Ion flow through biomembranes. Physical theory explains its high sensitivity.

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.     

K Channels Are Responsible for an Inwardly Rectifying Current in the Plasma Membrane of Mesophyll Protoplasts of Avena sativa

In whole-cell recording, the conductance of the plasma membrane of protoplasts isolated from mesophyll cells of leaves of oat (Avena sativa) was greater for inward than outward current. The inward current in both the whole-cell mode and with isolated patches was dependent on [K⁺]_o. When the membrane voltage was more positive than −50 millivolts, the membrane conductance in the whole-cell mode was low, and K⁺ channels in cell-attached or outside-out patches had a low probability of being open. At a membrane voltage more negative than −50 millivolts, the membrane conductance increased by sevenfold in the whole-cell mode, and the probability of the channels being open increased. The inward current was highly selective for K⁺ compared with Cs⁺, Na⁺, choline or Cl⁻. Low concentrations of [Cs⁺]_o or [Na⁺]_o blocked the inward current in a strongly voltage-dependent fashion. Comparison of single-channel with the macroscopic current yields an estimate of about 200 inwardly rectifying K⁺ channels per cell at a density of 0.035 per square micrometer. At physiological membrane voltages and [K⁺]_o about 10 millimolar, the influx through these channels is sufficient to increase the internal [K⁺] by 2 millimolar per minute. These K⁺ channels are activated by membrane voltages in the normal physiological range and could contribute to K⁺ uptake whenever the membrane is more negative than the K⁺ equilibrium potential.     

Chloride currents inChara—A patch-clamp study

Summary Ionic current steps were recorded with the patch-clamp technique from algal cells that had been prepared without enzyme treatment. Inward current steps with different conductance levels occurred, the lowest level being 7 pS. There were complex transitions between levels indicating either a lack of independence between single channels, or sublevels of a much larger conductance unit. The reversal potential was consistent with the permeant ion being Cl^–. Furthermore, when a different concentration of Cl^– was used in the patch electrode the reversal potential of the inward current shifted in a manner consistent with a Nernstian change in the Cl^– reversal potential. The frequency of the current steps was voltage dependent and suggestive of the hyperpolarization-activated Cl^– currents reported in voltage-clamp studies. Outward current steps, with conductances of 38 pS, were recorded when the membrane patch was depolarized by more than +120 mV. Their amplitude and frequency increased at more positive potentials. The current was probably carried by an efflux of cations through a different set of channels. The resting membrane potential, measured unambiguously without contamination from the tonoplast, was –190±5 mV.     

Stretch-activated chloride,potassium, and calcium channels coexisting in plasma membranes of guard cells of Vicia faba L.

Mechanosensitive ion channels in the plasma membrane of Vicia faba guard cell protoplasts were studied by use of the patch clamp technique. Stretch-activated (SA) channels in outside-out patches were analyzed for channel conductance, kinetics and ion selectivity. We found three distinct SA channels, permeable to Cl^–, K⁺ and Ca²⁺ and distinguishable from spontaneous (non-SA) channels for these ions on the basis of conductance, kinetics, and voltage-dependence, as well as sensitivity to membrane stretch. In the attached patch configuration, light suction (2 to 10 kPa) reversibly induced channel opening with multiple amplitudes and complex kinetics. The open probability for SA channels increased nonlinearly with pipette suction. In guard cells in situ, these SA channels may mediate ion transport across the plasma membrane directly, as well as influence the activity of non-SA channels via effects on membrane voltage and cytoplasmic calcium. Through such effects, SA channels likely influence volume and turgor regulation of guard cells, and thereby control of leaf gas exchange.Abbreviations EK equilibrium potential for potassium transport - E_Cl equilibrium potential for chloride transport - SA stretchactivated Dedicated to the 80. birthday of Franz HedrichSupported by a grant from the Deutsche Forschungsgemeinschaft to R.H. and a Department of Energy grant to D.J.C. gratefully acknowledges a John S. Guggenheim Fellowship and Fulbright Kommission Senior Professor Award. We thank Ingrid Baumann and Angela Schön for technical assistance, and Klaus Raschke and Heiner Busch for spirited discussions and support.     

Characterization of mechanosensitive channels in Escherichia coli cytoplasmic membrane by whole-cell patch clamp recording

Whole-cell patch clamp recordings were done on giant protoplasts of Escherichia coli. The pressure sensitivity of the protoplasts was studied. Two different unit conductance mechanosensitive channels, 1100 ± 25 pS and 350 ± 14 pS in 400 mm symmetric KCl solution, were observed upon either applying positive pressure to the interior of the cells or down shocking the cells osmotically. The 1100 pS conductance channel discriminated poorly among the monovalent ions tested and it was permeable to Ca²⁺ and glutamate^?. Both of the two channels were sensitive to the osmotic gradient across the membrane; the unit conductances of the channels remained constant while the mean current of the cell was increased by increasing the osmotic gradient. Both of the channels were voltage sensitive. Voltage-ramp results showed that the pressure sensitivity of protoplasts was voltage dependent: there were more channels active upon depolarization than hyperpolarization. The mech anosensitive channels were reversibly blocked by gadolinium ion. Also they could reversibly be inhibited by protons. Mutations in two of the potassium efflux systems, KefB and KefC, did not affect the channel activity, while a null mutation in the gene for KefA changed the channel activity significantly. This indicates a potential modulation of these channels by KefA.     

Stretch activated ion channels in ventricular myocytes

Patch-clamp recordings from ventricular myocytes of neonatal rats identified ionic channels that open in response to membrane stretch caused by negative pressures (1 to 6 cm Hg) in the electrode. The stretch response, consisting of markedly increased channel opening frequency, was maintained, with some variability, during long (>40 seconds) stretch applications. The channels have a conductance averaging 120 pS in isotonic KCl, have a mean reversal potential 31 mV depolarized from resting membrane potential, and do not require external Ca⁺⁺ for activation. The channels appear to be relatively non-selective for cations. Since they are gated by physiological levels of tension, stretch-activated channels may represent, a cellular control system wherein beat-to-beat tension and/or osmotic balance modulate a portion of membrane conductance.Abbreviations SACs stretch-activated channels - HEPES 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid     

The Nature of the Negative Resistance in Bimolecular Lipid Membranes Containing Excitability-Inducing Material

When sufficiently small amounts of excitability-inducing material (EIM) are added to a bimolecular lipid membrane, the conductance is limited to a few discrete levels and changes abruptly from one level to another. From our study of these fluctuations, we have concluded that the EIM-doped bilayer contains ion-conducting channels capable of undergoing transitions between two states of different conductance. The difference in current between the "open" and "closed" states is directly proportional to the applied membrane potential, and corresponds to a conductance of about 3 x 10^-10 ohm^-1. The fraction of the total number of channels that is open varies from unity to zero as a function of potential. The voltage-dependent opening and closing of channels explains the negative resistance observed for bimolecular lipid membranes treated with greater amounts of EIM.     

Kinetics of voltage- and Ca2+ activation and Ba2+ blockade of a large-conductance K+ channel fromNecturus enterocytes

Summary Potassium channels in membranes of isolatedNecturus enterocytes were studied using the patch-clamp technique. The most frequent channel observed had a conductance of 170 pS and reversal potential of 0 mV in symmetrical potassium-rich solutions. Channels were highly K⁺ selective. Channel activity was modulated by membrane potential and cytosolic Ca²⁺ concentration. Channel openings occurred in characteristic bursts separated by long closures. During bursts openings were interrupted by brief closures. Two gating modes controlled channel opening. The primary gate's sensitivity to intracellular Ca²⁺ concentration and membrane potential crucially determined long duration closures and bursting. In comparison, the second gate determining brief closures was largely insensitive to voltage and intracellular Ca²⁺ concentration. The channel was reversibly blocked by cytosolic barium exposure in a voltage-sensitive manner. Blockade reduced open-state probability without altering single-channel conductance and could be described, at relatively high Ca²⁺ concentration, by a three-state model where Ba²⁺ interacted with the open channel with a dissociation constant of about 10^–4 m at 0 mV.     

Ca++-induced fusion of fragmented sarcoplasmic reticulum with artificial planar bilayers

Summary Addition of fragmented sarcoplasmic reticulum (SR) vesicles to the aqueous phase of a black lipid membrane (BLM) causes a large increase in BLM conductance within 10 min. The conductance increase is absolutely dependent on three conditions: The presence of at least 0.5mm Ca⁺⁺, an acidic phospholipid such as phosphatidylserine or diphosphatidylglycerol in the BLM phospholipid mixture, and an osmotic gradient across the SR vesicle membrane, with the internal osmolarity greater than the external. These requirements are identical to conditions under which the fusion of phospholipid vesicles occurs.When the early part of the time course of conductance rise is examined at high sensitivity, the conductance is seen to increase in discrete steps. The probability of a step increases with the concentration of Ca⁺⁺ in the medium, with the fraction of acidic phospholipid in the BLM, and with the size of the osmotic gradient across the SR vesicle membrane. On the other hand, the average conductance change per step is independent of the above parameters, but varies with the type and concentration of ions present in the aqueous phase. For a given ion, the mean specific conductance per step is independent of the ion's concentration between 10 and 100mm.The probability distribution of the step-conductances agrees well with the distribution of SR vesicle surface areas, both before and after sonication of the vesicles.The evidence indicates that SR vesicles fuse with the BLM, thereby inserting SR membrane conductance pathways into it. Each discrete conductance jump appears to be the result of the fusion of a single SR vesicle with the BLM. This technique may serve as a general method for inserting membrane vesicles into an electrically accessible system.     

Surface charge near the cardiac inward-rectifier channel measured from single-channel conductance

Summary The conductance of a channel to permeable ions depends on the number of ions near the mouth of the pore. Surface charge controls the local concentration, and impermeable cations can modify this charge. Correlating channel conductance with the concentration of impermeable cations therefore determines the local charge near the open pore. This paper presents data from cell-attached patches on embryonic chick ventricle cells, and it uses the conductance of inward-rectifier channels in the patch (in 100mm K, with various concentrations of Na, Ca, Ba, and Mg) to estimate the local surface potential. The results indicate the presence of ionized residues near the mouth of the channel. Using the Boltzmann equation and the Gouy-Chapman relation, the surface potential due to these residues (in 100K/33Na/0Ca/0Ba/0Mg) is –40 mV, and the charge density is –0.25e/nm².     

Asymmetry in the Osmotic Response of a Rat Cortical Collecting Duct Cell Line: Role of Aquaporin-2

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride (Cl⁻) channel known to influence the function of other channels, including connexin channels. To further study potential functional interactions between CFTR and gap junction channels, we have co-expressed CFTR and connexin45 (Cx45) in Xenopus oocytes and monitored junctional conductance and voltage sensitivity by dual voltage clamp electrophysiology. In single oocytes expressing CFTR, an increase in cAMP caused by forskolin application induced a Cl⁻ current and increased membrane conductance; application of diphenylamine carboxylic acid (CFTR blocker) readily blocked the Cl⁻ current. With co-expression of CFTR and Cx45, application of forskolin to paired oocytes induced a typical outward current and increased junctional conductance (G_j). In addition, the presence of CFTR reduced the transjunctional voltage sensitivity of Cx45 channels without affecting the kinetics of junctional current inactivation. The drop in voltage sensitivity was further enhanced by forskolin application. The data indicate that CFTR influences cell-to-cell coupling mediated by Cx45 channels.     

Single Cl− channels in molluscan neurones: Multiplicity of the conductance states

Summary Properties of the single Cl^– channels were studied in excised patches of surface membrane from molluscan neurones using single-channel recording technique. These channels are controlled by Ca²⁺ and K⁺ acting on cytoplasmic and outer membrane surfaces, respectively, and by the membrane potential. The channels display about 16 intermediate conductance sublevels, each of them being multiples of 12.5 pS. The upper level of the channel conductance is about 200 pS. The channel behavior is consistent with an aggregation of channel-forming subunits into a cluster.     

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.     

ATP regulation of a large conductance voltage-gated cation channel in rough endoplasmic reticulum of rat hepatocytes

ATP-sensitive K⁺ channels play an important role in regulating membrane potential during metabolic stress. In this work we report the effect of ATP and ADP-Mg on a K⁺ channel present in the membrane of rough endoplasmic reticulum (RER) from rat hepatocytes incorporated into lipid bilayers. Channel activity was found to decrease in presence of ATP 100 μM on the cytoplasmic side and was totaly inhibited at ATP concentrations greater than 0.25 mM. The effect appeared voltage dependent, suggesting that the ATP binding site was becoming available upon channel opening. Channel activity was suppressed by the nonhydrolyzable ATP analog (ATPγS), ruling out a phosphorylation-based mechanism. Notably addition of 2.5 mM ADP-Mg to the cytosolic side increased the channel open probability at negative potentials. We conclude that the large conductance voltage-gated cation channel in RER of rat hepatocytes is an ATP and ADP sensitive channel likely to be involved in cellular processes such as Ca²⁺ signaling or control of membrane potential across the endoplasmic reticulum membrane.     

Glucose Deprivation Activates Diversity of Potassium Channels in Cultured Rat Hippocampal Neurons

1. Glucose is one of the most important substrates for generating metabolic energy required for the maintenance of cellular functions. Glucose-mediated changes in neuronal firing pattern have been observed in the central nervous system of mammals. K⁺ channels directly regulated by intracellular ATP have been postulated as a linkage between cellular energetic metabolism and excitability; the functional roles ascribed to these channels include glucose-sensing to regulate energy homeostasis and neuroprotection under energy depletion conditions. The hippocampus is highly sensitive to metabolic insults and is the brain region most sensitive to ischemic damage. Because the identity of metabolically regulated potassium channels present in hippocampal neurons is obscure, we decided to study the biophysical properties of glucose-sensitive potassium channels in hippocampal neurons.2. The dependence of membrane potential and the sensitivity of potassium channels to glucose and ATP in rat hippocampal neurons were studied in cell-attached and excised inside-out membrane patches.3. We found that under hypoglycemic conditions, at least three types of potassium channels were activated; their unitary conductance values were 37, 147, and 241 pS in symmetrical K⁺, and they were sensitive to ATP. For K⁺ channels with unitary conductance of 37 and 241, when the membrane potential was depolarized the longer closed time constant diminished and this produced an increase in the open-state probability; nevertheless, the 147-pS channels were not voltage-dependent.4. We propose that neuronal glucose-sensitive K⁺ channels in rat hippocampus include subtypes of ATP-sensitive channels with a potential role in neuroprotection during short-term or prolonged metabolic stress.     

Single-Channel Characteristics of Wild-Type IKs Channels and Channels formed with Two MinK Mutants that Cause Long QT Syndrome

I_Ks channels are voltage dependent and K⁺ selective. They influence cardiac action potential duration through their contribution to myocyte repolarization. Assembled from minK and KvLQT1 subunits, I_Ks channels are notable for a heteromeric ion conduction pathway in which both subunit types contribute to pore formation. This study was undertaken to assess the effects of minK on pore function. We first characterized the properties of wild-type human I_Ks channels and channels formed only of KvLQT1 subunits. Channels were expressed in Xenopus laevis oocytes or Chinese hamster ovary cells and currents recorded in excised membrane patches or whole-cell mode. Unitary conductance estimates were dependent on bandwidth due to rapid channel “flicker.” At 25 kHz in symmetrical 100-mM KCl, the single-channel conductance of I_Ks channels was ∼16 pS (corresponding to ∼0.8 pA at 50 mV) as judged by noise-variance analysis; this was fourfold greater than the estimated conductance of homomeric KvLQT1 channels. Mutant I_Ks channels formed with D76N and S74L minK subunits are associated with long QT syndrome. When compared with wild type, mutant channels showed lower unitary currents and diminished open probabilities with only minor changes in ion permeabilities. Apparently, the mutations altered single-channel currents at a site in the pore distinct from the ion selectivity apparatus. Patients carrying these mutant minK genes are expected to manifest decreased K⁺ flux through I_Ks channels due to lowered single-channel conductance and altered gating.     

Membrane transport parameters in frog corneal epithelium measured using impedance analysis techniques

Summary Active Cl^– transport in bullfrog corneal epithelium was studied using transepithelial impendance analysis methods, and direct-current (DC) measurements of membrane voltages and resistance ratios. The technique allows the estimation of the apical and basolateral membrane conductances, and the paracellular conductance, and does not rely on the use of membrane conductance-altering agents to obtain these measurements as was requisite in earlier DC equivalent-circuit analysis studies. In addition, the analysis results in estimates of the apical and basolateral membrane capacitances, and allows resolution of the paracellular conductance into properties of the tight junctions and lateral spaces. Membrane capacitances (proportional to areas) were used to estimate the specific conductances of the apical and basolateral membranes, as well as to evaluate coupling between the cell layers. We confirm results obtained from earlier studies: (1) apical membrane conductance is proportional to the rate of active Cl^– transport and is, highly Cl^– selective; (2) intracellular Cl^– activity is above electrochemical equilibrium, thereby providing a net driving force for apical membrane Cl^– exit; (3) the paracellular conductance is comparable to the transcellular conductance. We also found that: (1) the paracellular conductance is composed of the series combination of the junctional conductance and a nonnegligible lateral space resistance; (2) a small K⁺ conductance reported in the apical membrane may result from Cl^– channels possessing a finite permeability to K⁺; (3) the basolateral membrane areas is 36 times greater than the apical membrane area which is consistent with the notion of electrical coupling between the five to six cell layers of the epithelium; (4) the specific conductance of the basolateral membrane is many times lower than that of the apical membrane; (5) the net transport of Cl^– is modulated primarily by changes in the conductance of the apical membrane and not by changes in the net electrochemical gradient resulting from opposite changes in the electrical and chemical gradients; (6) the conductance of the basolateral membrane does not change with transport which implies that the net driving force for K⁺ exit increases with transport, possibly due to an increase in the intracellular K⁺ activity.     

Control of membrane potential by external H concentration in Bacillus subtilis as determined by an ion-selective electrode

The membrane potential of intact bacteria was monitored by measuring the tetraphenylphosphonium ion distribution across the membrane using poly-(vinyl chloride) matrix-type electrode selective to tetraphenylphosphonium ion. It was found that the tetraphenylphosphonium ion was not countertransported against H⁺ movement. The membrane potential of Bacillus subtilis was estimated to be 80–120 mV inside-negative at external pH 7. The effect of the external pH on the membrane potential was studied. It varied from 30 to 40 mV/decade change in the external [H⁺] in the pH region of greater than 6.5, increasing pH making it more inside-negative. The addition of carbonyl cyanide m-chlorophenylhydrazone depolarized the membrane, and the membrane potential approached the H⁺ equilibrium potential. The addition of N,N′-dicyclohexylcarbodiimide did not abolish the pH dependence of the membrane potential. Increasing the external [K⁺] did not affect the pH dependence. CN⁻ partially depolarized the membrane. A parallel conductance model for membrane potential could explain the results qualitatively.     

Inward rectifier potassium channels in plants differ from their animal counterparts in response to voltage and channel modulators

We have investigated the electrophysiological basis of potassium inward rectification of the KAT1 gene product from Arabidopsis thaliana expressed in Xenopus oocytes and of functionally related K⁺ channels in the plasma membrane of guard and root cells from Vicia faba and Zea mays. The whole-cell currents passed by these channels activate, following steps to membrane potentials more negative than –100 mV, with half activation times of tens of milliseconds. This voltage dependence was unaffected by the removal of cytoplasmic magnesium. Consequently, unlike inward rectifier channels of animals, inward rectification of plant potassium channels is an intrinsic property of the channel protein itself. We also found that the activation kinetics of KAT1 were modulated by external pH. Decreasing the pH in the range 8.5 to 4.5 hastened activation and shifted the steady state activation curve by 19 mV per pH unit. This indicates that the activity of these K⁺ channels and the activity of the plasma membrane H⁺-ATPase may not only be coordinated by membrane potential but also by pH. The instantaneous current-voltage relationship, on the other hand, did not depend on pH, indicating that H⁺ do not block the channel. In addition to sensitivity towards protons, the channels showed a high affinity voltage dependent block in the presence of cesium, but were less sensitive to barium. Recordings from membrane patches of KAT1 injected oocytes in symmetric, Mg²⁺-free, 100 mM-K⁺, solutions allowed measurements of the current-voltage relation of single open KAT1 channels with a unitary conductance of 5 pS. We conclude that the inward rectification of the currents mediated by the KAT1 gene product, or the related endogenous channels of plant cells, results from voltage-modulated structural changes within the channel proteins. The voltage-sensing or the gating-structures appear to interact with a titratable acidic residue exposed to the extracellular medium. Correspondence to: R. Hedrich     

Electrical properties of chloride transport across theNecturus proximal tubule

Summary The chloride conductance of the basolateral cell membrane of theNecturus proximal tubule was studied using conventional and chloride-sensitive liquid ion exchange microelectrodes. Individual apical and basolateral cell membrane and shunt resistances, transepithelial and basolateral, cell membrane potential differences, and electromotive forces were determined in control and after reductions in extracellular Cl^–. When extracellular Cl^– activity is reduced in both apical and basolateral solutions the resistance of the shunt increases about 2.8 times over control without any significant change in cell membrane resistances. This suggests a high Cl^– conductance of the paracellular shunt but a low Cl^– conductance of the cell membranes. Reduction of Cl^– in both bathing solutions or only on the basolateral side hyperpolarizes both the basolateral cell membrane potential difference and electromotive force. Hyperpolarization of the basolateral cell membrane potential difference after low Cl^– perfusion was abolished by exposure to HCO ₃ ^– -free solutions and SITS treatment. In control conditions, intracellular Cl^– activity was significantly higher than predicted from the equilibrium distribution across both the apical and basolateral cell membranes. Reducing Cl^– in only the basolateral solution caused a decrease in intracellular Cl^–. From an estimate of the net Cl^– flux across the basolateral cell membrane and the electrochemical driving force, a Cl^– conductance of the basolateral cell membrane was predicted and compared to measured values. It was concluded that the Cl^– conductance of the basolateral cell membrane was not large enough to account for the measured flux of Cl^– by electrodiffusion alone. Therefore these results suggest the presence of an electroneutral mechanism for Cl^– transport across the basolateral cell membrane of theNecturus proximal tubule cell.