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The Journal of Neuroscience, January 1, 1998, 18(1):119-127
Activation Kinetics of AMPA Receptor Channels Reveal the Number of Functional Agonist Binding Sites
John D. Clements1, 2, Anne Feltz1, 3, Yoshinori Sahara1, 4, and Gary L. Westbrook1 1 Vollum Institute, Oregon Health Sciences University, Portland, Oregon 97201, 2 John Curtin School of Medical Research, Australian National University, Canberra, New South Wales ACT 0200, Australia, 3 Laboratoire de Neurobiologie Cellulaire, Centre National de la Recherche Scientifique, 67084 Strasbourg, France, and 4 Department of Physiology, Faculty of Dentistry, Tokyo Medical and Dental University, Tokyo 113, Japan
AMPA and NMDA receptor channels are closely related molecules, yet they respond to glutamate with distinct kinetics, attributable to differences in ligand binding and channel gating steps (for review, see ). We used two complementary approaches to investigate the number of functional binding sites on AMPA channels on outside-out patches from cultured hippocampal neurons. The activation kinetics of agonist binding were measured during rapid steps into low concentrations of selective AMPA receptor agonists and during steps from a competitive AMPA receptor antagonist, 6-cyano-7-nitro-quinoxaline-2,3-dione, into a saturating concentration of agonist. Both approaches revealed sigmoidal kinetics, which suggests that multiple agonist binding steps or antagonist unbinding steps are needed for channel activation. A kinetic model with two independent binding sites gave a better fit to the activation phase than models with one or three independent sites. A more refined analysis incorporating cooperative interaction between the two binding sites significantly improved the fits to the responses. The affinity of the first binding step was two to three times higher than the second step. These results demonstrate that binding of two agonist molecules are needed to activate AMPA receptors, but the two binding sites are not identical and independent. Because NMDA receptors require four ligand molecules for activation (two glycine and two glutamate; ; ), it may be that some binding sites on AMPA receptors are functionally silent.
Key words: glutamate receptors; AMPA receptors; cyclothiazide; hippocampus; ion channels; patch clamp
Copyright © 1998 Society for Neuroscience 0270-6474/98/181119-09$05.00/0
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