGlu8a), [H-3]LY341495 binding to mGlu6, 7a and 7a was displaceable by compounds which interact functionally with group III mGlu receptors. For example, L-AP4 displaced [H-3]LY341495 with Ki values of 6.8+/-3.1 muM (mGlu6), 211+/-43 muM (mGlu7a) and 1.6+/-0.3 muM (mGlu8a). With L-glutamate, we obtained K-i values of 12.3+/-3.5, 869+/-154 and 4.5+/-0.83 muM, for mGlu6, mGlu7a and mGlu8a, respectively. K-i values for unlabelled LY341495 were 0.058+/-0.008, 0.22+/-0.05 and 0.029+/-0.008 muM, respectively. These studies demonstrated that [H-3]LY341495 is a useful radioligand for studying the pharmacology and expression of recombinant mGlu6, 7a and 8a receptors in cell lines.