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K L Magleby

Publications and source records attributed to K L Magleby.

At least 37 records · Page 2Linked to original sources
PubMed1996

Wanderlust kinetics and variable Ca(2+)-sensitivity of Drosophila, a large conductance Ca(2+)-activated K+ channel, expressed in oocytes.

Cloned large conductance Ca(2+)-activated K+ channels (BK or maxi-K+ channels) from Drosophila (dSlo) were expressed in Xenopus oocytes and studied in excised membrane patches with the patch-clamp technique. Both a natural variant and a mutant that eliminated a putative cyclic AMP-dependent protein kinase phosphorylation site exhibited large, slow fluctuations in open probability with time. These fluctuations, termed "wanderlust kinetics," occurred with a time course of tens of seconds to minutes and had kinetic properties inconsistent with simple gating models. Wanderlust kinetics was still observed in the presence of 5 mM caffeine or 50 nM thapsigargin, or when the Ca2+ buffering capacity of the solution was increased by the addition of 5 mM HEDTA, suggesting that the wanderlust kinetics did not arise from Ca2+ release from caffeine and thapsigargin sensitive internal stores in the excised patch. The slow changes in kinetics associated with wanderlust kinetics could be generated with a discrete-state Markov model with transitions among three or more kinetic modes with different levels of open probability. To average out the wanderlust kinetics, large amounts of data were analyzed and demonstrated up to a threefold difference in the [Ca2+]i required for an open probability of 0.5 among channels expressed from the same injected mRNA. These findings indicate that cloned dSlo channels in excised patches from Xenopus oocytes can exhibit large variability in gating properties, both within a single channel and among channels.

Animals↗
PubMed1996

Wanderlust kinetics and variable Ca(2+)-sensitivity of dSlo [correction of Drosophila], a large conductance CA(2+)-activated K+ channel, expressed in oocytes.

Cloned large conductance Ca2+-activated K+ channels (BK or maxi-K+ channels) from Drosophila (dSlo) were expressed in Xenopus oocytes and studied in excised membrane patches with the patch-clamp technique. Both a natural variant and a mutant that eliminated a putative cyclic AMP-dependent protein kinase phosphorylation site exhibited large, slow fluctuations in open probability with time. These fluctuations, termed "wanderlust kinetics," occurred with a time course of tens of seconds to minutes and had kinetic properties inconsistent with simple gating models. Wanderlust kinetics was still observed in the presence of 5mM caffeine or 50 nM thapsigargin, or when the Ca2+ buffering capacity of the solution was increased by the addition of 5 mM HEDTA, suggesting that the wanderlust kinetics did not arise from Ca2+ release from caffeine and thapsigargin sensitive internal stores in the excised patch. The slow changes in kinetics associated with wanderlust kinetics could be generated with a discrete-state Markov model with transitions among three or more kinetic modes with different levels of open probability. To average out the wanderlust kinetics, large amounts of data were analyzed and demonstrated up to a threefold difference in the [Ca2+]i required for an open probability of 0.5 among channels expressed from the same injected mRNA. These findings indicate that cloned dSlo channels in excised patches from Xenopus oocytes can exhibit large variability in gating properties, both within a single channel and among channels.

Animals↗
PubMed1994

Testing for microscopic reversibility in the gating of maxi K+ channels using two-dimensional dwell-time distributions.

An assumption usually made when developing kinetic models for the gating of ion channels is that the transitions among the various states involved in the gating obey microscopic reversibility. If this assumption is incorrect, then the models and estimated rate constants made with the assumption would be in error. This paper examines whether the gating of a large conductance Ca-activated K+ channel in skeletal muscle is consistent with microscopic reversibility. If microscopic reversibility is obeyed, then the number of forward and backward transitions per unit time for each individual reaction step will, on average, be identical and, consequently, the gating must show time reversibility. To look for time reversibility, two-dimensional dwell-time distributions of the durations of open and closed intervals were obtained from single-channel current records analyzed in the forward and in the backward directions. Two-dimensional dwell-time distributions of pairs of open intervals and of pairs of closed intervals were also analyzed to extend the resolution of the method to special circumstances in which intervals from different closed (or open) states might have similar durations. No significant differences were observed between the forward and backward analysis of the two-dimensional dwell-time distributions, suggesting time reversibility. Thus, we find no evidence to indicate that the gating of the maxi K+ channel violates microscopic reversibility.

Animals↗
PubMed1993

Opening and closing transitions for BK channels often occur in two steps via sojourns through a brief lifetime subconductance state.

Single channel currents were recorded with microsecond time resolution from large-conductance calcium-activated K+ channels to examine the details of the opening and closings transitions. Analysis of averaged closing transitions indicated that the initial average conductance step for closing was to the 90-95% closed channel current level. Averaged brief closings (approximately 50 microseconds) reopened from the initial 90-95% level, whereas averaged longer closings (> 300 microseconds) closed completely from this level over the next 50-100 microseconds. The 90-95% initial closed level in the averaged current records resulted typically from the average of both complete and partial closings. From 45-80% of the initial closings were complete and 20-55% were to brief lifetime (approximately 50 microseconds) subconductance levels at 65-90% of the completely closed level. Averaged opening transitions were typically mirror images of averaged closing transitions. To extend the analysis to the very brief conductance changes that underlie the flickers of the single channel current toward the closed current level, flickers, brief closings, and longer closings were averaged separately and their slopes compared. The slopes were similar (within the 3% resolution of the method), suggesting similar initial conductance steps. Similar initial closing properties for both the briefer and longer closings would be expected if the channel first passed through the kinetic and subconductance states associated with the briefer closings (including flickers) before entering the longer closed states. Such transitions would provide an explanation for the observation that openings and closings often occur in two steps.

Animals↗
PubMed1993

Preventing errors when estimating single channel properties from the analysis of current fluctuations.

The conductance, number, and mean open time of ion channels can be estimated from fluctuations in membrane current. To examine potential errors associated with fluctuation analysis, we simulated ensemble currents and estimated single channel properties. The number (N) and amplitude (i) of the underlying single channels were estimated using nonstationary fluctuation analysis, while mean open time was estimated using covariance and spectral analysis. Both excessive filtering and the analysis of segments of current that were too brief led to underestimates of i and overestimates of N. Setting the low-pass cut-off frequency of the filter to greater than five times the inverse of the effective mean channel open time (burst duration) and analyzing segments of current that were at least 80 times the effective mean channel open time reduced the errors to < 2%. With excessive filtering, Butterworth filtering gave up to 10% less error in estimating i and N than Bessel filtering. Estimates of mean open time obtained from the time constant of decay of the covariance, tau obs, at low open probabilities (Po) were much less sensitive to filtering than estimates of i and N. Extrapolating plots of tau obs versus mean current to the ordinate provided a method to estimate mean open time from data obtained at higher Po, where tau obs no longer represents mean open time. Bessel filtering gave the least error when estimating tau obs from the decay of the covariance function, and Butterworth filtering gave the least error when estimating tau obs from spectral density functions.

Analysis of Variance↗
PubMed1992

Dependency plots suggest the kinetic structure of ion channels.

Ion channels are integral membrane proteins that regulate ionic flux through cell membranes by opening and closing (gating) their pores. The gating can be monitored by observing step changes in the current flowing through single channels, and analysis of the observed open and closed interval durations has provided a window to develop kinetic models for the gating process. One difficulty in developing such models has been to determine the connections (transition pathways) among the various kinetic states involved in the gating. To help overcome this difficulty we present a transform (dependency plot) of the single-channel data that can give immediate insight into the connections. A dependency plot is derived by calculating a contingency table from a two-dimensional (joint density) dwell-time distribution of adjacent open and closed intervals by assuming that the two classified criteria are the open and closed durations of each pair of adjacent intervals. A three-dimensional surface plot of the fractional difference between the numbers of observed interval pairs and the numbers expected if the durations of adjacent intervals are independent then gives the dependency plot. An excess of interval pairs in the dependency plot suggests that the open and closed states (or compound states) that give rise to the interval pairs in excess are directly connected. A deficit of interval pairs suggests that the open and closed states (or compound states) that give rise to the interval pairs in deficit are either not directly connected or that there are additional open-closed transition pathways arising from the directly connected states.

Animals↗
PubMed1992

Ion channels. Preventing artifacts and reducing errors in single-channel analysis.

The power of single-channel analysis techniques has rapidly expanded during the past few years, giving investigators increased ability to identify models and estimate parameters while reducing error and artifacts. At present, however, there is no single best method, as even the most advanced techniques have various limitations which depend on the experimental data and models being examined. Consequently, for the examined models and experimental data, the most critical part of single-channel analysis is to estimate errors and evaluate the ability of the methods used to discriminate among possible gating mechanisms. The magnitudes of the errors and the ability to identify models and estimate parameters depend on the models being examined as well as the experimental conditions and data. Consequently, the evaluation of the errors associated with each method needs to be repeated when the experimental data and examined models change.

Artifacts↗
PubMed1992

Voltage-dependent gating mechanism for single fast chloride channels from rat skeletal muscle.

1. A voltage-dependent gating mechanism for the fast Cl- channel was developed from the analysis of single-channel current records obtained with the patch clamp technique from primary cultures of rat skeletal muscle. Up to 10(6) open and shut intervals were analysed from each of five different excised patches of membrane containing a single fast Cl- channel. 2. Rate constants for a kinetic scheme with six closed and two open states (scheme I) were estimated at a given voltage by maximum likelihood fitting of open and closed dwell-time distributions obtained at that voltage. This procedure was then repeated for data obtained at each of three to eight different membrane potentials for each channel. 3. Plots of the estimated rate constants against membrane potential typically appeared linear on semilogarithmic co-ordinates, consistent with rate constants that are exponentially dependent on voltage. 4. Regression analysis of these plots yielded two parameters for each rate constant: the value of the rate constant at -50 mV (B) and its voltage sensitivity (A). The dwell-time distributions predicted with these parameters and scheme I gave a good description of the experimental dwell-time distributions at all the studied voltages, lending further support for an exponential dependence of rate constants on membrane potential. 5. Estimates of A and B were also obtained by simultaneously fitting dwell-time distributions obtained at three to eight different voltages, in order to better define these parameters. Predicted dwell-time distributions obtained with these estimates and scheme I could approach the theoretical best description of the data for discrete-state Markov models. 6. Eight to twelve of the fourteen rate constants in scheme I appeared voltage sensitive, with effective gating charges ranging from about -1.5 to +1.0 units of electronic charge. 7. The estimated rate constants and their voltage sensitivities for the five analysed channels were generally similar, but showed some heterogeneity. 8. Gating mechanisms which had fewer kinetic states than scheme I, or equal and opposite effective gating charges over each transition barrier, or four or five identical and independent voltage-dependent subunits, all gave poorer descriptions of the data than scheme I. These simpler mechanisms were also ranked below scheme I by the Schwarz criterion, which applies a heavy penalty for additional free parameters. 9. These findings indicate that the voltage dependence of the fast Cl- channel is consistent with a kinetic scheme with six closed and two open states, in which a majority of transitions among the states are voltage dependent.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Accounting for the Ca(2+)-dependent kinetics of single large-conductance Ca(2+)-activated K+ channels in rat skeletal muscle.

1. The Ca(2+)-dependent kinetics of large-conductance Ca(2+)-activated K+ channels from cultured rat skeletal muscle were studied with the patch clamp technique. Data were collected in the absence of Na+ and Mg2+, which can alter the kinetics. About 2 x 10(5) open and shut intervals were analysed from each of five different excised membrane patches containing a single active channel. Analysis was restricted to activity in the normal mode, which includes 96% of the intervals. 2. The open probability (Popen) and dwell-time distributions of open and shut intervals were obtained at three to four different [Ca2+]i for each of the channels. Popen data were also obtained from some multichannel patches. 3. Increasing [Ca2+]i increased Popen. At a pH of 7.0 the Hill coefficient was 3.7 +/- 0.8 (range of 3.0-5.0) and a Popen of 0.5 occurred at 14 +/- 7 microM [Ca2+]i (K0.5) for data obtained at +30 mV (n = 6). At a pH of 7.2 the Hill coefficient was 3.0 +/- 0.5 (range of 2.2-3.7) and K0.5 was 9 +/- 6 microM-Ca2+ (n = 7). The large standard deviations for K0.5 reflect the observation that fourfold differences in K0.5 could be observed for different channels studied under the same experimental conditions. 4. Hill coefficients that can be greater than 3 suggest that the channel may bind four or more Ca2+ to become fully activated. The binding of four Ca2+ before opening would require a minimum of five shut states. This estimate of the minimum number of shut states is in general agreement with that obtained from the number of exponential components in the dwell-time distributions of shut intervals. Thus, two different methods give similar estimates of the minimum number of shut states. If the channel can open with different numbers of bound Ca2+, then this could give rise to the three to four open states suggested by the three to four exponential components in the open dwell-time distributions. 5. Kinetic schemes consistent with the Ca(2+)-dependent kinetics were developed by simultaneously fitting open and shut dwell-time distributions obtained at three to four different [Ca2+]i, using maximum likelihood techniques and corrections for missed events. Such simultaneous fitting can provide an increased ability to define models and rate constants.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Identifying kinetic gating mechanisms for ion channels by using two-dimensional distributions of simulated dwell times.

Ion channels are integral membrane proteins that regulate ionic flux through cell membranes by opening and closing (or gating) their pores. The gating can be monitored by observing step changes in the current flowing through single channels. Analysis of the durations of the open and closed intervals and of the correlations among the interval durations can give insight into the gating mechanism. Although it is well known that the correlation information can be essential to distinguish among possible gating mechanisms, it has been difficult to use this information because it has not been possible to correct the predicted correlations for the distortion of the single-channel data because of filtering and noise. To overcome this limitation we present a method based on a comparison of simulated and experimental two-dimensional dwell-time distributions constructed by analysing simulated and experimental single-channel currents in an identical manner. The simulated currents incorporate the true effects of filtering and noise, the two-dimensional distributions retain the correlation information, and the identical analysis allows direct maximum-likelihood comparison of the simulated and experimental two-dimensional distributions. We show that the two-dimensional simulation method has a greatly increased ability to distinguish among models, compared with methods that use one-dimensional distributions.

Animals↗
PubMed1990

Estimating kinetic parameters for single channels with simulation. A general method that resolves the missed event problem and accounts for noise.

Analysis of currents recorded from single channels is complicated by the limited time resolution (filtering) of the data which can prevent the detection of brief intervals. Although a number of approaches have been used to correct for the undetected intervals (missed events) when identifying kinetic models and estimating parameters, none of them provide a general method which takes into account the true effects of noise and limited time resolution. This paper presents such a method. The approach is to use simulated single-channel currents to incorporate the true effects of filtering and noise on missed events and interval durations. The simulated currents are then analyzed in a manner identical to that used to analyze the experimental currents. An iterative search process using likelihood comparison of two-dimensional dwell-time distributions obtained from the simulated and experimental single-channel currents then allows the most likely rate constants to be determined. The large errors and false solutions that can result from the more typically applied assumptions of no noise and an absolute dead time (idealized filtering) are excluded by the iterative simulation method, and the correlation information contained in the two-dimensional distributions should increase the ability to distinguish among different gating mechanisms. The iterative simulation method is generally applicable to channels which typically open to a single conductance level. For these channels the method places no restrictions on the proposed gating mechanism or the form of the predicted dwell-time distributions.

Ion Channel Gating↗
PubMed1990

Voltage dependence and stability of the gating kinetics of the fast chloride channel from rat skeletal muscle.

1. The voltage dependence and stability of the gating kinetics of the fast Cl-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 five different patches containing a single fast Cl-channel. 2. To test for kinetic stability, plots of the mean durations of consecutive groups of 5-500 open and shut intervals were examined at each voltage. After excluding infrequent entries into both an apparent subconductance state and a long-lived (inactive) shut state, there were no abrupt and sustained changes in the moving means, indicating the absence of obvious shifts to other kinetic modes. The moving means did, however, fluctuate about the overall mean. 3. A comparison of experimental and simulated data indicated that most, but not all, of the fluctuation in the moving means was due to the stochastic variation inherent in the gating process. The fluctuation not accounted for by stochastic variation was mainly expressed as a slow, low-amplitude, component of drift about the mean. This slow component was unlikely to have arisen from measurement errors. 4. To examine whether the slow drift reflected detectable changes in kinetic modes, the data were divided into consecutive groups of 50,000 intervals. The exponential components describing the distributions were remarkably similar among the different groups, with stochastic variation accounting for most of the observed differences. This finding implies a single kinetic mode throughout the experiment. Thus, any changes in channel activity associated with the slow drift would have little effect on the analysis of gating kinetics presented here. 5. Depolarization increased channel open probability, Popen, for all five channels. This increase had a voltage sensitivity of 17 +/- 4 mV per e-fold change (effective gating charge of 1.6 +/- 0.32 electronic charges at 23 degrees C). Popen was 0.5 at -31 +/- 4 mV. 6. The depolarization-induced increase in Popen typically arose from a decrease in the mean shut time (19 +/- 4 mV per e-fold change; effective gating charge of 1.3 +/- 0.3 at 23 degrees C) and an increase in the mean open time (109 +/- 61 mV per e-fold change; effective gating charge of -0.24 +/- 0.13). 7. Neither plots of Popen versus voltage nor plots of the mean open and mean shut time versus voltage were completely described by a single Boltzmann distribution, suggesting multiple voltage-sensitive steps in channel gating.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

Kinetic time constants independent of previous single-channel activity suggest Markov gating for a large conductance Ca-activated K channel.

Models for the gating of ion channels usually assume that the rate constants for leaving any given kinetic state are independent of previous channel activity. Although such discrete Markov models have been successful in describing channel gating, there is little direct evidence for the Markov assumption of time-invariant rate constants for constant conditions. This paper tests the Markov assumption by determining whether the single-channel kinetics of the large conductance Ca-activated K channel in cultured rat skeletal muscle are independent of previous single-channel activity. The experimental approach is to examine dwell-time distributions conditional on adjacent interval durations. The time constants of the exponential components describing the distributions are found to be independent of adjacent interval duration, and hence, previous channel activity. In contrast, the areas of the different components can change. Since the observed time constants are a function of the underlying rate constants for transitions among the kinetic states, the observation of time constants independent of previous channel activity suggests that the rate constants are also independent of previous channel activity. Thus, the channel kinetics are consistent with Markov gating. An observed dependent (inverse) relationship between durations of adjacent open and shut intervals together with Markov gating indicates that there are two or more independent transition pathways connecting open and shut states. Finally, no evidence is found to suggest that gating is not at thermodynamic equilibrium: the inverse relationship was independent of the time direction of analysis.

Animals↗
PubMed1989

Adjacent interval analysis distinguishes among gating mechanisms for the fast chloride channel from rat skeletal muscle.

1. The durations of adjacent open and shut intervals, obtained with the patch-clamp technique from fast Cl- channels in tissue-cultured rat skeletal muscle, were analysed to distinguish among eight previously considered gating mechanisms for the channel which differed in the connections among the states. 2. Open intervals were separated into groups based on the duration of the shut intervals which occurred before or after each open interval. Fitting these conditional open distributions with sums of exponentials indicated that they were described by two exponential components. 3. The time constants of the two components in the conditional open distributions were independent of the adjacent shut interval durations. The observation of invariant time constants is consistent with gating mechanisms in which the rate constants for transitions among the states remain constant with time (discrete Markov process). 4. In contrast to the invariant time constants, the areas of the two components in the conditional open distributions were dependent on the adjacent shut interval durations. The area of the fast open component increased, and the area of the slow open component decreased, as the duration of adjacent shut intervals increased. Thus, it is changes in areas, rather than time constants, which give rise to the observed inverse relationship between the durations of adjacent open and shut intervals. 5. The findings in summary statements 2-4 indicate that at least two open states are connected by independent pathways to different shut states; the open state associated with the fast open component is connected to a shut state (or compound shut state) of longer effective lifetime, and the open state associated with the slow open component is connected to a shut state (or compound shut state) of briefer effective lifetime. 6. Seven of the eight previously considered gating mechanisms were rejected because they did not account for the observed relationships between the durations of adjacent open and shut intervals, when analysed in terms of either conditional open distributions or conditional mean open interval durations. 7. The seven rejected gating mechanisms also did not account for the observed correlations between interval durations, when analysed in terms of correlation coefficients. Adjacent interval and correlation analysis thus provided a means to distinguish among the gating mechanisms.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

Gating scheme for single GABA-activated Cl- channels determined from stability plots, dwell-time distributions, and adjacent-interval durations.

To study the gating of a GABA-activated Cl- channel, currents from single channels activated by 1.0 microM GABA were examined in patches of membrane excised from cultured chick cerebral neurons. The distributions of open and shut interval durations were each described by the sum of 3 exponential components, suggesting that the channel normally enters at least 3 open and 3 shut states. Five different 6-state gating schemes were found that could describe, all equally well, the observed distributions of open and shut interval durations. Plots of the mean duration of open intervals adjacent to shut intervals of specified durations revealed that, on the average, openings of brief duration were adjacent to closings of long duration. This observation indicated 2 or more independent transition pathways between the open and shut states. Examination of the distributions of open intervals adjacent to shut intervals of specified durations revealed that the time constants of the exponential components describing these conditional open-interval distributions were independent of the durations of the adjacent shut intervals. In contrast, the areas changed in a manner consistent with open states of briefer mean lifetimes typically making transitions to shut states of longer mean lifetimes. Four of the 5 gating schemes considered were rejected because they did not predict the relationship between adjacent intervals or because they predicted that the channel should switch between 2 gating modes with markedly different mean open and shut times, which was not a characteristic of the experimental data. The single remaining kinetic scheme could account for the observed kinetic properties of the GABA channel.

Animals↗
PubMed1988

Fractal models are inadequate for the kinetics of four different ion channels.

The gating kinetics of single ion channels have been well described by models which assume that channels exist in a number of discrete kinetic states, with the rate constants for transitions among the states remaining constant in time. In contrast to such discrete Markov models, it has recently been considered whether gating might arise from transitions among a continuum of states, with the effective rate constants for leaving the collections of states given by a fractal scaling equation (Liebovitch, L.S., J. Fischbarg, J.P. Koniarek, I. Todorova, and M. Wang. 1987. Biochim. Biophys. Acta. 896:173-180; Liebovitch, L.S., and J.M. Sullivan. 1987. Biophys. J. 52:979-988). The present study compares discrete Markov with fractal continuum models to determine which best describes the gating kinetics of four different ion channels: GABA-activated Cl channels, ACh-activated end-plate channels, large conductance Ca-activated K (BK) channels, and fast Cl channels. Discrete Markov models always gave excellent descriptions of the distributions of open and shut times for all four channels. Fractal continuum models typically gave very poor descriptions of the shut times for all four channels, and also of the open times from end-plate and BK channels. The descriptions of the open times from GABA-activated and fast Cl channels by the fractal and Markov models were usually not significantly different. If the same model accounts for gating motions in proteins for both the open and shut states, then the Markov model ranked above the fractal model in 35 of 36 data sets of combined open and shut intervals, with the Markov model being tens to thousands of orders of magnitude more probable. We suggest that the examined fractal continuum model is unlikely to serve as a general mechanism for the gating of these four ion channels.

Animals↗