Analysis of the effect of medium and membrane conductance on the amplitude and kinetics of membrane potentials induced by externally applied electric fields.

The kinetics and amplitudes of membrane potential induced by externally applied electric field pulses are determined for a spherical lipid bilayer using a voltage-sensitive dye. Several experimental parameters were systematically varied. These included the incorporation of gramicidin into the membrane to alter its conductivity and the variation of the external electrolyte conductivity via changes in salt concentration. The ability of the solution to Laplace's equation for a spherical dielectric shell to quantitatively describe the membrane potential induced on a lipid bilayer could thus be critically evaluated. Both the amplitude and the kinetics of the induced potential were consistent with the predictions of this simple model, even at the extremes of membrane conductance or electrolyte concentration. The success of the experimental approach for this system encourages its application to more complex problems such as electroporation and the influences of external electric fields in growth and development.     

Electroporation of the photosynthetic membrane: A study by intrinsic and external optical probes

The study examines the relationship between electric field-induced conductivity and permeability changes in a biological membrane (electroporation) and the amplitude-duration parameters of the externally applied electric field. These reversible changes were characterized in giant photosynthetic membrane vesicles by means of the calibrated response of an intrinsic voltage-sensitive optical probe (electrophotoluminescence) and by the uptake studies of dextran-FITC fluorescent probes of different molecular weights. We quantitatively monitored electric field-induced conductivity changes by translating the electrophotoluminescence changes into conductivity changes. This was carried out by measuring the attenuation of the electrophotoluminescent signal after the addition of known amounts of gramicidin. The results demonstrate that electroporation involves the reversible formation of discrete holes in the membrane having radii <5.8 nm. The total area of the electric field-induced holes was 0.075% of the total surface of the vesicle. The formation of the electropores was affected differently by the electric field strength than by its duration. Increase in electric field strength caused increase in the total area of the vesicle that undergoes electroporation. Increase in the duration of the electric field increases the area of single electropores. Each of the two electric parameters can be rate limiting for the dynamics of electropore formation. These results are in accordance with the model of electroporation based on electric field-induced expansion of transient aqueous holes.     

Electrophotoluminescence and the electrical properties of the photosynthetic membrane. II. Electric field-induced electrical breakdown of the photosynthetic membrane and its recovery

Preilluminated suspensions of swollen thylakoid vesicles (‘blebs’) were exposed to uni- and bipolar pairs of identical electric field pulses of variable duration, intensity and spacing. The resulting field-stimulated luminescence (electrophotoluminescence) was used as an intrinsic, voltage-sensitive optical probe to monitor electrical phenomena at the membrane level. The application of a pair of voltage pulses of opposite polarity made it possible to produce electric changes in the membrane by the first pulse and to analyse these effects by a second pulse of opposite polarity. It was found that the relative amplitudes of the two electrophoto-luminescence signals depended on the intensity of the applied electric field and on the time interval (t*) between the two pulses. When t* varied from 0.4 to 12 ms, the second stimulated luminescence signal was at first much smaller than the first one and then increased exponentially until the two signals were equal for t* ≥ 3 ms. We analysed these differences between the two field-stimulated luminescence signals as a measure of the electrical breakdown of the membrane, induced during the first pulse. In this way a distinction between irreversible and reversible breakdown could be made with an estimation of the recovery kinetics of the reversible breakdown, which was found to be complete within 3 ms. Irreversible breakdown of the membrane was found to increase with lengthening the exposure time from 0.1 to 1.3 ms especially when applying high electric field of at least 2000 V/cm.     

Electroporation by subnanosecond pulses

Electropermeabilization of cell membranes by micro- and nanosecond-duration stimuli has been studied extensively, whereas effects of picosecond electric pulses (psEP) remain essentially unexplored. We utilized whole-cell patch clamp and Di-8-ANEPPS voltage-sensitive dye measurements to characterize plasma membrane effects of 500 ps stimuli in rat hippocampal neurons (RHN), NG108, and CHO cells. Even a single 500-ps pulse at 190 kV/cm increased membrane conductance and depolarized cells. These effects were augmented by applying brief psEP bursts (5–125 pulses), whereas the rate of pulse delivery (8 Hz–1 kHz) played little role. psEP-treated cells displayed large inward current at negative membrane potentials but modest or no conductance changes at positive potentials. A 1-kHz burst of 25 pulses increased the whole-cell conductance in the range (?100)–(?60) mV to 22–26 nS in RHN and NG108 cells (from 3 and 0.7 nS, respectively), but only to 5 nS in CHO (from 0.3 nS). The conductance increase was reversible within about 2 min. Such pattern of cell permeabilization, with characteristic inward rectification and slow recovery, was similar to earlier reported effects of 60- and 600-ns pulses, pointing to the similarity of structural membrane rearrangements in spite of a different membrane charging mechanism.     

Electroporation of the photosynthetic membrane: structural changes in protein and lipid-protein domains.

A biological membrane undergoes a reversible permeability increase through structural changes in the lipid domain when exposed to high external electric fields. The present study shows the occurrence of electric field-induced changes in the conductance of the proton channel of the H(+)-ATPase as well as electric field-induced structural changes in the lipid-protein domain of photosystem (PS) II in the photosynthetic membrane. The study was carried out by analyzing the electric field-stimulated delayed luminescence (EPL), which originates from charge recombination in the protein complexes of PS I and II of photosynthetic vesicles. We established that a small fraction of the total electric field-induced conductance change was abolished by N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of the H(+)-ATPase. This reversible electric field-induced conductance change has characteristics of a small channel and possesses a lifetime < or = 1 ms. To detect electric field-induced changes in the lipid-protein domains of PS II, we examined the effects of phospholipase A2 (PLA2) on EPL. Higher values of EPL were observed from vesicles that were exposed in the presence of PLA2 to an electroporating electric field than to a nonelectroporating electric field. The effect of the electroporating field was a long-lived one, lasting for a period > or = 2 min. This effect was attributed to long-lived electric field-induced structural changes in the lipid-protein domains of PS II.     

Membrane conductance of an electroporated cell analyzed by submicrosecond imaging of transmembrane potential.

Transmembrane potential was induced in a sea urchin egg by applying a microsecond electric pulse across the cell. The potential was imaged at a submicrosecond time resolution by staining the cell membrane with the voltage-sensitive fluorescent dye RH292. Under moderate electric fields, the spatial distribution of the induced potential as well as its time dependence were in accord with the theoretical prediction in which the cell membrane was regarded as an insulator. At higher field intensities, however, the potential apparently did not fully develop and tended to saturate above a certain level. The saturation is ascribed to the introduction of a large electrical conductance, in the form of aqueous openings, in the membrane by the action of the induced potential (electroporation). Comparison of the experimental and theoretical potential profiles indicates that the two regions of the membrane that opposed the electrodes acquired a high membrane conductance of the order of 1 S/cm2 within 2 microseconds from the onset of the external field. The conductance was similar in the two regions, although permeability in the two regions of the membrane long after the pulse treatment appeared quite different.     

Role of gangliosides in adhesion and conductance changes in large spherical model membranes

The formation of two spherical model membranes at the tips of two syringes has allowed us to study the role of gangliosides in membrane adhesion and look for changes in conductance between two such membranes during the process of adhesion. Membranes were formed in aqueous 100 mM NaCl, 10 mM KCl, 1 mM CaCl2 from 1% (w/v) egg phosphatidylcholine in n-decane, with or without mixed bovine brain gangliosides. After thinning to the 'black' bilayer state, two membranes were moved into contact. With gangliosides, the contact area and conductance increased colinearly with time over a 5 to 20 min period of adhesion. The role of electrostatic bridging by calcium was investigated. In the absence of calcium or in the presence of 2 mM EDTA, adhesion proceeded after a longer lag time at about one-half the normal rate. As the ganglioside concentration was increased from 0 to 15 mol%, the electrical conductance of individual membranes decreased 3-fold from 48 +/- 30 nS/cm2 to 17 +/- 13 nS/cm2. The conductance was pH dependent with a minimum at neutral values. At neutral pH, when two membranes containing 4.1 mol% gangliosides adhered, the region of adhesion had a specific conductance three times that of the nonadhering regions of membranes. Without gangliosides, the specific conductance of the contact region was the same as that of non-adhering regions of the membrane. These data suggest that mixed gangliosides can mediate an adhesion-dependent increase in conductance.     

Relaxation spectra of gramicidin dimerization in a lipid bilayer membrane

The kinetics of formation and dissociation of gramicidin dimers in a lipid bilayer membrane have been studied by pressure-jump and electric field-jump methods. The traditional AC-coupled pressure-jump apparatus has been modified so that a known DC-voltage drop is maintained across a Teflon cell divided by a septum with a hole for membrane formation. From the response of the amplified output voltage after the pressure release, information about the kinetics of channel (dimer) formation is obtained. In addition, using the same apparatus, electric field-jump measurements were performed on the gramicidin/membrane system. In asolectin/7-dehydrocholesterol (5:1) membranes at 25 +/- 0.1 degrees C, the best fit to the pressure-jump data gives a dimer dissociation rate constant of 0.5 +/- 0.3 s-1. The standard volume change for dimerization determined from the amplitude of the pressure-jump experiments is -66 +/- 35 cm3/mol. Rate data determined by the electric field-jump method are consistent with the pressure-jump values; results obtained with either technique are compatible with other determinations of the kinetics of dimerization on gramicidin/membrane systems.     

Electrophotoluminescence and the electrical properties of the photosynthetic membrane. I. Initial kinetics and the charging capacitance of the membrane

Preilluminated chloroplast membranes, and particularly hypotonically swollen vesicles (blebs), give rise to a strong characteristic luminescence (electrophotoluminescence, EPL; Ellenson and Sauer, 1976, Photochem. Photobiol., 23:113-123; Arnold and Azzi, 1971, Photochem. Photobiol., 14:233-240) during the application of a strong external electric field. A detailed kinetic study of EPL was carried out and the initial kinetics from the field onset are reported here. The fast rise time (less than 0.2 mus) of the applied external electric field together with a high instrumental time resolution allowed the observation of a characteristic delay (lag time) between the field onset and the appearance of the induced emission. The lag time decreased with increase in the applied field strength and/or the conductivity of the suspension and is interpreted to be a consequence of (a) the necessity to reach a threshold electrical potential difference in the bleb membrane, below which no emission can be triggered, and (b) the finite time required to attain such a transmembranal field during the charging process of the membrane. A quantitative analysis, connecting the lag time, the controllable experimental parameters, and the membrane electrical characteristics is presented. Its verification was carried out in both size-selected and heterogeneous bleb populations. In the latter, experiments were consistent with the assumption that the lag time reflects the charging of the largest blebs. The results indicate (a) the possibility of directly measuring the specific membrane capacitance, yielding an estimate of Cm = 1.2 +/- 0.3 microF/cm2 (the precision being particle size-homogeneity dependent); (b) A minimal transmembranal potential difference (of approximately 240 mV) is necessary to induce electrophotoluminescence; and (c) the lag duration depends on the time elapsed between the preillumination and the external field application. Correlated with the study of ionophore effects on the lag time, this suggests additivity of the light- and field-induced transmembrane potentials in attaining the threshold for emission.     

Dimerization constant and single-channel conductance of Gramicidin in thylakoid membranes

Summary The effect of the pore-forming antibiotic gramicidin on pure lipid membranes is well characterized. We studied its action in protein-rich thylakoid membranes that contain less than 25% (wt/wt) acyl lipids. A transmembrane voltage was induced by flashing light, and its decay was measured and interpreted to yield the distribution of gramicidin over thylakoids, its dimerization constant and its single-channel conductance in this membrane. The distribution of gramicidin over the ensemble of thylakoids was immediately homogeneous when the antibiotic was added under stirring, while it became homogeneous only after 20 min in a stirred suspension that was initially heterogeneous. The dimerization constant, 5×10¹⁴ cm²/mol, was about 10 times larger than in pure lipid membranes. This was attributed to the upconcentration of gramicidin in the small fractional area of protein free lipid bilayer and further by a preference of gramicidin for stacked portions of the membrane. The latter bears important consequences with regard to bioenergetic studies with this ionophore. As gramicidin was largely dimerized from a concentration of 1 nm (in the suspension) on, the membrane's conductance then increased linearly as a function of added gramicidin. When the negative surface potential at the thylakoid membrane was screened, the conductance of a single gramicidin dimer agreed well with figures reported for bilayers from neutral lipid (about 0.5 pS at 10 mm NaCl). The modulation of the conductance by the surface potential in spinach versus pea thylakoids and between different preparations is discussed in detail.We would like to thank Ms. H. Kenneweg for photographs. financial support by the DFG (SFB 171/B3) is gratefully acknowledged.This paper is dedicated to the Late Prof. Peter Läger.     

Time courses of cell electroporation as revealed by submicrosecond imaging of transmembrane potential.

Changes in the membrane conductance of sea urchin eggs, during the course of electroporation, were investigated over the time range of 0.5 microsecond to 1 ms by imaging the transmembrane potential at a submicrosecond resolution with the voltage-sensitive fluorescent dye RH292. When a rectangular electric pulse of moderate intensity was applied across an egg, a position-dependent potential developed synchronously with the pulse, as theory predicts for a cell with an insulating membrane. From the rise and fall times, the membrane capacitance of unfertilized eggs was estimated to be 0.95 microF/cm2 and the intracellular conductance 220 omega.cm. Under an electric pulse of much higher intensity, the rise of the induced potential stopped at a certain level and then slowly decreased on the microsecond time scale. This saturation and subsequent reversal of the potential development was ascribed to the introduction of finite membrane conductance, or permeabilization of the membrane, by the action of the intense pulse (electroporation). Detailed analysis indicated the following: already at 0.5 microsecond in the rectangular electric pulse, the two sides of the egg facing the positive and negative electrodes were porated and gave a high membrane conductance in the order of 1 S/cm2; the conductance on the positive side appeared higher. Thereafter, the conductance increased steadily, reaching the order of 10 S/cm2 by 1 ms. This increase was faster on the negative-electrode side; by 1 ms the conductance on the negative side was more than twice that on the positive side. The recovery of the porated membrane after the pulse treatment was assessed from the membrane conductance estimated in a second electric pulse of a small amplitude. At least two recovery processes were distinguished, one with a time constant of 7 microseconds and the other 0.5 ms, at the end of which the membrane conductance was already < 0.1 S/cm2.     

Study of conductance changes of bilayer lipid membrane induced by electric field

Changes in the bilayer lipid membrane (BLM) conductance induced by electric field were studied. BLMs were formed from diphytanoylphosphocholine (DPhPC) solution in squalene. Certain time after a constant voltage (200-500 mV) was applied to the BLM in the voltage-clamp mode, the BLM conductance started to grow up to approximately 10 nS until the BLM ruptured. The conductance often changed abruptly (with the front duration of less than 33 micros) and then stabilized for a relatively long time (up to 10; 300 ms on average) thus resembling the ion channel activity. The mean amplitude of conductance steps was 650 pS. However, in some cases a slow conductance drift was recorded. When N-methyl-D-glucamine/glutamate ions were used instead of KCl, the conductance changes became 5 times smaller. We suggest that formation in the BLM of single pores approximately 1 nm in diameter should result in the observed changes in BLM conductance. The BLM conductance growth was due to consecutive opening of several such pores. When the electric field amplitude was abruptly decreased (down to 50-100 mV), the conductance dropped rapidly to the background value. When we increased the voltage again, the BLM conductance right after the increase depended on the time BLM spent under "weak" electric field. If this time exceeded 500 ms, the conductance was at the background level, but when the time was diminished, the conductance reached the value recorded before the voltage decrease. These data imply that the closure of the pores should lead to the formation in BLM of small defects (prepores) that can be easily transformed into pores when the voltage is increased. The lifetimes of such prepores did not exceed 500 ms.     

Biological effects of oscillating electric fields: Role of voltage-sensitive ion channels

An alternating component of potential across the membrane of an excitable cell may change the membrane conductance by interacting with the voltagesensing charged groups of the protein macromolecules that form voltage-sensitive ion channels. Because the probability that a voltage sensor is in a given state is a highly nonlinear function of the applied electric field, the average occupancy of a particular state will change in an oscillating electric field of sufficient magnitude. This “rectification” at the level of the voltage sensors could result in conformational changes (gating) that would modify channel conductance. A simplified two-state model is examined where the relaxation time of the voltage sensor is assumed to be considerably faster than the fastest changes of ionic conductance. Significant changes in the occupancy of voltage sensor states in response to an applied oscillating electric field are predicted by the model.     

Angiotensin II-induced formation of ionic channels in bilayer lipid membranes.

The interaction of angiotensin II (ANG II) with membrane was studied by measuring conductance and current-voltage characteristics (IVC) of bilayer lipid membranes (BLM) prepared of a mixture of egg lecithin with cholesterol, and of gramicidin D-modified membranes of the same composition. Addition of physiological concentrations of ANG II (approx. 15 mumol/l) into the electrolyte (1 mol/l KCl, pH = 7) in contact with one side of BLM resulted in the appearance of discrete membrane conductance (symbol; see text) = (39.5 +/- 1.07) pS with a duration of the conductivity state tau = (52.15 +/- 6.44) s. Raising ANG II concentration to 75 mumol/l resulted in an additional conductance level of approx. 130 pS with a lifetime of approx. 1s. The electrolyte pH markedly influenced ANG II modified BLM conductance. A decrease of the electrolyte pH to 2.8 resulted in a reduction of the discrete conductance level to approx. 14 pS, whereas ANG did not induce any conductivity at pH = 11.5. The results obtained suggest that ion channels are formed consisting at least of two ANG II molecules. IVC of ANG II-modified BLM are superlinear within the range of electrolyte concentrations studied (between 0.01 and 3 mol/l KCl), i.e, the limiting stage of ion transport is the internal area of the conducting pore. ANG II affects in a cooperative manner the gramicidin D (GRD)-mediated transport, most likely by forming ANG II aggregates in the area of local inhomogeneities in the BLM structure of GRD channels.     

He-Ne激光对大鼠离体颈上神经节后神经元膜电导的影响

应用细胞内生物电记录技术 ,观测不同功率、不同照射时间的 He- Ne激光 (脉冲频率 1Hz)对大鼠离体颈上神经节后神经元快兴奋性突触后电位 (f- EPSP)期间膜电导的影响。功率密度为 2 m W/ cm2 的 He- Ne激光在照射初期 (1min～ 2 min)引起快兴奋性突触后电位 (f- EPSP)幅值增大 ,同时膜电导增大 ;而在激光照射后期 (后 3m in～8m in)引起节后神经元膜电导减少。功率密度为 5 m W/ cm2 的 He- Ne激光照射期膜电导无明显变化 .结果表明 :功率密度为 2 m W/ cm2 的 He- Ne激光照射初期引起膜电导 (Gl=34.6± 5 .4 n S)较照射前 (Gf=2 6 .8± 6 .2 n S)有明显增大 (P<0 .0 5 ) ,照射后期膜电导减少。提示 :He- Ne激光照射可能是通过两时相效应改变节后神经元膜电导来影响交感神经节内兴奋传递过程。这可能是低功率激光对神经细胞的一种作用机制。     

Leaks in the epithelial barrier caused by spontaneous and TNF-alpha-induced single-cell apoptosis.

Current opinion assumes epithelial integrity during spontaneous apoptotic cell death. We measured, for the first time, the local conductances associated with apoptoses and show leaks of up to 280 nS (mean 48 +/- 19 nS) in human intestinal epithelium. The results disprove the dogma that isolated cell apoptosis occurs without affecting the epithelial cell permeability barrier. After induction by tumor necrosis factor alpha (TNF-alpha) the apoptotic leaks were dramatically enhanced: not only was the frequency increased by threefold, but the mean conductance also increased by 12-fold (597+/-98 nS). Thus, apoptosis accounted for about half (56%) of the TNF-alpha-induced permeability increase whereas the other half was caused by degradation of tight junctions in nonapoptotic areas. Hence, spontaneous and induced apoptosis hollow out the intestinal barrier and may facilitate loss of solutes and uptake of noxious agents.     

Miniature-probe measurements of electric fields and currents induced by a 60-Hz magnetic field in rat and human models

A miniaturized probe was designed and built to provide detailed data on fields induced by a uniform 60-Hz magnetic field in homogeneous models of rat and human. The probe employed three silver wires twisted and potted in an 8-cm hypodermic needle. The exposed tips of the wires formed three sensing electrodes with a centered ground; highly sensitive voltage measurements were enabled by a lock-in amplifier. Tests were conducted in a 1-mT rms field that was uniform within +/- 5%. The models were made by casting 1.5% agar at 1-S/m conductivity into plastic-foam molds. The rat model was scaled 1:1 as an adult (22 cm length; mass about 640 g). The human model was scaled 1:4 as an adult (height = 46.5 cm; mass 1.4 kg). The probe was inserted into each model in several regions, and readings of induced fields were made under different exposure geometries. Maximal strengths of fields induced near the surface of the torso were as high as 120 microV/cm in the laterally exposed rat model. Data extrapolated from the quarter-scale human model revealed that an induced field as high as 700 microV/cm could occur at the torso of a frontally exposed human adult. An overall size-scale factor of about 5 appears to be appropriate for experimental exposures of rats that are intended to simulate currents induced in human beings by magnetic fields. The average strength of electric fields induced in the torso by a 1-mT magnetic field is comparable to that by a vertical electric-field at 60 kV/m and 28 kV/m, respectively, for the rat and human.     

Calcium waves induced by large voltage pulses in fish keratocytes.

Intracellular calcium waves in fish keratocytes are induced by the application of electric field pulses with amplitudes between 55 and 120 V/cm and full width at half-maximum of 65-100 ms. Calcium concentrations were imaged using two-photon excited fluorescence microscopy (Denk et al., 1990 Science. 248:73-76; Williams et al. 1994 FASEB J. 8:804-813) and the ratiometric calcium indicator indo-1. The applied electric field pulses induced waves with fast calcium rise times and slow decays, which nucleated in the lamellipodium at the hyperpolarized side of the cells and, less frequently, at the depolarized side. The effectiveness of wave generation was determined by the change induced in the membrane potential, which is about half the field strength times the cell width in the direction of the field. Stimulation of waves began at voltage drops across the cell above 150 mV and saturated at voltage drops above 300 mV, where almost all cells exhibited a wave. Waves were not induced in low-calcium media and were blocked by the nonselective calcium channel blockers cobalt chloride and verapamil, but not by specific organic antagonists of voltage-sensitive calcium channel conductance. Thapsigargin stopped wave propagation in the cell body, indicating that calcium release from intracellular stores is necessary. Thus a voltage pulse stimulates Ca2+ influx through calcium channels in the plasma membrane, and if the intracellular calcium concentration reaches a threshold, release from intracellular stores is induced, creating a propagating wave. These observations and the measured parameters (average velocity approximately 66 micron/s and average rise time approximately 68 ms) are consistent with a wave amplification model in which[equation, see text] determines the effective diffusivity of the propagating molecules, D approximately 300 micron2/s (Meyer, 1991. Cell. 64:675-678).     

Formation of large, ion-permeable membrane channels by the matrix protein (porin) of Escherichia coli.

One of the major proteins of the outer membrane of Escherichia coli, the matrix protein (porin), has been isolated by detergent solubilisation. When the protein is added in concentrations of the order 10 ng/cm3 to the outer phases of a planar lipid bilayer membrane, the membrane conductance increases by many orders of magnitude. At lower protein concentrations the conductance increases in a stepwise fashion, the single conductance increment being about 2 nS (1 nS = 10(-9) siemens = 10(-9) omega -1) in 1 MKCl. The conductance pathway has an ohmic current vs. voltage character and a poor selectivity for chloride and the alkali ions. These findings are consistent with the assumption that the protein forms large aqueous channels in the membrane. From the average value of the single-channel conductance a channel diameter of about 0.9 nm is estimated. This channel size is consistent with the sugar permeability which has been reported for lipid vesicles reconstituted in the presence of the protein.     

Gap junction channels: distinct voltage-sensitive and -insensitive conductance states.

All mammalian gap junction channels are sensitive to the voltage difference imposed across the junctional membrane, and parameters of voltage sensitivity have been shown to vary according to the gap junction protein that is expressed. For connexin43, the major gap junction protein in the cardiovascular system, in the uterus, and between glial cells in brain, voltage clamp studies have shown that transjunctional voltages (Vj) exceeding +/- 50 mV reduce junctional conductance (gj). However, substantial gj remains at even very large Vj values; this residual voltage-insensitive conductance has been termed gmin. We have explored the mechanism underlying gmin using several cell types in which connexin43 is endogenously expressed as well as in communication-deficient hepatoma cells transfected with cDNA encoding human connexin43. For pairs of transfectants exhibiting series resistance-corrected maximal gj (gmax) values ranging from < 2 to > 90 nS, the ratio gmin/gmax was found to be relatively constant (about 0.4-0.5), indicating that the channels responsible for the voltage-sensitive and -insensitive components of gj are not independent. Single channel studies further revealed that different channel sizes comprise the voltage-sensitive and -insensitive components, and that the open times of the larger, more voltage-sensitive conductance events declined to values near zero at large voltages, despite the high gmin. We conclude that the voltage-insensitive component of gj is ascribable to a voltage-insensitive substate of connexin43 channels rather than to the presence of multiple types of channels in the junctional membrane. These studies thus demonstrate that for certain gap junction channels, closure in response to specific stimuli may be graded, rather than all-or-none.