Barry W. Madsen

Evidence for Cooperativity Between Nicotinic Acetylcholine Receptors in Patch Clamp Records

It is often assumed that ion channels in cell membrane patches gate independently. However, in the present study nicotinic receptor patch clamp data obtained in cell-attached mode from embryonic chick myotubes suggest that the distribution of steady-state probabilities for conductance multiples arising from concurrent channel openings may not be binomial. In patches where up to four active channels were observed, the probabilities of two or more concurrent openings were greater than expected, suggesting positive cooperativity. For the case of two active channels, we extended the analysis by assuming that 1) individual receptors (not necessarily identical) could be modeled by a five-state (three closed and two open) continuous-time Markov process with equal agonist binding affinity at two recognition sites, and 2) cooperativity between channels could occur through instantaneous changes in specific transition rates in one channel following a change in conductance state of the neighboring channel. This allowed calculation of open and closed sojourn time density functions for either channel conditional on the neighboring channel being open or closed. Simulation studies of two channel systems, with channels being either independent or cooperative, nonidentical or identical, supported the discriminatory power of the optimization algorithm. The experimental results suggested that individual acetylcholine receptors were kinetically identical and that the open state of one channel increased the probability of opening of its neighbor.

Disopyramide kinetics in patients with acute myocardial infarction

The kinetic behavior of disopyramide was studied in 20 patients with supected myocardial infarction: in 13 of these, the diagnosis was subsequently confirmed. All received a 400‐mg oral loading dose of disopyramide base followed by an oral maintenance regimen of either 100 or 200 mg 4 times daily. The elimination half‐life (t½β) was longer (p < 0.05) in patients with confirmed infarction than in patients with unconfirmed infarction [38.0 ± 3.7 hr (mean ± SEM) compared to 24.3 ± 0.8 hr, and 21.2 ± 2.1 hr compared to 7.2 ± 2.4 hr for the 100‐ and 200‐mg maintenance dose regimens, respectively]. The t½β was dose dependent for infarct and noninfarct patients. Two of the patients with confirmed infarction failed to reach trough plasma levels equal to or exceeding the lower end of the manufacturers recommended therapeutic range (3.3 µg/ml) during the study. For the remaining 11 patients the time taken to achieve trough plasma levels of 3.3 µg/ml varied from 18 to 170 hr; hence plasma disopyramide concentration in these patients was suboptimal at a time when the risk of arrhythmias is high. Modification of existing oral loading dose regimens is therefore required for optimization of oral disopyramide therapy.

Protein binding of tolmetin.

The protein binding of the new nonsteroidal anti‐inflammatory agent tolmetin to human serum albumin (HSA) and to the plasma of 8 healthy subjects was studied by equilibrium dialysis at 37° and pH 7.4 with 14C‐tolmetin. Over the total concentration (Ct) range 3.0 to 28.7 µg/ml (therapeutic range), the fraction oftolmetin unbound to 4% HSA was largely invariant at 0.3%. At 100 µg/ml the unboundfraction rose to 0.8 and at 434 µg/ml to 3.6%. Within the therapeutic concentration range, tolmetin binding to 0.4% HSA was reduced in accordance with the law of mass action and at Ct = 26.2 µg/ml, 10.5% was free. Analysis of the 0.4% HSA data showed tolmetin had 3 classes of binding sites (n1 = 1, Kt = 8.3 × 105 M−1; n2 = 4, K2 = 2.4 × 104 M−1; n3 = 44, K1 = 7.9 × 101 M−1). By studying the binding to 0.4% HSA at 23°, it was established that the free energy change in binding for the first two classes of sites was entirely entropic in nature. Albumin accounted for almost all the binding oftolmetin in human plasma. The effect of other drugs, the tolmetin metabolite McN 2987 (5‐p‐carboxybenzoyl‐l‐methylpyrrole‐2‐acetic acid), tryptophan, and oleic acid on tolmetin binding to 4% HSA was studied using ultrafiltration and 14C‐tolmetin. Aspirin and salicyclic acid decreased tolmetin binding and a combination of aspirin and salicylic acid exerted a synergistic displacing effect. Indomethacin and ibuprofen had no effect while phenylhbutazone and acetaminophen increased tolmetin binding slightly. Tolmetin binding was decreased slightly by McN 2987 and tryptophan and markedly increased by oleic acid. McN 2987 was not bound as extensively as tolmetin. Binding of 14C‐tolmetin to the plasma of 4 arthritic patients was studied by ultrafiltration and found to be less than to normal plasma and 4% HSA. Distribution of tolmetin in the whole blood of 8 healthy subjects using a centrifugation technique showed that the drug was not taken up by red blood cells at therapeutic concentrations.

Fluorescein-labeled naloxone binding to mu opioid receptors on live Chinese hamster ovary cells using confocal fluorescent microscopy

A general method of confocal laser scanning microscopy was used to demonstrate specific binding of fluorescein-labeled naloxone (FNAL, 10-50 nM) to stably transfected mu opioid receptors on live Chinese hamster ovary cells. Nonspecific binding was visually indistinguishable from autofluorescence in cells with intact cell membranes. Fluorescent labeling of cell perimeters, not present in control nontransfected cells, reversed in transfected cells upon washout of FNAL or following the addition of either unlabeled naloxone (25 microM) or the mu specific antagonist CTOP (1 microM). The addition of the delta and kappa specific agonists DPDPE (1 microM) and U50488 (1 microM), respectively, failed to reverse the labeling. Further evidence of specific binding was obtained from kinetic experiments, where it was observed that only transfected cells showed a time-dependent exponential change in fluorescence that permitted estimation of association and dissociation binding rate constants of (5.8+/-0.5, mean+/-S.E.M.)x10(5) M(-1) s(-1) and (3.3+/-0.6)x10(-3) s(-1), respectively and a kinetically derived dissociation constant of 5.7+/-1.4 nM. These estimates were comparable to those obtained under similar conditions in radioligand binding experiments using [3H]-naloxone.

Estimation of single channel kinetic parameters from data subject to limited time resolution

The limited responsiveness of single-channel recording systems results in some brief events not being detected, and if this is ignored parameter estimation from the observed data will be biased. Statistical methods of correcting for this limited time resolution in a two-state Markov model have been proposed by Neher (1983. J. Physiol. (Lond.). 339:663-678) and by Colquhoun and Sigworth (1983. Single Channel Recording. 191-263). However, a numerical study by Blatz and Magleby (1986. Biophys. J. 49:967-980) indicated differences of 3-40% in the corrected values given by the two techniques. Here we explain why Nehers method produces biased results and the Colquhoun and Sigworth approach, which is no more difficult, provides reasonably accurate estimates.

Stochastic Modelling of a Single Ion Channel: An Alternating Renewal Approach with Application to Limited Time Resolution

Stochastic models of ion channels have been based largely on Markov theory where individual states and transition rates must be specified, and sojourn-time densities for each state are constrained to be exponential. This study presents an approach based on random-sum methods and alternating-renewal theory, allowing individual states to be grouped into classes provided the successive sojourn times in a given class are independent and identically distributed. Under these conditions Markov models form a special case. The utility of the approach is illustrated by considering the effects of limited time resolution (modelled by using a discrete detection limit, ξ) on the properties of observable events, with emphasis on the observed open-time (ξ-open-time). The cumulants and Laplace transform for a ξ-open-time are derived for a range of Markov and non-Markov models; several useful approximations to the ξ-open-time density function are presented. Numerical studies show that the effects of limited time resolution can be extreme, and also highlight the relative importance of the various model parameters. The theory could form a basis for future inferential studies in which parameter estimation takes account of limited time resolution in single channel records. Appendixes include relevant results concerning random sums and a discussion of the role of exponential distributions in Markov models.

Cooperative interaction of warfarin and phenylbutazone with human serum albumin

Abstract Phenylbutazone is commonly thought to displace warfarin from human serum albumin (HSA) by direct competition, and the aim of the present study was to confirm or deny this mechanism using standard equilibrium dialysis. A number of titration curves for [14C]warfarin (4–1000 μm) in the presence of fixed concentrations of phenylbutazone (0–2000 μM) were obtained at 25° in 0.1 M, pH 7.0 phosphate buffer. The first two site dissociation constants (K1 and K2) for each of these curves were estimated according to Adairs equation. Comparison of K1 with that predicted for a single site directly competitive model showed large discrepancies, indicating unacceptability of the model beyond experimental error. The data for the first warfarin site could be partly explained by a dual interaction mechanism where at lower phenylbutazone concentration (

Chlorphentermine binding in rat lung subcellular fractions and its displacement by desmethylimipramine

Abstract An examination of possible mechanisms of binding interactions between drugs accumulated in the lung has been made by equilibrium dialysis using [ 14 C]chlorphentermine (CP) in rat lung subcellular fractions (microsomal and 15,000 g and cytosol). By far the greatest CP binding was in the microsomal and 15,000 g fractions and with all isotherms, CP binding was shown to consist of a specific, saturable component and a non-specific partitioning component. Desmethylimipramine (DMI) was the most potent inhibitor of a series of compounds and was selected for detailed study. Generally, as DMI concentration was increased, CP binding decreased, however this relationship was not in accordance with simple direct competitive theory. At 5 × 10 −4 M DMI in the microsomal fraction, CP binding was initially increased with increasing CP concentration. In the 15,000 g fraction, the apparent CP affinity constant did not decrease regularly with increasing DMI concentration, the affinity constant of DMI increased with increasing DMI concentration, a Hill coefficient significantly less than unity was obtained and the binding model became increasingly untenable for higher DMI levels. It is likely that DMI and CP interact in rat lung via a cooperative mechanism involving binding-induced conformational transitions. The possible role of phospholipids in the binding of basic amines in lung is discussed.

Verapamil, neuromuscular transmission and the nicotinic receptor

The effect of verapamil on neuromuscular transmission was studied in the frog by analysing ionophoretic endplate current (iEPC) trains and the growth and decay phases of miniature endplate currents (mepcs). In addition, single channel data on the interaction of verapamil with the nicotinic acetylcholine receptor were obtained from cultured embryonic chick skeletal muscle cells. Verapamil caused both open and closed channel blockade in the iEPC trains. Mepc amplitude was decreased at low micromolar concentrations, and at higher concentrations there was also accelerated mepc decay indicating open channel blockade. The latter effect could not be explained by a sequential channel occlusion mechanism. Analysis of the mepc rising phase showed that low micromolar concentrations of the drug decreased the pool of receptors which could be activated. Single channel data confirmed the specific interaction of verapamil with the nicotinic receptor, showing closed channel blockade at low concentrations, and at higher levels the shortening of open channel lifetime. It is suggested that both forms of blockade may involve desensitization processes.

Superposition properties of interacting ion channels.

Quantitative analysis of patch clamp data is widely based on stochastic models of single-channel kinetics. Membrane patches often contain more than one active channel of a given type, and it is usually assumed that these behave independently in order to interpret the record and infer individual channel properties. However, recent studies suggest there are significant channel interactions in some systems. We examine a model of dependence in a system of two identical channels, each modeled by a continuous-time Markov chain in which specified transition rates are dependent on the conductance state of the other channel, changing instantaneously when the other channel opens or closes. Each channel then has, e.g., a closed time density that is conditional on the other channel being open or closed, these being identical under independence. We relate the two densities by a convolution function that embodies information about, and serves to quantify, dependence in the closed class. Distributions of observable (superposition) sojourn times are given in terms of these conditional densities. The behavior of two channel systems based on two- and three-state Markov models is examined by simulation. Optimized fitting of simulated data using reasonable parameters values and sample size indicates that both positive and negative cooperativity can be distinguished from independence.