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The apparent lifetimes of frog end-plate channels activated by several nicotinic agonists have been determined with voltage-jump and fluctuation analysis techniques. The agonists were monoquaternary, N-substituted derivatives of trimethylammonium (TMA). Methyl TMA activated channels which had apparent lifetimes about 3-4 times shorter than acetylcholine (ACh)-activated channels. This result was confirmed with single-channel recordings from embryonic chick skeletal muscle. Channel conductance and voltage dependence of channel lifetime were similar for methyl TMA- and ACh-activated channels. Methyl TMA showed no signs of blocking open end-plate channels. Ethyl TMA, acetylthiocholine, cholinethiol and carbamylcholine all activated channels similar to methyl TMA-activated channels with regard to lifetime. None of these agonists appeared to block end-plate channels in the employed concentrations. 4-ketopentyl TMA, which contains a methylene group in place of the ether oxygen of ACh, sometimes opened end-plate channels with similar apparent lifetimes as those opened by ACh. Single-channel recordings showed that bursts of current from channels activated by 4-ketopentyl TMA have similar durations as do those activated by ACh. Pentyl TMA and benzyl TMA block open end-plate channels even when delivered at doses which elicit very small currents. It is concluded that the ester moiety of ACh serves to stabilize the open conformation of the channel.

1. The electrical potential difference across the innervated membrane of the electroplaque of Electrophorus electricus was measured with an intracellular micro-electrode while an extracellular double-barrelled micropipette was used to apply acetylcholine and carbamylcholine iontophoretically very close to the point of insertion of the recording electrode. 2. The average depolarizing response to brief (several msec) pulses of carbamylcholine decayed 22 times more slowly than the response to acetylcholine. Treatment of the electroplaque with eserine or neostigmine prolonged the acetylcholine responses. 3. When a steady current of acetylcholine was applied for several seconds, the membrane first depolarized, then partially repolarized. Usually no repolarization was seen during long pulses of carbamylcholine or long pulses of acetylcholine in the presence of eserine or neostigmine. 4. During long conditioning pulses of acetylcholine or carbamylcholine, the responses to brief test pulses of acetylcholine showed a progressive decline in amplitude, but recovered after termination of the conditioning pulse. Desensitization half-times as short as 0-6 sec were observed, making these results similar to those obtained in the frog motor end-plate.

1. Progressive block of neuromuscular transmission in frog sartorius and gastrocnemius preparations by haemagglutinin-free crystalline Type A botulinum toxin (BTX) was investigated by in vitro application and by injection of the toxin into living animals.2. Neuromuscular block was characterized by (a) decline in amplitude of evoked twitch contractions, (b) decline in amplitudes of end-plate potentials (e.p.p.s) and (c) changes in statistical characteristics of spontaneous miniature end-plate potentials (m.e.p.p.s).3. Progress of the block was enhanced by nerve stimulation.4. A decrease in frequency to less than 0.1/sec and decreased average amplitudes of m.e.p.p.s preceded observable impairment of neuromuscular transmission. These changes occurred as early as 3 hr after injection of the toxin into dorsal lymph sacs.5. The amplitude distributions of m.e.p.p.s changed from a normal distribution to one that showed an increased skewness toward smaller amplitudes as the block progressed. These changes were first detectable as early as 75 min following addition of the toxin to the bath.6. At later stages of toxin action, e.p.p.s began to decrease in amplitude and eventually failed altogether. E.p.p.s showed a normal quantal variation at very early stages in the block in Mg(2+)-treated preparations. At later stages of the block, it was not possible to test the quantal make-up of the e.p.p.7. At all stages before complete failure it was possible to obtain normal or greater than normal degrees of synaptic facilitation with paired stimuli to the nerve. This aspect of the coupling of nerve terminal depolarization to transmitter release appears to be relatively unaffected by BTX.8. Electrical depolarization of nerve terminals in partially blocked preparations evoked a maintained discharge of m.e.p.p.s with an amplitude distribution similar to that of the spontaneous m.e.p.p.s; hyperpolarization of the terminals evokes a distinctly larger class of m.e.p.p.s. In fully blocked preparations, depolarization of the terminals does not evoke transmitter release whereas hyperpolarization continues to yield the larger class of m.e.p.p.s.9. It is proposed that the neuromuscular block caused by BTX is due to impairment of a process by which vesicles become charged with transmitter before release.