Frog Sartorius Muscle Research Articles

Electrophysiological studies of the action of ketamine in frog skeletal muscle

The effects of the dissociative analgesic anaesthetic, ketamine, on action potential production in frog sartorius muscle fibres, were studied with bipolar extracellular electrodes and intracellular microelectrode techniques. All concentrations of ketamine depressed the extracellularly recorded compound action potential. Low concentrations of ketamine (less than 1 × 10 −4 m) increased excitability by reducing the current needed to initiate an action potential. Higher concentrations of ketamine decreased excitability by inhibiting the maximum rate of rise, the overshoot, and the maximum amplitude of the action potential. The resting membrane potential was not significantly affected by any of the ketamine concentrations employed. The depressant effects of ketamine on the action potential were antagonized in an apparently competitive manner by sodium ions. Ketamine caused a shift in the relationship between rate of fall and amplitude to higher action potential amplitudes, indicating that the drug inhibited the increase in potassium conductance (gK) associated with the falling phase of the action potential. It can be concluded that ketamine decreases excitability in frog sartorius muscle by inhibiting the specific increase in sodium conductance, which normally follows a threshold stimulus and is responsible for the rising phase of the action potential.

An attempt to distinguish between the actions of neuromuscular blocking drugs on the acetylcholine receptor and on its associated ionic channel.

The effects of lobeline and tubocurarine on the voltage-clamped endplates of frog sartorius and cutaneous pectoris muscles were examined at room temperature (20-23 degrees C). Like tubocurarine, lobeline causes nondepolarizing neuromuscular blockade. The half-time of decay (t((1/2))) of endplate currents (e.p.c.s) recorded at a holding potential (V(m)) of -90 mV was significantly shorter in endplates treated with lobeline (50 muM; mean t((1/2)) +/- SEM = 0.41 +/- 0.02 ms) or tubocurarine (11.4 muM; t((1/2)) = 0.64 +/- 0.04 ms) than in those treated with Mg(2+) (13 mM; t((1/2)) = 1.39 +/- 0.11 ms) or a low concentration of tubocurarine (3 muM; t((1/2)) = 0.87 +/- 0.05 ms). Similarly, lobeline (10 muM) shortened the t((1/2)) of untreated miniature e.p.c.s by 35%; tubocurarine, however, abolished miniature e.p.c.s at the concentration required to observe its actions on e.p.c. decay kinetics. The t((1/2)) of e.p.c.s recorded from preparations treated with Mg(2+) (13 mM), tubocurarine at low concentrations (3 muM), or untreated miniature e.p.c.s was logarithmically related to V(m), being slower at more hyperpolarized values. By contrast, the t((1/2))s of e.p.c.s recorded in either lobeline (50 muM) or tubocurarine (11.4 muM) were independent of voltage in the range -150 to -80 mV. The ability of lobeline to shorten t((1/2)) and to remove the voltage dependence of t((1/2)) was partially antagonized by Mg(2+) (13 mM). As expected, when lobeline or tubocurarine was removed from the bath or when acetylcholine release from the motor nerve terminals was increased by 4-aminopyridine (20 muM) and Ca(2+) (10 mM) (in the presence of lobeline or tubocurarine), the amplitude of e.p.c.s increased as a function of time. However, the t((1/2)) of the decay phase of the e.p.c.s remained shortened (i.e., unaltered from the earlier treatment). These results suggest that both tubocurarine and lobeline have at least two distinct postjunctional actions including: (i) a block of the acetylcholine receptor and (ii) a block of the ionic channel associated with the acetylcholine receptor.

Three-dimensional structure of the vertebrate muscle A-band: II. The myosin filament superlattice

The three-dimensional arrangement of the myosin filaments in the A-band of frog sartorius muscle was studied using electron micrographs of very thin and accurately cut transverse sections through the bare region (on each side of the M-band) where the thick filament shafts are roughly triangular in shape. It was found that the orientations of these triangular profiles are arranged to give a superlattice of the same size and shape as that proposed by Huxley & Brown (1967) on the basis of X-ray diffraction evidence, but the contents of the superlattice may not be as they suggested. The results from detailed image analysis strongly suggest that myosin filaments (which have been shown to have 3-fold rotational symmetry, Luther, 1978; Luther, Munro and Squire, unpublished results) are arranged with one of two orientations which are 60 ° (or 180 °) apart. This arrangement of filaments with 3-fold symmetry is not that predicted for a superlattice with the symmetry suggested by Huxley & Brown. Two rules define the way in which the orientations of neighbouring filaments are defined. Rule (1): no three mutually adjacent filaments in the hexagonal array of filaments in the A-band can all have identical orientations; and rule (2): no three successive filaments along a 10 1 ̄ row in the filament array can have identical orientations. These two no-three-alike rules are sufficient to describe the observed arrangement of filament profiles in the frog bare region (except for some minor violations discussed in the text), and they lead automatically to the generation of the required superlattice. The A-band structure in fish muscle is different; there is no superlattice and the triangular bare region profiles have only one orientation. The frog superlattice and fish simple lattice are explained directly in terms of different interactions between the M-bridges in the M-bands of these muscles. The observed structures require that the myosin filament symmetry at the centre of the M-band is that of the dihedral point group 32. The two possible forms of interaction between filaments with this symmetry (apart from a completely random structure) give rise to the observed A-band lattices in frog and fish muscles. The 3-fold rotational symmetry of the myosin filaments required to explain the observed micrographs also requires that the myosin crossbridge arrangements around the actin filaments in frog and fish muscles will be different. It is suggested that the structure in the frog A-band (and in the A-bands of other higher vertebrates) has evolved from that in fish to improve the distribution of crossbridges around the aotin filaments. The X-ray diffraction evidence of Huxley & Brown (1967) will be accounted for in terms of the proposed A-band structure in a further paper in this series.

Fourier transform analysis of periodic variations of red cell velocity in capillaries of resting skeletal muscle in frogs

Red cell velocity in capillaries of resting sartorius muscle in frogs was measured by means of a television-computer method. The velocity, represented in a digital form, was transferred from the time domain into the frequency domain by means of the Fourier transform. In this way, both the periods and amplitudes of periodic components in the velocity could be measured accurately. The Fourier transform analysis revealed that, in the range of periods from 0.7 sec to 25 min, the only significant periodic variation of the velocity was that related to the heart beat. The period of this variation was defined as the pulsatility period and, in the range of 0.7 to 3.4 sec, this was practically identical to the heart beat period (measured independently of the velocity). The average amplitude of the pulsatile component was ±24% of the mean velocity. Even though the visual analysis of some velocity records suggested the presence of variations with longer periods, the Fourier analysis indicated that these periods were not constant. We conclude that, in the capillaries of resting sartorius muscle in frogs, two types of velocity variations occur: (i) pulsatile variations caused by the pumping action of the heart, and (ii) superimposed nonperiodic fluctuations. On the assumption that these fluctuations are caused by the activity of precapillary sphincters and/or arteriolar smooth muscle, our results indicate that such activity is irregular and nonperiodic.

A method for on-line measurements of red cell velocity in microvessels using computerized frame-by-frame analysis of television images

We have used a television signal as the input to a digital computer system to measure red cell velocity in capillaries in a sartorius muscle in frogs. In principle, the velocity was measured in terms of the displacement of red cells and plasma gaps along the capillary (represented here by the shift of an optical density waveform) that occurred in a fixed time interval. Instead of using the conventional spatial cross-correlation of the waveforms, the displacement was determined by means of the absolute differences between the waveforms. The quality of the velocity measurement depended on the optical contrast between the cells and plasma gaps and an index of this quality was determined. The velocity in the capillaries of the frog muscle did not exceed 0.7 mm/sec, but it was demonstrated that, in theory, the velocity could be measured over a range of 0–21 mm/sec. This method is simple, allows further computer analysis of the velocity data and is very useful for studies of the microcirculation.

Rubidium block and rubidium permeability of the inward rectifier of frog skeletal muscle fibres.

1. A three-electrode voltage clamp method was used to investigate the Rb block of inward rectification in frog sartorius muscle fibres. 2. In a solution containing 80 mM-K+, the potassium conductance increased with increasing hyperpolarization to 3.18 +/- 0.11 mS. cm-2 (n = 17) when (V - VK) was -150 mV. In the presence of Rb+, the conductance increased, fell and increased again with increasing hyperpolarization, i.e. the Rb block was first increased and then reduced by increasing hyperpolarization. Increasing [Rb]o increased the block at all voltages. 3. In a solution containing 80 mM-Rb+ (zero K+) inward currents were recorded when the membrane was hyperpolarized beyond about -60 mV. These currents, which were < 10% the amplitude of those in 80 mM-K solution, were blocked by tetraethylammonium ions. 4. Experiments were carried out in solutions either where both [K]o and [K]i were increased, or where [K]o only was increased. The form of the relation between K conductance and membrane potential appeared to depend on [K]o. The magnitude of the conductance appeared to depend on [K]o and on [K]i. 5. So far as the block by Rb+ is concerned, increasing [K]o appeared to enhance the release of Rb block under large hyperpolarizations. Increasing [K]o and [K]i reduced the Rb block at all membrane potentials. 6. The results of experiments in the presence of Rb+ and Cs+ suggest that these two ions do not compete with each other for a site at which they block inward rectification. Rather, over a range of membrane potentials from -25 to -65 mV, the presence of Cs+ enhances the Rb block and vice versa. 7. Single dissected muscle fibres (from semitendinosus) were used to measure sarcoplasmic resistivity in 80 mM-K solution and 160 mM-K (hyperosmotic) solution. The measured values were 163.2 +/- 11.7 omega x cm and 136.1 +/- 16.0 omega x cm, respectively (n = 7). 8. A semi-empirical model is presented, supposing that Rb interacts with a site in the membrane to produce its blocking effect, but is able to move on through into the sarcoplasm. Internal K+ is supposed to reduce the affinity of the site for Rb+; external K+ is able to enhance the moving on of Rb+ into the sarcoplasm. 9. The implications of our experiments for the nature of the permeability mechanism inward rectification are discussed.

Effect of length and caffeine on isometric tetanus relaxation of frog sartorius muscles

In an isometric tetanus of frog sartorius muscle the total relaxation time increased linearly with change in length from 0.7 to 1.4 times rest length. Maximal rate of relaxation, measured from the time derivative ( dp dt ) of tension decay, decreased with both decrease and increase from rest length in correlation with the generated tetanus tension. Stretching the muscle did not significantly affect the times to maximal rate, positive and negative inflexion points but greatly increased the time to total relaxation from the negative inflexion point. Caffeine at 2 mM, acting on muscles at rest length, also slowed the relaxation and decreased the maximal rate of tension decay. However, caffeine increased the times to maximal rate, positive and negative inflexion points without significantly affecting time to total relaxation from the negative inflexion point. These results suggest that caffeine slows an earlier step in relaxation, while stretch slows a later step. It is proposed that muscle relaxation is a two step process: an initial step that is regulated by the rate of Ca 2+ uptake by sarcoplasmic reticulum, and a later step that is mostly controlled by the speed of dissociation of remaining cross-bridges.

A model of myosin crossbridge structure consistent with the low-angle x-ray diffraction pattern of vertebrate muscle.

Low-angle X-ray diffraction patterns have been taken of relaxed frog sartorius muscles stretched to destroy the lattice sampling in the pattern and the observed layer line pattern has been interpreted with model-building studies. The modelling calculations indicate that each crossbridge cannot be represented by a single cylindrical shape, but that the two S-1 heads of the crossbridge are tilted in opposite directions along the filament while twisted the same way round it so that the crossbridge lies wrapped round the filament backbone. The radial position derived for the crossbridge depends on how many strands the filament is assumed to have. If the filament is 2, 3 or 4 stranded then the centre of mass of the crossbridge lies at about 9.5, 12.5, or 15.5 nm respectively from the filament axis.

Effect of pH on muscle fatigue in isolated frog sartorius muscle.

Isolated frog sartorius muscle was incubated for 3 h in Ringer solution (5 mM phosphate buffer) at pH 7 or pH 8. Each muscle was stimulated supramaximally at 2 per second continuously. Muscles at pH 7 fatigued more quickly than those at pH 8. Muscles at pH 8 released acid whereas those at pH 7 did not.

Voltage dependence of desensitization. Influence of calcium and activation kinetics.

The voltage dependence of carbachol-induced desensitization has been analyzed in potassium-depolarized frog sartorius muscle preparations with voltage clamp techniques over a wide voltage range (-120 to +40 mV). Desensitization developed exponentially at all voltages with tau, the time constant of desensitization onset, varying as a logarithmic function of membrane voltage. The voltage dependence of tau remained in calcium-deficient solutions and was not altered by elevating either the level of extracellular or intracellular calcium. We have analyzed our results according to a simple sequential kinetic scheme in which the rate-limiting step in the development of desensitization is a transition of the receptor channel complex from the activated conducting state to a desensitized, nonconducting state. We conclude (a) that the observed voltage sensitivity of desensitization primarily resides in the voltage dependence of this transition, and (b) the kinetics of activation appear to have a greater influence on the observed rate of desensitization than on its voltage dependence. The magnitude of the voltage dependence suggests that a greater change in free energy is required for the transition to the desensitized state than for the transition between the open and closed states of the receptor channel complex.

Effects of ammonium ions on endplate channels.

Miniature endplate currents, recorded from voltage-clamped toad sartorius muscle fibers in solutions containing ammonium ions substituted for sodium ions, were increased in amplitude and decayed exponentially with a slower time constant than in control (Na) solution. The peak conductance of miniature endplate currents was also greater in ammonium solutions. The acetylcholine null potential was -2.8 +/- 0.8 mV in control solution, and shifted to 0.9 +/- 1.6 mV in solutions in which NH4Cl replaced half the NaCl. In solutions containing NH4Cl substituted for all the NaCl, the null potential was 6.5 +/- 1.3 mV. Single channel conductance and average channel lifetime were both increased in solutions containing ammonium ions. The exponential relationship between the time constant of decay of miniature endplate currents or channel lifetime and membrane potential was unchanged in ammonium solutions. A slight but consistent increase in peak conductance during miniature endplate currents and single channel conductance was seen as membrane potential became more positive (depolarized) in both control and ammonium solutions. Net charge transfer was greater in ammonium solutions than in control solution, whether measured during a miniature endplate current or through a single channel. The results presented here are consistent with an endplate channel model containing high field strength, neutral sites.

The visualization of myosatellite cells in normal and denervated muscle: a new light microscopic staining technique.

A new light microscopic staining technique allows the visualization of satellite cells on the surface of myofibers. Either prior to or during fixation, whole frog sartorius muscles are bathed in an acidic buffered solution containing lead nitrate and subsequently exposed to ammonium sulfide. The staining of the satellite cells resulting from this procedure reveals their positions, and the outlines of their cell processes which occasionally branch. Electron microscopy shows that the staining is due to lead deposits localized between apposing membranes of satellite cells and associated myofibers. Prior exposure to N-ethyl-maleimide (NEM) does not alter the formation of the lead deposits on the satellite cell, but reduces the amount of Pb deposits on the muscle surface and connective tissue. This technique has been applied to determine the effects of denervation on the satellite cells of frog sartorius muscles. Four weeks after denervation, the number of satellite cells is essentially the same in both denervated muscles and the intact muscles of the contralateral side. However, denervation results in a subpopulation of satellite cells with altered shapes. They have elongated cytoplasmic processes which often branch. It is suggested that these supernumerary cytoplasmic processes represent an intermediate phase in the transition of satellite cells to myoblasts.

X-ray diffraction observations of chemically skinned frog skeletal muscle processed by an improved method

Whole frog sartorius muscles can be chemically skinned in approximately 2 h by relaxing solutions containing 0.5% Triton X-100. The intensity and order of the X-ray diffraction pattern from living muscle is largely retained after such skinning, indicating good retention of native structure in fibrils and filaments. Best X-ray results were obtained using a solution with (mM): 75 K acetate; 5 Mg acetate; 5 ATP; 5 EGTA; 15 K phosphate, 2% PVP, pH 7.0. Equatorial X-ray patterns showed that myofibrils swell after detergent skinning, as also observed after mechanical skinning. This swelling could be reversed by adding high molecular weight colloids (PVP or dextran) to the extracting solution. By finding the colloid osmotic pressure needed to restore the in vivo interfilament spacing (3% PVP, 4 X 10(4) mol wt) the swelling pressure was estimated as 35 Torr in a standard KCl-based relaxing solution. The swelling pressure and the extent of swelling were less than acetate replaced chloride as the major anion. Detergent-skinned muscle lost the constant-volume relation between sarcomere length and lattice spacing seen in intact muscle. Changes in A band spacing were paralleled by changes in I and band-Z line spacing at a constant sarcomere length. After detergent skinning, I1,0 rose while I1,1 fell, a change in the relaxing direction. Since raising the calcium ion concentrations from pCa 9 to PCa 6.7 was without effect on equatorial or axial X-ray patterns, we concluded that these intensity changes were not due to calcium-dependent cross-bridge movement but rather to disordering of thin filaments in the A band.

Central respiratory and circulatory depression caused by intravascular saxitoxin.

1 In cats anaesthetized with pentobarbitone and vagotomized, observations were made on the phrenic nerve action potential and the diaphragm electromyogram (EMG) at constant end-tidal Pco(2). Arterial blood pressure was stabilized by intravenous infusions of noradrenaline.2 Intravenous administration of saxitoxin (STX) initially abolished respiratory activity in the EMG and caused a slowing of oscillation in the central phrenic neurogram. Additional STX produced apneustic phrenic discharges followed by a progressive loss of nerve action potentials.3 The inspiratory centre in the medulla oblongata was stimulated electrically to evoke a sustained phrenic nerve discharge. STX, given intravenously, resulted in the elimination of spontaneous nerve activity without interfering with the evoked response.4 The cephalic intravascular infusion of STX into a carotid or vertebral artery depressed spontaneous respiratory activity while sparing EMG activity evoked by electrical stimulation of the intact phrenic nerve.5 Spontaneous respiratory discharge in the phrenic nerve was eliminated by smaller doses of STX administered intra-arterially than were required intravenously. In addition, onset of and recovery from neural silence occurred faster following intra-arterial injection of STX.6 Depressant effects on arterial blood pressure coincided with those on respiration when STX was given intra-arterially.7 An electrophysiological assay on frog sartorius muscle was used to measure STX in the cerebrospinal fluid. Levels of STX detected were proportional to amounts of the toxin infused intra-arterially.8 It is concluded that STX exchanges rapidly between blood and brain to bring about central depression and this adds to its peripheral paralytic actions.

Myotonia as a side effect of diuretic action.

1. Commonly used loop diuretics produce side effects in man which are similar to chemically induced myotonia. These diuretics have structural affinity with known myotonic agents. 2. We have observed EMG myotonia in vivo in leg muscles of rats treated with intravenous frusemide. 3. In the presence of several different diuretics, rat isolated diaphragm, soleus and extensor digitorum longus muscles as well as frog sartorius muscles produce typically myotonic contractions with relaxation times up to several seconds. 4. Intracellular recording of action potentials from diuretic-treated muscles reveals long lasting after-discharges following a brief electrical stimulus, again typical of chemically induced myotonia. 5. Having demonstrated a myotonic action of several diuretics we suggest a need for caution in using these drugs in persons with hereditary myotonia and a need to be aware of possible provocation of myotonia in subclinical cases. Myopathies and neuropathies which are known to result from chronic exposure to myotonic agents also need to be considered. 6. In our study, the diuretic, acetazolamide, unmasked subthreshold myotonia. This seems to be at variance with reports of its usefulness in the treatment of myotonia. 7. Diuretics should probably not be employed in the treatment of herbicide intoxication where their myotonic activity would be expected to add to the known myotonic activity of the herbicide.

The interaction between the effects of insulin and ouabain on the activity of Na transport system in frog skeletal muscle.

The effects of insulin and ouabain on 22Na efflux and net Na loss were examined in frog sartorius muscles. After removal of ouabain, 22Na efflux which had been inhibited by ouabain remained at the inhibited level for more than 2 hr. In ouabain-free solution, insulin stimulated the Na efflux from ouabain-preincubated muscles. The stimulating effect of insulin on 22Na efflux was completely blocked by the presence of ouabain. Insulin shifted the Na efflux vs. intracellular Na concentration relationship toward the region of lower Na concentrations, and made the slope of log (Na efflux) vs. log [Na]1 relationship less step. On the other hand, ouabain shifted the Na efflux vs. intracellular Na concentration relationship toward the region of higher Na concentration, and made the slope of the log (Na efflux) vs. log [Na]1 relationship steeper. The relationship between insulin-stimulated Na efflux and intracellular Na concentration obtained from ouabain-preincubated muscle was located in the region of higher Na concentrations than that obtained from control muscle. These findings suggest that insulin increases an apparent affinity of binding sites for Na ions, and the possibility that insulin increases the Vmax of Na transport system may be excluded. In contrast with the effect of insulin, ouabain may be considered to decrease the apparent affinity of binding site for Na ions.

Intracellular chloride concentration and evidence for the existence of a chloride pump in frog skeletal muscle.

Intracellular chloride concentration in frog sartorius muscle was determined at 20 degrees C utilizing ionic permeability change of the membrane for an ion-selective electrode. The membrane potential and external potassium concentration relation was obtained with changes in pH. Using the constant field equation with a condition given by the crossing point in the two relations obtained with the two different pH, the intracellular chloride concentration was estimated to be 3.7 +/- 0.18 mM (mean +/- S.E.M.) with a 0.76 of the activity coefficient for chloride in Ringer's solution. The chloride potential (-88.5 +/- 1.26 mV) was significantly positive to the resting potential (-94.8 +/- 1.09 mV), suggesting that a chloride pump may be working in the frog skeletal muscle. The existence of the chloride pump was supported further by the fact that the depolarization induced by the pH increase became smaller with cooling and finally disappeared when cooled below 5 degrees C.

The effect of anticholinesterase drugs on postjunctional potentials of skeletal muscle

1. 1. Quantitative detailed investigation of the effects of seven anticholinesterase (anti-ChE) drugs—organophosphorus inhibitors (OPI) (armin, GD-42, GD-7), carbamates (proserine or neostigmine, eserine), quaternary ammonium salts (ambenonium, edrophonium)—on postjunctional potentials in skeletal muscle was carried out with a reduced quantum content of end plate potentials (EPP). 2. 2. It was established by the method of elimination of other possible causes that the amplitude of miniature end plate potentials (MEPP) and the duration of EPP were increased by inhibitors as a result of their anti-ChE action only. 3. 3. Curare-like action of the inhibitors was analysed. It was found to be related neither to their effect on membrane potential or passive electrical properties of the muscle fibres nor to inhibition of cholinesterases (ChE). 4. 4. It was found that inhibitors, excepting OPI, failed to affect the EPP quantum content and MEPP frequency. OPI affected these parameters in experiments on the frog sartorius muscle but not on the rat diaphragm muscle and only at concentrations of 2 × 10 −5 M and higher. Under these conditions armin decreased the EPP quantum content and the MEPP frequency. GD-42 and GD-7 decreased the EPP quantum content as well but increased MEPP frequency. These variations of parameters were not related to inhibition of ChE activity and were reversible by washout.

Measurement of intracellular pH of bullfrog skeletal muscle and renal tubular cells with double-barreled antimony microelectrodes.

A pencil-type antimony microelectrode of double-barreled design with a tip of less than 1 to 2 microns in outside diameter was constructed and used to measure intracellular pH(pHi) on frog sartorius muscle and renal tubular cells. Simultaneous observations of membrane potential difference (EM) were made. The results obtained were as follows: (1) The in vivo pHi of frog sartorius muscle was 7.12 +/- 0.07 (SD) (n = 144); the simultaneously measured EM was -51.1 +/- 7.9 mV. The in vivo pHi of frog proximal tubule was 7.49 +/- 0.07 (n = 221) and the EM Peri across the peritubular membrane was -50.2 +/- 9.0 mV. (2) In proximal tubule in vivo, there was a negative correlation between pHi and EM (r = -.62, p < .05). On the other hand, in sartorius muscle in vivo, a positive correlation between the two was found (r = .85, p < .001). (3) In in vitro sartorius muscle, the pHi was 7.03 +/- 0.14 (n = 9) and EM was -62.4 +/- 4.4 mV within one hour after isolation. (4) Increasing the external potassium concentration in the preparations to 75 mM caused a progressive depolarization by 43.3 +/- 15.9 (m = 4) mV, while pHi changed in the alkaline direction by 0.22 +/- 0.03 pH unit. (5) These results indicate that the pHi in both tissues does not obey the Donnan rule.

Lifetime and conductance of acetylcholine-activated channels in normal and denervated toad sartorius muscle.

1. The average lifetime and conductance of acetylcholine-activated channels were measured in normal and denervated, voltage-clamped toad sartorius muscle fibres at 10 degrees C. 2. The null potential was -4 +/- 1 mV for subsynaptic channels in normal fibres and -6 +/- 3 mV for extrasynaptic channels in denervated fibres. 3. There was a linear relationship between variance of conductance fluctuations and mean conductance for acetylcholine-induced currents up to 50 nA, in denervated fibres clamped at -50 mV. The ratio gave a channel conductance of 14 pS. 4. At the same membrane potential, the average lifetime of extrasynaptic channels in denervated fibres was approximately double, whereas channel conductance was approximately half, that of subsynaptic channels in normal fibres: there was little difference in net charge transfer through the two types of channel under similar conditions. 5. Single channel conductance increased, whereas average channel lifetime decreased, as the membrane potential became more positive (depolarized). The effect of potential on channel lifetime and conductance was more pronounced in denervated than in normal fibres.