A Kinetic Model for the Action of Xylocaine on Receptors for Acetylcholine (original) (raw)
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Kinetic parameters for acetylcholine interaction in intact neuromuscular junction
Proceedings of the National Academy of Sciences, 1981
The dependency of miniature endplate current (mepc) rise time upon mepc amplitude and acetylcholine receptor site density was measured in lizard intercostal muscles and used to fit the rate constants in a simple kinetic scheme.The kinetic scheme included diffusion, two-step sequential binding of acetylcholine to receptor, and opening of the ion channel. Numerical simulation of the observed mepc behavior yielded the following kinetic constants: (i) diffusion constant, 4 X 10-6 cm2 sec'1; (ii) forward binding rates, 4.7 x 107 M-1 sec'; (iii) channel relaxation rate, 25 msec-. The value above for the forward binding rates assumed both rates to be equal. If they are different, the slower of the two is in the range of 2-5 X 107 MI sec-1. A radial profile of bound receptor indicated that activation of the receptor was very local, occurring essentially within a radius of about 0.3 Itm from the point of acetylcholine release.
The Journal of General Physiology, 1968
Xylocaine and its derivatives act specifically at the neuromuscular junction within the concentration range 0.05 to 2.0 mM. The charged form is the active form of the drugs. There is no correlation between "local anesthetic" activity and effect at the junction. Like d-tubocurarine, these drugs have little or no effect on quantum content, acetylcholinesterase activity, or the passive impedance of the muscle fiber. Yet they produce end plate potentials characterized by a brief, early component and a late, greatly prolonged component, as does procaine. Analysis of these changes in time course suggests that the drugs have little or no effect before receptors are activated by acetylcholine, but cause a decreased and often greatly prolonged response. Clear structure-activity relations indicate that the receptor to which the drugs bind to produce the prolonged response can be the receptor for acetylcholine. Comparison of the effects of the drugs on the end plate potential and on ...
Biophysical Journal, 1991
Outside-out patches of enzymatically dissociated adult and denervated mouse muscle fibers were superfused repetitively by pulses of acetylcholine (ACh) containing solution. Up to 300 channels opened simultaneously 300 ,us after the beginning of a 1,000 ,uM ACh pulse corresponding to a peak current i of almost-1 nA. Single responses to ACh were averaged and the concentration dependence of i and of the rise time t, from 0.1 i to 0.9 i was measured. In adult receptors, i increased proportional to the second to third power of ACh concentration, whereas in embryonic-type receptors it was proportional to the first to the second power. t, increased from-0.3 ms at 1,000 ,uM ACh to a plateau value of-5 ms for adult and of-10 ms for embryoniclike receptors at concentrations < 10 ,uM ACh. The concentration dependence of i and tr was simulated using the standard model of ACh binding with different combinations of rate constants and two and three binding sites for ACh. The calculated curves were compared to the measurements and a set of well fitting rate constants was determined for adult and embryoniclike receptors. Three binding sites for ACh were necessary to fit the dose response for i ' for adult receptors. A method for deriving rate constants in a model of ACh-receptor interaction is described that avoids analysis of open-closed kinetics of single channels, which in rapid systems, as the ones studied here, are at the limit of the frequency response of the current measurement.
Medical hypotheses, 1983
The recently reported amino acid sequence of the alpha subunit of Electrophorus indicates that a previous hypothesis presented of the molecular structure of the acetylcholine receptor at the neuromuscular junction based on the sequence of the alpha subunit of Torpedo is improbable: an "essential" glu (13) of Torpedo is replaced by ser (13) which cannot subsume the function for glu (13) postulated in the hypothesis. This paper presents a new hypothesis which is applicable to both Electrophorus and Torpedo. A mode of rigorously testing the hypothesis provided by the irreversible blocker, lophotoxin, is described.
The Journal of Physiology, 1985
1. 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). 2. 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'). 3. The mean duration of both short and intermediate gaps was dependent on the nature of the agonist. For short gaps the mean durations (us) 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. 4. 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. 5. The short gaps in CCh increased in frequency with agonist concentration and were mainly produced by channel blockages by CCh itself. 6. 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. 7. 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. 8. The frequency of channel openings increased slightly with hyperpolarization. 9. 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. D. COLQUHOUN AND B. SAKMANAN 10. Reduction of [Ca2+]0 to 1/10th of normal increased the single-channel conductance by 50 %, and considerably increased the number of intermediate gaps. 11. 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. 12. The component of brief openings behaves, at low concentrations, as though it originates from openings of singly occupied channels. However, the persistence of about 10 % of brief openings at high concentrations makes it unlikely that this is the only mechanism. 13. Short gaps within bursts behave in our preparation in a way that is consistent with the view that they originate from multiple openings of the doubly occupied channel, before dissociation occurs. The evidence for this interpretation is circumstantial, and the results are consistent with other mechanisms too. However, the values for opening (,8) and dissociation (k12) rate constants implied by this interpretation are consistent with the relative potencies of the agonists, the results of high concentration experiments, and, in the case of ACh, the phenomena of synaptic transmission. For ACh we find , = 30600 s-' and k2 = 8150 s-'; the mean length of a single opening (at-130 mV) was 1/a = 1-4 ms (about one-third ofthe burst length), so the conformational equilibrium constant was f8/ac = 43, and the equilibrium constant for binding was about 80 /M. The values are inconsistent with the hypothesis that binding is much faster than channel opening. The results suggest that ACh has a higher efficacy but a lower affinity than SubCh. were of smaller amplitude than the usual (full) open-channel current (Colquhoun & Sakmann, 1983). Qualitatively similar observations have been made on a number of other preparations, and with several different neurotransmitters; for example Cull-Candy & Parker (1982) who worked with glutamate and its analogues on locust muscle fibres, and Dionne & Leibowitz (1982) with acetylcholine-like agonists on snake muscle fibres. Similar phenomena have been observed with several cultured cell types (
Amphipathic analysis and possible formation of the ion channel in an acetylcholine receptor
Proceedings of the National Academy of Sciences, 1984
Fourier analysis of the hydrophobicities of the acetylcholine receptor subunit sequences reveals regions of amphipathic secondary structure. Prediction of a consensus secondary structure based on this analysis and on an empirical prediction method leads to a testable hypothesis about how the ion channel is formed and might function. Knowledge of the three-dimensional structure of acetylcholine receptors is consistent with features of the model proposed and provides some constraints.
Proceedings of the National Academy of Sciences, 1981
Acetylcholine-induced flux of inorganic ions across membranes and inactivation of the acetylcholine receptor were measured at pH 7.0, 1 degrees C, over a 5000-fold concentration range of acetylcholine. Receptor-containing electroplax membrane vesicles prepared from Electrophorus electricus and a quench-flow technique were used, allowing flux to be measured in the 2-msec to 1-min time region. Five different measurements were made: (i) rate of ion translocation with the active state of the receptor, (ii) rate of the slower ion translocation after equilibration of active and inactive receptor states, (iii) rate of inactivation, (iv) equilibrium between active and inactive forms of the receptor, and (v) reactivation of inactivated receptor. The kinetics of the steps in the receptor-controlled ion flux follow single-exponential rate laws, and simple analytical expressions for their ligand concentration dependence can be used. Thus, the rate and equilibrium constants in a scheme that rela...
The Journal of physiology, 1986
Single-channel currents were recorded through acetylcholine receptor channels of clonal BC3H-1 muscle cells activated by the curare-like compound, DMT binds selectively to the two alpha-neurotoxin-binding sites on these receptors, with apparent dissociation constants differing by about 1000-fold (Sine & Taylor, 1981). Receptor channels do not open with DMT bound only to the high-affinity site, but only at DMT concentrations at which both high- and low-affinity sites are occupied. Open-duration histograms are not single exponentials, but are described by the sums of two (or three) exponentials. Both brief- and long-duration openings are observed in the presence of 3 microM-DMT, and are seen at the same relative frequency up to 80 microM-DMT. Long-duration openings are interrupted by brief closures with a mean duration of 50 microseconds and which occur at a frequency of 50-60 per second of open time. These temporal characteristics closely parallel those of the brief closures observed...
Proceedings of The National Academy of Sciences, 1981
The specific reaction rate ({J}) of the acetylcholine receptor-controlled ion translocation has been determined. In eel Ringer's solution (pH 7.0) at 1 degrees C, {J}=3× 107 M-1 sec-1. {J} is an intrinsic constant that is characteristic of the receptor and independent of other properties of a receptor-containing cell that also determine the rates of ion translocation. Membrane vesicles (prepared from the electric organ of Electrophorus electricus) and a flow-quench technique that has a millisecond time resolution were used to measure the receptor-controlled ion translocation. Using the value of {J} and the molar concentrations of receptor sites and inorganic ions, we calculated that 6× 103 ions are translocated per msec per receptor. Analysis of electrical noise in frog muscle cells at temperatures above 8 degrees C [Neher, E. & Stevens, C.F. (1977) Annu. Rev. Biophys. Bioeng. 6, 345-381] gave a value of about 1× 104 ions msec-1 per channel. Thus, each technique gives essentially the same result. It is now possible, therefore, to correlate the results obtained when receptor function is measured in two different ways in membrane vesicles and in muscle cells: (i) chemical kinetic measurements, using membrane vesicles, which relate the ligand binding and ion translocation processes and (ii) analysis of acetylcholine noise in muscle cells [Katz, B. & Miledi, R. (1972) J. Physiol. (London) 224, 665-699], which allows one to measure elementary steps in the formation of ion channels through the cell membrane.