The kinetics of tubocurarine action and restricted diffusion within the synaptic cleft.
- 1 September 1979
- journal article
- research article
- Published by Wiley in The Journal of Physiology
- Vol. 294 (1), 365-386
- https://doi.org/10.1113/jphysiol.1979.sp012935
Abstract
The kinetics of tubocurarine inhibition were studied at the post-synaptic membrane of frog skeletal muscle fibers. Acetylcholine (ACh) and (+)-tubocurarine were ionophoresed from twin-barrel micropipettes, and the membrane potential of the muscle fiber was recorded intracellularly. Tubocurarine-receptor binding was measured by decreases in the response to identical pulses of ACh. The responses to both ACh and tubocurarine had brief latencies and reached their maxima rapidly. Apparently under these conditions the kinetics of tubocurarine action are not slowed by diffusion in the space outside the synaptic cleft. After a pulse of tubocurarine, recovery from inhibition proceeds along a roughly exponential time course with a rate constant, 1/.tau.off .simeq. 0.5/s. This rate constant does not depend on the maximal level of inhibition and varies only slightly with temperature (Q10 = 1.25). After a sudden maintained increase in tubocurarine release, the ACh responses decrease and eventually reach a new steady-state level. Inhibition develops exponentially with time and the apparent rate constant, 1/.tau.on, is greater than 1/.tau.off. When the steady-state inhibition reduces the ACh response to 1/n of its original level, the data are summarized by the relation, 1/.tau.on = n(1/.tau.off). When the ACh sensitivity is reduced with cobra toxin, both 1/.tau.on and 1/.tau.off increase. The kinetics of tubocurarine inhibition depend on the density of ACh receptors in the synaptic cleft. After treatment with collagenase, part of the nerve terminal is displaced and the post-synaptic membrane is exposed directly to the external solution. Under these circumstances, 1/.tau.off increases > 10-fold. Bath-applied tubocurarine competitively inhibits the responses to brief ionophoretic ACh pulses with an apparent Kd = 0.5 .mu.M. In denervated frog muscle fibers, extrasynaptic receptors have a lower apparent affinity for tubocurarine. After a pulse of tubocurarine, inhibition decays 10-fold more rapidly at these extrasynaptic sites than at the synapse. Apparently each tubocurarine molecule binds repeatedly to several ACh receptors before escaping from the synaptic cleft and that the probability of this repetitive binding is enhanced because the nerve terminal presents a physical barrier to diffusion out of the cleft. Consequently, the receptors transiently buffer the concentration of tubocurarine in the cleft, and the macroscopic kinetics of inhibition are much slower than the molecular binding rates for tubocurarine.This publication has 56 references indexed in Scilit:
- The time course of cellular responses to iontophoretically applied drugsJournal of Theoretical Biology, 1977
- A comparison of the effect of cholinesterase inhibitors on end-plate current and on cholinesterase activity in frog muscleNeuropharmacology, 1975
- The rate of action of tetrodotoxin on sodium conductance in the squid giant axonPhilosophical Transactions of the Royal Society of London. B, Biological Sciences, 1975
- Conductance increases produced by bath application of cholinergic agonists to Electrophorus electroplaques.The Journal of general physiology, 1975
- Density and dose-response curve of acetylcholine receptors in frog neuromuscular junctionNature, 1975
- Binding properties of acetylcholine receptors extracted from normal and from denervated rat diaphragmFEBS Letters, 1974
- The acetylcholine sensitivity in the vicinity of the neuromuscular junction of the frogPflügers Archiv - European Journal of Physiology, 1974
- Binding of acetylcholine and related compounds to purified acetylcholine receptor from Torpedocalifornica electroplaxBiochemical and Biophysical Research Communications, 1973
- Acetylcholine ReceptorsThe Journal of general physiology, 1972
- The kinetics of action of acetylcholine antagonists in smooth muscleProceedings of the Royal Society of London. B. Biological Sciences, 1966