Search

Key Points: Fast sarcomere-level mechanics in contracting intact fibres from frog skeletal muscle reveal an I-band spring with an undamped stiffness 100 times larger than the known static stiffness. This undamped stiffness remains constant in the range of sarcomere length 2.7-3.1 µm, showing the ability of the I-band spring to adapt its length to the width of the I-band. The stiffness and tunability of the I-band spring implicate titin as a force contributor that, during contraction, allows weaker half-sarcomeres to equilibrate with in-series stronger half-sarcomeres, preventing the development of sarcomere length inhomogeneity. This work opens new possibilities for the detailed in situ description of the structural-functional basis of muscle dysfunctions related to mutations or site-directed mutagenesis in titin that alter the I-band stiffness., Abstract: Force and shortening in the muscle sarcomere are due to myosin motors from thick filaments pulling nearby actin filaments toward the sarcomere centre. Thousands of serially linked sarcomeres in muscle make the shortening (and the shortening speed) macroscopic, while the intrinsic instability of in-series force generators is likely prevented by the cytoskeletal protein titin that connects the thick filament with the sarcomere end, working as an I-band spring that accounts for the rise of passive force with sarcomere length (SL). However, current estimates of titin stiffness, deduced from the passive force-SL relation and single molecule mechanics, are much smaller than what is required to avoid the development of large inhomogeneities among sarcomeres. In this work, using 4 kHz stiffness measurements on a population of sarcomeres selected along an intact fibre isolated from frog skeletal muscle contracting at different SLs (temperature 4°C), we measure the undamped stiffness of an I-band spring that at SL > 2.7 µm attains a maximum constant value of ∼6 pN nm -1 per half-thick filament, two orders of magnitude larger than expected from titin-related passive force. We conclude that a titin-like dynamic spring in the I-band, made by an undamped elastic element in-series with damped elastic elements, adapts its length to the SL with kinetics that provide force balancing among serially linked sarcomeres during contraction. In this way, the I-band spring plays a fundamental role in preventing the development of SL inhomogeneity., (© 2019 The Authors. The Journal of Physiology © 2019 The Physiological Society.)

In eukaryotic cells, a rise in cytoplasmic Ca(2+) can activate a plethora of responses that operate on time scales ranging from milliseconds to days. Inherent to the use of a promiscuous signal like Ca(2+) is the problem of specificity: how can Ca(2+) activate some responses but not others? We now know that the spatial profile of the Ca(2+) signal is important Ca(2+) does not simply rise uniformly throughout the cytoplasm upon stimulation but can reach very high levels locally, creating spatial gradients. The most fundamental local Ca(2+) signal is the Ca(2+) microdomain that develops rapidly near open plasmalemmal Ca(2+) channels like voltage-gated L-type (Cav1.2) and store-operated CRAC channels. Recent work has revealed that Ca(2+) microdomains arising from these channels are remarkably versatile in triggering a range of responses that differ enormously in both temporal and spatial profile. Here, I delineate basic features of Ca(2+) microdomains and then describe how these highly local signals are used by Ca(2+)-permeable channels to drive cellular responses.

There is evidence that reactive oxygen species (ROS) are produced and released during neuromuscular activity, but their role in synaptic transmission is not known. Using a two-electrode voltage-clamp technique, at frog neuromuscular junctions, the action H2O2 on end-plate currents (EPC) was studied to determine the targets for this membrane-permeable ROS. In curarized or cut muscles, micromolar concentrations of H2O2 increased the amplitude of EPCs. Higher (> 30 microM) doses inhibited EPCs and prolonged current decay. These effects were presynaptic since H2O2 did not change the amplitude or duration of miniature EPCs (although it reduced the rate of spontaneous release at high concentrations). Quantal analysis and deconvolution methods showed that facilitation of EPCs was due to increased quantal release, while depression was accompanied by temporal dispersion of evoked release. Extracellular recordings revealed prolonged presynaptic Ca2+ entry in the presence of high H2O2. Both low and high H2O2 increased presynaptic potentiation during high-frequency stimulation. Pro-oxidant Fe2+ did not affect facilitation by low doses of H2O2 but augmented the inhibition of EPCs by high H2O2, indicating involvement of hydroxyl radicals. High Mg2+ and the ROS scavenger N-acetylcysteine eliminated both the facilitatory and depressant effects of H2O2. The facilitatory effect of H2O2 was prevented by protein kinase C (PKC) inhibitors and 4beta-phorbol 12-myristate, 13-acetate (PMA), an activator of PKC. PKC inhibitors but not PMA also abolished the depressant effect of H2O2. Our data suggest complex presynaptic actions of H2O2, which could serve as a fast feedback modulator of intense neuromuscular transmission.

This study examined the relationship between the level of Ca2+ loading in the sarcoplasmic reticulum (SR) and the amount of Ca2+ released by an action potential (AP) in fast-twitch skeletal muscle fibres of the rat. Single muscle fibres were mechanically skinned and electric field stimulation was used to induce an AP in the transverse-tubular system and a resulting twitch response. Responses were elicited in the presence of known amounts (0-0.38 mM) of BAPTA, a fast Ca2+ buffer, with the SR Ca2+ pump either functional or blocked by 50 microM 2,5-di-tert-butyl-1,4-hydroquinone (TBQ). When Ca2+ reuptake was blocked, an estimate of the amount of Ca2+ released by an AP could be derived from the size of the force response. In a fibre with the SR loaded with Ca2+ at the endogenous level (approximately 1.2 mM, expressed as total Ca2+ per litre fibre volume; approximately one-third of maximal loading), a single AP triggered the release of approximately 230 microM Ca2+. If a second AP was elicited 10 ms after the first, only a further approximately 60 microM Ca2+ was released, the reduction probably being due to Ca2+ inactivation of Ca2+ release. When Ca2+ reuptake was blocked, APs applied 15 s apart elicited similar amounts of Ca2+ release (approximately 230 microM) on the first two or three occasions and then progressively less Ca2+ was released until the SR was fully depleted after a total of approximately eight APs. When the SR was loaded to near-maximal capacity (approximately 3-4 mM), each AP (or pair of APs 10 ms apart) still only released approximately the same amount of Ca2+ as that released when the fibre was endogenously loaded. Consistent with this, successive APs (15 s apart) elicited similar amounts of Ca2+ release approximately 10-16 times before the amount released declined, and the SR was fully depleted of Ca2+ after a total release calculated to be approximately 3-4 mM. When the SR was loaded maximally, increasing the [BAPTA] above 280 microM resulted in an increase in the amount of Ca2+ released per AP, probably because the greater level of cytoplasmic Ca2+ buffering prevented Ca2+ inactivation from adequately limiting Ca2+ release. These results show that the amount of Ca2+ released by AP stimulation in rat fast-twitch fibres normally stays virtually constant over a wide range of SR Ca2+ content, in spite of the likely large change in the electrochemical gradient for Ca2+. This was also found to be the case in toad twitch fibres. This constancy in Ca2+ release should help ensure precise regulation of force production in fast-twitch muscle in a range of circumstances.

The beta(1a) subunit, one of the auxiliary subunits of Ca(V)1.1 channels, was expressed in COS-1 cells, purified by electroelution and electrodialysis techniques and identified by Western blot using monoclonal antibodies. The purified beta(1a) subunit strongly interacted in vitro with the alpha interaction domain (AID) of Ca(V)1.1 channels. The actions of the purified beta(1a) subunit on Ca(V)1.1 channel currents were assessed in whole cell voltage clamp experiments performed in vesicles derived from frog and mouse adult skeletal muscle plasma membranes. L-type inward currents were recorded in solutions containing Ba(2+) (I(Ba)). Values of peak I(Ba) were doubled by the beta(1a) subunit in frog and mouse muscle vesicles and the amplitude of the slow component of tail currents was greatly increased. The actions of the beta(1a) subunit on Ca(V)1.1 channel currents reached a steady state within 20 min. The beta(1a) subunit had no effect on the time courses of activation or inactivation of I(Ba) or shifted the current-voltage relation. Non-linear capacitive currents were recorded in solutions that contained mostly impermeant ions. Charge movement depended on voltage with average Boltzmann parameters: Q(max) = 28.0 +/- 6.6 nC microF(-1), V = -58.0 +/- 2.0 mV and k = 15.3 +/- 1.1 mV (n = 24). In the presence of the beta(1a) subunit, these parameters remained unchanged: Q(max) = 29.8 +/- 3.5 nC microF(-1), V = -54.5 +/- 2.2 mV and k = 16.4 +/- 1.3 mV (n = 21). Overall, the work describes a novel preparation to explore in situ the role of the beta(1a) subunit on the function of adult Ca(V)1.1 channels.

Our previous mathematical model of solute-coupled water transport through the intestinal epithelium is extended for dealing with electrolytes rather than electroneutral solutes. A 3Na+-2K+ pump in the lateral membranes provides the energy-requiring step for driving transjunctional and translateral flows of water across the epithelium with recirculation of the diffusible ions maintained by a 1Na+-1K+-2Cl- cotransporter in the plasma membrane facing the serosal compartment. With intracellular non-diffusible anions and compliant plasma membranes, the model describes the dependence on membrane permeabilities and pump constants of fluxes of water and electrolytes, volumes and ion concentrations of cell and lateral intercellular space (lis), and membrane potentials and conductances. Simulating physiological bioelectrical features together with cellular and paracellular fluxes of the sodium ion, computations predict that the concentration differences between lis and bathing solutions are small for all three ions. Nevertheless, the diffusion fluxes of the ions out of lis significantly exceed their mass transports. It is concluded that isotonic transport requires recirculation of all three ions. The computed sodium recirculation flux that is required for isotonic transport corresponds to that estimated in experiments on toad small intestine. This result is shown to be robust and independent of whether the apical entrance mechanism for the sodium ion is a channel, a SGLT1 transporter driving inward uphill water flux, or an electroneutral Na+-K+-2Cl- cotransporter.

1. Ca2+ transients and Ca2+ release flux were determined optically in cut skeletal muscle fibres under voltage clamp. 'Decay' of release during a depolarizing pulse was defined as the difference between the peak value of release and the much lower steady level reached after about 100 ms of depolarization. Using a double-pulse protocol, the inactivating effect of release was measured by 'suppression', the difference between the peak values of release in the test pulse, in the absence and presence of a conditioning pulse that closely preceded the test pulse. 2. The relationship between decay and suppression was found to follow two simple arithmetic rules. Whenever the conditioning depolarization was less than or equal to the test depolarization, decay in the conditioning release was approximately equal to suppression of the test release. Whenever the conditioning depolarization was greater than that of the test, suppression was complete, i.e. test release was reduced to a function that increased monotonically to a steady level. The steady level was the same with or without conditioning. 3. These arithmetic rules suggest that inactivation of Ca2+ release channels is strictly and fatally linked to their activation. More than a strict linkage, however, is required to explain the arithmetic properties. 4. The arithmetic rules of inactivation result in three other properties that are inexplicable with classical models of channel gating: constant suppression, incremental inactivation and increment detection. These properties were first demonstrated for inositol trisphosphate (IP3)-sensitive channels and used to define IP3-induced release as quantal. In this sense, it can now be stated that skeletal muscle Ca2+ release is activated by membrane voltage in a quantal manner. 5. For both classes of intracellular Ca2+ channels, one explanation of the observations is the existence of subsets of channels with different sensitivities (to voltage or agonist dose). In an alternative explanation, channels are identical, but have a complex repertoire of voltage- or dose-dependent responses.

1. Inwardly rectifying ionic currents were studied using patch-clamp recording methods in oscillatory-type and spike-type hair cells and supporting cells dissociated from the goldfish sacculus. These cells had different types of inwardly rectifying currents. The biophysical properties of these currents were investigated. 2. A unique potassium current (Isc) was the sole ionic current recognized in supporting cells. Isc was active throughout the membrane potential range between +30 and -170 mV, but showed weak inward rectification and no inactivation. 3. In spike-type hair cells, inwardly rectifying current (Ik1) was selectively permeable to K+ (K+:Na+ permeability ratio, 1:0.0021). Ik1 could underlie the high negative resting potential of these hair cells because it is partially active at this potential. The strong inward rectification of Ik1 contributed to the low negative plateau potential seen in spike-type hair cells. 4. In oscillatory-type hair cells, hyperpolarization-activated potassium-sodium current (Ih), which had properties similar to that in photoreceptor and other neurons, was present instead of inwardly rectifying K+ current. 5. In the cell-attached and inside-out modes with 125 microM external K+ ([K+]o), IK1 channel had a unitary conductance of 27 pS and showed inactivation with increasing hyperpolarization. Putative Ih and Iso single channels had unitary conductances of 7 and 61 pS, respectively, in the cell-attached mode with 125 microM Ko+.

1. We have investigated the effects of microvascular pressures, high enough to rupture the vessel wall, upon the ultrastructure and permeability of mesenteric capillaries in pithed frogs. 2. The vessels were perfused via micropipettes with Ringer solutions containing bovine serum albumin (1 mg ml-1) and a few human red cells. After a perfused section of vessel had been closed downstream, pressure (applied via the micropipette) was raised in a series of steps of 10 mmHg, each lasting approximately 10 s, until the vessel ruptured. Fluid filtration through the vessel wall prior to rupture was estimated from the movements of the red cells. 3. Seven vessels were fixed in glutaraldehyde immediately after rupture and prepared for electron microscopy. The electron micrographs revealed openings in the vessel walls and thirty-six of these gaps were completely defined in runs of serial sections made on four of the vessels. Twenty-nine of these gaps passed through the endothelial cells (transcellular) and seven were intercellular. 4. The pressure at which a vessel ruptured, Pb, was measured in twenty-nine vessels and had a mean +/- S.E.M. value of 79.6 +/- 5.0 cmH2O. In ten of these vessels, which had a mean +/- S.E.M. Pb of 84.2 +/- 6.5 cmH2O, microvascular pressure was lowered immediately after the initial rupture and the vessel perfused at a pressure of 20 cmH2O. Pb was then remeasured and found to be 69.9 +/- 8.4 cmH2O, which was not significantly different from its initial value. 5. Hydraulic permeability (Lp) was measured in six vessels over the range of 15-30 cmH2O before and 10 min after the vessel wall ruptured at high pressure. Mean values were 5.5 x 10(-7) and 4.0 x 10(-7) cms-1 cmH2O-1 and were not significantly different. 6. At pressures equal to and 10 mmHg below Pb, small short-lived increases in filtration rate were observed. It is suggested that these may correspond to the increased permeability to fluid and macromolecules observed at high microvascular pressures in intact capillary beds.

1. The experiments described in this paper aimed to determine whether the gaps which develop in microvascular endothelium in association with increases in permeability are located in the intercellular clefts or are openings passing through the endothelial cells. 2. Hydraulic permeability (Lp) was estimated in frog mesenteric capillaries and single rat venules using a microperfusion-micro-occlusion technique before and during perfusion with solutions containing the ionophore A23187 at a concentration of 10 microM. When Lp was seen to have increased, the tissues were fixed in situ with 2.5% glutaraldehyde. 3. The increases in Lp varied considerably from vessel to vessel. In six frog vessels Lp increased from 2.6 +/- 0.9 x 10(-7) to 266 +/- 159 x 10(-7) cm s-1 cmH2O-1 and in three rat venules Lp rose from 0.94 +/- 0.09 x 10(-7) to 16.4 +/- 4.9 x 10(-7) cm s-1 cmH2O-1 (means +/- S.E.M.). 4. Forty openings or gaps were completely reconstructed from electron micrographs of serial ultrathin sections of the six frog vessels. Thirty-nine of these gaps passed through the endothelial cells and did not communicate with the intercellular clefts; one was intercellular. Similarly, fifteen out of sixteen gaps reconstructed from electron micrographs of the rat venules were transcellular and clearly separated from the intercellular clefts. 5. The increased Lp and associated ultrastructural changes induced by A23187 were reversed by perfusion with ionophore-free solutions.

1. We have measured changes in cell volume, membrane potential and ionic currents in distal nephron A6 cells following a challenge with hypotonic solutions (HTS). 2. The volume increase induced by HTS is compensated by a regulatory volume decrease (RVD), which is inhibited by both 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and quinine. Quinine (500 microM) completely blocked RVD, whereas 100 microM NPPB delayed and attenuated RVD. 3. The resting potential in A6 cells was -52.3 +/- 4.8 mV (n = 53), and shifted to -35.1 +/- 2.2 mV (n = 33) during HTS. 4. Resting membrane current in A6 cells was 0.35 +/- 0.12 pA pF-1 at -80 mV and 0.51 +/- 0.16 pA pF-1 at +80 mV (n = 5). During cell swelling these values increased to 11.5 +/- 1.1 and 29.3 +/- 2.8 pA pF-1 (n = 29), respectively. 5. Quinine (500 microM) completely blocked the HTS-activated current at -15 mV, the reversal potential for Cl- currents, but exerted only a small block at -100 mV (K+ equilibrium potential). NPPB (100 microM) inhibited the current at both potentials almost to the same extent. The HTS-induced net current reversed at -41 +/- 2.5 mV (n = 15), which is close to the measured resting potential during HTS. 6. The quinine-insensitive current reversed near the Cl- equilibrium potential. The quinine-sensitive current reversed near the K+ equilibrium potential. The respective conductances activated by HTS at the zero-current potential were 2.1 +/- 0.7 nS for K+ and 5.2 +/- 1.3 nS for Cl- (n = 15). 7. Single channel analysis unveiled activation of at least two different channels during HTS. A 36 pS channel reversing at the Cl- equilibrium potential showed increased open probability at depolarized potentials. HTS also activated a K+ channel with a 29 pS conductance in high-K+ extracellular solutions (130 mM) or 12 pS in 2.5 mM K+. 8. This coactivation of K+ and Cl- channels shifts the membrane potential towards a value between EK and ECl (the reversal potentials for K+ and Cl-), where a net efflux of Cl- (Cl- inward current) and K+ (K+ outward current) under zero-current conditions occurs. Block of either the K+ or the Cl- conductance will shift the zero-current potential towards the equilibrium potential of the unblocked channel, preventing net efflux of osmolytes and RVD. This coactivation of K+ and Cl- currents causes a shift of osmolytes out of the cells, which almost completely accounts for the observed RVD.

1. Fluxes studies were carried out to investigate the Na(+)-dependent outward movement of Ca2+ in intact frog sartorius muscle from Leptodactylus ocellatus, a preparation for which published data on the subject are sparse. 2. Under normal resting conditions the Na(+)-Ca2+ exchange was not readily detectable. 3. When muscles were exposed to 4 mM caffeine, the rate of fractional loss of Ca2+ (kCa,o) increased by about 50%. Most of this increase exhibits characteristics typical of the Na(+)-Ca2+ antiport working in the forward mode found in other cells. 4. The increase in kCa,o promoted by caffeine was decreased by: (a) 72% in the absence of external Na+ (Nao+); (b) 73% in Na(+)-loaded muscles ([Na+]i = 98 mM); (c) 70% when fibres were depolarized to -27 mV ([K+]o = 50 mM); and (d) 80% in the presence of 5 mM amiloride. 5. Ni2+ (5 mM), an inhibitor of the Na(+)-Ca2+ exchanger current, unexpectedly increased the caffeine-promoted rise in kCa,o. This effect of Ni2+ was associated with a concomitant caffeine-stimulated Ni2+ influx. In the absence of caffeine Ni2+ did not affect kCa,o. 6. It was concluded that: (a) under resting conditions the sarcolemmal Ca2+ pump suffices to handle the cytosolic calcium concentration ([Ca2+]i); (b) Na(+)-Ca2+ activity becomes apparent when [Ca2+]i is substantially increased by caffeine-induced Ca2+ release from the sarcoplasmic reticulum; and (c) the blocking effect of Ni2+ on the current generated by a Na(+)-Ca2+ exchange with a coupling ratio > 2 might actually represent a shift of the antiport mode toward an electroneutral 1 Ni(2+)-1Ca2+ exchange.

1. The requirement for extracellular Ca2+ in the process of evoked acetylcholine (ACh) release by nerve impulses was tested at endplates in frog skeletal muscle. Ca(2+)-containing lipid vesicles (Ca2+ liposomes) were used to elevate cytoplasmic Ca2+ concentrations under conditions in which Ca2+ entry from the extracellular fluid was prevented. 2. In an extracellular solution containing no added Ca2+ and 1 mM Mg2+ ('Ca(2+)-free' solution), Ca2+ liposomes promoted the synchronous release of ACh quanta, reflected electrophysiologically as endplate potentials (EPPs), in response to temporally isolated nerve impulses. 3. Motor nerve stimulation generated EPPs during superfusion with Ca2+ liposomes in Ca(2+)-free solutions containing the Ca2+ channel blocker Co2+ (1 mM), and the Ca2+ chelator EGTA (2 mM). As a physiological control for Ca2+ leakage from the liposomes to the extracellular fluid, the effect of Ca2+ liposomes on asynchronous evoked ACh release mediated by Ba2+ was examined. In contrast to the effects of 0.2-0.3 mM extracellular Ca2+, which generated EPPs but antagonized Ba(2+)-mediated asynchronous ACh release, Ca2+ liposomes generated EPPs but did not reduce asynchronous release mediated by Ba2+. The effects of Ca2+ liposomes were thus not due to leakage of Ca2+ from the liposome to the extracellular fluid. 4. Morphological studies using fluorescently labelled liposomes in conjunction with a confocal microscope demonstrate that lipid is transferred from the liposomes to nerve endings and liposomal contents are delivered to the nerve terminal cytoplasm. 5. The results suggest that when intracellular Ca2+ is elevated using liposomes as a vehicle, evoked ACh release can occur in the absence of Ca2+ entry via Ca2+ channels.

1. The stimulation of transepithelial Na+ transport caused by insulin in A6 cultured toad kidney cells was investigated by determination of membrane capacitance (Cm), short circuit current (Isc) and current fluctuation analysis. Values of Cm are proportional to membrane area while blocker-induced current fluctuation analysis provides an estimate of the number of active amiloride-sensitive Na+ channels in the apical membrane. 2. Insulin simultaneously increased Cm, Isc and Gt (transepithelial conductance) in epithelia incubated with Na(+)-containing solutions on both sides. 3. Analysis of 6-chloro-3,5-diaminopyrazine-2-carboxamide (CDPC)-induced noise showed that insulin increased the number of active Na+ channels in the apical membrane, without altering the single channel current. 4. When nystatin was used to permeabilize the apical membrane the impedance data revealed the presence of a second time constant. Analysis of these data indicated that the basolateral membrane capacitance (Cb) is much larger than the apical membrane capacitance (Ca). Insulin administered to nystatin-treated epithelia increased the values for both capacitances. 5. We suggest that the stimulation of transepithelial Na+ transport caused by insulin may be associated with the exocytotic delivery of transporters to the apical membrane.

1. The modulation by Ca2+ of the activation by inositol 1,4,5-trisphosphate (IP3) of Ca2+ channels present in native sarcoplasmic reticulum membranes from frog skeletal muscle was studied after channel incorporation into planar phospholipid bilayers in the presence of Ca2+ or Ba2+ as current carrier species. 2. Channel activity expressed as fractional open time (Po) was low (less than or equal to 0.15) in the presence of varying free Ca2+ concentrations bathing the myoplasmic face of the channel (cis side), and did not increase significantly between 0.01 and 30 microM-Ca2+. 3. Channel activation mediated by IP3 could be elicited from free Ca2+ levels similar to those of resting skeletal muscle (about 0.1 microM) and was found to be strongly regulated by the free Ca2+ concentration present at the myoplasmic moiety of the channel. 4. Channel activation by 10 microM-IP3 depended on the Ca2+ concentration on the cis side. Po reached a maximum between pCa 7.0 and 6.0, but decreased at higher concentrations of free Ca2+. Thus, Ca2+ exerted a modulatory influence on IP3-mediated activation in a concentration range where the channel was insensitive to Ca2+. 5. The results indicate that Ca2+ ions act as modulators of IP3 efficacy to open the channel. This could arise from an interaction of Ca2+ with the channel gating mechanism or with the agonist binding site.

1. In frog a variety of treatments have been shown to increase quantal size at the neuromuscular junction (NMJ), apparently by releasing more acetylcholine (ACh) per quantum. The present experiments were undertaken to see whether similar changes occur at the mouse NMJ. 2. None of the hormones tested, adrenaline, insulin or corticosteroids, significantly increased quantal size at the mouse NMJ. 3. Soaking diaphragms in hypertonic solution (about 475 mosmol/kg) for 15-30 min roughly doubled the size of miniature endplate potentials (MEPPs), miniature endplate currents (MEPCs), and uniquantal endplate potentials (EPPs). We will refer to these as 'large quanta'. Note that all of the measurements were made after returning the preparation to normal (Tyrode) solution. 4. In frog hypertonic solution made with sodium gluconate replacing NaCl increases quantal size four- rather than two-fold. In mouse there was little difference in the effects of solutions made with the two anions. In Cl(-)-free hypertonic solution, made with sodium gluconate and SO4(2-) solutions, quantal size increase is somewhat less, so there may be a role for Cl- in enlarging quantal size. 5. After hypertonic treatment, quantal size remained elevated for at least 1 h and then gradually declined back to usual levels. The data suggest a gradual decrease in mean quantal size, rather than the appearance of a new subpopulation of smaller quanta. 6. Hypertonic treatment did not change the endplate depolarization in response to ionophoretically applied ACh. This suggests that the increased quantal size is not due to a postsynaptic change. Large MEPP's disappear in the presence of tubocurarine and reappear when the drug is washed away. 7. Vesamicol is an inhibitor of active ACh uptake into isolated synaptic vesicles. 5 microM-vesamicol has no detectable postjunctional effect. However, when vesamicol was included in the Tyrode and the hypertonic solutions the increase in quantal size was blocked. This is further evidence that the large quanta are produced by the release of more ACh per quantum. 8. Even when added after the hypertonic treatment, vesamicol soon decreased quantal size back to the normal level. Two other inhibitors of active ACh uptake into vesicles, tetraphenylboron (TPB) and hexanitrodiphenylamine (HNPA) also decreased quantal size after hypertonic treatment, apparently by a presynaptic action. This suggests that the additional ACh that produces large miniatures may be incorporated into the quanta shortly before release.(ABSTRACT TRUNCATED AT 400 WORDS)

1. The three intracellular micro-electrode voltage-clamp technique was used to study the recovery of membrane charge movement in semitendinosus muscles of Rana pipiens. Muscles were placed in a hypertonic depolarizing solution to inactivate voltage dependent charge movement. Tetrodotoxin and tetraethylammonium ions (TEA+) were present to block voltage dependent ionic conductances. Rb+ and SO4(2-) were present to reduce inward rectification and leakage conductance. 2. The recovery ('repriming') of membrane charge movement was studied following hyperpolarizing pulses from a holding potential of -20 mV to membrane potentials from -30 to -140 mV for durations of 2--100 sec. The reprimed charge movement measured as the difference in membrane current required for identical voltage steps before and after long duration hyperpolarizing pulses was a linear function of membrane potential and symmetrical in shape. Reprimed charge is, therefore, simply the result of an increase in the linear capacitance of the fibre. 3. The mean value of the percent increase in capacitance for repriming at -100 mV was 12.3 +/- 1.7% (S.E. of mean) for 25 sec duration pulses and 27.8 +/- 2.9% for 100 sec duration pulses. If these data are corrected to the steady state and the surface contribution subtracted, the mean increase in 'volume' capacity is 40.3 +/- 3.6% (n = 21) for fibres with a mean diameter of 51 +/- 4 micron. 4. The increase in capacity can arise either by an increase in the transverse tubular length constant (lambdaT) or by gaining electrical access to additional linear capacitance within the fibre volume. If the capacitance arises solely from the transverse tubular system, the value of lambdaT before repriming can be no larger than 20 micron in order to explain the observed increase in volume capacity. A value of lambdaT as small as this seems unlikely. 5. The observation that reprimed charge is simply the result of an increase in linear capacitance is not consistent with the hypothesis that it is a gating mechanism for the activation of contraction.

1. The previously demonstrated K+-dependent short-circuit current through the skin of frog species Rana temporaria (Zeiske & Van Driessche, 1979), bathed with mucosal K+- and serosal Na+-Ringer solution, was investigated with current-fluctuation analysis. 2. The current-noise spectra were recorded in the frequency range from 1 to 800 Hz and showed a Lorentzian component with a mean plateau value S0 = (1.50 +/- 0.05).10(-20) A2.s.cm-2 and a corner frequency of fc=(81.0 +/- 3.4)Hz(n=14). 3. S0 increased with mucosal K+ concentration, [K]o, while fc remained almost unchanged. A decrease in S0 was observed when serosal Na+ was replaced by K+. 4. Mucosal Cs+ (10 mM) depressed, reversibly, the K+-dependent current noise to the level of the background noise. Moreover, a linear decrease in fc with increasing Cs+ concentration was observed. 5. Among the other tested alkali cations, Rb+ was the only blocker though less potent than Cs+. Tetraethylammonium, 4-aminopyridine, 2.4.6-triaminopyrimidine and amiloride had no effect. 6. Alterations in the transcellular transport of Na+ contained in a mucosal solution with high [K]o resulted in significant changes in K+ current noise. 7. The current-fluctuation intensities decreased with increasing contact time to high [K]o; these changes were concomitant with the previously reported time dependence of the short-circuit current (Zeiske & Van Driessche, 1979). 8. The K+-dependent fluctuations are thought to originate from K+-selective pathways in the apical cell membranes. The description of the K+-current noise by a single Lorentzian suggests that the "K+ channels" switch randomly between an open and closed state. 9. Assuming a two state model for the channel-kinetics, the single channel current i and the channel density M were calculated as i=(0.37 +/- 0.05)pA and M=(0.53 +/- 0.08) mu-2 (n=13).

1. The effect of nerve stimulation on spontaneous transmitter release was studied at the frog neuromuscular synapse which was bathed in a solution containing very low extracellular calcium concentration. Conventional methods for intracellular and extracellular recording were used and the pattern of quantal liberation following the nerve stimulus was determined. 2. Stimulation of the motor nerve (at rates between 0.09 and 2Hz) caused a reduction in the frequency of the miniature e.p.p.s in comparison to the prestimulation values. 3. The mean distribution of the time of occurrence of the miniature e.p.p.s during the interstimulus period showed periodic oscillations. 4. The quelling effect of nerve stimulation on transmitter release is explained by the hypothesis that a low [Ca]o a reversed electrochemical gradient for calcium occurs and nerve stimulation causes an increased calcium conductance leading to calcium efflux which in turn temporarily reduces [Ca]i and transmitter release.

1. In the present study changes in extracellular potassium, ([K]e), were recorded in the isolated spinal cord of the frog with glial cell recordings and K-selective micro-electrodes to test the hypothesis that elevations in [K]e during neuronal activity produce the dorsal root potential (d.r.p.). 2. Sucrose gap recording from the dorsal root (d.r.) was used to record responses to root stimulation and to exogenously applied K+. 3. Stimulation of the ventral root, which elicits a d.r.p. in the frog spinal cord, was not associated with any change in [K]e, suggesting that d.r.p.s produced by ventral root stimulation are not due to changes in [K]e. 4. The largest change in [K]e observed following single stimuli to the dorsal root was 0.4 mM. Such a change in [K]e, if evenly distributed, would depolarize the dorsal root by about 1 mV and yet the simultaneously recorded d.r.p. evoked by stimulating an adjacent dorsal root (d.r.-d.r.p.) was over 10 mV. 5. The time-to-peak of the glidal cell responses was 10 times that of the d.r.-d.r.p. Low frequency (1-10 Hz) d.r. stimulation caused a decremental summation of glial cell responses, while there was no summation in the d.r.-d.r.p. These results suggest that the d.r.p. produced by single d.r. stimulation is generated in large part by a mechanism other than a change in [K]e. 6. During high frequency d.r. stimulation, which evoked 6-8 mM increases in [K]e, the adjacent d.r. was depolarized to a greater extent than that produced by single stimuli. The magnitude of this depolarization was similar to that produced by applying a [K]e equivalent to that observed in the spinal cord during high frequency stimulation. Thus, a substantial component of the sustained d.r. depolarization during high frequency d.r. stimulation may result from changes in [K]e. 7. In the presence of magnesium, high frequency d.r. stimulation evoked a picrotoxin resistant depolarization of an adjacent d.r. whose magnitude correlated well with the changes in [K]e recorded in the spinal cord. 8. In the presence of picrotoxin a slow, long duration depolarization of the d.r. occurred following single stimuli to the adjacent d.r. and the appearance and time course of this response correlated well with the time course of changes in [K]e. 9. Addition of K+ to the Ringer solution in concentrations up to 12 mM had a facilitatory action on reflex activity in the frog spinal cord. 10 The present results suggest that although changes in [K]e play a relatively minor role in generating d.r.p.s. elicited by single d.r. stimulation, the sustained dorsal root depolarization evoked either by high frequency stimulation or by single stimuli in the presence of picrotoxin may be due to a considerable extent to [K]e.

1. Focal electrical stimulation of Schwann cells at the end-plates of denervated frog muscles elicited slow depolarizations of up to 30 mV in the muscle fibres. This response is referred to as a Schwann-cell end-plate potential (Schwann-e.p.p.).2. Repeated stimulation sometimes evoked further Schwann-e.p.p.s, but they were never sustained for more than 30 pulses. Successive e.p.p.s varied in amplitude and time course independently of the stimulus.3. The Schwann-e.p.p.s were reversibly blocked by curare, suggesting that they result from a release of acetylcholine (ACh) by the Schwann cells.4. ACh release by electrical stimulation did not seem to occur in quantal form and was not dependent on the presence of calcium ions in the external medium; nor was it blocked by tetrodotoxin.5. Stimulation which caused release of ACh also resulted in extensive morphological disruption of the Schwann cells, as seen with both light and electron microscopy.6. It is concluded that electrical stimulation of denervated Schwann cells causes break-down of the cell membrane and releases ACh, presumably in molecular form.

1. The relationship between the [Ca](o), the [Na](o) and the strength of the contracture evoked when the [Na](o) is reduced has been investigated in isolated frog atrial trabeculae.2. The strength of the contracture varies by the [Ca](o) (2) and by 4 radical([Na](o)) over the lower tension range.3. The contracture induced by reduction of [Na](o) is not sustained, but relaxes spontaneously. The rate of this relaxation is only dependent on the [Na](o) is the presence of strophanthidin.4. After the spontaneous relaxation of an Na-free contracture, the ability of the trabecula to develop tension upon a second challenge with Na-free solution returns in about 3 min if the muscle is perfused with Na-containing fluid. This recovery process is slowed if the [Na](o) is low during the recovery period, but the recovery is hastened by electrical stimulation of the preparation or by perfusion with K-free or strophanthidin containing sodium-Ringer.5. It is suggested that the influx of Ca(2+) which induces the Na-free contracture depends on the presence of Na(+) inside the cells. When the intracellular Na concentration falls, the Ca influx falls, and the muscle relaxes as a result of the activity of an intracellular relaxing structure.

1. Electrophysiological studies were made on regenerating motor end-plates in frog cutaneous pectoris muscle after crushing the motor nerve.2. The pattern of degenerative and regenerative changes is similar to that already described for the frog sartorius, although it occurs more quickly in the cutaneous pectoris. Resumption of synaptic transmission first occurs after 9 days, in muscle fibres closest to the nerve crush. During the subsequent 12 days synaptic contact is progressively re-established across the muscle.3. During the period of regeneration muscle fibres can be found which have been reinnervated, but which do not produce an end-plate potential in response to stimulation of the nerve trunk. This is referred to as the ;non-transmitting' stage of regeneration.4. Increases in extracellular potassium, addition of lanthanum to the bath and focal extracellular stimulation all indicate that the axon terminals at non-transmitting junctions do have a releasable supply of neurotransmitter. Focal stimulation occasionally sets up active responses in these nerve terminals.5. It is concluded that this ;non-transmitting' stage results from failure of action potential propagation in the regenerating neurone proximal to the end-plate region.

1. The abdominal frog skin was mounted between two chambers containing Ringer with 1 mM-Na on the outside and 115 mM-Na on the inside. When the Na concentration of the outer solution ([Na](o)) is instantaneously raised from 1 to 50 mM, the short circuit current (I) increases to a new value in less than a second, and becomes essentially time-independent. Only in a few experiments was it observed to increase further, although at a much slower rate.2. At a time t after this increase, the addition of 10(-4)M amiloride to the outer solution produces an exponential decrease of I. The area under this exponential curve is generally taken to reflect the existence of a Na- transporting compartment (NaTC).3. The amount of Na represented by NaTC is a function of t: it increases from 1.7 x 10(-9) mole. cm(-2), at t = 10 sec, to 22.8 x 10(-9) mole. cm(-2) at t = 10 min.4. In view of the fact that (a) I is not a function of the size of the ;NaTC' and (b) that whereas I reaches a steady value in a fraction of a second the size of NaTC keeps increasing for minutes, it is proposed that the ;NaTC' represents an amount of Na which is not located along the main route of transepithelial transport.5. On the assumption that the NaTC is located in a cellular compartment and that, in order to accumulate in this compartment Na should be accompanied by a permeable anion, a series of experiments were performed with Ringer in which Cl(-) was replaced by gluconate. It was observed as expected, that NaTC in gluconate is 164 times smaller than in Cl(-), but I only decreases to one half its value in Cl(-) Ringer.

1. Extension of a frog muscle spindle evoked a discharge of impulses in its sensory axon. The adaptation of the discharge after the dynamic phase of stretching occurred in two phases. At first the impulse train was almost regular and adapted quickly, but later this gave place to a phase of slower adaptation in which the variability of discharge was much increased. 2. The discharge of action potentials by the muscle spindle depressed the response of the receptor to a subsequent extension. This was true whether they were elicited antidromically by afferent stimulation or orthodromically by longitudinal vibration. This depression had its most marked effect on the first phase of adaptation where it greatly increased the rate of adaptation. The second, slower, phase of adaptation was little influenced. 3. The depression increased with the duration and with the frequency of afferent stimulation. It persisted for about 300 msec. 4. The results show that the properties of the spike generating mechanisms in the muscle spindle are modified by previous discharge and that this modification may influence the overall properties of the receptor. 5. The fact that afferent stimulation has different effects on the two phases of adaptation is consistent with the suggestion that the impulse train evoked by extension is derived from more than one spike generator within the muscle spindle.

1. The relationship between active force and velocity of shortening was studied during tetanic contraction of isolated semitendinosus muscle fibres of the frog (0.5-2.0 degrees C). Measurements were carried out with the fibre immersed in isotonic (1.00R) Ringer solution and in solutions that were made hypotonic by reduction of NaCl (osmolality 0.62 and 0.81 of normal Ringer) and hypertonic by addition of sucrose (osmolality 1.22 and 1.44 of normal Ringer).2. The force-velocity relation was hyperbolic at loads lower than 80% of measured isometric force (P(0)) but exhibited a reversed curvature between 0.8P(0) and P(0). The maximum velocity of shortening was determined in two different ways: (i) by extrapolation to zero load from force-velocity data truncated at 0.8P(0) (computer fitting of hyperbola, leastsquares method) and (ii) by recording the time required to take up the slack of the fibre after a quick release during tetanus.3. Isometric force and maximum speed of shortening both changed inversely with the tonicity of the extracellular medium. Immersion of the fibre in 0.81R hypotonic solution caused active tension and shortening velocity to increase by 10 +/- 1% (mean +/- S.E. of mean, n = 14) and 12 +/- 1%, respectively. Conversely, force and shortening velocity decreased by 12 +/- 1% (n = 13) and 22 +/- 2% when normal Ringer was replaced by 1.22R hypertonic solution. These changes doubled when the tonicity was altered from normal Ringer to 0.62R and 1.44R, respectively.4. Changes in fibre cross-sectional area equivalent to those obtained in the 0.81R and 1.22R solutions (+11% and -13%, respectively) were produced by varying the sarcomere length within the range 2.0-2.5 mum in the normal Ringer solution. Maximum velocity of shortening remained very nearly constant under these conditions, indicating that the shortening velocity, like the isometric force, is not critically dependent on changes in myofilament lattice width over the range considered.5. The results support the view that both shortening velocity and active force are modulated by changes of the intracellular ionic strength above and below the level that normally exists in the intact muscle fibre.

1. Previous work has shown that a frog sartorius muscle consists of parallel cells running all the way from one end of the muscle to the other and that amputation of one end of the muscle is not followed by regeneration of a new cell membrane. If now only the cut end of the amputated muscle is exposed to a Ringer solution in which the solutes 42K and 22Na act as radioactive labels and the rest of the cell is suspended in air, we have what is described as an effectively membraneless open-ended cell or EMOC preparation. In this case the only remaining anatomically intact plasma membrane and pumps are made nonfunctional by the removal of 'sources' for inward pumps and 'sinks' for outward pumps. 2. The healthy region of a frog sartorius muscle EMOC preparation continues to accumulate labelled K+ to a level higher than that in the Ringer solution and to exclude labelled Na+ to a level below that in the Ringer solution, much as a normal uncut muscle does in its normal environment. The differences were reduced by inclusion of ouabain in the medium. 3. The diffusion coefficient of Na+ in the normal muscle cytoplasm at 25 degrees C was measured using two methods. The average diffusion coefficient measured was 2.07 X 10(-6) cm2/sec, roughly 1/6 that of the diffusion coefficient of Na+ in a 0.1 N-NaCl solution. 4. The data obtained are discussed in terms of the association-induction hypothesis. In this theory asymmetrical solute distribution, basically an expression of a non-energy consuming metastable equilibrium state, is the result of specific combinations of two opposing mechanisms: adsorption which raises the level of the intracellular solute; and exclusion from cell water which tends to lower it.

1. A voltage-clamp method was used to examine the effects of zinc ions on the delayed potassium currents and also the slowly activating potassium currents that are turned on by depolarizing the membrane of frog sartorius muscle fibres. 2. In a control solution, the delayed potassium conductance had a maximum value of 17-8 +/- 2-5 mmho.cm-2. The reversal potential for the currents was -76-9 +/- 2-5 mV. The membrane potential where ninfinity had the value 0-5 was -49 mV. 3. The major effect of zinc ions was to slow the delayed potassium currents. The value of taun was increased approximately tenfold in 0-1 mM zinc. Zinc does not alter the effective valency of the gating particles of the potassium channel, but the conductance was shifted to more positive membrane potentials: in 0-1 mM zinc, the membrane potential where ninfinity had the value 0-5 was -32 mV. 4. Zinc ions, at a concentration of 0-1 mM, also reduced the maximum potassium conductance by about 60% to 7-3 +/- 0-8 mmho, cm-2; they did not alter the reversal potential of the currents, which had a value in 0-1 mM zinc of -74-6 +/- 1-5 mV. 5. Zinc ions had little or no effect on the rate of inactivation of the potassium currents. 6. Zinc ions had little effect on the conductance attributable to the slowly activating potassium system. In 0-01 mM zinc this conductance had a value at 0 to +10 mV of 1-25 +/- 0-29 mmho.cm-2. Zinc did not alter the reversal potential of the slow potassium currents from the value of -85 +/- 1-6 mV in the absence of zinc and had no effect on the time course of the turn-off of these currents at -60 mV. 7. The delayed potassium currents obtained in 0-002 and 0-01 mM zinc could not be fitted exactly with a simple fourth order equation, but were well fitted by a model proposing that zinc ions slow the opening and closing of the gating mechanism to one tenth the normal rate when they bind to the gating molecule. If the binding sites are not saturated, those gating molecules that do not bind zinc are assumed to be quite unaltered in their properties, though the potential dependence of their rate constants alphan and betan was assumed to be shifted to more positive levels. In one fibre in 0-01 mM zinc, the model fitted the currents best if 50% of the gating molecules bound zinc.

1. At rat and frog neuromuscular junctions, perhydrohistrionicotoxin (H12-HTX), at concentrations below 10(-6) M, blocked end-plate currents and potentials generated by ionophoretic application of ACh (extrinsic responses) more effectively than end-plate currents and potentials generated by neurotransmitter secreted from the motor nerve (intrinsic responses). 2. In contrast, (+)-tubocurarine affected both extrinsic and intrinsic responses in a parallel manner. 3. There was no change in the time course and little or no change in the amplitude of intrinsic end-plate currents when extrinsic currents were depressed by H12-HTX nor was there any change in the conductance or lifetime of channels activated by applied ACh. 4. The depressant effect of H12-HTX on extrinsic responses persisted both when carbachol was used as the agonist and when acetylcholinesterase was inhibited with diisopropylfluorophosphate. 5. Large end-plate currents elicited by nerve stimulation that presumably activate the whole end-plate area were not depressed by H12-HTX to the same degree as extrinsic end-plate currents generated by ionophoresis of ACh at the same end-plate. 6. Brief (50 microsec) pulses of ACh produced brief end-plate potentials which were depressed by concentrations of H12-HTX that had little or no effect on miniature end-plate potentials. 7. Extrinsic responses to ACh at extrajunctional regions of denervated fibres were also depressed by low concentrations of H12-HTX. 8. It was concluded that the differential effects of H12-HTX on intrinsic and extrinsic end-plate responses could be due to the existence of two populations of receptor-channel complexes or to protection of local receptor-channel complexes from the toxin by a substance secreted from motor nerve terminals.

1. The firing thresholds of single myelinated fibres of frog sciatic nerves were monitored as a function of impulse activity in the fibre. The threshold was given by the number of coulombs in current pulses that excited a particular fibre half the time when delivered to the whole nerve. Threshold was tracked by a device that incrementally decreased the number of coulombs in the current pulse whenever the fibre responded and increased the pulse if it did not respond. 2. There was a pattern to the after-oscillations of threshold following activity. The fibres were briefly refractory, transiently superexcitable for about 1-1.5 sec and then entered a phase of raised threshold or 'depression' that lasted for many minutes. 3. Activity produced little change in the threshold curve during the refractory period. Strong depressions following prolonged activity prevented the threshold from returning to the base-line level within the time associated with the refractory period for the same fibre at rest. 4. After an impulse, superexcitability reached a maximum within 7-20 msec. This peak was larger as the number of impulses in a preceding burst increased and as the intervals between the impulses became briefer. Each successive impulse of a burst contributed less to the growth of superexcitability, and after the burst had 6-10 impulses additional impulses contributed nothing. 5. The depression phase was marked by the interaction between build-up, which depended on the activity rate, and recovery, which required as long as an hour or more for the threshold to be completely restored to resting level. These two mechanisms, one causing build-up and the other recovery, led to formation of dynamic equilibria. The threshold level at equilibrium increased monotonically with the activity rate. 6. The processes associated with superexcitability interact with those producing depression. In active fibres showing raised thresholds, impulses are followed by a relative superexcitability that persists for at least as long as an absolute superexcitability (with threshold below the resting level) can be measured in the same fibre at rest. 7. The duration of the superexcitable phase interpreted as a relative change in excitability was roughly the same regardless of the level of depression. 8. The magnitude of the oscillation in threshold was give to ten times larger than the grey region (the range of stimuli for which response is probabilistic). It is concluded that at regions of low conduction safety such as axonal branches, where weak forces can influence whether an impulse will pass, such pronounced and long-lasting after-effects of firing can be expected to modulate conduction of nerve impulses.

1. The dependency of the transmembranal potential differences upon the [Na] of the mucosal bathing solution was investigated in frog skin. Semilogarithmic linear correlations were obtained between [na]0 and the transepithelial potential difference (slope: 28-4 +/- 1-8 mV/dec [Na]0), the potential differences across the outer and the inner membrane under open circuit conditions (slope: 33-4 +/- 1-8 and 4-8 +/- 1-5 mV/dec [Na]0, respectively) and the intracellular potential under short circuit conditions (slope: 33-3 +/- 3-4 mV/dec [Na]0). 2. No difference could be observed between skins incubated in Cl- or SO4-Ringer. 3. The results are not in accordance with the Koefoed-Johnsen & Ussing hypothesis. Neither the polarity of the postulated Na electrode at the outer border could be obtained nor the expected slope of the dependency between potential gradient and [Na] of the mucosal bathing solution. 4. Current recirculation through paracellular shunt pathways is suggested to explain the deviation, at least in part. In addition, the I-R-drop resulting from Na entry across the outer border might account for part of the changes of potential gradients. 5. Under all conditions, Na uptake occurs energetically downhill, but permeability changes of the outer border must be postulated to explain the observed dependency of net Na transport upon [Na]0.

1. The action of monoethylcholine (MECh) on neuromuscular transmission has been studied by electrophysiological methods. 2. End-plate potentials (e.p.p.s.) in curarized rat muscle were unaffected or slightly increased in amplitude by MECh (0-1-1 mM). Stimulation at 3 Hz for about 30 min in the presence of MECh caused a progressive decline in e.p.p. amplitude, and a shortening of the e.p.p. time course. These changes were reversed by addition of choline to the medium. Similar changes in amplitude, but no change in time course, occurred when the preparation was stimulated in the presence of hemicholinium or triethylcholine. 3. Extracellular recordings of miniature end-plate potentials in frog muscle showed that stimulation in the presence of MECh caused the time constant of the exponential decay of the m.e.p.p.s. to decrease by 42%. The amplitude of intracellular m.e.p.p.s. was reduced by 45%. These changes were maximal by the time about 3 X 10(5) quanta had been released. 4. Voltage clamp experiments in rat muscle in which miniature end-plate currents (m.e.p.c.s) were recorded showed that stimulation in the presence of MECh reduced the amplitude (by 33%) and the decay time constant (by 42%). 5. Analysis of end-plate current flucutations produced by local application of acetylcholine (ACh) and acetylmonoethycholine (AMECh) to voltage clamped rat end-plates showed that the amplitude of the elementary current events was the same for both compounds whereas the average channel lifetime was 44% shorter for AMECh than for ACh. 6. The voltage-sensitivity of the channel lifetime (measured from end-plate current fluctuations) was the same for ACh and AMECh. The voltage-sensitivity of the m.e.p.c. decay time constant was the same as that found from noise measurements. The shortened m.e.p.c.s. (false m.e.p.c.s.) occurring after stimulation in the presence of MECh also showed the same voltage-sensitivity. 7. Both normal and false m.e.p.c.s. were prolonged by neostigmine by almost the same factor; false m.e.p.c.s. were thus shorter than normal m.e.p.c.s. even when cholinesterase was inactivated. Experiments with progressive curarization of neostigmine-treated end-plates suggested that the fraction of transmitter molecules bound is smaller for false than for normal m.e.p.c.s. The difference implies that the false transmitter has one quarter of the affinity of ACh for the receptors. 8. It is concluded that stimulation in the presence of MECh gives rise to a false transmitter, presumably AMECh, which has a lower affinity for receptors than ACh, and gives rise to ionic channels with a shorter average lifetime than those activated by ACh.

1. Ion-sensitive electrodes (made with a chloride-sensitive ion-exchange resin) were used to measure the internal chloride activity (a(i) (Cl)) of frog sartorius fibres at 25 degrees C.2. The internal pH (pH(i)) of other sartorius fibres was measured with a recessed tip pH-sensitive electrode (made with pH-sensitive glass).3. In normal bicarbonate-free solution (containing 2.5 mM potassium), the average chloride equilibrium potential, E(Cl) (calculated from a(i) (Cl) and the measured chloride activity of the external solution (a(o) (Cl)) was 87.7 +/- 1.7 mV (mean +/- S.E.; n = 16) in fibres where the average membrane potential, E(m), was 88.3 +/- 1.5 mV (mean +/- S.E.; n = 16). In experiments where a(i) (Cl) was varied between about 1 and 10 mM (which corresponds to values of E(m) between about -105 and -50 mV) E(Cl) was within 1-3 mV of E(m) at equilibrium. These measurements of a(i) (Cl) were obtained from the potential difference between the chloride-sensitive electrode and an intracellular indifferent micro-electrode filled with potassium chloride. If a potassium sulphate-filled indifferent micro-electrode was used, then values of a(i) (Cl) below about 5 mM were erroneously high, probably due to interference from other sarcoplasmic ions at the indifferent electrode.4. In solutions containing 15 mM bicarbonate and gassed with 5% CO(2), pH(i) was 6.9, corresponding to an internal bicarbonate concentration of 7.6 mM. E(Cl) measured in this solution was some 4 mV positive to E(m). Most of the difference between E(Cl) and E(m) could be ascribed to interference by sarcoplasmic bicarbonate on the basis of selectivity measurements of chloride against bicarbonate made on the ion-exchange resin in the relevant range of a(Cl).5. If bicarbonate/CO(2) in the external solution was replaced by HEPES/pure O(2) at constant pH, then pH(i) rose from 6.88 +/- 0.02 (mean +/- S.E.) to 7.05 +/- 0.02. A change in external pH of 1 unit caused pH(i) to change by about 0.02 unit and the intracellular buffering power was calculated to be about 35.6. In solution made hypertonic by the addition of sucrose, E(m) changed little or depolarized and E(Cl) and E(m) remained close. In contrast, in solution made hypertonic by the addition of solid sodium chloride (high-chloride solution) E(Cl) became negative to E(m). Conversely in low chloride solution E(Cl) became positive to E(m).7. When the chloride permeability (P(Cl)) was reduced by the use of acid solution, E(Cl) moved positive to E(m) indicating an accumulation of internal chloride. When P(Cl) was increased again by returning to more alkaline solution, E(m) depolarized to E(Cl).8. The results are consistent with the existence of a small, active movement of chloride, the effects of which are normally obscured by large passive movements of chloride when P(Cl) is large.