The kinetics of curare action and restricted diffusion within the synaptic cleft of motor nerve terminals on frog skeletal muscle

Citation

Armstrong, David Lee (1979) The kinetics of curare action and restricted diffusion within the synaptic cleft of motor nerve terminals on frog skeletal muscle. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/g7kf-2m61. https://resolver.caltech.edu/CaltechETD:etd-08142007-073818

Abstract

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1. The kinetics of curare inhibition were measured at the postsynaptic membrane of frog sartorius and cutaneous pectoris muscle fibers. Acetyleholine (ACh) and d-tubocurarine (dTC) were iontophoresed from twin-barrel micropipettes, and the muscle fiber's membrane potential was recorded intracellularly. By itself dTC produced no change in membrane potential, but dTC-receptor binding was assayed by observing changes in the response to a constant pulse of ACh.

2. The responses to both ACh and dTC had brief latencies, reached their maxima rapidly, and were highly sensitive to the dose. Under these conditions, the kinetics of drug action are not slowed by access of the drugs to the synaptic cleft.

3. After a pulse of dTC, recovery from inhibition proceeds slowly along an exponential time course with a rate constant, [...]. The recovery rate does not depend on the maximal level of inhibition and varies only slightly with temperature (Q10 = 1.25).

4. After a sudden maintained increase in dTC release, inhibition develops approximately exponentially until a steady-state level of inhibition is reached. The apparent rate constant for the onset of inhibition, [...], is greater than [...]. When the steady-state inhibition reduces the ACh response to 1/n of its control value, [...] = [...].

5. When the ACh sensitivity is reduced with cobra toxin, both [...] and [...] increase. Thus, the kinetics of dTC inhibition depend on the density of acetyleholine receptors in the synaptic cleft. If the density of acetylcholinesterase is reduced in the cleft by collagenase, [...] increases only twofold.

6. When the nerve terminal is removed after collagenase action, and the drugs are iontophoresed directly onto the exposed postsynaptic membrane, [...] increases more than tenfold.

7. Bath-applied dTC competitively inhibits the responses to brief iontophoretic ACh pulses with an apparent equilibrium dissociation constant, KD = 0.5 [...]. This suggests that dTC molecules equilibrate with the receptors on a millisecond time scale.

8. On denervated frog muscle cells, extrasynaptic receptors have a lower apparent affinity for dTC. After a pulse of dTC, inhibition decays tenfold more rapidly at these extrasynaptic sites than at the nerve-muscle synapse.

9. It is suggested that dTC inhibits synaptic receptors more effectively because the nerve terminal restricts diffusion within the synaptic cleft and each dTC molecule binds repeatedly to several acetylcholine receptors before escaping from the cleft. Consequently, the receptors transiently buffer the concentration of dTC in the cleft, and the macroscopic kinetics of inhibition are much slower than the molecular rates of dTC binding.

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