A method for evaluating chemical selectivity of agonists for glutamate receptor channels incorporated in liposomes based on an agonist-induced ion flux measured by ion-selective electrodes

A new method for evaluating chemical selectivity of agonists for the NMDA s ubtype of glutamate receptor (GluR) channels is described. The method is ba sed on the magnitude of Ca2+ release from GluR-incorporated liposomes, whic h is measured by a Ca2+ ion-selective electrode with a thin-layer mode. The partially purified GluRs from rat whole brain were reconstituted into Ca2-loaded liposomes. Small aliquots (each 50 mu l) of the proteoliposomes, in the presence of an antagonist DNQX for blocking non-NMDA subtype, were sub jected to potentiometric measurements of Ca2+ release under stimulation by three kinds of agonists, i.e. NMDA, L-glutamate and L-CCG-IV. The amount of the Ca2+-ion flux through the GluR channel induced by the agonists was fou nd to increase in the order of NMDA < L-glutamate < L-CCG-IV, which was con sistent with that of binding affinity of the agonists toward the NMDA subty pe. However, the range of selectivity of the relevant agonists was much sma ller compared with results based on binding affinities. The present method provides physiologically more relevant values for the agonist selectivity o f GluRs as compared to that of the conventional binding assay in the sense that the selectivity is based on the very magnitude of Ca2+ flux through th e NMDA receptor, i.e. the extent of signal transduction by a given agonist. The evaluation of agonist selectivity based on Na+ release was also invest igated by using a Na+ ion-selective electrode, but agonist-induced Na+ rele ase was not detected, because of low permeability of Na+ through the NMDA s ubtype. (C) 1999 Elsevier Science B.V. All rights reserved.