A single-channel method for evaluation of very magnitudes of Ca2+ ion fluxes through epsilon4/zeta1 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-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 epsilon4/zeta1 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 epsilon4/zeta1 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 microM, where the integrated currents for all the agonists reached their saturated values. The integrated Ca2+ currents obtained 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, 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 approximately 10(5) Ca2+ ions/s.
Wakabayashi
M
M
Department of Chemistry, College of Humanities and Sciences, Nihon University, Tokyo, Japan.
Hirano
A
A
Sugawara
M
M
Uchino
S
S
Nakajima-Iijima
S
S
D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D006224:Cricetinae; D005069:Evaluation Studies as Topic; D017136:Ion Transport; D008051:Lipid Bilayers; D008564:Membrane Potentials; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D008051:Lipid Bilayers; D016194:Receptors, N-Methyl-D-Aspartate; D011994:Recombinant Proteins; D002118:Calcium