A single-channel method for evaluation of very magnitudes of Ca2+ ion fluxes through epsilon 4/zeta 1 N-methyl-D-aspartate receptor channels in bilayer lipid membranes

A single-channel method for evaluating agonist selectivity in terms of the very number of Ca2+ ions passed through the epsilon4/zeta1 N-methyl-D-aspar tate (NMDA) receptor ion channel in bilayer lipid membranes (BLMs) is descr ibed. The number of Ca2+ passed through the single-channel was obtained fro m single-channel recordings in a medium where the primary permeant ion is C a2+. The recombinant epsilon4/zeta1 NMDA channel was partially purified fro m Chinese hamster ovary cells expressing the channel and incorporated in BL Ms formed by the tip-dip method. It was found that the epsilon4/zeta1 chann el in BLMs is permeable to Ca2+ and Na+, but the number of Ca2+ passed thro ugh the channel is much fewer than that of Na+. The integrated Ca2+ current s induced by three typical agonists NMDA, L-glutamate and L-CCG-IV were obt ained at concentration of 50 muM, where the integrated currents for all the agonists reached their saturated values. The integrated Ca2+ currents obta ined are (3.1 +/- 0.21) x 10(-13) C/s for NMDA, (4.6 +/- 0.31) x 10(-13) C/ s for L-glutamate and (5.7 +/- 0.25) x 10(-13) C/s for L-CCG-IV, respective ly, suggesting that the three kinds of agonists have different efficacies t o induce permeation of Ca2+. The range of the agonist selectivity thus obta ined is much narrower than that of binding affinities for the NMDA receptor s from rat brain. The present method is able to detect Ca2+ permeation with a detection limit of approximate to 10(5) Ca2+ ions/s. (C) 2001 Elsevier S cience B.V. All rights reserved.