Functional properties of human skeletal muscle acetylcholine receptors expressed by the TE671 cell line.
Functional properties of acetylcholine receptors from intact TE671 human medulloblastoma cells were examined using tracer ion flux, ligand competition against 125I-labeled alpha-bungarotoxin binding, and single channel recording measurements. 125I-Labeled alpha-bungarotoxin binds to surface receptors with the forward rate constant 1.8 X 10(5) M-1 s-1 and dissociates with the rate constant 4.6 X 10(-5) s-1, at 21 degrees C; the apparent dissociation constant is 2.6 X 10(-10) M. alpha-Bungarotoxin binds to at least two sites/receptor, but blocks agonist-induced 22Na+ uptake when bound to only one site. The reversible antagonists, dimethyl-d-tubocurarine and gallamine, occupy two sites which exhibit nearly equivalent affinities, but block agonist-induced uptake by occupying only one site. Strong agonists activate rapid sodium uptake with relatively low affinity, but desensitize with a much higher affinity; among agonists, the ratio of low to high affinity dissociation constants ranges from 1600 to 4000. By using the estimated dissociation constants, the allosteric model of Monod, Wyman, and Changeux (MWC) can be fitted to the concentration dependencies of both steady-state agonist occupancy and desensitization. The fitting analysis discloses an allosteric constant of 3 X 10(-5), which is the ratio of activatable to desensitized receptors in the absence of agonist. The rate of recovery from desensitization can exceed the rate of onset of desensitization elicited by low concentrations of agonist, further supporting the general MWC framework. Single channel recordings show that the channel opening probability is greater than 0.7 at high agonist concentrations. Favorable channel opening is shown to only slightly oppose strong desensitization.
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Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510.
D001667:Binding, Competitive; D002038:Bungarotoxins / Q000378:metabolism; D002217:Carbachol / Q000378:metabolism; D002460:Cell Line; D003992:Dibucaine / Q000494:pharmacology; D006801:Humans; D007473:Ion Channels / Q000502:physiology; D007700:Kinetics; D008433:Mathematics; D008564:Membrane Potentials; D008962:Models, Theoretical; D009132:Muscles / Q000378:metabolism*; D011950:Receptors, Cholinergic / Q000187:drug effects / Q000378:metabolism*
D002038:Bungarotoxins; D007473:Ion Channels; D011950:Receptors, Cholinergic; D002217:Carbachol; D003992:Dibucaine