Open Channel Lifetime Research Articles

Ion-channels formed by hypelcins, antibiotic peptides, in planar bilayer lipid membranes

Ion-channel properties of native hypelcins (HP) A-I, A-V and B-V isolated from Hypocrea peltata and a synthetic analog, HP-A-Pheol, were studied in planar bilayer lipid membranes by a single-channel recording technique. The native and synthetic hypelcins formed ion-channels with three conductance levels for 3 mole dm −3 KCl: ≤0.09 nS at 225 mV (level 0, only detectable at voltages above 200 mV), ≈0.6 nS at 150 mV (level 1, most common level) and ≈3 nS at 150 mV (level 2). The effects of the C-terminal aminoalcohol on the channel properties were examined with HP-A-I, HP-A-V and HP-A-Pheol, whose C-termini are leucinol (Leuol), isoleucinol (Ileol) and phenylalaninol (Pheol), respectively. The substitution of Pheol for Leuol and Ileol prolonged the open channel lifetime. A comparison of HP-A-V (Gln18) and HP-B-V (Glu18) indicated that the carboxyl group at position 18 increased both the open channel lifetime and the magnitude of unitary channel conductance at each conductance level. The pores of level 1 showed poor ion-selectivity for K + over Cl −. The selectivity order of alkali metal cations was Rb≥Cs≥K>Na>Li for level 1 and Cs>Rb>K>Na>Li for level 0. The unitary current-voltage characteristics showed non-linear relationships, which were simulated by a Nernst-Planck approach with a simple barrier model.

Single-channel and whole-cell analysis of ethanol inhibition of NMDA-activated currents in cultured mouse cortical and hippocampal neurons

The effects of 0.1 to 500 mM ethanol on NMDA-activated currents were studied in primary cultures of mouse cortical and hippocampal neurons. In whole-cell recordings the IC 50S for inhibition of NMDA-activated currents by ethanol were 129 mM ± 20 mM in hippocampal neurons and 126 ± 18 mM in cortical neurons. In single-channel recordings from excised outside-out patches of cortical neurons, ethanol inhibited total charge per minute with an IC 50 of 174 ± 23 mM, which was not significantly different from the IC 50S for inhibition of whole-cell current. The reduction in mean open channel lifetime by ethanol was fit by the logistic equation with an apparent IC 50 of 340 ± 28 mM. Analysis of single-channel data indicated that ethanol inhibition of NMDA currents did not involve substantial changes in fast closed state kinetics, changes in open channel conductance, or block of the open channel. At the whole-cell IC 50, of ethanol, mean open channel lifetime would decrease by 28% and frequency of opening would decline by 31% to account for the reduction in current. Single-channel data were consistent with ethanol being an allosteric modulator of gating which reduces agonist efficacy.

Kinetic properties of NMDA receptors in embryonic Xenopus spinal neurons

1. The kinetic properties of embryonic Xenopus spinal cord neuron N-methyl-D-aspartate (NMDA)-activated receptors (NMDAR) were examined at the single-channel level. These receptors have a main conductance state of 50 pS and make occasional sojourns to a subconductance level of approximately 40 pS. The open channel lifetime is 1.3 ms at -80 mV and in 1 mM Ca(2+)-, Mg(2+)-free solution. Extracellular Mg2+ blocks the channel at a rate of 2.9 x 10(8) M-1 s-1 and with a Kd of approximately 20 microM at -80 mV. In patches with only one channel active, the closed interval duration distribution requires at least four exponentials to be fit. The time constant of one closed interval component decreases with increasing NMDA concentration. Kinetic modeling indicates that these NMDA receptors open at a rate of 230 s-1 and close at a rate of 167 s-1, that in the absence of desensitization the maximum probability of being open is approximately 0.5, and that this probability is half-maximal at approximately 150 microM NMDA.

Modulation of potassium channel gating by external divalent cations.

We have examined the actions of Zn2+ ions on Shaker K channels. We found that low (100 microM) concentrations of Zn2+ produced a substantial (approximately three-fold) slowing of the kinetics of macroscopic activation and inactivation. Channel deactivation was much less affected. These results were obtained in the presence of 5 mM Mg2+ and 4 mM Ca2+ in the external solution and so are unlikely to be due to modification of membrane surface charges. Furthermore, the action of 100 microM Zn2+ on activation was equivalent to a 70-mV reduction of a negative surface potential whereas the effects on deactivation would require a 15-mV increase in surface potential. External H+ ions reduced the Zn-induced slowing of macroscopic activation with an apparent pK of 7.3. Treatment of Shaker K channels with the amino group reagent, trinitrobenzene sulfonic acid (TNBS), substantially reduced the effects of Zn2+. All these results are qualitatively similar to the actions of Zn2+ on squid K channels, indicating that the binding site may be a common motif in potassium channels. Studies of single Shaker channel properties showed that Zn2+ ions had little or no effect on the open channel current level or on the open channel lifetime. Rather, Zn2+ substantially delayed the time to first channel opening. Thus, K channels appear to contain a site to which divalent cations bind and in so doing act to slow one or more of the rate constants controlling transitions among closed conformational states of the channel.

A study of the bovine adrenal chromaffin nicotinic receptor using patch clamp and concentration-jump techniques.

1. Voltage clamp records have been obtained from bovine adrenal chromaffin cells in the outside-out and whole-cell configurations, in response to step changes of acetylcholine (ACh) concentration. The concentrations used ranged from 50 nM to 20 mM. 2. At high acetylcholine concentrations, the activation and desensitization kinetics of the nicotinic receptor, as observed in outside-out patches, may be described by a model incorporating a single, fast agonist binding step, and relatively slow isomerization to the open state. The affinity of the closed receptor for ACh is 310 microM, the channel opening rate constant is 460 s-1, and the closing rate constant is 29 s-1. 3. Single channel events, observed when nanomolar ACh concentrations are applied to whole cells, have two distinct channel lifetimes: 0.6 ms and 11-15 ms. The variation of the frequencies of the events with ACh concentration, suggests that the short lifetimes are openings of a singly liganded receptor and the longer lifetimes are openings of a doubly liganded receptor. 4. Only a single exponential associated with receptor desensitization is seen with outside-out patches, but two are seen with whole cells. It is postulated that there are two nicotinic receptor types present on adrenal chromaffin cells. 5. The rate of desensitization (9 s-1 and 26 s-1, whole cells; 24 s-1, patches), is fast enough to be significant in determining the open channel lifetime. 6. A sudden increase in current (rebound) is observed when a high concentration of ACh is abruptly removed from outside-out patches. This is evidence for a blocked state. The affinity of the blocking site for ACh is 1400 microM (outside-out patches). 7. The total number of activatable nicotinic channels per whole cell is estimated to be 2600.

ATP dependence of KATP channel kinetics in isolated membrane patches from rat ventricle

The dependence of KATP channel activity on [ATP] has been examined in isolated membrane patches from rat ventricular myocytes. The steady-state [ATP] dependence of channel open probability could be described by a sigmoidal relationship with the ki ([ATP] causing half-maximal inhibition of open probability) = 25 microM and Hill coefficient of 2. Description of channel open- and closed-time distributions required at least 2, and 3, time constants, respectively. Long open-channel lifetimes decreased with [ATP]; unconditional mean channel closed-times increased with [ATP]. Step decrease (jump) in bathing [ATP] resulted in a delay (of up to hundreds of milliseconds) followed by a pseudo-exponential rise of current (with a time constant of up to hundreds of milliseconds). The time course of channel current after changes of [ATP] (or the ATP-analogue AMP-PNP) was shown to be predominantly determined by the time course of diffusion into the tip of the electrode and to the membrane. This time course of diffusion of ATP into the pipette tip had to be taken into account when analyzing the current response to [ATP] steps. Several possible kinetic models of the ATP-dependent regulation of channel activity were considered. Adequate explanation of the data required a model with sequential ATP-binding sites. The model can account for the time course of channel opening after steps of [ATP], as well as for the steady-state dependence of P0 on [ATP]. The model predicts [ATP]-dependent closed and open lifetimes as were observed experimentally.

Synthetic analogues of alamethicin: Effect of C-terminal residue substitutions and chain length on the ion channel lifetimes

In a previous study, a synthetic analogue of the peptaibol alamethicin, in the sequence of which all α-aminoisobutyric acid (Aib) were substituted by leucine residues and the C-terminal residue modified, was shown to display the same single-channel behaviour as alamethicin in planar lipid bilayer, except that the sublevel lifetimes were much reduced. New analogues differing in their C-terminal residue (Phe-NH 2, Pheol, Trp-NH 2) have now been tested for their single channel properties in neutral lipid bilayers. The conductance amplitudes and open channel lifetimes do not differ significantly from the previous analogue. Thus, the nature of the last residue, which may be located near the membrane interface, does not seem to play an important role in the destabilisation of the conducting aggregate observed after the Aib substitution by Leu. Since the deletion of one residue (Glu 18) in the 14–20 moiety induces a slight decrease of the increment between the conductance levels, but has no effect upon the channel lifetimes, this residue and the length of this segment do not interfer much with the channel lifetime of peptaibols. In conclusion the factors influencing the aggregate stability may be sought in the helix-helix interactions.

The influence of the trichorzianin C-terminal residues on the ion channel conductance in lipid bilayers

Four natural trichorzianin analogues, channel-forming peptaibols, differing in their C-terminal residues (Gln or Glu, Trpol or Pheol) were tested for their macroscopic and single-channel conductances in planar lipid bilayers. The results indicate that, as regards to the voltage threshold, the most efficient analogue is the charged Trpol-bearing one. In addition, Trpol brings about a drastic lengthening of the open channel life-times. This behaviour is attributed to the dipole moment of the end residues and to the bulkiness and hydrogen bonding ability of Trpol.

Rate of opening of a population of Na channels in frog skeletal muscle fibers

Linear Systems convolution analysis of muscle sodium currents was used to predict the opening rate of sodium channels as a function of time during voltage clamp pulses. If open sodium channel lifetimes are exponentially distributed, the channel opening rate corresponding to a sodium current obtained at any particular voltage, can be analytically obtained using a simple equation, given single channel information about the mean open-channel lifetime and current.

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 oould 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.

Modulation of gramicidin A open channel lifetime by ion occupancy

The hypothesis that the gramicidin A channel stability depends on the level of ion occupancy of the channel was used to derive a mathematical model relating channel lifetime to channel occupancy. Eyring barrier permeation models were examined for their ability to fit the zero-voltage conductance, current-voltage, as well as lifetime data. The simplest permeation model required to explain the major features of the experimental data consists of three barriers and four sites (3B4S) with a maximum of two ions occupying the channel. The average lifetime of the channel was calculated from the barrier model by assuming the closing rate constant to be proportional to the probability of the internal channel sites being empty. The link between permeation and lifetime has as its single parameter the experimentally determined averaged lifetime of gramicidin A channels in the limit of infinitely dilute solutions and has therefore no adjustable parameters. This simple assumption that one or more ions inside the channel completely stabilize the dimer conformation is successful in explaining the experimental data considering the fact that this model for stabilization is independent of ion species and configurational occupancy. The model is used to examine, by comparison with experimental data, the asymmetrical voltage dependence of the lifetime in asymmetrical solutions, the effects of blockers, and the effects of elevated osmotic pressure.

Interaction of narcotic antagonist naltrexone with nicotinic acetylcholine receptor

The interactions of naltrexone with the nicotinic acetylcholine receptor were studied electrophysiologically using the frog sciatic nerve-sartorius muscle and biochemically using membranes from the electric organ of Torpedo ocellata. At nanomolar concentrations naltrexone increased the peak amplitude of endplate currents with little change in the decay time constant. At micromolar concentrations there was a concentration-dependent depression of endplate current and miniature endplate current amplitudes and decay time constants. Decay time constant depression was enhanced with hyperpolarization. Only marginal curvature was induced in peak endplate current amplitude versus membrane voltage plots by naltrexone. Naltrexone had no effect on single channel conductance but decreased open channel lifetime, according to fluctuation analysis. Naltrexone alone (⩽ 3 μM) did not impair binding of [ 125I]α-bungarotoxin to the receptor in a fast pre-equilibration assay, but increased the ability of acetylcholine to displace [ 125I]α-bungarotoxin. The drug displaced the agonist-stimulated binding of [ 3H]perhydrohistrionicotoxin to the channel site. Biphasic functional changes in neuromuscular transmission can be attributed to an allosteric mechanism with increased agonist binding to the nicotinic receptor at nanomolar concentrations and caused a non-competitive blockade of the ionic channel at micromolar concentrations.

N-bromoacetamide removes a calcium-dependent component of channel opening from calcium-activated potassium channels in rat skeletal muscle.

Calcium-activated potassium channels from cultured rat skeletal muscle were treated with the protein-modifying reagent N-bromoacetamide (NBA) (0.3-1 mM) and studied in excised patches using patch-clamp techniques. After NBA treatment, channels opened only occasionally, and, in contrast to untreated channels, the open probability was no longer sensitive to intracellular surface calcium ions (1 nM to 100 microM). Channel activity did, however, exhibit a voltage dependence similar in direction and magnitude to that shown before NBA treatment (increasing e-fold with 19 mV depolarization). Distributions of open channel lifetimes revealed that NBA treatment virtually abolished openings of long duration, which suggests that this class of openings requires calcium sensitivity. These effects were not reversed by subsequent washing. Quantitatively similar open probability, voltage dependence, and open-interval distributions were observed in untreated channels in calcium-free medium. These results suggest that NBA removed a calcium-dependent component of channel opening, and that normal channels are able to open in the absence of significant intracellular calcium concentrations.

Gamma-Aminobutyric acid- and piperazine-activated single-channel currents from Ascaris suum body muscle.

gamma-Aminobutyric acid (GABA)- and piperazine-activated single-channel currents were recorded from the bag region of the somatic muscle of the nematode parasite Ascaris suum. Cell-attached and outside-out patch-clamp techniques were used. Clean membranes were routinely prepared using collagenase. GABA (concentrations greater than 1 microM) or piperazine (concentrations greater than 200 microM) applied to the extracellular surface of the patches brought about the opening of channels producing rectangular shaped current pulses of varying duration but essentially constant amplitude. The I/V relationships of the single-channel currents for both agonists were linear and had conductances in the region of 22 pS (in symmetrical 170 mM Cl-). The reversal potential was near 0 mV when Cl- was equally distributed on both sides of the membrane. Occasionally two subconductance states were seen. The mainstate single-channel permeability was estimated to be 4 X 10(-14) cm3 s-1. At low concentrations of GABA (3-4 microM), the effective mean channel open time was in the region of 32 ms (-75 mV, 22 degrees C, cell-attached patches). At low concentrations of piperazine (500 microM) the effective mean open channel lifetime was shorter, in the region of 14 ms (-75 mV, 22 degrees C cell-attached patches). For each agonist the channel open lifetime distributions were best described by the sum of two exponentials suggesting two open mainstates. Channel openings occurred as single events and in bursts with brief closed periods within bursts. The channel closed time histograms at these concentrations were best described by the sum of up to three exponentials, suggesting the presence of three closed states. Channel open times showed no appreciable voltage sensitivity. Before desensitization, increases in agonist concentration produced an increase in the probability of the channel being open. The increased probability was associated with an increase in the frequency of channel opening, an increase in the effective mean channel open time, an increase in burst duration, an increase in the number of openings per burst, together with a reduction in the proportion of brief openings. Desensitization was seen as a decline in the probability of the channel being opened during prolonged applications of agonist. It was associated with the appearance of very long (seconds) closed periods. The distributions of the closed channel times were then best described by up to four exponentials.

Role for mouse macrophage IgG Fc receptor as ligand-dependent ion channel.

The interaction of ligands with the mouse macrophage Fc receptor which binds IgG2b and IgG1 immune complexes (FcR gamma 2b/gamma 1) triggers phagocytosis and secretion of various mediators of inflammation. FcR gamma 2b/gamma 1 has been purified using a monoclonal anti-FcR antibody, 2.4G2, and seems to be an integral membrane glycoprotein of molecular weight (Mr) 47,000-60,000 (ref. 6). Monoclonal antibody 2.4G2 is suitable as a tool for functional studies of FcR because it binds to a functional site of the receptor and induces cellular responses that are normally associated with the occupied receptor. We reported previously that binding of ligands to the macrophage FcR resulted in Na+/K+ ion fluxes through the plasma membrane, and that similar ion fluxes were observed in proteoliposomes containing reconstituted FcR. We have now incorporated FcR into planar lipid bilayers and report here that FcR gamma 2b/gamma 1 forms ligand-dependent cation-selective ion channels, with a conductance of 60 pS in 1 M KCl and an average open channel lifetime of 250 ms. The conductance decays to baseline levels within a few minutes. These results suggest a receptor-ionophore model for the signalling of phagocytosis and inflammatory responses.

Properties of miniature post-synaptic currents at the Torpedo marmorata nerve-electroplate junction.

The post-synaptic conductance changes induced by spontaneous release of neurotransmitter on the Torpedo marmorata electroplates were studied by focal extracellular recording methods. It was found that miniature post-synaptic currents (m.p.s.c.s) recorded on the innervated and non-innervated faces of the electrocyte are opposite in sense and the distribution of amplitudes is bimodal. The bimodal distribution could reflect the presence of two populations of m.p.s.c.s (smaller and giant m.p.s.c.s). In the smaller m.p.s.c.s the distribution of the ratio (amplitude/decay time constant) shows two populations that probably represent areas with different receptor densities. The value of the mean open-channel lifetime estimated from the decay time constant of the m.p.s.c. is 0.92 +/- 0.67 ms (n = 319, temperature 20 +/- 1 degree C). Prostigmine (10 microM) increases the amplitude, decay time constant and time to peak of the m.p.s.c. A decrease of temperature increases m.p.s.c. decay time, the activation energies found ranging from 8.0 to 12.6 kcal x mol-1. Increasing the external K+ concentration produces a decrease in the decay time constant. High concentrations of divalent ions, particularly Ca2+, increase the decay time constant of the m.p.s.c. Alkaline or acid bathing solution produces a decrease in the decay time constant. An almost total substitution of external Cl- by non-permeant SO42- does not modify the time course of the m.p.s.c.

Effect of N-bromoacetamide on single sodium channel currents in excised membrane patches.

We have studied the effect of N-bromoacetamide (NBA) on the behavior of single sodium channel currents in excised patches of rat myotube membrane at 10 degree C. Inward sodium currents were activated by voltage steps from holding potentials of about -100 mV to test potentials of -40 mV. The cytoplasmic-face solution was isotonic CsF. Application of NBA or pronase to the cytoplasmic face of the membrane irreversibly removed sodium channel inactivation, as determined by averaged single-channel records. Teh lifetime of the open channel at -40 mV was increased about 10-fold by NBA treatment without affecting the amplitude of single-channel currents. A binomial analysis was used both before and after treatment to determine the number of channels within the excised patch. NBA was shown to have little effect on activation kinetics, as determined by an examination of both the rising phase of averaged currents and measurements f the delay between the start of the pulse and the first channel opening. Our data support a kinetic model of sodium channel activation in which the rate constant leading back from the open state to the last closed state is slower than expected from a strict Hodgkin-Huxley model. The data also suggest that the normal open-channel lifetime is primarily determined by the inactivation process in the voltage range we have examined.

Diazepam and (--)-pentobarbital: fluctuation analysis reveals different mechanisms for potentiation of gamma-aminobutyric acid responses in cultured central neurons.

Diazepam and (--)-pentobarbital each potentiate the increase in chloride ion conductance produced by gamma-aminobutyric acid (GABA) i voltage-clamped mouse spinal neurons grown in culture. Fluctuation analysis was used to compare the properties of elementary ion-channel events underlying the chloride conductance produced by GABA alone and during potentiation by the two drugs. Neither drug altered the conductance of an open ion channel, but both drugs affected the kinetics of channel activity. Diazepam increased the frequency of channel openings and either did not affect or slightly increased the average open-channel lifetime, whereas (--)-pentobarbital decreased the frequency of channel openings and increased average open-channel lifetime. These changes in the kinetics of GABA-activated ion channels can quantitatively account for the potentiation of GABA responses observed with the drugs. Thus, the drugs each increase the response to GABA but do not act on channel kinetics in the same manner.

THE NATURE OF GENTAMICIN-INDUCED NEUROMUSCULAR BLOCK

Gentamicin in concentrations of 0.05--0.2 mmol litre-1 blocked neuromuscular transmission in the toad sartorius nerve--muscle preparation by decreasing the release of transmitter. In concetnrations of 0.5--1 mmol litre-1 it decreased the amplitude of the miniature end-plate current by a curare-like action, without affecting the mean open channel lifetime.

Relaxation and fluctuations of membrane currents that flow through drug-operated channels.

The theoretical background is presented for ( a ) the relaxation towards equilibrium of drug-induced membrane currents, and ( b ) the fluctuations of membrane current about its equilibrium value that originate in the opening and closing of membrane ion channels. General expressions are given that relate the relaxation current, autocovariance function, spectral density function, fluctuation variance and mean open channel lifetime to the rate constants and single channel conductances for any theory of drug action based on the law of mass action. The question of how much can be validly inferred from experimental spectra that appear to have only one component is discussed. The equations are illustrated by their application to some simple theories of drug action that are currently under consideration.