Time course of TEA(+)-induced anomalous rectification in squid giant axons

Changes in the voltage clamp currents of squid giant axons wrought by low axoplasmic TEA+ (tetraethylammonium chloride) concentrations (0.3 mM and above) are described. They are: (a) For positive steps from the resting potential in sea water, the K+ current increases, decreases, then increases, instead of increasing monotonically. (b) For positive steps from the resting potential in 440 mM external K+, the current has an exponentially decaying component, whose decay rate increases with axoplasmic [TEA+]. The control currents increase monotonically. (c) For negative steps from the resting potential in 440 mM external K+, the current record has a peak followed by a decay that is slow relative to the control. The control record decreases monotonically. Qualitatively these findings can be described by a simple kinetic model, from which, with one assumption, it is possible to calculate the rate at which K+ ions move through the K+ channels. An interesting conclusion from (c) is that the channels cannot be closed by the normal voltage-sensitive mechanism (described by Hodgkin and Huxley) until they are free of TEA+.     

Properties of channels in the mitochondrial outer membrane

Patch-clamping studies with native outer mitochondrial membranes show a complex behavior. In the range of potentials in which the polarity of the pipette is positive, the behavior resembles that of VDAC incorporated into bilayers. Accordingly, there is a decrease in conductance with voltage. An involvement of VDAC is also supported by responses of the patches to the presence of polyanion or treatment with succinic anhydride, both of which affect VDAC. In contrast, in the negative range of potential, the conductance of the patches generally increases with the magnitude of the voltage. The increase in conductance shows a biphasic time course which is consistent with a model in which channels are first activated (first phase) and then assembled into larger high-conductance channels (second phase). A variety of experiments support the notion that an assembly takes place. The time course of the conductance increase is consistent with formation of the second-phase channels from 6±1 subunits.     

Ion channels in Madin-Darby canine kidney cells.

Ion channels in Madin-Darby canine kidney cells serve transepithelial chloride transport and probably cell volume regulation. Three distinct potassium channels and one anion channel have been revealed by patch clamp studies in Madin-Darby canine kidney cells. The potassium channels are activated by an increase in intracellular calcium activity. A number of hormones activate the potassium channels by an increase in intracellular calcium activity. However, under certain conditions the hormones hyperpolarize the cell membrane without increasing intracellular calcium activity sufficiently to activate the calcium-sensitive potassium channels. Thus, the hormones may activate potassium channels via another, as yet undefined, intracellular mechanism. The anion channel is stimulated by cAMP. Another factor modifying channel activity is cell volume: cell swelling leads probably to subsequent activation of potassium and anion channels. The net result is a variable transient hyperpolarization followed by a sustained depolarization of the cell membrane.     

A model of calcium dynamics in cardiac myocytes based on the kinetics of ryanodine-sensitive calcium channels.

The ryanodine-sensitive calcium channels are pivotal to signal transduction and cell function in many cell types, including cardiac myocytes. In this paper a kinetic model is proposed for these channels. In the model there are two Ca regulatory sites on the channel protein, one positive and the other negative. Cytoplasmic Ca binds to these regulatory sites independently It is assumed that the binding of Ca to the positive site is a much faster process than binding to the negative site. At steady state, the channel opening as a function of the Ca concentration is a bell-shaped curve. The model predicts the adaptation of channels to constant Ca stimulus. When this model is applied to cardiac myocytes, it predicts excitability with respect to Ca perturbations, smoothly graded responses, and Ca oscillations in certain pathological circumstances. In a spatially distributed system, traveling Ca waves in individual myocytes exist under certain conditions. This model can also be applied to other systems where the ryanodine-sensitive channels have been identified.     

Synaptic rectification model equivalent to the correlation-type movement detector

Neural models which are equivalent to the correlation-type movement detector are described. The models involve contrast-coding channels which comprise bandpass linear filters followed by synaptic rectifiers. Linear, one-directional lateral interactions are assumed among the contrast-coding channels. Synaptic rectifiers convert linear spatial interaction into a multiplication-like (quadratic) interaction, which is the core of the correlation-type movement detector. One of the neural models (E-I model) well approximates the correlation model in both time-averaged and dynamic (instantaneous) responses. Possible applicability of the model to movement detection by insects is discussed.     

Mechanical influence and cAMP injection evoke the same reaction of neuron ionic channels.

Intracellular cAMP injection and negative pressure in the patch-electrode increase the interburst closed time of the same potassium ionic channels in the snail neuron membrane. Sodium channels which were registered as change of background noise are activated both by cAMP injection and by negative pressure. These results are considered in connection with data about the unusual biochemistry of the neuron reaction to cAMP.     

Mechanism of excitation and contraction uncoupling in frog and guinea pig myocardial fibers during block of slow sodium-calcium channels by compound D-600]

In the experiments performed on the strips of frog's atria and ventricle it was found that rhythmic stimulation facilitated the dissociation of excitation--contraction coupling caused by D-600 compound through the block of calcium channels, i.e. D-600 prevented the increase of tension in the series of contractions or even converted positive staircase into negative one. The first contraction was affected by D-600 to a lesser degree than the subsequent ones. Guinea pig's left auriculus depressing action of D-600 was found to be strongly dependent on the frequency of stimulation. The data obtained were analysed by the model of excitation--contraction coupling. It was concluded that in amphibians as well as in mammalians tension amplitude during rhythmic activity of myocardial cells is determined mainly by Ca inflow through the excitable calcium channels. On the other hand the amplitude of the first contraction depends on the storage content determined by Ca inflow through the Ca channels open at rest. These latter channels have low sensitivity to D-600 and are better presented in atrium than in ventriculum. An increase of contraction tension observed in low sodium medium took place also under the action of D-600; in this case Ca storage was replenished by the Na-Ca exchange diffusion.     

Use-dependent block of sodium channels in frog myelinated nerve by tetrodotoxin and saxitoxin at negative holding potentials

Na+ currents were measured in myelinated frog nerve fibres in the presence of nanomolar concentrations of tetrodotoxin (TTX) or saxitoxin (STX) in the extracellular solution. The Na+ currents declined during a train of depolarizing pulses if the fibre was held at hyperpolarizing potentials between the pulses. At a pulse frequency of 0.8 Hz, the peak Na+ currents were reduced to 70 or 60% of the initial value in 9.3 nM TTX and 3.5 nM STX solutions, respectively. A decline of Na+ currents was also observed in two-pulse experiments. The peak Na+ current during a second test pulse did not depend on the duration (0.2 to 12 ms) of the first pulse. It decreased with increasing interval between the pulses, reached a minimum and increased again. The results are interpreted with a use-dependent blockage of Na+ channels by TTX or STX at negative holding potentials. The effects were described quantitatively, assuming a fast affinity increase of toxin receptors at Na+ channels triggered by Na+ activation followed by slow toxin binding to channels and relaxation of the receptor affinity.     

Effect of electrolyte composition of various solutions on potential-sensitive ion channels, formed by syringomycin E in lipid bilayers]

Using the planar lipid bilayer technique, the dependence of properties of ion channels formed by syringomycin E on bath ion composition (electrolyte concentration and pH) and on the applied potential was studied. Time courses of the membrane current in field reversal experiments with 1M NaCl, pH 6 in the bathing solution were similar to the time courses observed with a bathing solution of 0.1 M NaCl, pH 2: there was no increase (decrease) of the membrane current at positive (negative) values of the transmembrane potential. However, kinetic curves were different at 0.1 M. NaCl, pH 6 and pH 9. At pH 6 there was an abrupt increase (decrease) of the membrane current over the time at the positive (negative) values of the applied potential. At pH 9 there was a reversed current response. The results indicate that charged groups are likely to be responsible for opening or closing the channel facing the water phase. Based on these data, a model is proposed describing the potential dependent opening and closing of ion channels formed by syringomycin E.     

Expression of the amiloride-blockable Na+ channel by RNA from control versus aldosterone-stimulated tissue.

The amiloride-blockable Na+ channel was expressed in Xenopus oocytes injected with total RNA isolated from the toad urinary bladder. This system was used to investigate mechanisms that mediate the natriferic action of aldosterone. Incubation of the epithelium with aldosterone for 3 h doubled its channel activity but did not increase the ability of isolated RNA to express functional channels in oocytes. A 20-h incubation with the hormone produced an additional increase of Na+ transport across the intact epithelium and also augmented the channel activity expressed in oocytes by nearly 10-fold. The data are in agreement with our model that aldosterone enhances the apical Na+ permeability of tight epithelia by a short term activation of pre-existing channels, followed by chronic induction of new channel protein. Blocking methyl transfer reactions, previously shown to inhibit the natriferic action of aldosterone in tight epithelia, did not alter the basal or aldosterone-induced response in oocytes.     

Mechanism of beta4 subunit modulation of BK channels

Large-conductance (BK-type) Ca(2+)-activated potassium channels are activated by membrane depolarization and cytoplasmic Ca(2+). BK channels are expressed in a broad variety of cells and have a corresponding diversity in properties. Underlying much of the functional diversity is a family of four tissue-specific accessory subunits (beta1-beta4). Biophysical characterization has shown that the beta4 subunit confers properties of the so-called "type II" BK channel isotypes seen in brain. These properties include slow gating kinetics and resistance to iberiotoxin and charybdotoxin blockade. In addition, the beta4 subunit reduces the apparent voltage sensitivity of channel activation and has complex effects on apparent Ca(2+) sensitivity. Specifically, channel activity at low Ca(2+) is inhibited, while at high Ca(2+), activity is enhanced. The goal of this study is to understand the mechanism underlying beta4 subunit action in the context of a dual allosteric model for BK channel gating. We observed that beta4's most profound effect is a decrease in P(o) (at least 11-fold) in the absence of calcium binding and voltage sensor activation. However, beta4 promotes channel opening by increasing voltage dependence of P(o)-V relations at negative membrane potentials. In the context of the dual allosteric model for BK channels, we find these properties are explained by distinct and opposing actions of beta4 on BK channels. beta4 reduces channel opening by decreasing the intrinsic gating equilibrium (L(0)), and decreasing the allosteric coupling between calcium binding and voltage sensor activation (E). However, beta4 has a compensatory effect on channel opening following depolarization by shifting open channel voltage sensor activation (Vh(o)) to more negative membrane potentials. The consequence is that beta4 causes a net positive shift of the G-V relationship (relative to alpha subunit alone) at low calcium. At higher calcium, the contribution by Vh(o) and an increase in allosteric coupling to Ca(2+) binding (C) promotes a negative G-V shift of alpha+beta4 channels as compared to alpha subunits alone. This manner of modulation predicts that type II BK channels are downregulated by beta4 at resting voltages through effects on L(0). However, beta4 confers a compensatory effect on voltage sensor activation that increases channel opening during depolarization.     

Cadmium block of squid calcium currents. Macroscopic data and a kinetic model

The mechanism of Cd2+ block of Ca2+ currents (ICa) was explored in squid neurons using whole-cell patch clamp. Control currents activated sigmoidally, more rapidly at more positive potentials, and did not inactivate significantly. External Cd2+ up to 250 microM reduced ICa reversibly. For small depolarizations, the current for a step of 10 ms increased to a maintained value, resembling the control; but for Vm greater than 0 mV, the increase was followed by a decrease, as Cd2+ block became greater. Final block was greater for larger depolarizations. At 0 mV the half-blocking concentration was 125 microM. Tail currents, measured as channels close, had an initial "hook" when recorded in Cd2+: currents increased transiently, then decreased. This suggests that Cd2+ escapes from some channels, which then conduct briefly before closing. Analysis of tail currents shows that Cd2+ does not slow channel closing. The data can be explained if Cd2+ is a permeant blocker of Ca2+ channels and if channels can close when occupied by Cd2+. Cd2+ permeates the channels, but binds transiently to a site in the pore, obstructing the passage of other ions (e.g., Ca2+). Dwell time depends on the transmembrane potential, becoming shorter for more negative internal potentials. A five-state model was used to simulate the steady-state and kinetic features. It combines a Hodgkin-Huxley type m2 gating scheme and a one-site Woodhull ionic blockage model for a permeant blocker and includes a closed blocked state. To fit the data, the binding site for Cd2+ had to be near the outer end of the pore, with a well depth of -12.2 RT, and with a barrier at each end of the pore. The model predicts that the Cd2+ entry rate is nearly voltage independent, but the exit rate is steeply voltage dependent (e-fold/17 mV). Analysis further suggests that the channel closes at a normal rate with Cd2+ in the pore.     

Properties of Voltage-gated Ion Channels Formed by Syringomycin E in Planar Lipid Bilayers

Using the planar lipid bilayer technique we demonstrate that the lipodepsipeptide antibiotic, syringomycin E, forms voltage-sensitive ion channels of weak anion selectivity. The formation of channels in bilayers made from dioleoylglycerophosphatidylserine doped with syringomycin E at one side (1–40 μg/ml) was greatly affected by cis-positive voltage. A change of voltage from a positive to a negative value resulted in (i) an abrupt increase in the single channel conductance (the rate of increase was voltage dependent) simultaneous with (ii) a closing of these channels and an exponential decrease in macroscopic conductance over time. The strong voltage dependence of multichannel steady state conductance, the single channel conductance, the rate of opening of channels at positive voltages and closing them at negative voltages, as well as the observed abrupt increase of single channel conductance after voltage sign reversal suggest that the change of the transmembrane field induces a significant rearrangement of syringomycin E channels, including a change in the spacing of charged groups that function as voltage sensors. The conductance induced by syringomycin E increased with the sixth power of syringomycin E concentration suggesting that at least six monomers are required for channel formation. Received: 3 April 1995/Revised: 24 August 1995     

Gating kinetics of batrachotoxin-modified sodium channels in neuroblastoma cells determined from single-channel measurements

We have observed the opening and closing of single batrachotoxin (BTX)-modified sodium channels in neuroblastoma cells using the patch-clamp method. The conductance of a single BTX-modified channel is approximately 10 pS. At a given membrane potential, the channels are open longer than are normal sodium channels. As is the case for normal sodium channels, the open dwell times become longer as the membrane is depolarized. For membrane potentials more negative than about -70 mV, histograms of both open-state dwell times and closed-state dwell times could be fit by single exponentials. For more depolarized potentials, although the open-state histograms could still be fit by single exponentials, the closed-state histograms required two exponentials. This data together with macroscopic voltage clamp data on the same system could be accounted for by a three-state closed-closed-open model with transition rates between these states that are exponential functions of membrane potential. One of the implications of this model, in agreement with experiment, is that there are always some closed BTX-modified sodium channels, regardless of membrane potential.     

Voltage-clamp experiments on oxidized cholesterol membranes modified with excitability-inducing material and comparison with a model

The conductance of oxidized cholesterol membranes modified with excitability-inducing material was observed in membranes containing either single conductance channels or 100–1000 channels. Membranes containing single channels have several conductance states for each voltage polarity, and the current through membranes containing many channels decays with at least two, and probably three, time constants following a step change in voltage (voltage-clamp). The time constants differ by about an order of magnitude. The multi-state behavior seems more pronounced in membranes made from highly oxidized cholesterol. Although a given conductance state could occur at either positive or negative voltages, each state was much more frequent at one polarity or the other. The most frequently observed single-channel conductance states in 0.1 M NaCl are about 0.3, 0.1, 0.03, 0.0 nΩ^-1 for negative voltages and 0.25, 0.05, 0.03, and 0.0 nΩ^-1 for positive voltages. The current following a voltage clamp decays to a quasi-steady state within 1 min for positive voltages and 1–15 min for negative voltages. When the holding voltage is −20 mV, the decay constants and quasi-steady state conductances as functions of clamping voltage are reasonably well described by either a three-state model of the conductance or a two-state model applied independently at negative and positive voltages. However, for high voltages, the quasi-steady state does not appear to approach a state in which all the channels are in a low conductance state.     

A model of binocular brightness and binaural loudness perception in humans with general applications to nonlinear summation of sensory inputs

A single neural model is proposed to account for how responses of the two eyes and two ears combine to form the perception of binocular brightness and binaural loudness respectively. It involves nonlinear reciprocal feedback inhibition between left and right channels, followed by linear summation between the channels. Local circuit synaptic interactions are an important source of nonlinearity. The model combines inputs in a manner that approximates vector magnitude models in general. This suggests that the model can be applied to a variety of circumstances beyond the visual and auditory data discussed here.     

Calcium currents in the A7r5 smooth muscle-derived cell line. An allosteric model for calcium channel activation and dihydropyridine agonist action

We have investigated the gating kinetics of calcium channels in the A7r5 cell line at the level of single channels and whole cell currents, in the absence and presence of dihydropyridine (DHP) calcium channel agonists. Although latencies to first opening and macroscopic currents are strongly voltage dependent, analysis of amplitude histograms indicates that the primary open-closed transition is voltage independent. This suggests that the molecular mechanisms for voltage sensing and channel opening are distinct, but coupled. We propose a modified Monod-Wyman-Changeux (MWC) model for channel activation, where movement of a voltage sensor is analogous to ligand binding, and the closed and open channels correspond to inactive (T) and active (R) states. This model can account for the activation kinetics of the calcium channel, and is consistent with the existence of four homologous domains in the main subunit of the calcium channel protein. DHP agonists slow deactivation kinetics, shift the activation curve to more negative potentials with an increase in slope, induce intermingled fast and slow channel openings, and reduce the latency to first opening. These effects are predicted by the MWC model if we make the simple assumption that DHP agonists act as allosteric effectors to stabilize the open states of the channel.     

Ionic channels induced by sea nettle toxin in the nodal membrane.

Toxin isolated from nematocysts of the sea nettle Chrysaora quinquecirrha (SNTX) is known to depolarize nerve and muscle membranes and to increase the miniature end-plate potentials' frequency. To investigate its mode of action at the membrane level, we have studied the toxin's effects on the frog myelinated nerve fibre. We show that SNTX creates large cation-selective channels that open and close spontaneously. The conductance of these channels, almost constant in the voltage range - 100 to + 50 mV, is 760 pS. The SNTX-induced channels are almost equally permeable to Na+, Li+, K+, and Cs+, but are impermeable to Ca++. The open and closed times of SNTX-induced channels are voltage dependent, the open probability increasing with increased negative membrane potentials. To our knowledge, this is the first demonstration of the production of single-channel currents by a toxin, in a biological membrane.     

Dynamic simulation of the effect of calcium-release activated calcium channel on cytoplasmic Ca2+ oscillation

A mathematical model is proposed to illustrate the activation of STIM1 (stromal interaction molecule 1) protein, the assembly and activation of calcium-release activated calcium (CRAC) channels in T cells. In combination with De Young-Keizer-Li-Rinzel model, we successfully reproduce a sustained Ca(2+) oscillation in cytoplasm. Our results reveal that Ca(2+) oscillation dynamics in cytoplasm can be significantly affected by the way how the Orai1 CRAC channel are assembled and activated. A low sustained Ca(2+) influx is observed through the CRAC channels across the plasma membrane. In particular, our model shows that a tetrameric channel complex can effectively regulate the total quantity of the channels and the ratio of the active channels to the total channels, and a period of Ca(2+) oscillation about 29 s is in agreement with published experimental data. The bifurcation analyses illustrate the different dynamic properties between our mixed Ca(2+) feedback model and the single positive or negative feedback models.     

Ionic current through batrachotoxin-modified sodium channels of the ranvier node membrane at high positive and negative potentials

Ionic current through batrachotoxin (BTX)-modified sodium channels within a wide range of membrane potentials were measured by the voltage clamp method on the membrane of a myelinated frog nerve fiber. At high positive voltages (above +80 mV) the current decreased with time; with an increase in voltage the steady-state level of the currents fell. The results of measurement of "instant" currents showed that this phenomenon is connected with a decrease in overall conductivity of the modified channels. Scorpion toxin had no significant effect on the kinetics of decline of the currents. This indicates that they are due to processes which differ from ordinary inactivation. In the presence of procaine, at high positive voltages slow (tens of milliseconds) potential-dependent blocking of BTX-modified channels was observed. An increase in negative potentials above ?100 mV caused a decrease in "instant" currents, connected with rapid potential-dependent blocking of BTX-modified sodium channels by calcium ions.