Acetylcholine-induced Current Fluctuations Research Articles

Nicotinic acetylcholine receptor-ion channels involved in synaptic currents in bullfrog sympathetic ganglion cells and effects of atropine

The nicotinic acetylcholine receptor-ion channels (AChR channels) of the bullfrog sympathetic ganglion cells were studied with a two-electrode voltage clamp technique. The decay phase of the fast excitatory postsynaptic current (fast e.p.s.c.) in B-type neurones followed a double exponential function whose time constants were 3.2 and 8.0 ms at -60 mV and increased with membrane hyperpolarization. Likewise, the decay phase of the fast e.p.s.c. in C-type neurones was double-exponential with time constants of 4.4 and 12.3 ms (at -60 mV). The miniature e.p.s.c. in B-type neurones also decayed with a double exponential function (2.7 and 15.4 ms at -100 mV). Analysis of acetylcholine-induced current fluctuations revealed the power spectral density distribution of a double Lorentzian function which yielded the time constants of elementary events [tau noise(f) and tau noise(s): 1.7 and 29.7 ms, respectively, at -100 mV] and the averaged elementary conductance (gamma: 7.8 pS). The amplitude of fast e.p.s.c. and the time constant of the fast component of its decay phase decreased during the initial ("acute") phase (within 15 min) of the action of atropine (3 microM), but recovered during the later ("chronic") phase (more than 30 min after application) of the action. The slow component was affected by atropine in a manner similar to the fast component during the "acute" phase. During the "chronic phase", however, the slow time constant recovered and exceeded the control value. Furthermore, this prolongation remained for at least 1 h after the removal of atropine.(ABSTRACT TRUNCATED AT 250 WORDS)

On the effect of muscle activity on the end-plate membrane in denervated mouse muscle.

1. Mouse soleus muscles were denervated and some of them were chronically stimulated. Sixteen to twenty-one days later, the number of junctional acetylcholine receptors (AChR) and their metabolic stability were examined by measuring binding of 125I-alpha-bungarotoxin, their gating properties by analysis of acetylcholine-induced current fluctuations and the ultrastructure of the end-plate membrane by electron microscopy. 2. In agreement with other studies on inactive muscles, no effect of denervation on junctional AChR number could be resolved. However, some of the fast-gating 'adult' AChR channels had been replaced by slowly gating fetal AChRs, their half-life was lowered to 38 h and the folding of the end-plate membrane was reduced. 3. These changes were prevented in denervated but stimulated active muscles: the junctional AChR population remained homogeneously 'adult', the half-life of junctional AChRs was 13 days and folding of the end-plate membrane remained comparable to that in control muscles. 4. The significance of these results is discussed with respect to the role of muscle activity in end-plate development.

Factors determining the duration of a single postsynaptic response in neuromuscular junctions

The miniature end-plate currents (MEPCs) and acetylcholine-induced current fluctuations were compared in experiments on fast and slow muscle fibres of lamprey, frog, chicken and rat. Simultaneous analysis of MEPCs decay time constant and mean life-time of single ionic channels suggests that the former is the main but not the sole factor determining the MEPCs duration. Additional factors, in particular, the relative insufficiency of acetylcholinesterase activity (AChE) are more important for the generation of the usual MEPSs in slow muscle fibres or the "giant" MEPCs in fast ones. Low activity of AChE favours the prominent asynchrony of interaction between acetylcholine molecules and cholinoreceptors causing prolongation of the time course of synaptic response. The mechanisms of MEPCs decay shortening induced by alpha-bungarotoxin or d-tubocurarine and the nonuniformity of different active spots of junctions are discussed.

Analysis of fast excitatory postsynaptic currents in bullfrog parasympathetic ganglion cells

Excitatory postsynaptic currents (EPSCs), miniature excitatory postsynaptic currents (MEPSCs), and acetylcholine-induced current fluctuations (noise) have been studied in voltage-clamped bullfrog parasympathetic ganglion cells of the atrial septum. EPSCs were also recorded from voltage-clamped sympathetic B cells and, in general, it was found that the basic properties of the EPSCs are similar in both parasympathetic and sympathetic B cells. For parasympathetic cells, the EPSC reached a peak amplitude of several nanoamperes within 3 msec and decayed exponentially. For 31 cells voltage-clamped to -50 mV (22 to 23 degrees C), peak amplitude was -4.3 +/- 1.4 nA (mean +/- SD) and the decay time constant, tau, was 5.6 +/- 1.0 msec. tau was independent of EPSC amplitude at a set voltage but increased with hyperpolarization, the coefficient of voltage dependence being -0.0070 +/- 0.0025 mV-1 in 13 cells (21 to 23 degrees C). The EPSC amplitude-voltage relationship was linear between -30 and -90 mV. The reversal potential, determined by interpolation, was -4.0 +/- 6.7 mV (n = 11). The EPSC tau had a Q10 equal to 2.9. Blocking the acetylcholinesterase with methane sulfonyl fluoride (MSF) pretreatment prolonged EPSC decay but decreased EPSC amplitude. In addition, EPSC decay after MSF treatment deviated from a single exponential function. MEPSCs exhibited decay characteristics very similar to those of EPSCs recorded at the same voltage and temperature. Acetylcholine-induced current fluctuations were well described by a single Lorentzian function with the estimated mean channel open time (tau noise) very similar to the EPSC decay time constant.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of divalent cations on toad end-plate channels.

Miniature end-plate currents (MEPCs) and acetylcholine-induced current fluctuations were recorded in voltage-clamped, glycerol-treated toad sartorius muscle fibers in control solution and in solutions with added divalent cations. In isosmotic solutions containing 20 mM Ca or Mg, MEPCs had time constants of decay (tau D) which were about 30% slower than normal. In isotonic Ca solutions (Na-free), greater increases in both tau D and channel lifetime were seen; the null potential was -34 mV, and single-channel conductance decreased to approximately 5 pS. Zn or Ni, at concentrations of 0.1-5 mM, were much more effective in increasing tau D than Ca or Mg, although they did not greatly affect channel conductance. The normal temperature and voltage sensitivity of tau was not significantly altered by any of the added divalent cations. Surface potential shifts arising from screening of membrane fixed charge by divalent cations cannot entirely explain the observed increases in tau, especially when taken together with changes in channel conductance.

Neuromuscular block produced by polymyxin B: Interaction with end-plate channels

The effect of polymyxin B on the amplitude and kinetics of miniature end-plate currents and acetylcholine-induced current fluctuations was studied in voltage-clamped fibers of the snake. Polymyxin B produced a voltage and concentration dependent reduction in both mean peak MEPC amplitude and the time constant of MEPC decay. A concomitant decrease in mean channel lifetime and single channel conductance indicated that a major mechanism for the neuromuscular block produced by polymyxin B is endplate channel blockade.

A re-examination of curare action at the motor endplate.

Recent evidence indicates that curare, in addition to its competitive' interference with endplate receptors, can block open ionic channels by a 'non-competitive' action on the activated acetylcholine-receptor complex. These findings called for further study of the kinetic behaviour of endplate channels and their modification by curare. Examining impulse-evoked endplate currents and acetylcholine-induced current fluctuations, it is found that the lifetime of the open channel is shortened by relatively high concentrations of curare (greater than 5 micrometer), an effect which shows up most strikingly at hyperpolarized levels of membrane potential (-130 mV and above). No shortening of this kind is observed when a neuromuscular block of equal or greater intensity is produced by a dose of alpha-bungarotoxin. Two other neuromuscular blocking agents, gallamine and pancuronium are shown to have an action on channel kinetics which cannot be explained by competitive receptor binding, but conforms to the hypothesis of rapidly repeated blocking and unblocking of individual ion channels, which had been proposed originally to account for the endplate action of local anaesthetics.

Acetylcholine-induced current fluctuations in tissue-cultured muscle cells under voltage clamp

Acetylcholine applied ionophoretically to chick skeletal muscle cells grown in tissue culture produces membrane current fluctuations. Cells treated with vinblastine are transformed to a roughly spherical shape. Such transformed cells can be voltage-clamped with microelectrodes. The frequency spectrum of the current fluctuations at fixed voltage obeys a relation of the Lorentz form. From analysis of the current noise, the conductance of a single ionic channel is estimated to be 39 pmho at a temperature of 28 degrees C, and increases with increasing temperature, exhibiting a Q10 of 1.7. The relaxation time for the channel conductance is more sharply temperature dependent, showing a Q10 of approximately 5. These results are in agreement with the picture of acetylcholine-activated ionic channels determined from experiments on frog end plate (Anderson and Stevens, 1973). The relaxation time for carbachol activation is shorter than for acetylcholine, and appears to be more temperature sensitive.