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D Colquhoun

Publications and source records attributed to D Colquhoun.

At least 91 records · Page 5Linked to original sources
PubMed1982

The effect of tubocurarine competition on the kinetics of agonist action on the nicotinic receptor.

1 The rates at which tubocurarine associates with, and dissociates from, the nicotinic receptor, while exerting its classical competitive effect, are still in doubt. We have investigated this problem by observing the effect of low concentrations of tubocurarine on the re-equilibration rate, following a step change in membrane potential, of the current produced by carbachol in voltage-clamped endplates of frog muscle. 2 It is expected, and observed, that in order to see the effects of competition (as opposed to ion channel block), sufficiently high agonist concentrations must be used so that the relaxation rate becomes faster than that seen at low agonist concentrations. 3 Small concentrations of tubocurarine were found to reduce this relaxation rate, towards a value appropriate to a lower agonist concentration. 4 The results suggest that tubocurarine equilibrates very rapidly with the nicotinic receptor. 5 Some of the possible technical problems of this sort of experiment are discussed. The results are similar to those already published for nicotinic receptors in eel electric tissue.

Animals↗
PubMed1982

The action of ganglionic blocking drugs on the synaptic responses of rat submandibular ganglion cells.

1 The effects of tubocurarine, hexamethonium and trimetaphan on the synaptic currents of rat submandibular ganglion cells have been measured at 20 degrees C by means of a two-microelectrode voltage-clamp system. The aim was to distinguish between the receptor-blocking and channel-blocking actions of those drugs, and to test for possible selectivity of action on the ;fast' and ;slow' acetylcholine-operated channels.2 Tubocurarine had no effect on the amplitude of evoked synaptic currents (e.s.cs) or miniature synaptic currents (m.s.cs), except at concentrations exceeding 20 muM. The slow component of the e.s.c. was shortened by tubocurarine, this effect becoming more marked as the cell was hyperpolarized. The timecourse of m.s.cs, which have no slow component, was unaffected.3 Hexamethonium (2-30 muM) caused a voltage-dependent reduction of e.s.c. amplitude, and voltage-dependent shortening of both fast and slow components of the e.s.c. M.s.cs were also shortened.4 Trimetaphan (2-10 muM) reduced the amplitude of e.s.cs and m.s.cs. Neither component of the e.s.c. was shortened by trimetaphan; however, the slow component was reduced in amplitude more than the fast component, so that the overall duration of the e.s.c. appeared to be reduced. At higher concentrations (15-25 muM) trimetaphan clearly shortened the fast component.5 It is concluded that tubocurarine acts selectively on the slow ionic channels, the association rate constant being 2.8 x 10(6) M(-1) s(-1) at -80 mV. Hexamethonium acts on both fast and slow channels, the association rate constants, at -80 mV, being respectively 5.3 x 10(6) M(-1) s(-1) and 1.3 x 10(7) M(-1) s(-1). With both drugs, the association rate constant increases if the cell is hyperpolarized, this effect being more pronounced with hexamethonium than with tubocurarine.6 The marked voltage-dependent reduction of e.s.c. amplitude by hexamethonium cannot be accounted for by open channel block, and requires an additional mechanism, the nature of which is discussed.7 Trimetaphan, at low concentrations, acts in a way consistent with receptor block, and shows a degree of selectivity for the slow component of the e.s.c.8 In an appendix, the effect of temporal dispersion of the time of opening of ionic channels on the amplitude and time-course of the composite synaptic response is analysed. It is concluded that the shortening of the time-constant of the e.s.c. decay by hexamethonium cannot, by itself, account for the drug's effect on e.s.c. amplitude.

Animals↗
PubMed1981

The modes of action of gallamine.

The action of gallamine, a classical competitive neuromuscular blocking agent, has been examined on voltage-clamped endplates of frog skeletal muscle fibres. Gallamine produces a parallel shift of the equilibrium log (concentration)--response curves in concentrations of up to about 40 microM. At a membrane potential of -70 mV the Schild plot of the dose ratios so measured has a gradient of slightly less than the theoretical value, for a competitive antagonist, of unity. The apparent equilibrium constant for 'competitive' block is about 2 microM, and is approximately independent of the membrane potential. Fluctuation analysis of the endplate current shows two components in the presence of gallamine. The results can be fitted, over the range tested, by a mechanism that involves block of open ion channels by gallamine in a manner similar to that by procaine or quaternary local anaesthetic analogues. The rate constants for this action are strongly dependent on the membrane potential. At -100 mV the association rate constant is about 4 x 10(7) M-1S-1, the dissociation rate constant is about 600 s-1, and the equilibrium constant about 15 microM. Other kinetic measurements (voltage-jump relaxation, and nerve-evoked endplate currents) give results consistent with this conclusion, but apparently these results are valid over a range of conditions narrower than that for fluctuation analysis.

Animals↗
PubMed1981

On the stochastic properties of single ion channels.

It is desirable to be able to predict, from a specified mechanism, the appearance of currents that flow through single ion channels (a) to enable interpretation of experiments in which single channel currents are observed, and (b) to allow physical meaning to be attached to the results observed in kinetic (noise and relaxation) experiments in which the aggregate of many single channel currents is observed. With this object, distributions (and the means) are derived for the length of the sojourn in any specified subset of states (e.g. all shut states). In general these are found to depend not only on the state in which the sojourn starts, but also on the state that immediately follows the sojourn. The methods described allow derivation of the distribution of, for example, (a) the number of openings, and total length of the burst of openings, that may occur during a single occupancy, and (b) the apparent gap between such bursts. The methods are illustrated by their application to two simple theories of agonist action. The Castillo-Katz (non-cooperative) mechanism predicts, for example, that the number of openings per occupancy, and the apparent burst length, are independent of agonist concentration whereas a simple cooperative mechanism predicts that both will increase with agonist concentration.

Animals↗
PubMed1981

Block of acetylcholine-activated ion channels by an uncharged local anaesthetic.

It is now thought that amine local anaesthetic compounds (procaine, lignocaine and related molecules) depress electrical activity in nerve and muscle cells by binding to sites within ion channels and blocking current flow. Such mechanisms have been proposed to account for the effects of these local anaesthetics on both the voltage-dependent sodium current and the postsynaptic actylcholine (ACh)-activated ionic current. Recently, strong evidence for block of ion channels by cationic drug molecules has been obtained by recording current from single ACh-activated channels in the presence of permanently charged quaternary derivatives of lignocaine. Most amine local anaesthetic compounds are, however, weak bases, present in both charged and uncharged forms at physiological pH, and some question remains as to whether a charged group is essential for blockade of ion channels. To resolve this question, we studied the action of the uncharged local anaesthetic benzocaine (ethyl-4-aminobenzoate) on postsynaptic ACh-activated endplate current and extrajunctional single channel current of frog muscle. We report here evidence that strongly suggests that benzocaine blocks ACh-activated ion channels.

Acetylcholine↗
PubMed1979

The actions of tubocurarine at the frog neuromuscular junction.

1. The action of tubocurarine on voltage-clamped frog muscle end-plates has been re-examined by means (a) equilibrium dose-ratio measurements, (b) current fluctuation measurements and (c) voltage-jump relaxation measurements. 2. The equilibrium measurements can be interpreted as implying that tubocurarine has (a) a competitive blocking action, with a dissociation constant of 0.34 microM, which is not dependent on membrane potential, and (b) an additional voltage-dependent blocking action. 3. In the presence of tubocurarine two kinetic components can be seen. The faster one is similar to, but rather faster than, the normal ion channel closing rate. The other is much slower (1--3 sec), and, in relaxation experiments it is in the opposite direction to the fast relaxation. 4. A number of alternative explanations for the results are discussed. The mechanism that fits them best appears to be a combination of competitive block (or block of shut channels), with a strongly voltage-dependent block of open ion channels by tubocurarine. Estimates of the rate constants for channel blocking (and their voltage dependence) are derived. From these estimates the dissociation constant for the binding of tubocurarine to open channels appears to be roughly 0.12 microM at --70 mV and 0.02 microM at --12 mV. 5. Several potential sources of error in the experiments, and in their interpretation, are discussed. The most serious of these are problems associated with diffusion in the small volume of the synaptic cleft, viz. (a) changes in cleft concentration consequent on changes in binding, and (b) ionophoretic flux of antagonist and agonist into the synaptic cleft.

Animals↗
PubMed1977

An analysis of the action of a false transmitter at the neuromuscular junction.

1. The action of monoethylcholine (MECh) on neuromuscular transmission has been studied by electrophysiological methods. 2. End-plate potentials (e.p.p.s.) in curarized rat muscle were unaffected or slightly increased in amplitude by MECh (0-1-1 mM). Stimulation at 3 Hz for about 30 min in the presence of MECh caused a progressive decline in e.p.p. amplitude, and a shortening of the e.p.p. time course. These changes were reversed by addition of choline to the medium. Similar changes in amplitude, but no change in time course, occurred when the preparation was stimulated in the presence of hemicholinium or triethylcholine. 3. Extracellular recordings of miniature end-plate potentials in frog muscle showed that stimulation in the presence of MECh caused the time constant of the exponential decay of the m.e.p.p.s. to decrease by 42%. The amplitude of intracellular m.e.p.p.s. was reduced by 45%. These changes were maximal by the time about 3 X 10(5) quanta had been released. 4. Voltage clamp experiments in rat muscle in which miniature end-plate currents (m.e.p.c.s) were recorded showed that stimulation in the presence of MECh reduced the amplitude (by 33%) and the decay time constant (by 42%). 5. Analysis of end-plate current flucutations produced by local application of acetylcholine (ACh) and acetylmonoethycholine (AMECh) to voltage clamped rat end-plates showed that the amplitude of the elementary current events was the same for both compounds whereas the average channel lifetime was 44% shorter for AMECh than for ACh. 6. The voltage-sensitivity of the channel lifetime (measured from end-plate current fluctuations) was the same for ACh and AMECh. The voltage-sensitivity of the m.e.p.c. decay time constant was the same as that found from noise measurements. The shortened m.e.p.c.s. (false m.e.p.c.s.) occurring after stimulation in the presence of MECh also showed the same voltage-sensitivity. 7. Both normal and false m.e.p.c.s. were prolonged by neostigmine by almost the same factor; false m.e.p.c.s. were thus shorter than normal m.e.p.c.s. even when cholinesterase was inactivated. Experiments with progressive curarization of neostigmine-treated end-plates suggested that the fraction of transmitter molecules bound is smaller for false than for normal m.e.p.c.s. The difference implies that the false transmitter has one quarter of the affinity of ACh for the receptors. 8. It is concluded that stimulation in the presence of MECh gives rise to a false transmitter, presumably AMECh, which has a lower affinity for receptors than ACh, and gives rise to ionic channels with a shorter average lifetime than those activated by ACh.

Animals↗