THE ionic channel associated with the acetylcholine (ACh) receptor at the neuromuscular junction of skeletal muscle fibres is probably the best described channel in biological membranes. Nevertheless, the properties of individual channels are still unknown, as previous studies were concerned with average population properties. Macroscopic conductance fluctuations occurring in the presence of ACh were analysed to provide estimates for single channel conductance and mean open times1–3. The values obtained, however, depended on assumptions about the shape of the elementary conductance contribution—for example, that the elementary contribution is a square pulse-like event2. Clearly, it would be of great interest to refine techniques of conductance measurement in order to resolve discrete changes in conductance which are expected to occur when single channels open or close. This has not been possible so far because of excessive extraneous background noise. We report on a more sensitive method of conductance measurement, which, in appropriate conditions, reveals discrete changes in conductance that show many of the features that have been postulated for single ionic channels.

Single-channel currents recorded from membrane of denervated frog muscle fibres

The physiology of excitatory amino acids in the vertebrate central nervous system.

Distinct classes of acetylcholine receptor channels are formed when Xenopus oocytes are injected with combinations of the bovine α-, β-, γ- and δ- or the α-, β-, γ- and e-subunit-specific messenger RNAs. The conductance and gating properties of the two classes of channels, in conjunction with the developmental changes in the muscular contents of the mRNAs, suggest that replacement of the γ-subunit by the e-subunit is responsible for the functional alteration of the receptor during muscle development.

Molecular distinction between fetal and adult forms of muscle acetylcholine receptor

1. Single channel currents through acetylcholine receptor channels (ACh channels) were recorded at chronically denervated frog muscle extrajunctional membranes in the absence and presence of the lidocaine derivatives QX-222 and QX-314. 2. The current wave forms due to the opening and closing of single ACh channels (activated by suberyldicholine) normally are square pulses. These single pulses appear to be chopped into bursts of much shorter pulses, when the drug QX-222 is present in addition to the agonist. 3. The mean duration of the bursts is comparable to or longer than the normal channel open time, and increases with increasing drug concentration. 4. The duration of the short pulses within a burst decreases with increasing drug concentration. 5. It is concluded that drug molecules reversibly block open end-plate channels and that the flickering within a burst represents this fast, repeatedly occurring reaction. 6. The voltage dependence of the reaction rates involved, suggested that the site of the blocking reaction is in the centre of the membrane, probably inside the ionic channel.

Local anaesthetics transiently block currents through single acetylcholine-receptor channels.

1 A suction electrode was used to record the membrane current of single rod outer segments in pieces of toad retina During dim illumination the membrane current showed pronounced fluctuations 2 Amplitude histograms of responses to dim flashes of fixed intensity exhibited two discrete peaks, one at 0 pA and one near 1 pA, suggesting that the response was quantized By setting a criterion amplitude level, flash responses could be classed as 'failures' (no response) or as 'successes' (at least one quantal event) 3 The variation of fraction of successes with flash intensity was consistent with the hypothesis that each quantal electrical event resulted from a single photoisomerization 4 The quantal event had a mean amplitude of about 1 pA (5% of the standing dark current) and a standard deviation of 02 pA Dispersion in the event amplitude prevented identification of histogram peaks corresponding to two or more photoisomerizations 5 Individual quantal responses exhibited a smooth shape very similar to that of the average quantal response This suggests that a single photoisomerization releases many particles of transmitter and that radial diffusion of internal transmitter is not a major source of delay in the light response 6 The 'quantum efficiency' with which an absorbed photon generated an electrical event was measured as 05 +/- 01 (SE of mean, n = 4) This is slightly lower than the quantum efficiency of photoisomerization obtained previously for rhodopsin in solution 7 At wavelengths between 420 and 700 nm the quantal event was invariant in size, although the cell's sensitivity varied over a range of 10(5) 8 The power spectrum of the fluctuations in dim steady light was predicted by assuming that a random series of quantal events occurred independently 9 In brighter light the fluctuations were faster, and the response to an incremental flash was reduced in size and duration The power spectrum could be predicted by assuming random superposition of events with the shape of the incremental flash response

https://physoc.onlinelibrary.wiley.com/doi/pdf/10.1113/jphysiol.1979.sp012716

Responses of retinal rods to single photons.

1. Acetylcholine produced end-plate current (e.p.c.) noise is shown to be the results of statistical fluctuations in the ionic conductance of voltage clamped end-plates of Rana pipiens.

2. These e.p.c. fluctuations are characterized by their e.p.c. spectra which conform to a relation predicted from a simple model of end-plate channel gating behaviour.

3. The rate constant of channel closing α is determined from e.p.c. spectra and is found to depend on membrane potential V according to the relation α = BeAV (B = 0·17 msec−1±0·04 S.E., A = 0·0058 mV−1±0·0009 S.E. at 8° C) and to vary with temperature T with a Q10 = 2·77, at −70 mV. A and B in this expression both vary with T and therefore produce a membrane potential dependent Q10 for α.

4. Nerve-evoked e.p.c.s and spontaneous miniature e.p.c.s decay exponentially in time with a rate constant which depends exponentially on V. The magnitude and voltage dependence of this decay constant is exactly that found from e.p.c. spectra for the channel closing rate α.

5. The conductance γ of a single open end-plate channel has been estimated from e.p.c. spectra and is found not to be detectibly dependent on membrane potential, temperature and mean end-plate current. γ = 0·32±0·0045 ( S.E.) × 10−10 mhos. Some variation in values for γ occurs from muscle to muscle.

6. It is concluded that the relaxation kinetics of open ACh sensitive ionic channels is the rate limiting step in the decay of synaptic current and that this channel closing has a single time constant. The relaxation rate is independent of how it is estimated (ACh produced e.p.c. fluctuations, e.p.c., m.e.p.c.), and is consistent with the hypothesis that individual ionic channels open rapidly to a specific conductance which remains constant for an exponentially distributed duration.

7. The voltage and temperature dependence of the channel closing rate constant agree with the predictions of a simple dipole-conformation change model.

C. R. Anderson

Papers

Voltage clamp analysis of acetylcholine produced end-plate current fluctuations at frog neuromuscular junction