A single-channel method for evaluating agonist selectivity in terms of the very number of Ca2+ ions passed through the ε4/ζ1 N-methyl-D-aspartate (NMDA) receptor ion channel in bilayer lipid membranes (BLMs) is described. The number of Ca2+ passed through the single-channel was obtained from single-channel recordings in a medium where the primary permeant ion is Ca2+. The recombinant ε4/ζ1 NMDA channel was partially purified from Chinese hamster ovary cells expressing the channel and incorporated in BLMs formed by the tip-dip method. It was found that the ε4/ζ1 channel in BLMs is permeable to Ca2+ and Na+, but the number of Ca2+ passed through the channel is much fewer than that of Na+. The integrated Ca2+ currents induced by three typical agonists NMDA, L-glutamate and L-CCG-IV were obtained at concentration of 50 μM, where the integrated currents for all the agonists reached their saturated values. The integrated Ca2+ currents obtained are (3.1 ± 0.21) × 10-13 C/s for NMDA, (4.6 ± 0.31) × 10-13 C/s for L-glutamate and (5.7 ± 0.25) × 10-13 C/s for L-CCG-IV, respectively, suggesting that the three kinds of agonists have different efficacies to induce permeation of Ca2+. The range of the agonist selectivity thus obtained is much narrower than that of binding affinities for the NMDA receptors from rat brain. The present method is able to detect Ca2+ permeation with a detection limit of ≈ 105 Ca2+ ions/s.
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