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D Colquhoun

Publications and source records attributed to D Colquhoun.

At least 73 records · Page 4Linked to original sources
PubMed1990

Stochastic properties of ion channel openings and bursts in a membrane patch that contains two channels: evidence concerning the number of channels present when a record containing only single openings is observed.

If a single ion channel record is observed in which two ion channels are never simultaneously open, then it is often of interest to know whether the observations indeed arose from the activity of only one ion channel. This question can be answered if it is possible to calculate the distribution of the duration of runs of single openings in a membrane patch that contains two active channels. If the observed run of single openings is much longer than that expected for a patch with two channels it is likely that only one channel was active. An approximate method is presented for calculating the distribution of the duration of runs of single openings in a patch with two active channels; this method has the advantage that it can be calculated from observable quantities, and requires no knowledge of the details of the ion-channel mechanism or its rate constants. The accuracy of this approximation is tested by exact calculations of the properties of runs of single openings, and of single bursts, for two specific mechanisms and a large range of rate constants. The approximation is good in all cases in which openings occur singly, or in closely spaced bursts. If, as is common in practice, openings occur in clusters that are separated by long shut periods, then overlap of clusters from two different channels may be detected, if no double opening is produced, as a period in the middle of a cluster in which the probability of being open doubles. The results derived here can be applied to such a period to test whether it results from the simultaneous activity of two channels, rather than from a change in the properties of a single channel.

Animals↗
PubMed1990

Rectification of currents activated by nicotinic acetylcholine receptors in rat sympathetic ganglion neurones.

1. The inward rectification of the whole-cell current evoked by acetylcholine (ACh) and other nicotinic agonists in rat sympathetic ganglion neurones has been studied using patch-clamp recording techniques. The selective nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) (20 microM) induced an average peak current of -367 pA at -50 mV but no detectable outward current at +50 mV. Similar observations were made with ACh and carbachol. 2. The current-voltage relation of the whole-cell response induced by DMPP was linear in the negative voltage range; however, there was no detectable outward current in the voltage range 0 to about +70 mV. Above +70 mV an outward current became clearly detectable. Rapid depolarizing jumps in the holding potential failed to reveal any rapidly decaying outward current. 3. The rectification was not alleviated by changing the main permeant cation, by removal of divalent cations from the intracellular or extracellular solutions or by altering the pH buffer in the extracellular solution from HEPES to Tris. 4. Intracellular magnesium ions can block the channel. This effect increases with depolarization, but dissociation outwards (i.e. permeation by Mg2+) appears to relieve the block at more extreme positive potentials. This effect alone, or in combination with the voltage dependence of the burst length, is unlikely to be able to account for the whole-cell rectification in intact cells, much less that seen in cells perfused with Mg2(+)-free intracellular medium. 5. When the reversal potential was shifted to approximately -50 mV (by the use of impermeant cations) nicotinic agonists produced small outward currents in the membrane potential range -20 to +10 mV while shifting it to about +40 mV produced small inward currents in the potential range 0 to +20 mV. The rectification therefore appears to be independent of the direction of current flow and is maximum at a potential positive to 0 mV. 6. At positive potentials the receptors desensitized much less than at negative potentials in the continued presence of agonist. Thus, exposure of the cells to a steady application of 30 microM-ACh produced no detectable response if the cell was at a positive potential, but when the cell was stepped to a negative potential in the continued presence of ACh (at a time when much of the ACh current would be expected to have desensitized), ACh induced a large inward current. The onset of the ACh current had a time constant of 10 ms. It then decayed with a time constant of 790 ms as desensitization developed.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholine↗
PubMed1990

The actions of suxamethonium (succinyldicholine) as an agonist and channel blocker at the nicotinic receptor of frog muscle.

1. Patch clamp methods were used to study the equilibrium and kinetic properties of the acetylcholine analogue, succinyldicholine (suxamethonium), which is used clinically as a neuromuscular blocking agent. 2. The equilibrium concentration-response curve, corrected for desensitization was estimated by measuring as the response the probability of being open of single ion channels during clusters of activity that occur between long desensitized periods. Suxamethonium (Sux) was about 7.6-fold less potent than acetylcholine (ACh) (at low concentrations), partly because of 2.9-fold lower affinity for the resting receptor, and partly because of a lower ability to activate the receptor once bound. 3. Sux was a more potent blocker of the open ion channel than ACh (equilibrium constant about 200 microM); this limited the maximum open probability to about 0.36 (at 12 degrees C and -120 mV). Individual channel blockages lasted about 65 microseconds on average. They appeared to get longer at high agonist concentration; however, a simulation method was used to show that this effect could be accounted for by the fact that at higher concentrations there are more openings that are too brief to be detected. Over the concentration range tested the effects were described by a simple open channel block mechanism. 4. No component of brief shut times could be detected other than those resulting from channel blockages. However, the results suggest that multiple channel openings (the nachschlag phenomenon) should be rare, so this is not inconsistent with previous results with other agonists. 5. Sux differed from ACh and carbachol in that it had a somewhat lower efficacy and a greater channel blocking action. However, in clinical practice channel block is unlikely to contribute to neuromuscular block to any significant extent; the main mechanism of paralysis, at least in the early stages, is probably a result of prolonged depolarization of the region of membrane surrounding the motor endplate leading to inactivation of the sodium channels therein.

Animals↗
PubMed1989

Desensitization of the acetylcholine receptor of frog end-plates measured in a Vaseline-gap voltage clamp.

1. Desensitization of the nicotinic acetylcholine receptor of the frog end-plate was investigated in dissociated frog muscle fibres using the Vaseline-gap clamp method so that a wide range of well-defined agonist concentrations could be used without having to use alpha-bungarotoxin to reduce currents, and so that the intracellular medium could be controlled. 2. Acetylcholine (ACh) concentrations between 1 and 1000 microM were used, after inactivation of acetylcholinesterase. The intracellular calcium concentration was usually kept near zero by using 80 mM-K2EGTA as the intracellular solution. 3. When using the low intracellular calcium solution, desensitization proceeded as a biphasic process with estimates of fast and slow time constants of about 8 and 80 s at 4 degrees C and 20 microM-ACh (the rates increased with concentration). In contrast, only one (fast) component of desensitization was detected when the intracellular calcium concentration was allowed to increase during ACh application. 4. Despite rapid application of ACh the time to peak response was 0.2 s (with 400 microM-ACh) to 2 s (with 1 microM-ACh); this slow rise was shown to result from diffusion delays. Nevertheless the peak current with 200 microM-ACh corresponded to opening of most of the channels present, so there is probably not much desensitization in the millisecond time range. 5. Both fast and slow time constants for onset of desensitization showed only slight dependence on membrane potential when [Ca2+]i was buffered with 80 mM-K2EGTA. 6. Increasing the intracellular cyclic AMP concentration directly, or indirectly with forskolin and IBMX, had no effect on the time course of desensitization. 7. Intracellular application of submicromolar concentrations of phorbol-12,13-dibutyrate (PDBu) and phorbol-12-myristate-13-acetate (PMA) yielded a small but reproducible reduction of the peak response to ACh. The time course of desensitization was, however, not modified by these substances. 8. The implications of these observations for the mechanism of desensitization, and their relationship to single-channel observations, are discussed.

Acetylcholine↗
PubMed1988

On the kinetics of large-conductance glutamate-receptor ion channels in rat cerebellar granule neurons.

Ion channels activated by glutamate, aspartate, and N-methyl-D-aspartate (NMDA) have been investigated in outside-out patches from cultured cerebellar granule neurons of the rat. Openings of these channels occur in bursts, within which the individual openings are separated by brief shuttings or gaps. The shut-time distributions obtained with each agonist were fitted with four exponential components. The briefest two components were considered as 'gaps within bursts'. Their mean time-constants were: glutamate, 58.0 microseconds and 592 microseconds; aspartate, 31.3 microseconds and 644 microseconds; NMDA, 40.5 microseconds and 903 microseconds. Distributions of burst durations were fitted with three exponential components. The mean time-constants obtained for the longest two components were: glutamate, 1.33 ms and 10.5 ms; aspartate, 2.15 ms and 10.3 ms; NMDA, 2.42 ms and 10.5 ms. Evidence is given that these two components of burst duration reflect the gating kinetics of 50 pS openings and not the fact that each agonist produces openings to more than one conductance level. Not only do openings occur in bursts, but these bursts were observed to occur in clusters, which can be hundreds of milliseconds long. We discuss the relation between the kinetics of single-channel openings observed in patches and the spectral components detected in whole-cell current noise.

Animals↗
PubMed1988

Activation of ion channels in the frog end-plate by high concentrations of acetylcholine.

1. The equilibrium relationship between acetylcholine (ACh) concentration and response (fraction of channels open), corrected for the effects of desensitization, has been estimated by single-ion-channel recording at the adult frog skeletal neuromuscular junction. At high ACh concentration channel openings occur in well-defined clusters separated by long desensitized intervals. The response, po, was estimated as the proportion of time for which a single channel was open during a cluster. 2. At negative membrane potential (-120 mV) po reached a maximum value of 0.9 at 100 microM-ACh and was half-maximum at 15 microM with a Hill slope of 1.6 at this point. At concentrations higher than 200 microM-ACh, po declined as a result of open-channel block by free ACh itself. 3. At positive membrane potentials (+100 mV) there was little channel block by ACh; po reached a maximum value of 0.41 at 500 microM-ACh, with half-maximum activation at 50 microM and Hill slope of 1.2 at this point. 4. Particular mechanisms for channel activation by ACh were fitted to the data by the method of least squares. Fits were fully determinate only if the two binding sites for ACh were assumed to be equivalent with no co-operativity in the ACh binding reactions. At negative potential the microscopic equilibrium constant for binding was K1 = K2 = 77 microM and the equilibrium constant for channel opening (opening/closing rates, beta/alpha) was 32. At positive potential the affinity was slightly higher, K = 32 microM, which confirms the view that the binding sites for ACh are outside the membrane electric field. The equilibrium constant for channel opening was reduced to 0.7 mainly as a result of the much shorter open lifetime (increased closing rate alpha) at positive potentials. 5. The data were also fitted well by very high values of beta/alpha together with a high degree of negative co-operativity or non-equivalence in ACh binding affinity (K2 much greater than K1). A good fit could also be obtained with moderate positive co-operativity combined with non-equivalence of the binding sites. 6. A mechanism that postulates a receptor with two independent gating subunits provided a poor fit to the data at negative potential. 7. The rate constants for channel opening and ACh dissociation were estimated by constraining the fitted parameters so that the burst length for channel opening was equal to its observed value at low concentrations of ACh.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholine↗
PubMed1987

Single-channel and whole-cell currents evoked by acetylcholine in dissociated sympathetic neurons of the rat.

Single acetylcholine-activated channels have been recorded from neurons dissociated from the sympathetic chain of 17-21 day old rats. The mean single channel conductance is 35 pS in normal medium containing 1 mM calcium, and 51 pS in the absence of calcium. The measured current amplitudes are about five times more variable than at the frog endplate, at least in part because the current, while the channel is open, is much noisier than when it is shut. Single activations of the receptor by acetylcholine (ACh) produce a burst of openings; the distribution of the burst length has two components, the longer of which is of primary importance in synaptic transmission. Whole-cell currents, in response to ACh (up to 30 microM), show strong inward rectification with no outward current being detectable. This phenomenon is similar whether the intracellular ion is sodium or cesium, whether or not divalent cations are present, and whether or not atropine is present. Nevertheless, outward single-channel currents (of normal conductance) are detectable in isolated outside-out patches.

Acetylcholine↗
PubMed1987

A note on correlations in single ion channel records.

General expressions are derived for the correlation coefficients between the length of an opening and that of the nth subsequent opening for a single ion channel. Analogous results are given for the correlation between shut times, and between an open time and subsequent shut times. An alternative derivation of the results of Fredkin et al. (in Proc. Berkeley Conf. in honor of Neyman & Kiefer, vol. 1, pp. 269-289 (1985] is given, and their results are extended to the case where openings occur in bursts. Expressions are given for the correlation between the first and nth opening in a burst, between the lengths of bursts, and between the number of openings per burst. Each of these sorts of correlation can give information about the connections that exist between the various states of the system; interpretations of the correlations are discussed. Expressions are derived for the distributions of the nth open time, shut time, burst length, etc. following the application of a perturbation (e.g. a voltage jump or a concentration jump). It is shown that these distributions will all be the same (namely the equilibrium distribution) only in the case where the openings, burst lengths, etc. are not correlated. Certain reaction schemes predict a component in the distribution of the number of openings per burst that has a unit mean (i.e. a component of isolated single openings). For some schemes this component is predicted to have zero amplitude, in principle, whereas in others it may be quite prominent. The presence or absence of this component can give information about the way in which the various states of the system are connected. The interpretation in terms of mechanism is discussed.

Animals↗
PubMed1985

Ion channel block by acetylcholine, carbachol and suberyldicholine at the frog neuromuscular junction.

Three nicotinic agonists, suberyldicholine, acetylcholine and carbachol, have been investigated by single channel recording at the endplates of adult frog muscle fibres. All three agonists can block the channels that they open. Suberyldicholine is the most potent blocker; it has an equilibrium constant for binding to the open channel of about 6 microM and blockages last for about 5 ms on average, at -105 mV. A plot of the mean number of blockages per unit open time against concentration ('blockage frequency plot') suggests that suberyldicholine does not produce long-lived blocked states such as might occur, for example, if it could be trapped within a shut channel. The characteristics of the 'blockage frequency plot' are analysed in Appendix 2. Block by acetylcholine and carbachol has much lower affinity (the equilibrium constants being a few millimolar for both), and blockages are much briefer, so that blockage appears to produce noisy single channel currents of reduced amplitude. A method based on the spectral density of the excess 'open' channel noise has been used to investigate the rate of blocking and unblocking. The basis of this method is discussed in Appendix 1. It is estimated that the mean duration of a blockage is about 18 microseconds for acetylcholine and 9 microseconds for carbachol.

Acetylcholine↗
PubMed1985

Fast events in single-channel currents activated by acetylcholine and its analogues at the frog muscle end-plate.

The fine structure of ion-channel activations by junctional nicotinic receptors in adult frog muscle fibres has been investigated. The agonists used were acetylcholine (ACh), carbachol (CCh), suberyldicholine (SubCh) and decan-1,10-dicarboxylic acid dicholine ester (DecCh). Individual activations (bursts) were interrupted by short closed periods; the distribution of their durations showed a major fast component ('short gaps') and a minor slower component ('intermediate gaps'). The mean duration of both short and intermediate gaps was dependent on the nature of the agonist. For short gaps the mean durations (microseconds) were: ACh, 20; SubCh, 43; DecCh, 71; CCh, 13. The mean number of short gaps per burst were: ACh, 1.9; SubCh, 4.1; DecCh, 2.0. The mean number of short gaps per burst, and the mean number per unit open time, were dependent on the nature of the agonist, but showed little dependence on agonist concentration or membrane potential for ACh, SubCh and DecCh. The short gaps in CCh increased in frequency with agonist concentration and were mainly produced by channel blockages by CCh itself. Partially open channels (subconductance states) were clearly resolved rarely (0.4% of gaps within bursts) but regularly. Conductances of 18% (most commonly) and 71% of the main value were found. However, most short gaps were probably full closures. The distribution of burst lengths had two components. The faster component represented mainly isolated short openings that were much more common at low agonist concentrations. The slower component represented bursts of longer openings. Except at very low concentrations more than 85% of activations were of this type, which corresponds to the 'channel lifetime' found by noise analysis. The frequency of channel openings increased slightly with hyperpolarization. The short gaps during activations were little affected when (a) the [H+]o or [Ca2+]o were reduced to 1/10th of normal, (b) when extracellular Ca2+ was replaced by Mg2+, (c) when the [Cl-]i was raised or (d) when, in one experiment on an isolated inside-out patch, the normal intracellular constituents were replaced by KCl. Reduction of [Ca2+]O to 1/10 of normal increased the single-channel conductance by 50%, and considerably increased the number of intermediate gaps. No temporal asymmetry was detectable in the bursts of openings. Positive correlations were found between the lengths of successive apparent open times at low SubCh concentrations, but no correlations between burst lengths were detectable. The component of brief openings behaves, at low concentrations, as though it originates from openings of singly occupied channels.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholine↗
PubMed1984

Kinetics of acetylcholine activated ion channels in chick ciliary ganglion neurones grown in tissue culture.

The acetylcholine activated conductance of chick ciliary ganglion neurones grown in tissue culture was studied by the patch clamp method. Single channel currents at 30 degrees C had a conductance of 38-42 pS, a reversal potential near + 10 mV and an average open lifetime of 1.08 ms (range 0.74 - 1.54 ms) at the resting potential. The presence of a single component in the distributions of amplitudes and open lifetimes, and also in the noise spectrum of voltage clamp currents, suggests that acetylcholine channels have uniform characteristics in these cells. Evidence of a desensitised state of the receptor was obtained from the distribution of gap intervals and the decline of voltage clamp current. These properties are similar to those of acetylcholine channels at the vertebrate neuromuscular junction. However, two important differences were found. (a) The acetylcholine concentrations used here were 10-25 times higher than those required to produce a similar degree of channel activation at the endplate. (b) When the membrane was hyperpolarised the mean open lifetime of the channel showed no change or a slight reduction.

Acetylcholine↗
PubMed1983

The efficacy of agonists at the frog neuromuscular junction studied with single channel recording.

The efficacy of acetylcholine, carbachol and suberyldicholine at the frog neuromuscular junction was estimated by single channel recording. The probability of channel opening at high concentration was found to be greater than 0.9 for each, thus showing (a) that their efficacy is high and (b) that the opening rate for the fully-liganded channel is higher than previously thought.

Acetylcholine↗