Biomedical subjects

D J Maconochie

Publications and source records attributed to D J Maconochie.

8 recordsLinked to original sources

PubMed1998

The channel opening rate of adult- and fetal-type mouse muscle nicotinic receptors activated by acetylcholine.

1. In this paper, we examine acetylcholine (ACh)-induced currents in quail fibroblast cell lines expressing either the fetal (Q-F18) or the adult (Q-A33) complement of nicotinic acetylcholine receptor subunits derived from mouse skeletal muscle. Pulses of ACh were applied to outside-out patches of cell membrane by means of a fast perfusion system at concentrations from 100 nM to 10 mM. We obtained current records with intracellular potentials of -60 and +40 mV. The goal of this study was to estimate the channel opening rate. 2. By fitting sums of exponentials to averaged responses, we estimated the rate of development of the current on the application of acetylcholine. The rate constant of the predominant exponential component (the on-rate) ranges over 3 orders of magnitude, from around 100 s-1 (fetal) at low concentrations ACh to over 100,000 s-1 (fetal and adult) at the highest concentrations. 3. We establish that our measurement of the on-rate is not limited by technical constraints, and can therefore be related to the rate constants of a kinetic scheme. Our observations are consistent with a model having a rate-limiting channel opening step with a forward rate constant (beta) of 80,000 s-1 on average for adult receptors and 60,000 s-1 for fetal receptors, and a minimum opening to closing ratio (beta/alpha) of around 33 (adult) or 50 (fetal). The channel opening rate, beta, varies from around 30,000 s-1 to well over 100,000 s-1 for different patches. The large variation cannot all be ascribed to errors of measurement, but indicates patch to patch variation.

Acetylcholine↗

PubMed1995

The conductance of the muscle nicotinic receptor channel changes rapidly upon gating.

We have recorded single-channel currents through fetal-type muscle nicotinic receptor channels at recording bandwidths of approximately 50 and 75 kHz. The time course of the rising phase of aligned and averaged openings can be entirely accounted for if it is assumed that the conductance of the single channel changes instantaneously, and that alignment and averaging introduce a dispersion of 2-3 microseconds. We conclude that we find no evidence for a gradual change in conductance as a channel opens or closes. The shapes of averaged power spectra are consistent with this conclusion, insofar as they exclude an exponential relaxation in the transition with a time constant of 10 microseconds or more.

Animals↗

PubMed1995

Block by acetylcholine of mouse muscle nicotinic receptors, stably expressed in fibroblasts.

We have measured the concentration and voltage dependence of block by acetylcholine (ACh) of fetal- and adult-type mouse muscle nicotinic receptors, expressed in a fibroblast cell line. Data, obtained at a transmembrane potential of -60 mV and with ACh concentrations of 1 mM and above, are broadly consistent with the occlusion of an open channel with a single ACh+ ion (simple open channel block). The rate of recovery from block is approximately 40,000s-1 and has only a weak voltage dependence. This is in contrast to the strong voltage dependence observed for the degree of block. Deviations from the predictions of the simple model are seen in data collected at positive transmembrane potentials and at negative potentials for ACh concentrations < 1 mM. Less concentration dependence is observed than expected. Of a number of models tested, we demonstrate that two models incorporating both a high and a low affinity blocking site can predict our data.

Acetylcholine↗

PubMed1994

A novel use of differential equations to fit exponential functions to experimental data.

A procedure for fitting multi-exponential functions to experimental data is described. It is fast, requires no initial parameter estimates and is particularly suited to sums of several closely spaced exponentials. The method comprises the application of three well tried numerical techniques: (i) the signal is smoothed by representing it as an abbreviated Legendre series; (ii) the coefficients of a certain kind of differential equation are determined such that it's solution is the closest fit to the smoothed signal; and (iii) the amplitudes of the exponential components are determined, given the calculated values of the exponential rate constants. The method is computationally efficient, since determination of amplitudes and exponents involves the use of linear techniques, and therefore does not require multiple iterations, and the smoothed signal is contained in a handful of coefficients rather than as a lengthy time series. The severe ill-conditioning that is unavoidable in this problem is contained within the well-understood procedures of inverting a matrix and determining the roots of a polynomial. This method is particularly appropriate for analysis of data that may be modelled by a scheme of linked first-order reactions, describing for example the stochastic behaviour of ion channels, a chemical reaction, or the uptake and distribution of a drug within body compartments.

Data Interpretation, Statistical↗

PubMed1994

How quickly can GABAA receptors open?

We have examined GABAA receptor activation by making rapid applications of GABA to outside-out patches excised from cultured postnatal rat cerebellar neurons. The rate of development of current increases with increasing GABA concentration from a low to a high concentration asymptote. The low concentration asymptote is about 10 s-1 for patches taken from granule cells and 4 s-1 for patches from Purkinje cells. The high concentration asymptote is about 6000 s-1 for patches taken from either granule cells or Purkinje cells. The high concentration asymptote gives an estimate of the fastest rate at which these channels can open and indicates that agonist binding steps are not rate limiting. The concentration dependence of the development of current indicates that more than one GABA molecule is bound to most receptors with open channels and that the final binding step is of low affinity (about 500 microM). A comparison with GABA-mediated postsynaptic currents suggests that the properties of the GABAA receptor play a major role in determining the shape of inhibitory synaptic responses and that the cleft concentration of GABA reaches at least 500 microM.

Animals↗

PubMed1992

A study of the bovine adrenal chromaffin nicotinic receptor using patch clamp and concentration-jump techniques.

1. Voltage clamp records have been obtained from bovine adrenal chromaffin cells in the outside-out and whole-cell configurations, in response to step changes of acetylcholine (ACh) concentration. The concentrations used ranged from 50 nM to 20 mM. 2. At high acetylcholine concentrations, the activation and desensitization kinetics of the nicotinic receptor, as observed in outside-out patches, may be described by a model incorporating a single, fast agonist binding step, and relatively slow isomerization to the open state. The affinity of the closed receptor for ACh is 310 microM, the channel opening rate constant is 460 s-1, and the closing rate constant is 29 s-1. 3. Single channel events, observed when nanomolar ACh concentrations are applied to whole cells, have two distinct channel lifetimes: 0.6 ms and 11-15 ms. The variation of the frequencies of the events with ACh concentration, suggests that the short lifetimes are openings of a singly liganded receptor and the longer lifetimes are openings of a doubly liganded receptor. 4. Only a single exponential associated with receptor desensitization is seen with outside-out patches, but two are seen with whole cells. It is postulated that there are two nicotinic receptor types present on adrenal chromaffin cells. 5. The rate of desensitization (9 s-1 and 26 s-1, whole cells; 24 s-1, patches), is fast enough to be significant in determining the open channel lifetime. 6. A sudden increase in current (rebound) is observed when a high concentration of ACh is abruptly removed from outside-out patches. This is evidence for a blocked state. The affinity of the blocking site for ACh is 1400 microM (outside-out patches). 7. The total number of activatable nicotinic channels per whole cell is estimated to be 2600.

Acetylcholine↗

PubMed1992

Markov modelling of ensemble current relaxations: bovine adrenal nicotinic receptor currents analysed.

1. A general approach to the analysis of ensemble currents of ligand-gated channels is presented, with a variety of examples that include single and multiple agonist binding steps, desensitization and several blocking pathways. 2. The use of matrix methods to describe model reaction schemes leads to a simplification if the reaction scheme is irreversible: the product of the exponential relaxation rate constants is exactly equal to the product of the forward reaction steps. 3. This method of analysis applied to the bovine adrenal nicotinic receptor suggests that in the range of acetylcholine concentrations from 1 microM to 2 mM, a model with a single kinetically relevant agonist binding step is appropriate. 4. Complex models, to explain the presence of two desensitizing components in currents recorded from whole cells, may be discounted in favour of two distinct receptor types. 5. A simple model of open channel block is discounted, and desensitization of the blocked state proposed.

Acetylcholine↗

PubMed1989

A method for making solution changes in the sub-millisecond range at the tip of a patch pipette.

A method is described here for making multiple fast external solution changes at the tip of a patch pipette. The time for the change, 0.2 ms, has been established by measuring changes in liquid junction potential at the tip of an open patch pipette. This technique of producing an abrupt change in solution allows agonist/receptor reactions to be studied under non-equilibrium conditions. We have applied this technique to the nicotinic receptors in outside-out patches from skeletal muscle cell line C2 (Jaffe and Saxel 1979) and from bovine adrenal chromaffin cells. The application of step changes in acetylcholine concentration produces current traces with a characteristic shape, which may be compared with the predictions of established models for the activation and desensitisation of the nicotinic receptor. The results of making single steps and also short pulses in acetylcholine concentration are demonstrated. The direct comparison of two different cholinergic agonists is demonstrated.

Acetylcholine↗