Differences in conductance of M2 proton channels of two influenza viruses at low and high pH.

The M2 protein of influenza A viruses forms a proton channel involved in modifying virion and trans Golgi pH during infection. Previous studies of the proton current using whole-cell patch clamp of mouse erythroleukaemia (MEL) cells expressing the M2 protein of the "Weybridge" strain provided evidence for two protonation sites, one involved in permeation, the other in activation by acid pH. The present report compares the M2 channels of two different strains of influenza virus, "Weybridge" (WM2) and "Rostock" (RM2). Whereas with external acid pH the current-voltage relations showed similar small degrees of inward rectification, a similar apparent K(d) of approximately 10 microM for proton permeation and a high selectivity for protons over Na(+), the two M2 proteins differed in whole-cell conductance at low and high pH. The proton conductance of unit membrane area was on average 7-fold greater in RM2- than WM2-expressing MEL cells. At high external pH WM2 was shown previously to have small conductance for outward current at positive driving potential. In contrast, RM2 shows high conductance for outward current with high external pH, but shows small conductance for inward current with high internal pH, conditions in which WM2 shows high conductance for inward current. The different properties of the conductances due to the two channels at high pH were determined by three amino acids in their transmembrane domains. All intermediate mutants possessed one or other property and transformation of the WM2 phenotype into that of RM2 required substitution in all three residues V27I, F38L and D44N; single substitutions in RM2 effected the opposite phenotypic change. The significance of this difference for virus replication is not clear and it may be that the higher proton flux associated with RM2 is the main factor determining its increased ability to dissipate pH gradients. It is apparent, however, from the specific differences in the sidedness of the pH-induced changes in voltage dependence of the whole-cell current that this is an intrinsic property of the virus proton channel which may have parallels with regulation of other proton channels.

Selective proton permeability and pH regulation of the influenza virus M2 channel expressed in mouse erythroleukaemia cells.

1. The M2 protein of influenza A virus is implicated in transmembrane pH regulation during infection. Whole-cell patch clamp of mouse erythroleukaemia cells expressing the M2 protein in the surface membrane showed a conductance due to M2 which was specifically blocked by the anti-influenza drug rimantadine. 2. The ion selectivity of the rimantadine-sensitive current through M2 was determined. Reversal potentials were close to equilibrium potentials for transmembrane pH gradients and not to those for Na+, K+ or Cl- concentration gradients. M2 permeability to Na+ relative to H+ was estimated to be less than 6 x 10(-7). 3. The M2 conductance increased as external pH decreased below 8.5 and approached saturation at an external pH of 4, effects attributable to increased permeability due to increased driving potential and to activation by low external pH. Both activation and permeation could be described by interaction of protons with sites on M2, with apparent dissociation constants of approximately 0.1 microM and 1 microM, respectively, under physiological conditions. 4. The M2 protein can transfer protons selectively across membranes with the H+ electrochemical gradient, properties consistent with its role in modifying virion and trans-Golgi pH during virus infection.

Kinetics of cytosolic Ca2+ concentration after photolytic release of 1-D-myo-inositol 1,4-bisphosphate 5-phosphorothioate from a caged derivative in guinea pig hepatocytes.

The influence of 1-D-myo-inositol 1,4,5-trisphosphate (InsP3) breakdown by InsP3 5-phosphatase in determining the time course of Ca2+ release from intracellular stores was investigated with flash photolytic release of a stable InsP3 derivative, 5-thio-InsP3, from a photolabile caged precursor. The potency and Ca(2+)-releasing properties of the biologically active D isomers of 5-thio-InsP3 and InsP3 itself were compared by photolytic release in guinea pig hepatocytes. After a light flash, cytosolic free calcium concentration ([Ca2+]i) showed an initial delay before rising quickly to a peak and declining more slowly to resting levels, with time course and amplitude generally similar to those seen with photolytic release of InsP3. Differences were a three- to eightfold lower potency of 5-thio-InsP3 in producing Ca2+ release, much longer delays between photolytic release and Ca2+ efflux with low concentrations of 5-thio-InsP3 than with InsP3, and persistent reactivation of Ca2+ release, producing periodic fluctuations of cytosolic [Ca2+]i with high concentrations of 5-thio-InsP3 but not InsP3 itself. The lower potency of 5-thio-InsP3 may be a result of a lower affinity for closed receptor/channels or a lower open probability of liganded receptor/channels. The longer delays with 5-thio-InsP3 at low concentration suggest that metabolism of InsP3 by 5-phosphatase may reduce the concentration sufficiently to prevent receptor activation and may have a similar effect on InsP3 concentration during hormonal activation. The maximal rate of rise of [Ca2+]i, the duration of the period of high Ca2+ efflux, and the initial decline of [Ca2+]i are similar with5-thio-lnsP3 and lnsP3, indicating that lnsP3 breakdown is not important in terminating Ca2+ release. The second activation ofInsP3 receptors with 5-thio-lnsP3 and particularly the sustained periodic fluctuations of [Ca2+]i indicate persistence of 5-thio-lnsP3,suggesting that InsP3 breakdown prevents reactivation of InsP3 receptors. The photochemical properties of 1-(2-nitrophenyl)-ethyl caged 5-thio-lnsP3 are photolytic quantum yield = 0.57 (cf. 0.65 for caged InsP3) and rate of photolysis = 87 s-I (half-life approximately 8 ms; cf. 3 ms for caged lnsP3; pH7.1; ionic strength, 0.2 M; 21 OC). Caged 5-thio-lnsP3 at concentrations up to 360 pM did not activate lnsP3 receptors to produce Ca2+ release or block Ca2+ release by free 5-thio-lnsP3.

Mechanisms of intracellular calcium release during hormone and neurotransmitter action investigated with flash photolysis.

To understand the complex time course of cytosolic Ca2+ signalling evoked by hormones and neurotransmitters, it is necessary to know the kinetics of steps in the second-messenger cascade, particularly cooperative and inhibitory interactions between components that might give rise to periodic fluctuations. In the case of inositol trisphosphate (InsP3)-evoked Ca2+ release, fast perfusion studies with subcellular fractions or permeabilised cells can be made if sufficient homogeneous tissue is available. Single-cell studies can be made by combining whole-cell patch-clamp techniques and microspectrofluorimetry with flash photolytic release of InsP3 to give quantitative, time-resolved data of Ca2+ release from stores. A technical description is given here of flash photolysis of caged InsP3, and the results of fast perfusion and flash photolytic experiments are reviewed. Studies of kinetics of Ca2+ release have shown that the InsP3 receptor/channel is regulated first by positive and then by negative feedback by free cytosolic Ca2+ concentration, producing a pulse of Ca2+ release having properties that may be important in the spatial propagation of Ca2+ signals within and between cells. The properties of InsP3-evoked Ca2+ release in single cells differ between peripheral tissues, such as the liver, and Purkinje neurones of the cerebellum. Purkinje neurones need 20-50 times higher InsP3 concentrations and release Ca2+ to change the free cytosolic concentration 30 times faster and to higher peak concentrations than in liver. The InsP3 receptors in the two cell types appear to differ in apparent affinity, and the greater Ca2+ efflux from stores in Purkinje cells is probably due to a high receptor density.

Postsynaptic activation at the squid giant synapse by photolytic release of L-glutamate from a 'caged' L-glutamate.

1. Pharmacological evidence suggests L-glutamate is a strong candidate as a transmitter at the giant synapse of the squid. Postsynaptic activation at the giant synapse cannot be effected by conventional application of putative neurotransmitters by iontophoresis or perfusion, apparently because the complex structure of the synapse prevents a sufficiently rapid change in concentration at the postsynaptic membrane. Flash photolytic release of L-glutamate from a pharmacologically inert 'caged' L-glutamate pre-equilibrated in the stellate ganglion of Alloteuthis or Loligo was used to determine whether L-glutamate can produce postsynaptic activation when released rapidly in the synaptic clefts. 2. The preparation, reaction mechanism and properties of the caged L-glutamate, N-1-(2-nitrophenyl)ethoxycarbonyl-L-glutamate, are described. The product quantum yield on photolysis was 0.65 (+/- 0.05). On flash photolysis glutamate release followed a single exponential time-course in the pH range 5.5-7.8. The rate constant was proportional to [H+] and was 93 s-1 at pH 5.5 and 16 degrees C in artificial sea water (ionic strength, I = 0.68 M). 3. At pH 7.8 flash photolysis of caged glutamate pre-equilibrated in the synapse caused only a slow depolarization. A second photolytic release of L-glutamate or transsynaptic activation produced no further depolarization, suggesting desensitization and inactivation of postsynaptic mechanisms by the initial pulse of L-glutamate. 4. Synaptic transmission in the giant synapse was normal at pH 5.5. Flash photolysis at pH 5.5 caused rapid production of L-glutamate within the synaptic cleft and a fast postsynaptic depolarization which generated postsynaptic action potentials.(ABSTRACT TRUNCATED AT 250 WORDS)

The actions of suxamethonium (succinyldicholine) as an agonist and channel blocker at the nicotinic receptor of frog muscle.

1. Patch clamp methods were used to study the equilibrium and kinetic properties of the acetylcholine analogue, succinyldicholine (suxamethonium), which is used clinically as a neuromuscular blocking agent. 2. The equilibrium concentration-response curve, corrected for desensitization was estimated by measuring as the response the probability of being open of single ion channels during clusters of activity that occur between long desensitized periods. Suxamethonium (Sux) was about 7.6-fold less potent than acetylcholine (ACh) (at low concentrations), partly because of 2.9-fold lower affinity for the resting receptor, and partly because of a lower ability to activate the receptor once bound. 3. Sux was a more potent blocker of the open ion channel than ACh (equilibrium constant about 200 microM); this limited the maximum open probability to about 0.36 (at 12 degrees C and -120 mV). Individual channel blockages lasted about 65 microseconds on average. They appeared to get longer at high agonist concentration; however, a simulation method was used to show that this effect could be accounted for by the fact that at higher concentrations there are more openings that are too brief to be detected. Over the concentration range tested the effects were described by a simple open channel block mechanism. 4. No component of brief shut times could be detected other than those resulting from channel blockages. However, the results suggest that multiple channel openings (the nachschlag phenomenon) should be rare, so this is not inconsistent with previous results with other agonists. 5. Sux differed from ACh and carbachol in that it had a somewhat lower efficacy and a greater channel blocking action. However, in clinical practice channel block is unlikely to contribute to neuromuscular block to any significant extent; the main mechanism of paralysis, at least in the early stages, is probably a result of prolonged depolarization of the region of membrane surrounding the motor endplate leading to inactivation of the sodium channels therein.

Kinetics of the conductance evoked by noradrenaline, inositol trisphosphate or Ca2+ in guinea-pig isolated hepatocytes.

1. Guinea-pig hepatocytes respond to noradrenaline (NA, 5-10 microM) with a large membrane conductance increase to K+ and Cl-. The response has a long initial delay (range 2-30 s). Following the delay, the K+ conductance (studied in Cl(-)-free solutions) rises quickly to a peak in 1-2 s and is maintained in the continued presence of NA, though often with superimposed oscillations of conductance. The roles of intracellular Ca2+ and D-myo-inositol 1,4,5-trisphosphate (InsP3) in this complex response have been investigated by rapid photolytic release of intracellular Ca2+ (from Nitr5-Ca2+ buffers) or InsP3 from 'caged' InsP3. 2. A rapid increase of intracellular [Ca2+] produced an immediate membrane conductance increase which rose approximately exponentially to a new steady level, consistent with a direct activation of Ca2(+)-dependent ion channels. 3. Following a pulse of InsP3, conductance rose after a brief delay (range 70-1500 ms) which was shortest at high [InsP3] or if the initial cytosolic [Ca2+] had been raised above normal levels. The maximum conductance produced by InsP3 was similar in each cell to the peak recorded with NA and could be evoked by InsP3 concentrations of 0.5-1 microM. 4. The rates of rise of conductance increased with InsP3 concentration in the range 0.25-12.5 microM (range 10-90%, rise times 90-1000 ms), indicating that InsP3-evoked Ca2(+)-efflux from stores increases with InsP3 concentration in this range. 5. Photochemically released InsP3 and Ca2+ activate at physiological concentrations the same membrane conductances as NA. The results indicate that the long initial delay in NA action occurs prior to or during generation of InsP3. The mechanism of the delay and the subsequent apparently all-or-none conductance increase during NA action are discussed in terms of the high co-operativity in InsP3 and Ca2+ actions and an additional positive feedback step. 6. Evidence was found of a negative interaction between [Ca2+] and InsP3-evoked Ca2+ release. The time course of the recovery of InsP3-evoked Ca2+ release following a rise of cytosolic [Ca2+] suggests that this interaction may be important in regulating oscillatory responses of [Ca2+] during hormonal stimulation of guinea-pig hepatocytes.

Properties of membrane ion conductances evoked by hormonal stimulation of guinea-pig and rabbit isolated hepatocytes.

Membrane conductance changes evoked in isolated guinea-pig or rabbit hepatocytes by hormonal stimulation were studied with the whole-cell patch clamp technique. In Cl-containing solutions, noradrenaline (NA), ATP or angiotensin II (AII) evoked an increase of conductance to both K (GK) and Cl (GCl) ions. Activation of GK occurred after a delay of several seconds and was sustained in the presence of hormone. Activation of GCl was transient, lasting several seconds, and arose either at the same time or shortly after the increase in GK. Conductances showed an initial rapid rise and slow oscillatory changes during maintained hormone application. The NA-induced current reversed at -19 mV in Cl solutions, between the equilibrium potentials for chloride (ECl = 0 mV) and potassium ions (EK = -85 mV), and at -75 mV, near EK, in Cl-free solution. In both conditions whole-cell current-voltage curves were linear in the range -100 mV to +40 mV. The conductance increase produced by NA to Cl- ions was about 50 nS, that to K+ ions was 6 nS. The potassium conductance increase was abolished by the polypeptide toxin apamin (50 nM). An increase in membrane current noise was associated with NA-evoked outward K+ current and blocked by apamin. Spectral analysis gave estimates of the elementary K channel conductance of 1.7 pS. Power spectra were fitted by two Lorentzian components, with average half-power frequencies of 2 and 190 Hz. These results are discussed in relation to the single-channel properties and indicate that the open probability of K channels during the NA response is high. In Cl solutions, with apamin to block the K conductance, no increase in current noise was detected during the large Cl conductance evoked by NA. This suggests either that Cl channels are of very low unitary conductance (less than 1 pS) or that Cl transport is due to a membrane carrier. The complex time-course of hormonally evoked conductances is not due to the properties of ion conductances per se but probably to underlying changes of intracellular second-messenger concentration.

The properties of calcium-activated potassium ion channels in guinea-pig isolated hepatocytes.

1. Unitary currents due to calcium-activated potassium ion channels were studied in inside-out or outside-out excised membrane patches from guinea-pig hepatocytes. 2. Potassium ion channels were identified which were activated by internal calcium ions and blocked by external apamin (50 nM) or (+)-tubocurarine (10 microM). These properties are characteristic of the whole-cell potassium conductance increase evoked in guinea-pig hepatocytes by hormonal stimulation. 3. The single-channel conductance was 20 pS in inside-out or outside-out patches with external and internal K+ ion concentrations of 150 and 135 mM respectively and gluconate anion. Reducing external K+ concentration to 5 mM reduced the unitary conductance for outward current to 6 pS. 4. The calcium sensitivity was investigated with buffered internal Ca2+ ion concentrations in the range 0.3-2.2 microM. Tubocurarine-sensitive channels had an open probability of less than 0.05 at 0.3 microM-internal Ca2+. This increased steeply to a maximum of 0.85 at concentrations of 1.1 microM-Ca2+ or higher. 5. In patches with a single channel active, analysis of open and closed intervals showed that openings occurred in bursts. The increase of open probability at high internal Ca2+ concentration was associated with prolonged bursts of channel opening. 6. Comparison of these results with data from whole-cell conductance changes and with published levels of intracellular Ca2+ ion concentration (Woods, Cuthbertson & Cobbold, 1987) suggests that a large proportion, more than 40%, of potassium ion channels in guinea-pig hepatocytes are activated by hormonal stimulation.

Noise and single channels activated by excitatory amino acids in rat cerebellar granule neurones.

1. Glutamate-receptor ion channels in rat cerebellar granule cells maintained in explant cultures have been investigated with patch-clamp methods. Properties of these channels were determined from noise analysis of whole-cell currents and from noise and single-channel currents recorded in outside-out membrane patches. 2. Glutamate (10-20 microM) evoked two types of response. Some granule cells gave small inward currents accompanied by clear increases in current noise ('large noise' responses), whereas other cells gave larger inward currents and small noise increases ('small noise' responses). 3. A mean single-channel conductance (gamma) of 46.6 pS was estimated for glutamate from four 'large noise' cells. A mean gamma value of 8.4 pS was estimated for seven other 'large noise' cells. The results suggest that in these latter cells glutamate activated both large (approximately equal to 50 pS) and small conductance (approximately equal to 140 fS) channels. 4. Applications of aspartate (10-30 microM) or N-methyl-D-aspartate (NMDA, 10-30 microM) produced small inward currents and large increases in noise; gamma noise = 48.5 pS (aspartate) and 46.7 pS (NMDA). 5. Large single-channel currents were evoked by glutamate, aspartate and NMDA in outside-out patches. The mean conductance values obtained for the largest amplitude openings were: gamma(glutamate) = 49.5 pS, gamma(aspartate) = 51.5 pS, and gamma(NMDA) = 53.0 pS. For each agonist, these 50 pS openings comprised 75-85% of the completely resolved currents in each patch. Openings to 40 and 30 pS conductance levels accounted for 10-15% and 3-7% of the total, and the presence of apparently direct transitions between these levels and the 50 pS level suggests they are sublevels of the same multi-conductance channels. 6. A mean channel conductance of 22.9 pS was estimated from noise evoked by quisqualate (10-30 microM). Single-channel currents were examined in four patches. In two, quisqualate evoked predominantly small currents of two amplitudes, gamma = 8.4 pS and 16.5 pS; some 50 pS openings were also present. In the other two patches, most openings were 50 pS events. 7. Granule cells gave inward currents to kainate (10-30 microM), and a mean conductance of 3.1 pS was estimated from kainate noise. In patches in which aspartate or NMDA produced mainly 50 pS openings, more than 74% of the single-channel currents evoked by kainate were of smaller amplitude, with mean conductances of gamma = 8.1 and 15.1 pS.(ABSTRACT TRUNCATED AT 400 WORDS)

Activation of ion channels in the frog end-plate by high concentrations of acetylcholine.

1. The equilibrium relationship between acetylcholine (ACh) concentration and response (fraction of channels open), corrected for the effects of desensitization, has been estimated by single-ion-channel recording at the adult frog skeletal neuromuscular junction. At high ACh concentration channel openings occur in well-defined clusters separated by long desensitized intervals. The response, po, was estimated as the proportion of time for which a single channel was open during a cluster. 2. At negative membrane potential (-120 mV) po reached a maximum value of 0.9 at 100 microM-ACh and was half-maximum at 15 microM with a Hill slope of 1.6 at this point. At concentrations higher than 200 microM-ACh, po declined as a result of open-channel block by free ACh itself. 3. At positive membrane potentials (+100 mV) there was little channel block by ACh; po reached a maximum value of 0.41 at 500 microM-ACh, with half-maximum activation at 50 microM and Hill slope of 1.2 at this point. 4. Particular mechanisms for channel activation by ACh were fitted to the data by the method of least squares. Fits were fully determinate only if the two binding sites for ACh were assumed to be equivalent with no co-operativity in the ACh binding reactions. At negative potential the microscopic equilibrium constant for binding was K1 = K2 = 77 microM and the equilibrium constant for channel opening (opening/closing rates, beta/alpha) was 32. At positive potential the affinity was slightly higher, K = 32 microM, which confirms the view that the binding sites for ACh are outside the membrane electric field. The equilibrium constant for channel opening was reduced to 0.7 mainly as a result of the much shorter open lifetime (increased closing rate alpha) at positive potentials. 5. The data were also fitted well by very high values of beta/alpha together with a high degree of negative co-operativity or non-equivalence in ACh binding affinity (K2 much greater than K1). A good fit could also be obtained with moderate positive co-operativity combined with non-equivalence of the binding sites. 6. A mechanism that postulates a receptor with two independent gating subunits provided a poor fit to the data at negative potential. 7. The rate constants for channel opening and ACh dissociation were estimated by constraining the fitted parameters so that the burst length for channel opening was equal to its observed value at low concentrations of ACh.(ABSTRACT TRUNCATED AT 400 WORDS)

Internal perfusion of guinea-pig hepatocytes with buffered Ca2+ or inositol 1,4,5-trisphosphate mimics noradrenaline activation of K+ and Cl- conductances.

External application of noradrenaline to voltage-clamped guinea-pig isolated hepatocytes evoked membrane conductance increases to K+ and Cl-. This effect was reproduced by internal perfusion of the cells with 2 microM buffered Ca2+ and with 20 microM inositol 1,4,5-trisphosphate (IP3). The kinetic properties of the K+ conductance and its selective block by the toxin apamin were the same in each case. Cyclical fluctuations of conductance observed with noradrenaline were reproduced by internal IP3 but not by Ca2+ perfusion, indicating that oscillations of intracellular free Ca2+ may arise from properties of the Ca2+ sequestration mechanism at constant IP3 concentration.

Ion channel block by acetylcholine, carbachol and suberyldicholine at the frog neuromuscular junction.

Three nicotinic agonists, suberyldicholine, acetylcholine and carbachol, have been investigated by single channel recording at the endplates of adult frog muscle fibres. All three agonists can block the channels that they open. Suberyldicholine is the most potent blocker; it has an equilibrium constant for binding to the open channel of about 6 microM and blockages last for about 5 ms on average, at -105 mV. A plot of the mean number of blockages per unit open time against concentration ('blockage frequency plot') suggests that suberyldicholine does not produce long-lived blocked states such as might occur, for example, if it could be trapped within a shut channel. The characteristics of the 'blockage frequency plot' are analysed in Appendix 2. Block by acetylcholine and carbachol has much lower affinity (the equilibrium constants being a few millimolar for both), and blockages are much briefer, so that blockage appears to produce noisy single channel currents of reduced amplitude. A method based on the spectral density of the excess 'open' channel noise has been used to investigate the rate of blocking and unblocking. The basis of this method is discussed in Appendix 1. It is estimated that the mean duration of a blockage is about 18 microseconds for acetylcholine and 9 microseconds for carbachol.

Ion channels activated by L-glutamate and GABA in cultured cerebellar neurons of the rat.

Glutamate and GABA-receptor channels were investigated in explants of rat cerebellum grown in cell culture. The patch-clamp technique was used to examine neurons under whole cell clamp and the properties of channels were derived by analysis of glutamate and GABA-evoked current noise. In addition, single channel currents activated by glutamate were recorded from isolated outside-out patches of membrane. We found evidence for at least two types of glutamate receptor-channels in cerebellar cells. Some neurons exhibited a channel of 50 pS conductance with a Lorentzian noise spectrum of 5.9 ms time constant. Single channels were readily resolved both in whole cell clamp and excised patches. Other neurons possessed low conductance channels which produced two component spectra. Estimates of the single channel conductance gave a value of about 140 fS. GABA channel noise obtained from these cells was also fitted by two component spectra which gave single channel conductance of 16 pS.

Conductances of single ion channels opened by nicotinic agonists are indistinguishable.

Hypotheses concerning the mechanism by which acetylcholine-like agonists cause ion channels to open often suppose that the receptor-ionophore complex can exist in either of two discrete conformations, open and shut. On the basis of noise analysis it has been reported that certain agonists open ion channels of lower conductance than usual, though many potent agonists give similar conductances, and hence that differences in the conductance of ion channels opened by different agonists may contribute to differences in efficacy. Here we have reinvestigated this question by recording single ion channel currents evoked by acetylcholine-like agonists on embryonic rat muscle in tissue culture and on adult frog muscle endplate. Ten different agonists (Fig. 1) were tested, including several that noise analysis has suggested have a low conductance. The single-channel conductance was found to be the same, within a few per cent, for all 10 agonists. It seems that noise analysis has given erroneously low conductances in some cases. Therefore efficacy differences do not depend on differences in single-channel conductance evoked by various agonists but presumably on the position of the open-shunt equilibrium of the agonist-channel complexes.

Kinetics of acetylcholine activated ion channels in chick ciliary ganglion neurones grown in tissue culture.

The acetylcholine activated conductance of chick ciliary ganglion neurones grown in tissue culture was studied by the patch clamp method. Single channel currents at 30 degrees C had a conductance of 38-42 pS, a reversal potential near + 10 mV and an average open lifetime of 1.08 ms (range 0.74 - 1.54 ms) at the resting potential. The presence of a single component in the distributions of amplitudes and open lifetimes, and also in the noise spectrum of voltage clamp currents, suggests that acetylcholine channels have uniform characteristics in these cells. Evidence of a desensitised state of the receptor was obtained from the distribution of gap intervals and the decline of voltage clamp current. These properties are similar to those of acetylcholine channels at the vertebrate neuromuscular junction. However, two important differences were found. (a) The acetylcholine concentrations used here were 10-25 times higher than those required to produce a similar degree of channel activation at the endplate. (b) When the membrane was hyperpolarised the mean open lifetime of the channel showed no change or a slight reduction.

The efficacy of agonists at the frog neuromuscular junction studied with single channel recording.

The efficacy of acetylcholine, carbachol and suberyldicholine at the frog neuromuscular junction was estimated by single channel recording. The probability of channel opening at high concentration was found to be greater than 0.9 for each, thus showing (a) that their efficacy is high and (b) that the opening rate for the fully-liganded channel is higher than previously thought.

Block of acetylcholine-activated ion channels by an uncharged local anaesthetic.

It is now thought that amine local anaesthetic compounds (procaine, lignocaine and related molecules) depress electrical activity in nerve and muscle cells by binding to sites within ion channels and blocking current flow. Such mechanisms have been proposed to account for the effects of these local anaesthetics on both the voltage-dependent sodium current and the postsynaptic actylcholine (ACh)-activated ionic current. Recently, strong evidence for block of ion channels by cationic drug molecules has been obtained by recording current from single ACh-activated channels in the presence of permanently charged quaternary derivatives of lignocaine. Most amine local anaesthetic compounds are, however, weak bases, present in both charged and uncharged forms at physiological pH, and some question remains as to whether a charged group is essential for blockade of ion channels. To resolve this question, we studied the action of the uncharged local anaesthetic benzocaine (ethyl-4-aminobenzoate) on postsynaptic ACh-activated endplate current and extrajunctional single channel current of frog muscle. We report here evidence that strongly suggests that benzocaine blocks ACh-activated ion channels.