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K L Magleby

Publications and source records attributed to K L Magleby.

At least 55 records · Page 3Linked to original sources
PubMed1988

Kinetic states and modes of single large-conductance calcium-activated potassium channels in cultured rat skeletal muscle.

1. Kinetic states and modes of a large-conductance Ca2+-activated K+ channel in excised patches of membrane from cultured rat skeletal muscle were studied with the patch clamp technique. Up to 10(6) open and shut intervals were analysed from each of seven different excised membrane patches containing a single channel. 2. Plots of the mean durations of consecutive groups of ten to fifty open and shut intervals were made to assess kinetic stability of the channel. Occasional abrupt decreases in the mean open interval duration from normal to different distinct levels, which were maintained for hundreds to thousands of consecutive intervals, indicated entry of the channel into different modes. 3. Four different kinetic modes were identified: normal mode, which included 96% of the intervals; intermediate open mode with 3.2% of the intervals; brief open mode with 0.5% of the intervals; and buzz mode with 0.1% of the intervals. The mean open interval durations were 61% of normal during the intermediate open mode, 12% of normal during the brief open mode, and 2.6% of normal during the buzz mode. 4. Most mode transitions were observed from the normal mode to one of the other modes and then back to normal. Sojourns in the normal mode lasted 5-1000 s. Sojourns in the intermediate open, brief open, and buzz modes lasted 1.5-150, 1-7, and 0.01-1 s, respectively. 5. During normal activity the distributions of interval durations were typically described by the sum of three to four exponential components for the open intervals and six to eight exponential components for the shut intervals, and this was the case for data obtained over a wide range of open channel probability resulting from different Ca2+i. These observations suggest that the channel can enter at least three to four open and six to eight shut states during normal activity. 6. The numbers of detected states for data sets of different sample sizes drawn from normal activity agreed with theoretical predictions, and were essentially independent of the segment of normal activity from which the data sets were drawn. These observations are consistent with relative stability of channel kinetics during normal activity. Detection of each additional open or shut state after the first was found to require a 3- to 10-fold increase in the number of analysed events. 7. The intermediate open mode differed from the normal mode in that the longest open component of the four normal open components was absent.

Action Potentials↗
PubMed1987

Sampling, log binning, fitting, and plotting durations of open and shut intervals from single channels and the effects of noise.

(1) Analysis of the durations of open and shut intervals measured from single channels currents provides a means to investigate the mechanisms of channel gating. Durations of open and shut intervals are conveniently measured from single channel data by using a threshold level to indicate transitions between open and shut states. This paper presents a detailed characterization of sampling, binning, and noise errors associated with 50% threshold analysis, provides criteria to reduce these errors, methods to correct for them, and presents an efficient means of data handling for binning and plotting interval durations. (2) Measuring interval durations by sampling at a fixed rate introduces two types of errors, (a) the number of intervals of a given measured duration are increased (promoted) over that expected in the absence of sampling, producing a sampling promotion error, (b) sampling decreases the total fraction of true intervals that are detected, producing a sampling detection error. Sampling errors can be reduced to negligible levels if the actual or effective (after interpolation) sampling period is less than 10-20% of both the dead time and fastest time constant in the distribution of intervals. Dead time is given by the duration of a true interval that has a filtered amplitude equal to 50% of the true amplitude. (3) Methods are presented to correct for sampling promotion error during least squares and maximum likelihood fitting. Sampling detection error is more difficult to correct, but an empirical description of the sampling detection error can be used to calculate the effective fraction of detected events with sampling. (4) Noise in the single channel current record can produce two types of error. (a) If noise peaks in the absence of channel activity exceed the threshold for detection, then false channel events of brief duration are produced. Sufficient filtering will prevent this type of error. (b) Noise can also increase the total fraction of true intervals that are detected, producing a noise detection error. Increased filtering over that required to prevent false events is not necessarily the best method for reducing noise detection error, as increased filtering can prevent detection of the faster exponential components. (5) Noise detection error can be reduced in two ways: (a) an empirical description of the noise detection error can be used to calculate the effective fraction of detected events in the presence of noise. (b) The sampling period can be selected so that the sampling detection error cancels the noise detection error.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1986

Correcting single channel data for missed events.

Interpretation of currents recorded from single ion channels in cellular membranes or lipid bilayers is complicated by the necessarily limited time resolution of the recording and detection systems. All intervals less than a certain duration, depending on the frequency response of the system, are not detected. Such missed events produce increases in the durations of observed open and shut intervals. In order to obtain the true kinetic scheme and rate constants underlying the observed activity, it is necessary to take into account missed events. We develop methods to correct for missed events for models with two or more states, including models with multiple open and shut states, compound states, and loops. Our methods can be used in a forward direction to predict observed distributions of open and shut intervals for a given kinetic scheme and time resolution. They can also be used in a backwards direction with iterative methods to determine rate constants consistent with the observed distributions. While a given kinetic scheme with rate constants predicts unique observed distributions of open and shut intervals, rate constants determined from observed distributions are not necessarily unique. Using these correction methods, we examine the effects of missed events for a five-state model consistent with some properties of large conductance Ca-activated K channels.

Ion Channels↗
PubMed1986

Quantitative description of three modes of activity of fast chloride channels from rat skeletal muscle.

The steady-state kinetic properties of single Cl- channels with fast kinetics active at resting membrane potentials in cultured rat skeletal muscle were studied using the patch-clamp technique. Membrane patches containing single active Cl- channels were often observed, and binomial analysis of the percentage open time in membrane patches containing several Cl- channels indicated that the channels did not occur as obligatory dimers and that they gated independently of one another. Channel activity could be divided into three categories: normal, which included about 99% of the openings and closings; buzz mode, which included about 1% and consisted of bursts of about 50 brief open and shut intervals; and inactivated shut states which included about 0.01% of the shut intervals and lasted for seconds, and occasionally minutes. The method of maximum likelihood was used to determine the number of significant exponential components required to fit the distributions of open and shut intervals during normal activity. Open interval distributions required at least two components, with time constants of 0.52 and 1.5 ms at -40 mV and 7.6 degrees C. Shut interval distributions required at least five exponential components, with time constants of 0.064, 0.72, 1.9, 12.3 and 350 ms. Kinetic reaction schemes were developed for the normal and buzz mode using maximum likelihood techniques to determine the most likely models and rate constants. In developing these models the effects of limited time resolution and missed events were taken into account. Each model tested typically had two or more sets of equally likely rate constants. Incorrect sets of rate constants resulting from the effect of missed events could be eliminated by analysis of the data with different time resolutions. Normal activity could be accounted for by several different seven-state models with two open and five shut states. As different models could be found that gave identical descriptions of the data, the distributions of open and shut intervals were not sufficient to define a unique model. It was established that no other seven-state models would be found that describe the distributions of open and shut intervals during normal activity better than the most likely presented models.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1985

Single chloride-selective channels active at resting membrane potentials in cultured rat skeletal muscle.

The patch-clamp technique was used to characterize channels that could contribute to the resting Cl-conductance in the surface membrane of cultured rat skeletal muscle. Two Cl- -selective channels, in addition to the Cl- -selective channel of large conductance described previously (Blatz and Magleby, 1983), were observed. One of these channels had fast kinetics and a conductance of 45 +/- 1.8 pS (SE) in symmetrical 100 mM KCl. The other had slow kinetics and a conductance of 61 +/- 2.4 pS. The channel with fast kinetics typically closed within 1 ms after opening and flickered between the open and shut states. The channel with slow kinetics typically closed within 10 ms after opening and displayed less flickering. Both channels were active in excised patches of membrane held at potentials similar to resting membrane potentials in intact cells, and both were open a greater percentage of time with depolarization. Under conditions of high ion concentrations, both channels exhibited nonideal selectivity for Cl- over K+ with the permeability ratio PK/PCl of 0.15-0.2. Additional experiments on the fast Cl- channel indicated that its activity decreased with lowered pHi and that SO2-4 and CH3SO-4 were ineffective charge carriers. These findings, plus the observation that the fast Cl- channel was also active in membrane patches on intact cells, suggest that the fast Cl- channel provides a molecular basis for at least some of the resting Cl- conductance. The extent to which the slow Cl- channel contributes is less clear as it was typically active only after excised patches of membrane had been exposed to high concentrations of KCl at the inner membrane surface.

Animals↗
PubMed1984

Ion conductance and selectivity of single calcium-activated potassium channels in cultured rat muscle.

The conductance and selectivity of the Ca-activated K channel in cultured rat muscle was studied. Shifts in the reversal potential of single channel currents when various cations were substituted for Ki+ were used with the Goldman-Hodgkin-Katz equation to calculate relative permeabilities. The selectivity was Tl+ greater than K+ greater than Rb+ greater than NH4+, with permeability ratios of 1.2, 1.0, 0.67, and 0.11. Na+, Li+, and Cs+ were not measurably permeant, with permeabilities less than 0.05 that of K+. Currents with the various ions were typically less than expected on the basis of the permeability ratios, which suggests that the movement of an ion through the channel was not independent of the other ions present. For a fixed activity of Ko+ (77 mM), plots of single channel conductance vs. activity of Ki+ were described by a two-barrier model with a single saturable site. This observation, plus the finding that the permeability ratios of Rb+ and NH+4 to K+ did not change with ion concentration, is consistent with a channel that can contain a maximum of one ion at any time. The empirically determined dissociation constant for the single saturable site was 100 mM, and the maximum calculated conductance for symmetrical solutions of K+ was 640 pS. TEAi+ (tetraethylammonium ion) reduced single channel current amplitude in a voltage-dependent manner. This effect was accounted for by assuming voltage-dependent block by TEA+ (apparent dissociation constant of 60 mM at 0 mV) at a site located 26% of the distance across the membrane potential, starting at the inner side. TEAo+ was much more effective in reducing single channel currents, with an apparent dissociation constant of approximately 0.3 mM.

Ammonia↗
PubMed1983

Single voltage-dependent chloride-selective channels of large conductance in cultured rat muscle.

Single-channel currents of an anion-selective channel in the plasma membrane of cultured rat muscle cells (myotubes) were recorded with the patch-clamp technique (Hamill, O.P., A. Marty, E. Neher, B. Sakmann, and F.J. Sigworth, 1981. Pfluegers Arch. Eur. J. Physiol., 391:85-100). The channel is selective for Cl- over cations, and has an unusually large single-channel conductance of approximately 430 pS in symmetrical 143 mM KCl. The channel is often active at 0 mV, opening and closing spontaneously. When active, steps from 0 mV to either negative or positive membrane potentials close the channel to an apparent inactivated state. The mean effective time that a channel is open before it inactivates is approximately 1.19 s for steps to -30 mV and 0.48 s for steps to +30 mV. Returning the membrane potential to 0 mV results in recovery from inactivation. Calcium ions are not required for channel activity.

Animals↗
PubMed1983

Calcium dependence of open and shut interval distributions from calcium-activated potassium channels in cultured rat muscle.

The stochastic properties of single Ca-activated K channels in excised patches of surface membrane from cultured rat muscle cells were studied using the patch-clamp technique. The distribution of all open intervals was described by the sum of two exponential distributions of short and long mean open time, suggesting at least two major open-channel states. Increasing the concentration of Ca at the inner membrane surface, [Ca]i, increased the mean duration of the long open distribution, while having little effect on the mean duration of the short open distribution. The frequency of openings to each distribution increased with [Ca]i. The rate of increase was a much steeper function of [Ca]i for openings in the long open distribution than for openings in the short open distribution; about 80% of the openings were to the long open distribution with 0.1 microM-Cai, increasing to 97% with 1 microM-Cai (+ 30 mV). These results suggest that openings in both open distributions are Ca-dependent, with openings in the long open distribution requiring the binding of more Ca ions than openings in the short open distribution. The distribution of all shut intervals at 0.5 microM-Cai and + 30 mV was described by the sum of three exponential distributions with mean durations of: 0.21 msec (short shut distribution), 1.90 msec (intermediate shut), and 44 msec (long shut). These results indicate that the channel typically enters at least three closed channel states during normal channel activity. In addition, a few longer shut intervals not accounted for by the above distributions suggested that there was a fourth infrequently occurring inactivated closed-channel state. The mean duration of the distribution of long shut intervals decreased with a power of about 2 with increasing [Ca]i under conditions where most openings were to the long open state (+ 30 mV, 0.25-1 microM-Cai). This observation suggests that openings to the long open distribution typically require the binding of two or more Ca ions. The mean intermediate shut interval appeared to increase slightly with increasing [Ca]i while the mean short shut interval was relatively Cai-independent. The percentage of all shut intervals that were short shut intervals increased with increasing [Ca]i while the percentage of long shut intervals decreased.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1983

Burst kinetics of single calcium-activated potassium channels in cultured rat muscle.

Burst kinetics of single Ca-activated K channels in excised patches of surface membrane from cultured rat muscle were studied using the patch-clamp technique. Channel activity was separated into bursts using a calculated gap derived from the distribution of shut intervals. Shut intervals greater than the calculated gap were taken as gaps between bursts. The distribution of burst duration was described as the sum of two exponentials with mean durations of about 0.8 and 24 msec (1 microM-Cai, + 20 mV), suggesting two classes of bursts (short and long). The composition of short and long bursts was determined from comparisons of the distributions of open intervals, unit bursts (bursts of single openings), and openings/burst. Short bursts consisted mainly of single openings to the open channel state of short mean lifetime. Long bursts consisted of one or more openings to the (compound) open-channel state of long mean lifetime, plus, in fewer than 70% of the long bursts, one or more openings to the short open-channel state. The frequency of occurrence of bursts from each class first increased and then decreased with increasing [Ca]i, with the number of long bursts increasing at a greater rate than the number of short bursts. The number of openings/short burst was relatively independent of [Ca]i, while the number of openings/long burst increased, often more than linearly, with increasing [Ca]i. This increase arose almost entirely from an increase in openings to the long open state. These results suggest that openings to the long open state typically require the binding of three or more Ca ions, and openings to the short open state typically require the binding of at least one Ca ion. This is the case whether the openings occur in isolation as bursts of single openings or in bursts composed of both types of openings. An obvious burst of channel activity would occur when the channel opens and closes several times without losing all its bound Ca. The power relationship between [Ca]i and the percentage of time spent in the open state is accounted for in terms of the effects of [Ca]i upon mean channel open time, openings/burst, and burst rate. A model is presented that describes quantitatively many features of the burst kinetics of the Ca-activated K channel for constant [Ca]i.

Animals↗
PubMed1982

Augmentation and facilitation of transmitter release. A quantitative description at the frog neuromuscular junction.

Endplate potentials were recorded from frog and toad sartorius neuromuscular junctions under conditions of greatly reduced quantal contents. The magnitudes of augmentation increased with the duration and frequency of stimulation, often increasing at an accelerating rate during 10-20-s conditioning trains. The magnitudes of the first and second components of facilitation also increased, but reached apparent steady state values within the first few seconds of stimulation. These observations could be accounted for by assuming (a) that augmentation and the first and second components of facilitation arise from underlying factors in the nerve terminal that act to increase transmitter release; (b) that each nerve impulse adds an increment to each of the underlying factors; (c) that the magnitude of the increment typically increases during the train for augmentation but remains constant for the components of facilitation; and (d) that the underlying factors decay with first-order kinetics with time constants of approximately 7 s for augmentation and 60 and 500 ms for the first and second components of facilitation, respectively. The increments of facilitation added by each impulse were about twice as large in the toad as in the frog. Facilitation was described better by assuming a power relationship between the underlying factor and the observed facilitation than by assuming a linear relationship. Augmentation was described by assuming either a linear or power relationship.

Animals↗
PubMed1982

A quantitative description of stimulation-induced changes in transmitter release at the frog neuromuscular junction.

Endplate potentials were recorded from frog sartorius neuromuscular junctions under conditions of greatly reduced quantal contents to develop a quantitative description of stimulation-induced changes in transmitter release. Four general models relating potentiation, augmentation, and the first and second components of facilitation to transmitter release were developed. These models were then tested by incorporating equations for the kinetic properties of the four components of increased transmitter release and examining the ability of the resulting sets of equations to predict stimulation-induced changes in transmitter release. Three of the models were essentially consistent with the observation that augmentation had a multiplicative type relationship to facilitation. These models could also predict the effect of frequency and duration of stimulation on endplate potential (EPP) amplitude during and after prolonged (40 s) trains including the response to step changes in stimulation rate. These models extend by about two orders of magnitude the duration of stimulation-induced changes in transmitter release that can be accounted for, and show that the combined kinetic properties of potentiation, augmentation, and the two components of facilitation are generally sufficient to account for these changes.

Animals↗
PubMed1982

Properties of single calcium-activated potassium channels in cultured rat muscle.

1. Properties of the Ca-activated K channel were studied in excised patches of surface membrane from cultured rat muscle cells using single channel recording techniques.2. Increasing the concentration of calcium at the intracellular membrane surface [Ca](i), increased both the frequency and effective duration of channel openings. Ca at the extracellular membrane surface was not sufficient to activate the channels.3. An approximate third power relationship (slope = 2.7) was observed between [Ca](i) and the percentage of time the channels spent in the open state.4. Both the frequency and effective duration of channel openings increased as the intracellular membrane surface was made more positive; the percentage of time spent in the open state increased e-fold for a 15 mV depolarization for low levels of activity.5. The percentage of time spent with 1, 2,...n channels open in membrane patches with n channels was described by the binomial distribution, suggesting that the channels opened and shut independently of one another.6. Single channel conductance (144 mM-K on both sides of the membrane) was essentially independent of membrane potential (-50 to +50 mV) and [Ca](i) (0.1 muM -1 mM), but did increase with temperature, from 100 pS at 1 degrees C to 300 pS at 37 degrees C.7. Channel activity occurred in apparent bursts, with the duration of the apparent bursts increasing with increasing [Ca](i).8. Two exponentials were required to describe the distribution of observed channel open times, suggesting two different open channel states of apparently normal conductance. The observed mean channel open time of these states at +30 mV was 0.34 and 2.2 msec with 0.1 muM-Ca(i) and was 0.47 and 6.9 msec with 0.5 muM-Ca(i).9. The channel occasionally entered an apparent third open channel state with a single channel current amplitude about 40% the amplitude of the normally observed single channel currents. The reduced conductance state was immediately preceded and followed by a normal conducting state.10. While the kinetics of the Ca-activated K channel appear complex, its large conductance and high Ca and voltage sensitivity suggest that it is uniquely suited to resist depolarizations of the cell membrane potential that are accompanied by increases in intracellular Ca.

Animals↗
PubMed1981

Changes in miniature endplate potential frequency during repetitive nerve stimulation in the presence of Ca2+, Ba2+, and Sr2+ at the frog neuromuscular junction.

Miniature endplate potentials (MEPPs) were recorded from frog sartorious neuromuscular junctions under conditions of reduced quantal contents to study the effect of repetitive nerve stimulation on asynchronous (tonic) quantal transmitter release. MEPP frequency increased during repetitive stimulation and then decayed back to the control level after the conditioning trains. The decay of the increased MEPP frequency after 100-to 200-impulse conditioning trains can be described by four components that decayed exponentially with time constants of about 50 ms, 500 ms, 7 s, and 80 s. These time constants are similar to those for the decay of stimulation-induced changes in synchronous (phasic) transmitter release, as measured by endplate potential (EPP) amplitudes, corresponding, respectively, to the first and second components of facilitation, augmentation, and potentiation. The addition of small amounts of Ca2+ or Ba2+ to the Ca2+-containing bathing solution, or the replacement of Ca2+ with Sr2+, led to a greater increase in the stimulation-induced increases in MEPP frequency. The Sr-induced increase in MEPP frequency was associated with an increase in the second component of facilitation of MEPP frequency; the Ba-induced increase with an increase in augmentation. These effects of Sr2+ and Ba2+ on stimulation-induced changes in MEPP frequency are similar to the effects of these ions on stimulation-induced changes in EPP amplitude. These ionic similarities and the similar kinetics of decay suggest that stimulation induced changes in MEPP frequency and EPP amplitude have some similar underlying mechanisms. Calculations are presented which show that a fourth power residual calcium model for stimulation-induced changes in transmitter release cannot readily account for the observation that stimulation-induced changes in MEPP frequency and EPP amplitude have similar time-courses.

Animals↗
PubMed1981

Is the quantum of transmitter release composed of subunits? A critical analysis in the mouse and frog.

1. Miniature end-plate potentials (m.e.p.p.s) were recorded from mouse diaphragm and frog cutaneous pectoris muscles and miniature end-plate currents (m.e.p.c.s) were recorded from frog cutaneous pectoris to investigate the proposal that the m.e.p.p. is built up of one to thirty subunits. Evidence for this hypothesis is drawn mainly from the observations that there is a class of m.e.p.p.s smaller than the classical m.e.p.p.s, and that histograms of m.e.p.p. amplitudes display multiple peaks which often appear to be regularly spaced and which extend throughout the histograms; in terms of the subunit hypothesis each successive peak results from an increasing integral number of subunits per m.e.p.p. (Kriebel, Llados & Matteson, 1976; Wernig & Stirner, 1977).2. Histograms of m.e.p.p. amplitudes and m.e.p.c. areas confirmed the existence of two classes of m.e.p.p.s as reported by Kriebel & Gross (1974): a larger class (well described by a Gaussian curve) which consists of the classical m.e.p.p.s, and a smaller class with amplitudes considerable less than the classical m.e.p.p.s.3. Histograms of m.e.p.p. amplitudes and m.e.p.c. areas showed multiple peaks that extended throughout the histograms.4. Autocorrelations of the histograms, an unbiased method used to test for regularity in data, showed that the multiple peaks were not regularly spaced, as required by the subunit hypothesis.5. A series of computer simulations demonstrated that, for expected levels of base-line noise in the recording system, multiple peaks that extend throughout histograms of m.e.p.p. amplitudes could arise from subunits only if the standard deviation of the subunit amplitude were less than 2-5% of the mean subunit amplitude and the standard deviation of the variability in post-synaptic sensitivity were less than 2% of the mean post-synaptic sensitivity. It seems unlikely that the variability in post-synaptic sensitivity and in proposed subunit amplitude would be as small as this.6. Taking more realistic estimates for the standard deviation of the subunit amplitude of 12% of the mean subunit amplitude and standard deviation of the variation in post-synaptic sensitivity of 4% of the mean sensitivity, it was found that at most three to four regularly spaced peaks would be apparent in m.e.p.p. amplitude histograms due to subunits.7. On the basis of these theoretical considerations it seems doubtful that the multiple peaks observed to extend throughout histograms of m.e.p.p. amplitudes could arise from subunits; therefore, the experimental data that have been used to support the subunit hypothesis are unlikely to have arisen from a subunit mechanism.8. We suggest that there are few, if any, data that directly support the subunit hypothesis. The multiple peaks observed to extend throughout m.e.p.p. amplitude histograms most likely arise from random variation in the data, although additional factors cannot be ruled out.

Animals↗
PubMed1981

The effect of (+)-tubocurarine on neuromuscular transmission during repetitive stimulation in the rat, mouse, and frog.

1. The effect of tubocurarine on amplitudes of end-plate currents in response to trains of repetitive stimulation (50-150/sec) was investigated in voltage-clamped muscle fibres of the rat, mouse and frog. 2. In rat and mouse muscle, the presence of tubocurarine led to a more rapid decline (rundown) in the amplitudes of successive end-plate currents during trains of impulses. In frog, tubocurarine caused an increase in apparent facilitation of end-plate current amplitudes during the first few impulses of repetitive stimulation; this increase was followed by a more rapid rundown of end-plate current amplitude. 3. These effects of tubocurarine appear not to be an artifact resulting from inadequate control of membrane potential in voltage-clamped fibres. 4. The more rapid rundown during trains of end-plate currents in the presence of tubocurarine showed little variation with membrane potential indicating that voltage-sensitive channel blockade by tubocurarine was not a major factor contributing to the rundown. 5. The effect of tubocurarine on the apparent facilitation and rundown of end-plate current amplitudes was typically decreased by reducing the frequency of stimulation. 6. These results suggest that tubocurarine affects transmitter release at neuromuscular junctions during repetitive stimulation.

Animals↗
PubMed1981

A study of desensitization of acetylcholine receptors using nerve-released transmitter in the frog.

1. Desensitization of acetylcholine (ACh) receptors was studied at the frog neuromuscular junction under voltage clamp.2. ACh was applied directly to junctional receptors by stimulating the motor nerve with trains of impulses. End-plate currents (e.p.c.s) were used to estimate the total number of channel openings by the junctional ACh receptors, and miniature end-plate currents (m.e.p.c.s) were used to measure changes in post-synaptic sensitivity. Under the conditions of these experiments the changes in m.e.p.c. amplitudes were shown to be post-synaptic in origin and thus provided a measure of desensitization.3. When the acetylcholinesterase was inhibited with diisopropylfluorophosphate, neostigmine, or collagenase treatment to prolong the duration of the nerve-released ACh in the synaptic cleft, desensitization developed during repetitive stimulation of 1000 impulses at 5-33 impulses/sec and then recovered after the conditioning trains, with a time constant of about 25 sec.4. When the acetylcholinesterase was active so that the duration of ACh in the synaptic cleft resulting from each nerve impulse was brief (< 300 musec), desensitization developed in response to 300-500 pairs of nerve stimuli if the interval between the impulses of each pair was 25 msec or less. When the interval was 30 msec or greater, however, measurable desensitization did not occur, even if the total number of channel openings was many times greater than in the experiments with shorter intervals or inhibited esterase where desensitization readily occurred.5. The desensitization observed to pairs of impulses was enhanced by chlorpromazine and decreased when the post-synaptic membrane was depolarized, properties similar to those described previously for desensitization to bath and ionophoretic application of ACh.6. These results indicate that desensitization to nerve-released transmitter is not a simple consequence of receptor activation, is not due to blockade of the open receptor channels by ACh, and does not result from ACh binding directly to desensitized receptors with a resulting shift in the receptor population towards the desensitized state.7. We suggest that the desensitization observed to nerve-released transmitter is a two-step process with both steps initiated by ACh. In the first step ACh converts some receptors into a desensitizable state which has an apparent lifetime of less than 30 msec; in the second step ACh desensitizes the desensitizable state.

Acetylcholine↗
PubMed1980

Differential effects of Ba2+, Sr2+, and Ca2+ on stimulation-induced changes in transmitter release at the frog neuromuscular junction.

Endplate potentials (EPP) were recorded from the frog sartorius neuromuscular junction under conditions of low quantal content to study the effect of Ba2+, Sr2+, and Ca2+ on the changes in evoked transmitter release that occur during and after repetitive stimulation. The addition of 0.1-1 mM Ba2+ or Sr2+ to the Ca2+-containing bathing solution, or the replacement of Ca2+ with 0.8-1.4 mM Sr2+, led to a greater increase in EPP amplitudes during and immediately after repetitive stimulation. These changes in release were analyzed in terms of the four apparent components of increased transmitter release that have previously been distinguished on the basis of their kinetic properties. The Ba2+-induced increase in EPP amplitudes was associated with an increase in the magnitude but not the time constant of decay of augmentation. Ba2+ had little effect on potentiation or the first and second components of facilitation. The Sr2+-induced increase in EPP amplitudes was associated with an increase in the magnitude and the time constant of decay of the second component of facilitation. Sr2+ had little effect on potentiation, augmentation, or the first component of facilitation. The selective effects of Ba2+ on augmentation and of Sr2+ on the second component of facilitation were reversible and could be obtained in the presence of the other ion. The addition of 0.1-0.3 mM Ca2+ to the bathing solution had little effect on potentiation, augmentation, or the two components of facilitation. These results provide pharmacological support for the proposal that there are four different components of increased transmitter release associated with repetitive stimulation and suggest that the underlying factors in the nerve terminal that give rise to these components can act somewhat independently of one another.

Animals↗