Decay Of End-plate Currents Research Articles

Characteristics of the acetylcholine-operated channel at twitch and slow fibre neuromuscular junctions of the garter snake.

1. Synaptic responses to acetylcholine were recorded from garter snake (sp. Thamnophis) neuromuscular junctions with the voltage clamp. 2. In the following respects the responses were identical in twitch and slow fibres: exponential miniature end-plate current decay, reversal potential (approximately -5 mV), permeant ionic species (Na+, K+, Ca2+, but not Cl-), two component miniature end-plate current decay in the presence of procaine, insensitivity to tetrodotoxin, alpha-bungarotoxin and leiurus toxin. 3. The responses differed in several important ways: miniature end-plate current decay rate was only half as sensitive to voltage at slow fibre end-plates as at twitch; while all twitch fibre current fluctuation spectra were single component, about 60% of the slow fibre spectra were not. The latter could be fitted with two Lorentzian components. In all cases the mean end-plate current was proportional to total induced noise variance. 4. Two mechanisms which might account for the response differences were identified and tested in a fashion independent of specific molecular kinetics. The different responses do not arise from separate ion-selective channels for the permeant ions at slow fibre end-plates and seem unlikely to derive from separate populations of synaptic and extrasynaptic channels.

Effects of McN-A-343, a cholinomimetic drug, on endplate currents in the frog.

The muscarinic ganglion stimulating agent, McN-A-343 has unusual blocking actions on endplate currents (EPCs) at frog neuromuscular junctions. McN-A-343 caused depolarization by a curare-sensitive process, blocked neuromuscular transmission, depressed EPCs and reduced the time for EPC decay. These results are explained best by a nicotinic agonist action of McN-A-343 on the acetylcholine receptor to cause ion flow and the blockade by McN-A-343 of the open ion channels. The actions of McN-A-343 are similar to those of decamethonium (C-10) described by others. Unlike C-10, however, McN-A-343 did not alter the exponential character of the EPC or alter the voltage dependency of the EPC. The prototypical nicotinic agonist, dimethylphenylpiperazinium had no effect on EPC parameters of endplate clamped at -90mV.

Phencyclidine interactions with the ionic channel of the acetylcholine receptor and electrogenic membrane.

The effects of phencyclidine (PCP) were studied on the electrogenic and chemosensitive properties of the neuromuscular junction of skeletal muscle as well as on the binding sites on the acetylcholine (AcCho) receptor and its ionic channel in the electric organ membranes of the electric ray. The directly elicited muscle twitch was markedly potentiated by prolonging the falling phase of the muscle action potential and blocking delayed rectification. The indirectly elicited muscle twitch was transiently potentiated and then blocked by PCP at concentrations below 60 muM. PCP blocked miniature endplate potentials and AcCho sensitivities at the junctional region of innervated muscle, blocked the extrajunctional sensitivity of the chronically denervated muscle, and significantly depressed the peak amplitude of the endplate current (EPC) in a voltage- and time-dependent manner. PCP also caused acceleration of the time course of EPC decay and shortening of the mean life-time of the open ionic channel. The effects of PCP were not due to inhibition of AcCho receptor sites because PCP did not protect against the quasi-irreversible inhibition of receptor sites by alpha-bungarotoxin, nor did it inhibit binding of [(3)H]AcCho or [(125)I-labeled alpha-bungarotoxin to the receptor sites. On the other hand, PCP blocked the binding of [(3)H]perhydrohistrionicotoxin to the sites of the ionic channel of the AcCho receptor. The data suggest that PCP reacts with the electrogenic K(+) channel and the ionic channel associated with the AcCho receptor in the open as well as the closed conformation.

Clindamycin and lincomycin alter miniature endplate current decay

Antibiotic-induced muscle paralysis has frequently been found in both experimental animals and man with three distinct classes of antibiotic: (1) streptomycin and related aminoglycoside compounds, (2) polymyxins and (3) tetracyclines. Recently lincomycin and its chemical congener, clindamycin, have been reported to produce muscle paralysis which has different characteristics from those seen with other classes of antibiotic. Although closely related in chemical structure, lincomycin and clindamycin also seem to produce muscle paralysis by different mechanisms. Clindamycin is considered to exert a direct depressant action on muscle contractility whereas the action of lincomycin is considered to be primarily a depression of neuromuscular transmission. We report here that each of these antibiotics had a significant but different influence on endplate channel behaviour. Clindamycin increased the rate of miniature endplate current (m.e.p.c.) decay and reduced its voltage sensitivity without altering its exponential nature. Lincomycin split m.e.p.c. decay into an initial rapid phase followed by a prolonged phase.

Tetraethylammonium: Voltage-dependent action on endplate conductance and inhibition of ligand binding to postsynaptic proteins

Tetraethylammonium (Et(4)N(+)) ions depressed the amplitude and accelerated the decay rate of spontaneously occurring and nerve-evoked endplate currents (EPCs) in frog sartorius muscle. The relationship between peak EPC amplitude and membrane potential became nonlinear in the presence of 100 muM Et(4)N(+), and with drug concentrations of 250 muM or greater the current-voltage relationship exhibited negative conductance in the hyperpolarized region. Et(4)N(+) modified the exponential dependence of the EPC decay on membrane potential such that the decays between -150 and -50 mV were abbreviated and voltage independent but remained near control levels at more positive membrane potentials. The minimal effective concentration of Et(4)N(+) for altering the EPC time course was 10, and maximal effects were attained with 100 muM. Little additional shortening in the EPC decay phase was detected on raising the drug concentration to 1000 muM. Acetylcholine noise analysis revealed a voltage-dependent reduction in the mean channel open time, which was comparable in magnitude to the shortening in the EPC decay, and a depression of single-channel conductance. In concomitant biochemical studies, Et(4)N(+) was found to inhibit the binding of both [(3)H]acetylcholine and [(3)H]perhydrohistrionicotoxin to receptor-rich membranes from the electric organ of Torpedo ocellata with K(i) values of 200 muM and 280 muM, respectively. These results suggest that Et(4)N(+) interacts with both the acetylcholine receptor and its associated ionic channel. The voltage-dependent actions of Et(4)N(+) are attributed to blockade of the ionic channel in closed as well as open conformation.

Effects of dithiothreitol on end‐plate currents.

1. End-plate currents have been studied in frog cutaneus pectoris nerve-muscle preparations mounted in continuously flowing solution, using the voltage clamp technique. 2. Exposure of the muscle to 1 mM-dithiothreitol reduced the amplitude of end-plate currents by a factor of 2.7 (mean; range 1.6-3.4; twelve fibres). 3. 1 mM-dithiothreitol also caused a 2.7-fold (2.3-3.1) increase in the rate of decay, and a 1.4-fold (1.3-1.6) decrease in the time to peak of end-plate currents. During the onset of action of dithiothreitol, there was little or no indication of departure of end-plate current decay from a simple exponential. 4. Dithiothreitol actions on amplitude and decay of end-plate currents developed with similar time courses and both effects were slower in onset at pH 7.2 than at pH 8.5. 5. The actions of dithiothreitol were reversed by exposure of the muscle to 1 mM-5,5'-dithio-bis-(2-nitrobenzoic acid). 6. Following dithiothreitol treatment, the rates of decay of end-plate currents continued to depend on membrane potential; there was little or no change in the slope of the relation between in (rate of decay) and membrane potential, consistent with little or no change in the dipole moment of a gating molecule for ion channels. 7. Dithiothreitol changed the relation between peak end-plate current and membrane potential, so that peak conductance increased at more negative membrane potentials; this finding could be accounted for in terms of the closure of ion-channel gates becoming faster though remaining voltage-sensitive after exposure to dithiothreitol. 8. It is concluded that dithiothreitol causes changes in the kinetics of gating of ion channels associated with receptors and that these changes accompany changes in the binding of ACh to receptors.

Octanol reduces end‐plate channel lifetime

1. Post-synaptic effects of n-octanol at concentrations of 0.1-1 mM were examined in toad sartorius muscles by use of extracellular and voltage-clamp techniques.2. Octanol depressed the amplitude and duration of miniature end-plate currents and hence depressed neuromuscular transmission.3. The decay of miniature end-plate currents remained exponential in octanol solutions even when the time constant of decay (tau(D)) was decreased by 80-90%.4. The lifetime of end-plate channels, obtained by analysis of acetylcholine noise, was also decreased by octanol. The average lifetime measured from noise spectra agreed reasonably well with the time constant of decay of miniature end-plate currents, both in control solution and in octanol solutions.5. Octanol caused a reduction in the conductance of end-plate channels. Single channel conductance was on average about 25 pS in control solution and 20 pS in octanol.6. In most cells the normal voltage sensitivity of the decay of miniature end-plate currents was retained in octanol solutions. The lifetime of end-plate channels measured from acetylcholine noise also remained voltage-sensitive in octanol solutions. In some experiments in which channel lifetime was exceptionally reduced the voltage sensitivity was less than normal.7. In octanol solutions, tau(D) was still very sensitive to temperature changes in most cells although in some the temperature sensitivity of tau(D) was clearly reduced. Changes in tau(D) with temperature could generally be fitted by the Arrhenius equation suggesting that a single step reaction controlled the decay of currents both in control and in octanol solutions. In some cells in which tau(D) became less than 0.3 ms, the relationship between tau(D) and temperature became inconsistent with the Arrhenius equation.8. As the decay of end-plate currents in octanol solutions remains exponential, and the voltage and temperature sensitivity can be unchanged even when tau(D) is significantly reduced, it seems likely that octanol decreases tau(D) by increasing the rate of the reaction which normally controls the lifetime of end-plate channels.

Effects of [Ca2+] and [Mg2+] on the decay of miniature endplate currents

THE endplate current at the neuromuscular junction is initiated by the combination of acetylcholine (ACh) with its receptor. The current rises rapidly to a maximum, and then decays exponentially1. The rate of decay of the endplate current (e.p.c.) depends on membrane potential; hyperpolarisation lengthens the decay phase2,3. A plausible hypothesis for this is that the rate of disassociation of the ACh from the receptor depends on the voltage gradient through the membrane4. The membrane potential consists of three components: the voltage gradient through the membrane itself and the surface potentials at the two membrane–solution interfaces. The surface potentials are thought to be negative, owing to an excess of negatively-charged groups on the membrane phospholipids. An elevation in the divalent ion concentration in the extracellular solutions reduces the negativity of the external surface potential, either by binding to the phospholipids or by screening the charges. A lowering of the negativity of the external surface potential leads to a corresponding increase in the voltage gradient within the membrane (over shorter periods than for phospholipid flip-flop to occur). This leads to the prediction that alterations in the extracellular [Ca2+] or [Mg2+] are likely to change the time course of the e.p.c. Our experiments were designed to test this hypothesis.

Ethanol reduces excitatory postsynaptic current duration at a crustacean neuromuscular junction

ETHANOL potentiates the postsynaptic voltage response to acetylcholine at mammalian and amphibian neuromuscular junctions1–4, by prolonging the duration of the postsynaptic conductance change5–7. At these synapses, hyperpolarisation also increases the time constant of decay (τ) of end-plate currents8–14. Because of this voltage sensitivity, it was proposed15 that the rate-limiting reaction in the decay of end-plate currents involves the relaxation of membrane proteins, which undergo a change in dipole moment normal to the field direction as they change conformation. The magnitude and direction of the change in dipole moment then determines the effect of electric field on α, the reaction rate, α ( = τ−1) would be proportional to exp (Vμ) (ref. 15) where V is membrane potential and μ the dipole moment change normal to the field direction. It has been suggested that the prolonged decay of end-plate currents produced by ethanol is due to increase by ethanol of the dielectric constant of the environment of receptors6, which lowers the rate of reactions involving a decrease in dipole moment16 and hence increases τ. In some arthropods, including crayfish, crab and locust, there is evidence that glutamate acts as an excitatory transmitter at neuromuscular junctions17,18. Also, the decay of excitatory postsynaptic currents in crayfish19 and locusts20 has the opposite voltage sensitivity to that found at amphibian neuromuscular junctions: hyper-polarisation reduces τ, suggesting that the rate-limiting reaction in the decay of conductance at these ‘glutamate’ synapses could be associated with a change in dipole moment which is opposite to that seen at amphibian neuromuscular junctions. Ethanol could then have an opposite effect at these two types of synapse, and depress synaptic transmission at glutamate synapses. We report here results of our study of spontaneous miniature excitatory junctional currents (MEJCs) in the crab, which confirm this hypothesis.

Endplate current fluctuations reveal only one channel type at frog neuromuscular junction

SIGNAL transduction between nerve and muscle is mediated by a chemical process at the neuromuscular junction. In the nerve ending electrical activity causes the release of acetylcholine (ACh) into the synaptic cleft separating the nerve and muscle cells. ACh can bind to specific chemical receptors in the postjunctional muscle membrane; these receptors control a gating mechanism that can increase membrane conductance locally. Normal nervous activity triggers an action potential and contraction in twitch muscle fibres. At constant membrane potential the membrane conductance change induces a current that is carried largely by Na+ and K+ in frog1, moving through ACh-gated membrane channels. There has been a controversy regarding the nature of the chemically gated endplate channels; are there kinetically-independent channels for Na+ and K+ , or are these ions transported by the same channel? Maeno2 initially proposed separate endplate channels, arguing that the two component decay of endplate potentials in procaine-treated cells resulted from a selective effect on Na+ channel kinetics. Kordas3,4 and Magleby and Stevens5 argued that Maeno's two-channel hypothesis could not account for the unique reversal potential observed or the monotonic voltage dependence of endplate current (EPC) decay. The analysis below of iontophoretically-induced EPC fluctuations in procaine-treated and untreated preparations indicates that the ions which constitute the EPC are not carried by kinetically distinguishable endplate channels.

A voltage-clamp study of the effect of two lidocaine derivatives on the time course of end-plate currents.

1. Voltage-clamped end-plate currents (e.p.c.s) have been studied in the glycerol-treated Rana pipiens sartorius nerve-muscle preparations in normal Ringer solution and in the presence of QX-222 and QX-314. 2. Both QX-222 and QX-314, the trimethyl and triethyl analogues, respectively, of lidocaine, greatly modify end-plate current kinetics. The altered e.p.c.s still show a true reversal potential, which is essentially the same as the reversal potential before drug treatment. The time course of the altered end-plate currents varies with both membrane potential and drug concentration. 3. In the presence of 0-1-1-0 mM QX-222, end-plate currents decay as the sum of three exponentials: I(t) =I1(0)e-k1t+I2(0)e-k2t+I3(0)e-k3t, where the subscipts 1, 2 and 3 refer to the rapidly, intermediately, and slowly decaying components, respectively. Both the amplitudes, Ij(0), and the decay rates, kj, depend upon membrane potential. 4. Hyperpolarization increases the relative size of the first and third components, i.e. I1(0) and I3(0) increase relative to I2(0). Depolarization increases the relative size of the second component. 5. Hyperpolarization causes a decrease in the decay rates k2 and k3 and causes a slight increase in the decay rate k1. Dependence of the three decay rates on membrane potential is well described by: kj=bjeajv. 6. The Q10 of each of the kj is about 3. 7. Raising QX-222 concentration, at any given membrane potential, augments I1(0) and I3(0) at the expense of I2(0). Raising concentration increases k1 and decreases k3; their voltage-dependence is little affected. 8. At all QX-222 concentrations tested the decay rate k2 is nearly the same as the decay rate of a normal e.p.c. recorded at an equivalent holding potential from the same fibre before drug exposure. 9. End-plate currents in the presence of 0-1 mM-QX-314 show a "major" or rapidly decaying phase and a very small, slowly decaying phase or "tail", but no intermediate component. Only the major component is discernible for end-plate currents in 0-5 mM-QX-314. 10. Voltage- and concentration-dependence of the decay rate of the major component in QX-314 is similar to k1 and QX-222. Voltage-dependence of the tails decay rate appears to be similar to k3. It is hypothesized that the second component in QX-222 represents currents of unaltered or normal conductance kinetics, and that the first and third components in QX-222, as well as the major component and tail in QX-314, represent current of "QX-altered conductance kinetics".

Local anesthetic alteration of miniature endplate currents and endplate current fluctuations

The effect of the local anesthetic QX222 on the kinetics of miniature endplate currents and acetylcholine induced endplate current fluctuations was studied in voltage clamped cutaneous pectoris muscle of Rana pipiens. Both the endplate current fluctuation spectra and the miniature endplate current decay consisted of two or three components depending upon the holding potential and local anesthetic concentration. The cutoff frequency of each spectral component was equal to the decay rate of its corresponding constituent of the miniature endplate current. Comparison of the relative amplitudes of the spectral and miniature endplate components indicated that QX222 did not act by creating two kinetically distinct populations of acetylcholine receptors. QX222 action could be explained by alteration of the acetylcholine receptors such that they sequentially change conformation form one open state to another. A specific case in which QX222 binds to the open state of the acetyl-choline receptor creating a blocked state, was found to account for the observed relationship between the relative amplitudes of the miniature endplate current and spectral components, as well as the previously observed voltage and concentration sensitivity of the decay rates of endplate current components.

Factors affecting the time course of decay of end‐plate currents: a possible cooperative action of acetylcholine on receptors at the frog neuromuscular junction.

1. End-plate currents have been studied in gylcerol-treated frog sartorius nerve-muscle preparations with the voltage-clamp technique. 2. Adding the anticholinesterase prostigmine (3 muM) to the solution bathing the muscle caused a 2-7 (mean 3-3) times increase in the time constant of decay of end-plate currents. The anticholinesterase edrophonium (15 muM) also prolonged the time course of end-plate currents. 3. Pre-treatment of the preparation with collagenase, which leads to the removal of acetylcholinesterase in the synaptic cleft, prolongs the time course of end-plate currents. 4. Curare (1-2 muM), cobratoxin (0-13 muM), or alpha-bungarotoxin (0-13-0-26 muM) decreased the time constant of decay of end-plate currents in the presence of prostigmine. 5. These observations are consistant with the suggestion that repeated binding of acetylcholine (ACh) molecules to receptors as the ACh escapes from the synaptic cleft can contribute to the prolongation of end-plate currents which occurrs when acetylcholinesterase activity is eliminated. 6. Increasing the amount of transmitter released from the presynaptic nerve terminal leads to a prolongation of end-plate currents in the presence of prostigmine. 7. In the presence of prostigmine, the second of two end-plate currents (interval 2-10 msec) decays more slowly than the first. 8. ACh (1-40 muM) or carbachol (40 muM) applied in the solution bathing the muscle prolongs end-plate currents in the presence of prostigmine. 9. It is suggested on the basis of the observations described in paragraphs 6 to 8 that the time constant of decay of end-plate currents in the presence of prostigmine increases with increasing concentrations of ACh in the synaptic cleft. In the absence of prostigmine, increasing the concentration of ACh in the synaptic cleft did not change the time constant for decay of end-plate currents. 10. We interpret these results to suggest that ACh can have a cooperative action on receptors such that the association of ACh with one receptor (defined as binding a single ACh molecule) favours the binding or retention of ACh at other receptors. This implies that receptors can interact.

The effect of voltage on the time course of end-plate currents.

1. End-plate currents have been studied in glycerol-treated frog sartorius nerve-muscle preparations with the voltage clamp technique.2. End-plate currents follow a simple exponential time course over most of their declining phase.3. The rate constant alpha that characterizes this exponential decay depends upon membrane potential V according to the relationship alpha (V) = Be(AV), with A = 0.00795 +/- 0.00043 (S.E.) mV(-1) and B = 1.67 +/- 0.04 (S.E.) msec(-1).4. Voltage sensitivity decreases (that is, A in the above equation becomes smaller) as the recording and current-passing electrodes are moved away from the end-plate region.5. The voltage sensitivity of alpha is decreased by decreasing the gain of the voltage clamp amplifier.6. Changing the end-plate current amplitude by curare treatment, by increased calcium ion concentration, and by facilitation and depression has essentially no effect on end-plate current time course.7. When membrane potential is changed step-wise during the decaying phase of the end-plate conductance change, currents begin to decline with a rate constant alpha appropriate to the new membrane potential in less than 0.2 msec.8. Treatment with prostigmine methylsulphate in concentrations up to 50 mug/ml. slows end-plate current decay but has little effect on voltage sensitivity. That is, B in the above equation is decreased by prostigmine treatment, but A is relatively unaffected.