Ion flow through biomembranes

Numerous biomembranes exhibit a sensitivity to changes in electrical potential greater than predicted as possible from the classical application of the Boltzmann relation, a phenomenon which has long defied explanation, the actual sensitivity of some Na⁺ channels being many times greater than the classical limit. This paper explains, using a minimum of mathematics, how the very rapid gating effect of adsorbed Ca²⁺ (or other impermeable divalent cations) can directly affect the conductance of channels, and thus interact with the electric field within the channel to produce a change in the potential across the channel’s gate much greater than the change in the membrane potential, with a corresponding change in the fraction of open conformational gates and change in conductance. These results are not in conflict with the Boltzmann relation, the necessary energy being made available from the total potential difference across the membrane by a long unrecognized stochastic process; the full mathematical theory is given in cited references.     

Characterization of ion channels on the surface membrane of adult rat skeletal muscle.

The channels present on the surface membrane of isolated rat flexor digitorum brevis muscle fibers were surveyed using the patch clamp technique. 85 out of 139 fibers had a novel channel which excluded the anions chloride, sulfate, and isethionate with a permeability ratio of chloride to sodium of less than 0.05. The selectivity sequence for cations was Na+ = K+ = Cs+ greater than Ca++ = Mg++ greater than N-Methyl-D-Glucamine. The channel remained closed for long periods, and had a large conductance of approximately 320 pS with several subconductance states at approximately 34 pS levels. Channel activity was not voltage dependent and the reversal potential for cations in muscle fibers of approximately 0 mV results in the channel's behaving as a physiological leakage conductance. Voltage activated potassium channels were present in 65 of the cell attached patches and had conductances of mostly 6, 12, and 25 pS. The voltage sensitivity of the potassium channels was consistent with that of the delayed rectifier current. Only three patches contained chloride channels. The scarcity of chloride channels despite the known high chloride conductance of skeletal muscle suggests that most of the chloride channels must be located in the transverse tubular system.     

Batrachotoxin-modified sodium channels from squid optic nerve in planar bilayers. Ion conduction and gating properties

Squid optic nerve sodium channels were characterized in planar bilayers in the presence of batrachotoxin (BTX). The channel exhibits a conductance of 20 pS in symmetrical 200 mM NaCl and behaves as a sodium electrode. The single-channel conductance saturates with increasing the concentration of sodium and the channel conductance vs. sodium concentration relation is well described by a simple rectangular hyperbola. The apparent dissociation constant of the channel for sodium is 11 mM and the maximal conductance is 23 pS. The selectivity determined from reversal potentials obtained in mixed ionic conditions is Na+ approximately Li+ greater than K+ greater than Rb+ greater than Cs+. Calcium blocks the channel in a voltage-dependent manner. Analysis of single-channel membranes showed that the probability of being open (Po) vs. voltage relation is sigmoidal with a value of 0.5 between -90 and -100 mV. The fitting of Po requires at least two closed and one open state. The apparent gating charge required to move through the whole transmembrane voltage during the closed-open transition is four to five electronic charges per channel. Distribution of open and closed times are well described by single exponentials in most of the voltage range tested and mean open and mean closed times are voltage dependent. The number of charges associated with channel closing is 1.6 electronic charges per channel. Tetrodotoxin blocked the BTX-modified channel being the blockade favored by negative voltages. The apparent dissociation constant at zero potential is 16 nM. We concluded that sodium channels from the squid optic nerve are similar to other BTX-modified channels reconstituted in bilayers and to the BTX-modified sodium channel detected in the squid giant axon.     

Voltage-gated potassium channels in brown fat cells

We studied the membrane currents of isolated cultured brown fat cells from neonatal rats using whole-cell and single-channel voltage-clamp recording. All brown fat cells that were recorded from had voltage-gated K currents as their predominant membrane current. No inward currents were seen in these experiments. The K currents of brown fat cells resemble the delayed rectifier currents of nerve and muscle cells. The channels were highly selective for K+, showing a 58-mV change in reversal potential for a 10-fold change in the external [K+]. Their selectivity was typical for K channels, with relative permeabilities of K+ greater than Rb+ greater than NH+4 much greater than Cs+, Na+. The K currents in brown adipocytes activated with a sigmoidal delay after depolarizations to membrane potentials positive to -50 mV. Activation was half maximal at a potential of -28 mV and did not require the presence of significant concentrations of internal calcium. Maximal voltage-activated K conductance averaged 20 nS in high external K+ solutions. The K currents inactivated slowly with sustained depolarization with time constants for the inactivation process on the order of hundreds of milliseconds to tens of seconds. The K channels had an average single-channel conductance of 9 pS and a channel density of approximately 1,000 channels/cell. The K current was blocked by tetraethylammonium or 4-aminopyridine with half maximal block occurring at concentrations of 1-2 mM for either blocker. K currents were unaffected by two blockers of Ca2+-activated K channels, charybdotoxin and apamin. Bath-applied norepinephrine did not affect the K currents or other membrane currents under our experimental conditions. These properties of the K channels indicate that they could produce an increase in the K+ permeability of the brown fat cell membrane during the depolarization that accompanies norepinephrine-stimulated thermogenesis, but that they do not contribute directly to the norepinephrine-induced depolarization.     

Ca2+ channels from the sea urchin sperm plasma membrane

Ca2+ influx across the sea urchin sperm plasma membrane is a necessary step during the egg jelly-induced acrosome reaction. There is pharmacological evidence for the involvement of Ca2+ channels in this influx, but their presence has not been directly demonstrated because of the small size of this cell. Sea urchin sperm Ca2+ channels are being studied by fusing isolated plasma membranes into planar lipid bilayers. With this strategy, a Ca2+ channel has been detected with the following characteristics: (a) the channel exhibits a high mainstate conductance (gamma MS) of 172 pS in 50 mM CaCl2 solutions with voltage-dependent decaying to smaller conductance states at negative Em; (b) the channel is blocked by millimolar concentrations of Cd2+, Co2+, and La3+, which also inhibit the egg jelly-induced acrosome reaction; (c) the gamma MS conductance sequence for the tested divalent cations is the following: Ba2+ greater than Sr2+ greater than Ca2+; and (d) the channel discriminates poorly for divalent over monovalent cations (PCa/PNa = 5.9). The sperm Ca2+ channel gamma MS rectifies in symmetrical 10 mM CaCl2, having a maximal slope conductance value of 94 pS at +100 mV applied to the cis side of the bilayer. Under these conditions, a different single-channel activity of lesser conductance became apparent above the gamma MS current at positive membrane potentials. Also in 10 mM Ca2+ solutions, Mg2+ permeates through the main channel when added to the cis side with a PCa/PMg = 2.9, while it blocks when added to the trans side. In 50 mM Ca2+ solutions, the gamma MS open probability has values of 1.0 at voltages more positive than -40 mV and decreases at more negatives potentials, following a Boltzmann function with an E0.5 = -72 mV and an apparent gating charge value of 3.9. These results describe a novel Ca2(+)-selective channel, and suggest that the main channel works as a single multipore assembly.     

The nature of the conductance increase induced by filipin in cholesterol-containing planar lipid bilayers

The effects of the polyene antibiotic filipin on the conductance and permeability of planar lipid bilayers were investigated under voltage-clamp conditions. The membrane conductance of lipid bilayers containing no cholesterol was not affected by filipin. In the presence of cholesterol containing lipid bilayers, filipin induced a 10(4)-10(5)-fold increase in transmembrane conductance. This conductance increase was dependent on the ionic species present in solution, decreasing in the following order: GCsCl greater than GNaAc greater than GKCl greater than GNaCl greater than CaCl2 greater than GNa2SO4 greater than GBaCl2 greater than GMgCl2. Reversal potential measurements in simple biionic conditions revealed the following relative permeability sequence: PK greater than PCl greater than PNa approximately Pac approximately PBa greater than PCs greater than PMg approximately PCa greater than Psulphate. The filipin-sterol mediated increase in membrane conductance was independent of the membrane potential. The increase in membrane current following a step alteration in membrane potential occurred instantaneously and had no dependence on the previous value of the holding membrane potential. We propose that the filipin-sterol complex forms ion channels in lipid membranes. These channels are found in a single configuration (open state) and select preferentially monovalent cations or anions over divalent ions. Our experimental results are discussed in relation to the effects of other polyene antibiotics on the membrane permeability, and also in relation to experimental problems previously reported with the use of filipin in planar lipid bilayers.     

Voltage-dependent block of anthrax toxin channels in planar phospholipid bilayer membranes by symmetric tetraalkylammonium ions. Single-channel analysis

Previous studies have shown that symmetric tetraalkylammonium ions affect, in a voltage-dependent manner, the conductance of membranes containing many channels formed by the PA65 fragment of anthrax toxin. In this paper we analyze this phenomenon at the single-channel level for tetrabutylammonium ion (Bu4N+). We find that Bu4N+ induces a flickery block of the PA65 channel when present on either side of the membrane, and this block is relieved by large positive voltages on the blocking-ion side. At high frequencies (greater than 2 kHz) we have resolved individual blocking events and measured the dwell times in the blocked and unblocked states. These dwell times have single-exponential distributions, with time constants tau b and tau u that are voltage dependent, consistent with the two-barrier, single-well potential energy diagram that we postulated in our previous paper. The fraction of time the channel spends unblocked [tau u/(tau u + tau b)] as a function of voltage is identical to the normalized conductance-voltage relation determined from macroscopic measurements of blocking, thus demonstrating that these single channels mirror the behavior seen with many (greater than 10,000) channels in the membrane. In going from large negative to large positive voltages (-100 to +160 mV) on the cis (PA65-containing) side of the membrane, one sees the mean blocked time (tau b) increase to a maximum at +60 mV and then steadily decline for voltages greater than +60 mV, thereby clearly demonstrating that Bu4N+ is driven through the channel by positive voltages on the blocking-ion side. In other words, the channel is permeable to Bu4N+. An interesting finding that emerges from analysis of the voltage dependence of mean blocked and unblocked times is that the blocking rate, with Bu4N+ present on the cis side of the membrane, plateaus at large positive cis voltages to a voltage-independent value consistent with the rate of Bu4N+ entry into the blocking site being diffusion limited.     

Characterization of a chloroplast inner envelope K+ channel.

A K(+)-conducting protein of the chloroplast inner envelope was characterized as a K+ channel. Studies of this transport protein in the native membrane documented its sensitivity to K+ channel blockers. Further studies of native membranes demonstrated a sensitivity of K+ conductance to divalent cations such as Mg2+, which modulate ion conduction through interaction with negative surface charges on the inner-envelope membrane. Purified chloroplast inner-envelope vesicles were fused into an artificial planar lipid bilayer to facilitate recording of single-channel K+ currents. These single-channel K+ currents had a slope conductance of 160 picosiemens. Antibodies generated against the conserved amino acid sequence that serves as a selectivity filter in the pore of K+ channels immunoreacted with a 62-kD polypeptide derived from the chloroplast inner envelope. This polypeptide was fractionated using density gradient centrifugation. Comigration of this immunoreactive polypeptide and K+ channel activity in sucrose density gradients further suggested that this polypeptide is the protein facilitating K+ conductance across the chloroplast inner envelope.     

Angiotensin II blocks ATP-sensitive K+ channels in porcine coronary artery smooth muscle cells.

ATP-sensitive K+ channels with small conductance (30 pS in symmetrical K(+)-rich solutions) in porcine coronary artery smooth muscle cells were highly active at physiological concentrations of Ca2+ (greater than 10(-4) M) even in the presence of physiological ATP levels, suggesting that these channels contribute to the generation of the resting membrane potential in vascular smooth muscle cells and their modulation is important in controlling vasomotor tone. Angiotensin II, applied from outside the membrane, blocked these channels in a concentration-dependent manner. This would be expected to cause depolarization and result in vasoconstriction.     

Anion permeation in an apical membrane chloride channel of a secretory epithelial cell

Single channel currents though apical membrane Cl channels of the secretory epithelial cell line T84 were measured to determine the anionic selectivity and concentration dependence of permeation. The current-voltage relation was rectified with single channel conductance increasing at positive potentials. At 0 mV the single channel conductance was 41 +/- 2 pS. Permeability, determined from reversal potentials, was optimal for anions with diameters between 0.4 and 0.5 nm. Anions of larger diameter had low permeability, consistent with a minimum pore diameter of 0.55 nm. Permeability for anions of similar size was largest for those ions with a more symmetrical charge distribution. Both HCO3 and H2PO4 had lower permeability than the similar-sized symmetrical anions, NO3 and ClO4. The permeability sequence was SCN greater than I approximately NO3 approximately ClO4 greater than Br greater than Cl greater than PF6 greater than HCO3 approximately F much greater than H2PO4. Highly permeant anions had lower relative single channel conductance, consistent with longer times of residence in the channel for these ions. The conductance sequence for anion efflux was NO3 greater than SCN approximately ClO4 greater than Cl approximately I approximately Br greater than PF6 greater than F approximately HCO3 much greater than H2PO4. At high internal concentrations, anions with low permeability and conductance reduced Cl influx consistent with block of the pore. The dependence of current on Cl concentration indicated that Cl can also occupy the channel long enough to limit current flow. Interaction of Cl and SCN within the conduction pathway is supported by the presence of a minimum in the conductance vs. mole fraction relation. These results indicate that this 40-pS Cl channel behaves as a multi-ion pathway in which other permeant anions could alter Cl flow across the apical membrane.     

Stretch-activated ion channels modulate the resting membrane potential during early embryogenesis

By using the patch-clamp technique, stretch-activated ionic channels were found in the membrane of cleaving freshwater fish embryos at the early stages of embryogenesis (2-256 cells). The application of negative pressure to the pipette increased the frequency of activation and the duration of bursts. This type of channel has a preferential K+ selectivity. When bathed on both membrane surfaces with 140 mM KCl the channel conductance was 71 pS. The kinetic behaviour did not depend markedly on either membrane potential (in the range from -70 to +70 mV) or calcium concentration on the cytoplasmic side of the membrane. On continuous recording, the probability of the channel being open was found to change periodically over a 5- to 20-fold range for different cells. These variations correlated with changes in resting potential and membrane conductance during the cell cycle. These results suggest that the oscillation of resting potential within the cell cycle is associated with the operation of stretch-activated ion channels.     

Ion-transporting properties and ATPase activity of (Na+ + K+)-ATPase large subunit incorporated into bilayer lipid membranes

A purified (Na+ + K+)-ATPase large subunit obtained from microsomes by water-alcohol extraction was incorporated into a bilayer lipid membrane. The protein formed in the membrane conductance channels which were sensitive to ouabain and selective for monovalent cations. ATP activated these channels in the presence of sodium and potassium ions. When sodium ions were eliminated ATP did not change the conductance of the modified membrane whereas p-nitrophenyl phosphate increased it. The (Na+ + K+)-ATPase large subunit incorporated into bilayer lipid membrane possessed an ATPase activity. The presence of a potential on the membrane was a necessary condition for the enzyme incorporated into a bilayer lipid membrane to show high ATPase activity. Increasing the potential above 100 mV resulted in the closing of conductance channels.     

Regulation of potassium conductance in the cellular membrane at early embryogenesis.

At the early stages of development of the fresh water fish loach (Misgurnus fossilis) the resting membrane potential (Er) of cleaving cells oscillates periodically with an amplitude of 8-12 mV. Er oscillation correlates with the cell cycle and is accompanied by changes of K+ conductivity. Two types of K(+)-selective ionic channels with conductance of approximately 70 and 25 pS in symmetrical (150 mM KCl) solution were observed in the membrane of cleaving loach embryos. 'High' conductance and 'low' conductance channels were recorded in approximately 90% and 10% of patches investigated (n = 275), respectively? The activity of 'high' conductance channels was regulated by the application of pressure to the membrane, ie these channels were stretch-activated (SA). The activity of SA channels changes dramatically during the cell-cleavage cycle. At the beginning of interphase the probability of SA channels being in the open state (P0) was minimal, while at prometaphase the probability was increased 10-100-fold. Application of ATP to the cytoplasmic inside-out patches induced a reversible elevation of stretch sensitivity of the SA channels in 50% of the patches, while the non-hydrolyzable analogue of ATP was not effective. Combined application of ATP, cAMP and cAMP-dependent protein kinase (PK) induced a reversible elevation in the SA channel activity while inhibitors of PK prevented its activating effects. Phosphatase inhibitors prolonged the activating effect of PK on SA channels. We propose that oscillations of the resting potential during the cell-cleavage cycle arise due to modulation of SA channel sensitivity to stretch through cAMP-dependent phosphorylation.     

Gating and permeation properties of two types of calcium channels in neuroblastoma cells.

The gating and permeation properties of two types of calcium channels were studied in the neuroblastoma cell line N1E-115. Calcium channel currents as carried by Ba2+ (50 mM) were recorded using the whole-cell variation of the patch electrode voltage-clamp technique. The two types of calcium channels showed similar membrane potential dependence with respect to the steady-state activation and inactivation gating properties. However, the properties of the long-lasting type II channels were shifted approximately 30 mV in the depolarizing direction compared with those of the transient type I channels. Activation of type I channels developed with a sigmoidal time course which was described by m2 kinetics, whereas the activation of type II channels was described by a single exponential function. Tail current upon repolarization followed an exponential decay in either type of calcium channels. In comparison to type I channels, the activation process of type II channels was shifted approximately 30 mV in the positive direction, while the deactivation process showed a 60 mV shift in the positive direction. The rate constants of activation obtained from the activation and deactivation processes indicated that under comparable membrane potential conditions, type II channels close 2.4 times faster than type I channels upon repolarization. When external 50 mM Ba2+ was replaced with Ca2+ or Sr2+ on the equimolar basis, the amplitudes of transient and long-lasting currents were altered without a significant change in their time courses. The ion permeability ratios determined from the maximum amplitude of the inward current were as follows: Ba2+ (1.0) = Sr2+ (1.0) greater than Ca2+ (0.7) for type I channels, and Ba2+ (1.0) greater than Sr2+ (0.7) greater than Ca2+ (0.3) for type II channels. Replacement of Ba2+ with Ca2+ caused a 10-12 mV positive shift in the current-voltage relation for type II channels. However, the shift for type I channels was much less. This suggests that negative surface charges are present around type II channels. After correction for the surface charge effect on the ion permeation, there was no significant difference between the permeability ratios of these cations for the two channel types. It was concluded that the two types of calcium channels have many common properties in their gating and permeation mechanisms despite their differential voltage sensitivity and ion selectivity.     

Permeation characteristics of gramicidin conformers.

To investigate the molecular origin of decreased conductance in variant gramicidin channels, we examined the current-voltage (IV) characteristics of single Val1-gramicidin A channels. Unlike standard channels, all variant channels showed pronounced rectification even though bathing solutions were symmetrical. Moreover, channels of lower conductance consistently showed more pronounced rectification. Analysis within the framework of a three-barrier, two-site, single-filing model indicates that the shape of the variant channel IVs could be best explained by an increase in binding affinity near one of the two channel entrances. This conclusion was further tested by characterizing single channel IVs in bi-ionic solutions having different cationic species at each channel entrance. In Cs/Na bi-ionic solutions, reversal potentials of variant channels often differed by a small but significant amount from those of standard channels. When a membrane potential was applied, the ionic currents tended to be reduced more when flowing from the Na+ side than the Cs+ side. These observations support the conclusion that variant channels have increased binding affinity at one end of the channel. Furthermore, H+ currents were increased while Ag+ currents were unaltered for most variant channels exhibiting decreased Na+ or Cs+ currents. The increased H+ conductance argues against long-range coulombic forces as the basis for decreased Na+ or Cs+ conductance while the normal Ag+ conductance suggests that the binding site field strength increases by a change in carbonyl geometry at the channel entrance.     

Nonselective cation channels in intact human T lymphocytes.

Nonselective cation channels were found in single channel recordings from cell-attached patches on human T lymphocytes. These channels were active under conditions that should lead to cell swelling (hypotonic bath solutions with NaCl or KCl); however, a definite dependence of activity on cell swelling has not been proven. Under these conditions similar channels were found in 20 of 23 patches from 11 different blood donors. The current-voltage relation was approximately linear for outward current (11-14 pS) and inwardly rectifying (to 23 pS) when the intact cells were depolarized with high KCl in the bath. The voltage dependence of channel activity is consistent with closing at hyperpolarized membrane potentials (Vm less than or equal to -50 mV) and block of open channels at strongly depolarized membrane potentials (Vm greater than 0 mV). Reversal potentials under all ionic gradients tested are consistent with a channel that is poorly selective between Na+ and K+ ions. Active channels in cell-attached patches were rapidly blocked by bath addition of the membrane-permeant inhibitor quinine. Channels that were active in cell-attached became quiescent after patch excision; however, two patches remained active long enough to obtain current-voltage relations. These were linear with a slope conductance for outward current of 8-11 pS. Because of the clustering of single-channel openings, detailed voltage dependence of kinetics and probability of opening were not studied.     

Calcium- and voltage-activated potassium channels in human macrophages.

Single calcium-activated potassium channel currents were recorded in intact and excised membrane patches from cultured human macrophages. Channel conductance was 240 pS in symmetrical 145 mM K+ and 130 pS in 5 mM external K+. Lower conductance current fluctuations (40% of the larger channels) with the same reversal potential as the higher conductance channels were noted in some patches. Ion substitution experiments indicated that the channel is permeable to potassium and relatively impermeable to sodium. The frequency of channel opening increased with depolarization and intracellular calcium concentration. At 10(-7) M (Ca++)i, channel activity was evident only at potentials of +40 mV or more depolarized, while at 10(-5) M, channels were open at all voltages tested (-40 to +60 mV). In intact patches, channels were seen at depolarized patch potentials of +50 mV or greater, indicating that the ionized calcium concentration in the macrophage is probably less than 10(-7) M.     

Fatty acid modifies Ca2+-dependent potassium channel activity in smooth muscle cells from the human aorta

By using the patch-clamp technique the effect of 2-decenoic acid (DA) on Ca2+-activated potassium (K+) channels in the membrane of smooth muscle cells from the human aorta was studied. In the presence of 0.5 microM Ca2+ and 2 mM Mg2+ on the cytoplasmic side of the membrane, a more than tenfold elevation in the probability of the channels being open (po) was observed under the effect of DA. With divalent cation concentrations of less than 1 nM DA caused a more than twofold elevation in po. In the DA-treated membranes Mg2+ ions, which normally fail to activate the channels, brought about a nearly threefold increase in the channel activity when applied to the inner membrane surface. Channel sensitivity to the activating effect of cytoplasmic Ca2+ ions did not increase with the application of DA. Single-channel conductance was unchanged by DA exposure. We suggest that DA alters the Ca2+-binding mechanism of the channel, increasing its sensitivity to Mg2+ ions, presumably owing to membrane fluidization.     

Diphtheria toxin at low pH depolarizes the membrane, increases the membrane conductance and induces a new type of ion channel in Vero cells.

Receptor-dependent translocation of diphtheria toxin across the surface membrane of Vero cells was studied using patch clamp techniques. Translocation was induced by exposing cells with surface-bound toxin to low pH. Whole cell current and voltage clamp recordings showed that toxin translocation was associated with membrane depolarization and increased membrane conductance. The conductance increase was voltage independent, with a reversal potential of approximately 15 mV. This value was unaffected by changing the Cl- gradient across the membrane and microfluorometric measurements showed that the cytosolic Ca2+ concentration was only marginally elevated by the translocation. The conductance increase is thus mainly due to monovalent cations. Exposing outside-out and cell-attached patches with bound toxin to low pH induced a new type of ion channel in the membrane. The channel current was inward at negative membrane potentials and the single channel conductance was approximately 30 pS. This value is about three times larger than for receptor-independent channels induced by diphtheria toxin or toxin fragments in artificial lipid membranes.     

Sensitivity of membranes to their environment. Role of stochastic processes

Ionic flow through biomembranes often exhibits a sensitivity to the environment, which is difficult to explain by classical theory, that usually assumes that the free energy available to change the membrane permeability results from the environmental change acting directly on the permeability control mechanism. This implies, for example, that a change delta V in the trans-membrane potential can produce a maximum free energy change, delta V X q, on a gate (control mechanism) carrying a charge q. The analysis presented here shows that when stochastic fluctuations are considered, under suitable conditions (gate cycle times rapid compared with the field relaxation time within a channel), the change in free energy is limited, not by the magnitude of the stimulus, but by the electrochemical potential difference across the membrane, which may be very much greater. Conformational channel gates probably relax more slowly than the field within the channel; this would preclude appreciable direct amplification of the stimulus. It is shown, however, that the effect of impermeable cations such as Ca++ is to restore the amplification of the stimulus through its interaction with the electric field. The analysis predicts that the effect of Ca++ should be primarily to affect the number of channels that are open, while only slightly affecting the conductivity of an open channel.