Membrane potential modulates the activation of GABA-gated channels

1. The activity of single gamma-aminobutyric acid (GABA)-gated Cl- channels (GABA = 0.5-2.0 microM) was recorded in inside-out patches of membrane from cultured chick cerebral neurons. 2. The distribution of open intervals of the GABA channel was described by the sum of two exponentials, which suggests the presence of at least two open states of the channel. The time constants of these two components were 0.39 +/- 0.1 and 2.1 +/- 0.9 ms (+/- SD, n = 9). 3. The distribution of shut intervals was described by the sum of either three (n = 5) or four (n = 3) exponentials. This suggests the presence of at least three or four shut states. 4. At all GABA concentrations examined, the activity of the GABA channel decreased over time. This decline in activity was most likely the result of desensitization of the GABA channels. 5. The distribution of open intervals was unchanged during desensitization of the GABA channel. Thus desensitization is not associated with an alteration in either the mean lifetime of the two open states or the relative number of transitions to these two states. Rather, desensitization results from a decrease in the probability of channel opening. 6. There was an e-fold increase in the probability of finding a GABA channel open for every 80 +/- 43 mV (n = 4) of depolarization. The degree of voltage dependence decreased as the GABA channels desensitized. 7. The depolarization-induced increase in open channel probability was not associated with any change in the distribution of open intervals. Thus depolarization does not affect the mean open time of the channel but rather increases the likelihood that it will open. 8. A simple model with three or four shut and two open states is considered for the gating of the GABA channel by the agonist. Possible sites for the voltage dependence within this proposed model are discussed.     

Temporal clustering of ion channel openings incorporating time interval omission

Ion channel gating mechanisms can be satisfactorily modelled by a time-reversible continuous-time Markov chain on a finite state space. The complete process is not observable, but rather the state space is partitioned into 'open' and 'closed' states corresponding to the receptor channel being open or closed, and it is only possible to observe whether the process is in an open or a closed state. Previous studies of locust muscle glutamate receptor channels have revealed single channel openings to be highly clustered in time. This clustering can be described by the ratio of the variance var N(t) to the mean E[N(t)] of the number of channel openings in a time interval of length t. In this paper we obtain expressions for (formula; see text) for the above aggregated Markov process. Applications of these expressions to a model for the locust muscle glutamate receptor channel show this aspect of the model to be reasonably consistent with experimental data. In practice very short sojourns in either the open or closed states will fail to be detected, a phenomenon known as time interval omission. Using a semi-Markov approach, we outline a general theoretical framework for analysing dynamic properties of aggregated Markov processes incorporating time interval omission. We illustrate the applicability of this framework by using it to find limt----infinity [[var N(t)]/E[N(t)]] theoretically, when time interval omission is incorporated. This allows us to study the robustness of limt----infinity [[var N(t)]/E[N(t)]] to time interval omission, as a measure of temporal clustering.     

Calcium-activated potassium channels in goldfish hair cells.

1. Characteristics of Ca(2+)-activated K+ channels in the basolateral membrane of hair cells isolated from the caudal part of the goldfish saccular macula were studied mainly with the inside-out mode of the patch clamp method. 2. Several types of Ca(2+)-activated K+ channels differing in unitary conductance were identified. The conductances (n = 156) ranged from 130 to 320 pS (when measured in symmetrical 125 mM KCl) and could be roughly separated into four groups, centred on values of 150, 200, 250 and 300 pS. The pharmacological profile, assessed by, for example, tetraethylammonium blockade, and the relatively large conductance indicated that these channels can be classified as large-conductance Ca(2+)-activated K+ channels (BK channels). The relative permeability of these channels to different ion species was in the order K+ (1.0) > Rb+ (0.8) > NH4+ (0.14) > Na+, Cs+ (< 0.05). 3. Curves relating open state probability to [Ca2+]i, for membrane potentials between -50 and +50 mV, were similar to those observed for BK channels of rat muscle. However, the maximum open state probability (100-1000 microM [Ca2+]i and 50 mV membrane potential) was 0.4-0.9, and always less than 1. 4. These channels had a short arithmetic mean open time ranging from 0.08 to 1.2 ms (0.08-0.5 ms in 88% of cases) and an arithmetic mean shut time ranging from 0.24 to 1.2 ms (10 microM [Ca2+]i and 50 mV membrane potential). The shut intervals were more sensitive to changes in [Ca2+]i and membrane potential than were the open intervals. 5. The distribution of individual open and shut intervals was fitted with the sum of exponential functions. Except for the slowest shut component, which only accounted for less than 1% of shut events, all other components had time constants shorter than 1 ms. As a result of these short open and shut intervals, the current trace had a flickery pattern rather than a burst-interburst pattern. 6. There was a rough correlation between unitary conductance and mean open time, i.e. channels with a large unitary conductance had a longer mean open time. 7. The sensitivity to [Ca2+]i of the Ca(2+)-activated K+ channel in goldfish hair cells was one to two orders of magnitude lower than that of BK channels in rat muscle. Channels with a longer mean open time had a higher Ca2+ sensitivity. 8. The stability of the single Ca(2+)-activated K+ channel kinetics was studied by measuring the 'moving' mean duration of open and shut intervals.(ABSTRACT TRUNCATED AT 400 WORDS)     

Deterministic Model of Ion Channel Flipping with Fractal Scaling of Kinetic Rates

The flipping of ion channels in biological membranes has usually been modeled in terms of Markov transitions between open and closed states. The basic assumption of this approach is that channel flipping between open and closed states is an inherent stochastic process, due to random thermal fluctuations of units forming the channel protein. In this paper, we propose a different view of channel flipping, one not involving external stochastic causes. We consider the channel as a physical dynamic system, the unpredictable flipping of which is due to a deterministic mechanism which sustains a chaotic dynamics. In particular, we presume the changes in the channel conformation are due to delayed interaction between the ionic flow through the channel and the protein forming the channel. The model proposed here describes the channel by means of macroscopic physical quantities such as conductance, current, membrane, and reversal potentials and predicts open and closed dwell time distributions consisting of multiple exponential components and exhibiting power-law scaling over a wide range of time scales. The effective kinetic rate computed through use of simulation data shows fractal properties in good agreement with those seen experimentally. This mathematical model of the ion channel is physically consistent in terms of a plausible real system and may provide a novel key to understanding the complex behavior of the flipping process. © 1999 Biomedical Engineering Society. PAC99: 8716Uv, 8710+e, 8717Aa, 0545Df, 8716Dg, 8719Nn, 8714Ee     

Desensitization of diliganded mouse muscle nicotinic acetylcholine receptor channels

Nicotinic ACh receptor channels (AChRs) exposed to high concentrations of ACh adopt 'desensitized' conformations that have a high affinity for the transmitter and no measurable ion conductance. Single-channel currents elicited by 0.1 or 1 m m ACh were recorded from human embryonic kidney (HEK) cells that had been transiently transfected with mouse α, β, δ, and ε subunits. On the time scale of ∼0.1 ms to ∼1 h, apparent open intervals are described by a single exponential component, and shut intervals associated with desensitization are described by the sum of four or five exponential components. The kinetic behaviour appeared to be stationary and homogeneous. Desensitization rate constants were estimated by kinetic modelling of currents from cell-attached and outside-out patches (where the number of channels in the patch was measured). A single AChR recovered from the longest-lived desensitized state only after ∼5 min. The occupancy of an AChR for each of the desensitized states was calculated as a function of time after the continuous application of a pulse of saturating ACh. The longest-lived desensitized state accounted for 90 % of the total only after several seconds. The fractional recovery from desensitization (during a 200 ms wash period) decreased as the duration of the desensitizing pulse increased, suggesting that recovery is slower from the longer-lived desensitized states. The free energy landscape for the AChR desensitization reaction in cell-attached patches exhibited an initial destabilization, followed by a plateau region of gradually increasing stability, followed by a deep well.     

Terminal distributions along a 'knight's line' for a stochastic epidemic

The model under consideration relates to the spread of a disease in a finite population where removal of infectives is allowed to occur. It is equivalent to a restricted random walk over a discrete grid of points interior to a trapezium. We consider an artificial barrier on the grid at which the epidemic ceases. The barrier is a semi-diagonal resembling the path of the knight in chess and allows some simplification of the analysis. In particular, we investigate the probability distribution along this barrier when time is infinite. Recursive relations are obtained for these terminal distributions and are used to develop an exact numerical method for their computation. In certain cases, a slight modification of the binomial distribution provides a good approximation to the actual distribution. A direct recursive method for calculating moments of the distribution is given and we show how the whole distribution may be recovered from these moments.     

A two-type model for the Cuban national programme on HIV/AIDS

We study deterministic and stochastic versions of a birth anddeath process for a two-type population with immigration forboth types. For the stochastic model we consider the case wherethe rates are time dependent, and also when they are constant,as is the case in our AIDS application. We derive the probabilitygenerating function of the bivariate process and the expectationsof the marginal processes. We also study the marginal behaviourof the bivariate process in a particular case where we supposethat the first event is an immigration, and examine the behaviourof the marginal processes divided by their expectations. Finally,we apply some of these results to a sexual-partner notificationsystem, as in the Cuban national programme on HIV/AIDS.     

Regulation of single NMDA receptor channel activity by alpha-actinin and calmodulin in rat hippocampal granule cells

The NMDA receptor is modulated by changes in the intracellular calcium concentration, through activation of various intracellular calcium-dependent proteins. We have investigated regulation of single NMDA receptor channel activity by the calcium-sensing proteins alpha-actinin and calmodulin. Both of these proteins bind to the NMDA receptor NR1 subunit C-terminus at the C0 region where they compete for occupation of the C0 site and contribute to calcium-dependent inactivation of NMDA receptor-mediated whole-cell currents. Calmodulin has also been shown to bind to the neighbouring C1 region where it has been shown to reduce single channel open time. To investigate regulation of single NMDA channel activity by alpha-actinin and calmodulin, we selected concentrations of these two proteins that would result in maximal binding to the C0 region and/or the C1 region in the case of calmodulin. Alpha-actinin binding was found to predominantly decrease single channel shut time, resulting in an increased open probability ( P _open), whereas calmodulin binding reduced single channel mean open time, resulting in an overall reduction in P _open. The physiological implications of these findings are discussed.     

Temperature-dependent subconducting states and kinetics of deltamethrin-modified sodium channels of neuroblastoma cells

The effects of temperature on the properties of sodium channels from mouse neuroblastoma cells modified by the pyrethroid insecticide deltamethrin were investigated using the patch-clamp technique. The study was aimed at determining various states of modified channels which were expected to be revealed by raising the temperature as a result of an increase in channel activity. After exposure to 10 M deltamethrin, the decay of whole cell sodium current at –30 mV was drastically slowed. It is expressed by two exponential functions at 11°C and by three exponential functions at room temperature (22±1° C). Thus, raising the temperature reveals a new process. Whole cell sodium tail currents associated with step repolarization from –30 mV to –100 mV were best fit by the sum of two exponential functions both at 11° C and at room temperature. The decay of the summed modified single sodium channel currents at –30 mV was expressed by a single exponential function at 11° C, and by two exponential functions at room temperature. In keeping with these results, the open time histograms show the single (11° C) and double (room temperature) exponential distributions. Thus, raising the temperature allows a new single channel process to be revealed. Other modified open states observed previously at 11° C were also found at room temperature including a flickering state and a subconducting state. In addition, several new subconducting states were found at room temperature. Furthermore, while at 11° C only a single state exists in which channels open with some delay at –100 mV after the termination of a depolarizing pulse, at room temperature, two such states having different amplitudes were found. The results show that more deltamethrin-modified channel states can be observed at room temperature than at 11° C. Even with this larger variety of channel states, similar states can be observed with normal channels or channels modified by other unrelated agents. This suggests that deltamethrin prolongs a variety of normal channel states.     

Multiple conductance states of newly formed single gap junction channels between insect cells

Two cells of an insect cell line (Aedes albopictus, clone C6/36) were pushed together to form a cell pair while the intercellular current flow was monitored. This approach enabled us to study the formation of gap junction channels and explore their electrical properties. We found that the single channels exhibit multiple conductance states. The conductance of a fully open channel was 365 pS; the subconductance steps were 1/7 to 1/5 of the maximal conductance. The voltage gradient across the junction did not influence the conductance of fully open channels, but affected the dwell time at particular conductance states. The latter provides an explanation for the voltage-dependent conductance of gap junction membranes seen in these cells. The very first channel opening always was slow (15–50 ms), suggesting the involvement of a mechanism different from conventional channel gating.     

Jung's Platypus and Hamlet's Complaint: A Place for Wonder in the Consulting Room

When Guildenstern is confronted with Hamlet's dubious invitation to ‘play upon this pipe’ (a recorder) a constrictive attitude is cruelly but artfully exposed. At another time and place Jung imagines an incredulous response to a creature so absurd that it should not exist (but does): the duck-billed platypus. Would the platypus, like Hamlet, have a complaint to make too? In this paper I use these examples, from very different kinds of literature, as touchstones to explore the ways in which the psychotherapeutic encounter can open up or, alternatively, shut down the way to ‘psychological understanding’, as Jung terms it. The paper addresses both the therapist's and patient's attitude to knowledge and in particular its relation to wonder which, I argue, can function in a way that can fruitfully open up the field for generative therapeutic change.     

The use of lectins in histopathology

Lectins are proteins and glycoproteins extracted predominantly from plants which have the capacity to bind sugars specifically. This property makes them of interest for histopathology since they will bind to saccharides forming parts of glycoproteins and glycolipids of tissue constituents. Lectins have and can be used as reagents for mucin histochemistry, to identify specific cells, in the recognition of glycoprotein alterations in disease states, in studies of infectious diseases, and in the assessment of glycoconjugate alterations occurring with malignancy. They can be used for both light microscopic and ultrastructural localisation and various methods are available. It is important though, to consider the nature of the glycoconjugates under study and select lectins appropriately because of their varying specificities and binding characteristics. A panel of lectins should be used to study a particular configuration. Care should be taken with tissue fixation and processing. It must be remembered that an open and critical mind should be kept concerning interpretation of results. At the present time lectins have a limited value diagnostically, but the binding of Ulex europeus agglutinin to endothelium is certainly of value.     

Single-channel analysis of a point mutation of a conserved serine residue in the S2 ligand-binding domain of the NR2A NMDA receptor subunit

We have examined the function of a conserved serine residue (Ser670) in the S2 ligand-binding region of the NR2A N -methyl- d -aspartate (NMDA) receptor subunit, using recombinant NR1/NR2A receptors expressed in Xenopus laevis oocytes. Mutation of Ser670 to glycine (S670G) in NR2A reduced the potency of glutamate by 124-fold. Single-channel conductance and the duration of apparent open periods of NR2A(S670G) receptor mutants were, however, indistinguishable from wild-type NMDA receptors. NR1/NR2A(S670G) shut-time distributions were best described by a mixture of six exponential components, and the four shortest shut intervals of each distribution were considered to occur within a channel activation (burst). Bursts of single-channel openings were fitted with a mixture of four exponential components. The longest two components carried the majority of the charge transfer and had mean durations of 9.6 ± 0.5 and 29.6 ± 1.5 ms. The overall channel open probability during a burst was high (mean, 0.83 ± 0.06). Consistent with a shortening of NMDA receptor-channel burst lengths was the observation of an increased deactivation rate of macroscopic currents evoked by brief applications of glutamate to outside-out membrane patches. Correlations between shut times and adjacent open times were observed in all data records. Noticeably, shorter than average openings tended to occur next to long closed periods, whereas longer than average openings tended to occur next to short closings. Our single-channel data, together with modelling using a kinetic scheme to describe channel activations, support our hypothesis that the S670G point mutation reduces the dwell time of glutamate in its binding site.     

Self-replicating machines in continuous space with virtual physics

JohnnyVon is an implementation of self-replicating machines in continuous two-dimensional space. Two types of particles drift about in a virtual liquid. The particles are automata with discrete internal states but continuous external relationships. Their internal states are governed by finite state machines, but their external relationships are governed by a simulated physics that includes Brownian motion, viscosity, and springlike attractive and repulsive forces. The particles can be assembled into patterns that can encode arbitrary strings of bits. We demonstrate that, if an arbitrary seed pattern is put in a soup of separate individual particles, the pattern will replicate by assembling the individual particles into copies of itself. We also show that, given sufficient time, a soup of separate individual particles will eventually spontaneously form self-replicating patterns. We discuss the implications of JohnnyVon for research in nanotechnology, theoretical biology, and artificial life.     

States of mind and metacognitive dysfunctions are different in the various personality disorders: a reply to Ryle (2005)

Our reply to Ryle concentrates on the following points: (1) we are not upholding a purely cognitive idea of the schema concept, but rather that individuals are driven by expectations that are open to being rewritten in the course of interpersonal relationships; (2) metacognition is not a purely theoretical activity, but includes an ability to simulate the other's mind and is subject to variations in the course of interpersonal relationships; (3) there is clinical and research evidence supporting the idea that metarepresentative malfunctioning differs between one disorder and another and that it is not a case of a generic difficulty in self-reflection; (4) the individual states of minds are not special features of particular personality disorders (PDs) and do not distinguish a patient from a person without problems. The pathological element is that in the PDs the set of states is limited and an individual is unable to switch into states that are adaptive or free of suffering. Copyright © 2005 John Wiley & Sons, Ltd.     

Kinetic properties of single sodium channels modified by fenvalerate in mouse neuroblastoma cells

(1) The kinetic properties of single sodium channels modified by the pyrethroid fenvalerate have been analyzed by patch clamp techniques using the cultured mouse neuroblastoma cells. (2) Fenvalerate drastically prolonged the open time of single sodium channels from the normal value of 5 ms to several hundred milliseconds during a depolarizing pulse. The channels remained open after termination of a depolarizing pulse for as long as several seconds. (3) The channel lifetime varied with the membrane potential, attained a maximum at –70 mV, and decreased with hyperpolarization and depolarization from –70 mV. (4) Prolonged openings of the modified channels allowed a current-voltage curve for a single channel to be plotted by sweeping a ramp pulse. The single channel conductance had a value of 11 pS and was linear over potentials ranging from 0 to –100 mV. (5) Power density spectral analysis of the open channel current noise indicated a single Lorentzian curve with a cut-off frequency at 90 Hz, indicating that the increase in noise during channel opening resulted from a relatively slow kinetic process. (6) The probability of the channel being modified by fenvalerate was independent of the length of time during which the channel was opened. This observation suggests that channel modification had taken place before the channel opened. This study of the prolonged opening at the single channel level provides a new insight into open channel properties and the kinetics of channel modification.     

Substantial reduction of noradrenaline in kitten visual cortex by intraventricular injections of 6-hydroxydopamine does not always prevent ocular dominance shifts after monocular deprivation

Summary Ten kittens had cannulas inserted into their lateral ventricles for daily injections of 6-hydroxydopamine (6-OHDA). At 5–6 weeks of age one eye was sutured shut, and one week later recordings were made from the visual cortex to assay the ocular dominance of a sample of cells. In six kittens the injections of 6-OHDA were continued until the day before recording, while in four kittens the injections were stopped around the time of eye suture, on the assumption that continued injections of 6-OHDA over several days has effects that are not specific to the noradrenaline (NA) system and that the two procedures might show different results. In all animals the concentration of NA in the visual cortex near the site of recording was reduced by approximately 90%. In all animals the ocular dominance histograms recorded from the visual cortex were shifted so that the majority of cells (83 ± 13%) were dominated by the open eye. There were no substantial differences between the two groups of experimental animals or between the experimental animals and two control animals that had cannulas implanted and ascorbate alone injected without 6-OHDA. We conclude that the concentration of NA in the visual cortex can be reduced substantially by injections of 6-OHDA into the lateral ventricle without preventing the shift in ocular dominance that usually occurs after suturing shut the eyelids of one eye.     

Different chemical reaction times between normal and solid cancer cells

Entropy generation approach has been developed in order to use it for the analysis of complex systems with particular regards to biological systems in order to evaluate their stationary states. The entropy generation is related to the transport processes related to energy flows. Moreover, cancer can be described as an open complex dynamic and self-organizing system. Using the entropy generation approach it is possible to point out different chemical reaction time between normal and solid cancer cells.