Calcium-activated conductance in skate electroreceptors: voltage clamp experiments

Voltage clamp experiments allow further characterization of the calcium-dependent repolarizing process in skate electroreceptor epithelium. Four current components are described: a prolonged capacity current, a leakage current, an early active current which flows inward across the lumenal membranes of the receptor cells, and a late current which flows outward. The leakage and capacity currents are linear and may be substracted from the total current, giving net active currents. The early active current is carried by calcium and does not undergo inactivation for at least several seconds. When large stimuli exceed the reversal potential for the early calcium current, the late current is suppressed. Reduction of the ionized calcium concentration in the lumen lowers the reversal potential for the early current and the suppression potential for the late current by the same amount. We conclude that the late current is initiated by a calcium influx into the cytoplasm. During pulses of moderate duration, activation of the late current does not begin until a fixed amount of calcium has entered the receptor cells. The required amount of calcium is reduced if a recent calcium influx has occurred. We suggest that the calcium-activated outward current is mediated by a distinct macromolecule that is insensitive to voltage. Such macromolecules are likely to have an important role in the regulation of electrical activity in excitable cells.     

非洲爪蟾卵细胞膜去极化后由超极化激活的瞬间内向电流

以双微电极电压钳制技术研究了未成熟非洲爪蟾卵细胞膜的离子流,发现在较长时间的去极化（－３０ｍｖ,５秒）前脉冲后由超极化引出的一个内向电流,其潜伏期约为０．５秒,经过０．４秒左右到达高峰,随近经０．８秒左右完全回复。其幅值随超极化程度增强而增大,其翻转电位接近氯平衡电位,并随胞外ＣＬ－浓度改变而变化。降低胞外ＣＬ－浓度使其幅值增大。降低胞外Ｎａ＾＋浓度对其无明显影响,提示此内几电流可能是氯离子流。以     

The Ventral Photoreceptor Cells of Limulus : III. A voltage-clamp study

In the dark, the ventral photoreceptor of Limulus exhibits time-variant currents under voltage-clamp conditions; that is, if the membrane potential of the cell is clamped to a depolarized value there is an initial large outward current which slowly declines to a steady level. The current-voltage relation of the cell in the dark is nonlinear. The only ion tested which has any effect on the current-voltage relation is potassium; high potassium shifts the reversal potential towards zero and introduces a negative slope-conductance region. When the cell is illuminated under voltage-clamp conditions, an additional current, the light-induced current, flows across the cell membrane. The time course of this current mimics the time course of the light response (receptor potential) in the unclamped cell; namely, an initial transient phase is followed by a steady-state phase. The amplitude of the peak transient current can be as large as 60 times the amplitude of the steady-state current, while in the unclamped cell the amplitude of the peak transient voltage never exceeds 4 times the amplitude of the steady-state voltage. The current-voltage relations of the additional light-induced current obtained for different instants of time are also nonlinear, but differ from the current-voltage relations of the dark current. The ions tested which have the greatest effect on the light-induced current are sodium and calcium; low sodium decreases the current, while low calcium increases the current. The data strongly support the hypothesis that two systems of electric current exist in the membrane. Thus the total ionic current which flows in the membrane is accounted for as the sum of a dark current and a light-induced current.     

Effects of conditioning polarization on the membrane ionic currents in rat myometrium

Summary Membrane ionic currents were measured in pregnant rat uterine smooth muscle under voltage clamp conditions by utilizing the double sucrose gap method, and the effects of conditioning pre-pulses on these currents were investigated. With depolarizing pulses, the early inward current was followed by a late outward current. Cobalt (1mm) abolished the inward current and did not affect the late outward currentper se, but produced changes in the current pattern, suggesting that the inward current overlaps with the initial part of the late outward current. After correction for this overlap, the inward current reached its maximum at about +10 mV and its reversal potential was estimated to be +62 mV. Tetraethylammonium (TEA) suppressed the outward currents and increased the apparent inward current. The increase in the inward current by TEA thus could be due to a suppression of the outward current. The reversal potential for the outward current was estimated to be –87 mV. Conditioning depolarization and hyperpolarization both produced a decrease in the inward current. Complete depolarization block occurred at a membrane potential of –20 mV. Conditioning hyperpolarization experiments in the presence of cobalt and/or TEA revealed that the decrease in the inward current caused by conditioning hyperpolarization was a result of an increase in the outward current overlapping with the inward current. It appears that a part of the potassium channel population is inactivated at the resting membrane potential and that this inactivation is removed by hyperpolarization.     

High-gain, low-noise amplification in olfactory transduction.

It is desirable that sensory systems use high-gain, low-noise amplification to convert weak stimuli into detectable signals. Here it is shown that a pair of receptor currents underlying vertebrate olfactory transduction constitutes such a scheme. The primary receptor current is an influx of Na+ and Ca2+ through cAMP-gated channels in the olfactory cilia. External divalent cations improve the signal-to-noise properties of this current, reducing the mean current and the current variance. As Ca2+ enters the cilium, it gates Cl- channels, activating a secondary depolarizing receptor current. This current amplifies the primary current, but introduces little additional noise. The system of two currents plus divalent cations in the mucus produces a large receptor current with very low noise.     

The wave of activation current in the Xenopus egg

A ring-shaped wave of inward current, the activation current, propagates across the Xenopus egg from the site of activation during the positive phase of the activation or fertilization potential. This activation current wave is due to an increased chloride conductance and reflects the propagated of the ionic channels responsible for the fertilization potential. These channels are present in the animal and vegetal hemispheres; however, the magnitude of the activation current is 6-7 times greater in the animal hemisphere. Outward current of a smaller magnitude and spread out over a larger area precedes and follows the inward current except at the point of activation where the current is first inward. The inward current wave is detected in all eggs activated by sperm and in eggs activated by pricking with a sharp needle, by application of the Ca2+ ionophore, A23187, and by intracellular iontophoresis of Ca2+ or inositol 1,4,5-trisphosphate. Reduction of the inward current by TMB-8, which blocks intracellular calcium release in some cells, suggests that the activation current channels are calcium sensitive and that the current wave is concomitant with a wave of increased intracellular calcium initiated by sperm-egg interaction. The wave of cortical granule exocytosis and two or more contraction waves follow the current wave.     

Properties of an endogenous steady current in rat muscle

A vibrating probe was used to study a steady electric current generated by isolated, whole lumbrical muscles of the rat. Spatial mapping showed that current leaves the muscle in the synaptic region and re-enters in the flanking extrajunctional regions. The point of maximum outward current coincided precisely with the endplate region. As the probe was moved radially away from the endplate region, the current declined monotonically, and the results could be fit with a simple model. As the probe was moved axially away from the endplate region, the current declined and became inward over a distance of approximately 0.5 mm. The physiological mechanism by which the current is generated was also studied. alpha-Bungarotoxin and tetrodotoxin had no significant effect on the current, which suggests that acetylcholine channels and gated sodium channels are not involved in the generation of the current. Ouabain produced a slowly developing, partial inhibition of the current, reducing it by approximately 40% over a period of 30-40 min. Carbachol produced a large inward current at the endplate region. After the carbachol action was terminated with alpha-bungarotoxin, an outward current reappeared, and a transient "overshoot" developed. During the overshoot, which lasted approximately 30-40 min, the outward current was approximately doubled. This overshoot was completely abolished by ouabain. The overshoot is interpreted as reflecting the increased activity of electrogenic sodium pumping in the endplate region, caused by the influx of Na ions during carbachol application. Because of the very different actions of ouabain on the normal current and on the overshoot after carbachol application, we concluded that the normal outward current is not produced by electrogenic sodium pumping in the endplate region.     

Fine structure of the intracochlear potential field. I. The silent current.

Field potentials were recorded along radial tracks in scala tympani and scala vestibuli of the guinea-pig cochlea. A current density analysis revealed standing current density profiles that were qualitatively similar between animals and between the second and third cochlear turns. Radial standing current densities were greatest at or near the spiral ligament. All the scala vestibuli current density profiles were scaled versions of one another while the scala tympani current density profiles showed more variability. Acoustic stimuli modulated the standing current and there was a cochlear microphonic current density peak in scala tympani near the organ of Corti. The results are summarized with a current-density field line model, the key element of which is a constant current pumped into scale media by the stria vascularis. The standing potential gradients drive current from each perilymphatic chamber into the spiral ligament en route to the lateral surface of the stria vascularis. The strial current is divided between the receptor cell pathway and leakage pathways. The standing current through the leakage pathways is indirectly modulated by acoustic stimulation through the modulation of the endocochlear potential. The reciprocal modulation of current between hair cell and leakage pathways suggests that the stria vascularis maintains a constant current during acoustic stimulation. The cochlear standing current is similar to the retinal dark current in its importance for sensory transduction but the fact that the silent current is generated by the stria vascularis and not the receptor cells provides significant benefits for the detection of mechanical stimuli.     

Effect of GLG-V-13, a class III antiarrhythmic agent, on potassium currents in rabbit ventricular myocytes

The effects of a new Class III antiarrhythmic drug, GLG-V-13, on the 4-aminopyridine sensitive transient outward current, on the inward rectifier potassium current, on the ATP sensitive potassium current and on the rapid and slow components of the delayed rectifier potassium current were studied in single rabbit ventricular myocytes using the whole-cell voltage-clamp technique. GLG-V-13 blocked the rapid component of the delayed rectifier potassium current in a dose-dependent manner, with an estimated EC50 value of 0.36 microM. At high concentration, the slow component of the delayed rectifier potassium current was also depressed by the drug (40% effect at 10 microM concentration). The transient outward current, the inward rectifier potassium current and the ATP sensitive potassium current were not influenced by GLG-V-13, even at 10 microM concentration. Thus, GLG-V-13 blocks predominantly the rapid component of the delayed rectifier potassium current which may play a significant role in the prolongation of repolarization by the drug in ventricular tissue.     

Constitutive expression of a Mg2+-inhibited K+ current and a TRPM7-like current in human erythroleukemia cells

Whole cell patch-clamp experiments were undertaken to define the basal K(+) conductance(s) in human erythroleukemia cells and its contribution to the setting of resting membrane potential. Experiments revealed a non-voltage-activated, noninactivating K(+) current. The magnitude of the current recorded under whole cell conditions was inhibited by an increase in free intracellular Mg(2+) concentration. Activation or inactivation of the Mg(2+)-inhibited K(+) current (MIP) was paralleled by activation or inactivation of a Mg(2+)-inhibited TRPM7-like current displaying characteristics indistinguishable from those reported for molecularly identified TRPM7 current. The MIP and TRPM7 currents were inhibited by 5-lipoxygenase inhibitors. However, inhibition of the MIP current was temporally distinct from inhibition of TRPM7 current, allowing for isolation of the MIP current. Isolation of the MIP conductance revealed a current reversing near the K(+) equilibrium potential, indicative of a highly K(+)-selective conductance. Consistent with this finding, coactivation of the nonselective cation current TRPM7 and the MIP current following dialysis with nominally Mg(2+)-free pipette solution resulted in hyperpolarized whole cell reversal potentials, consistent with an important role for the MIP current in the setting of a negative resting membrane potential. The MIP and TRPM7-like conductances were constitutively expressed under in vivo conditions of intracellular Mg(2+), as judged by their initial detection and subsequent inactivation following dialysis with a pipette solution containing 5 mM free Mg(2+). The MIP current was blocked in a voltage-dependent fashion by extracellular Cs(+) and, to a lesser degree, by Ba(2+) and was blocked by extracellular La(3+) and 2-aminoethoxydiphenyl borate. MIP currents were unaffected by blockers of ATP-sensitive K(+) channels, human ether-à-go-go-related gene current, and intermediate-conductance Ca(2+)-activated K(+) channels. In addition, the MIP current displayed characteristics distinct from conventional inwardly rectifying K(+) channels. A similar current was detected in the leukemic cell line CHRF-288-11, consistent with this current being more generally expressed in cells of leukemic origin.     

Calcium currents in a fast-twitch skeletal muscle of the rat

Slow ionic currents were measured in the rat omohyoid muscle with the three-microelectrode voltage-clamp technique. Sodium and delayed rectifier potassium currents were blocked pharmacologically. Under these conditions, depolarizing test pulses elicited an early outward current, followed by a transient slow inward current, followed in turn by a late outward current. The early outward current appeared to be a residual delayed rectifier current. The slow inward current was identified as a calcium current on the basis that (a) its magnitude depended on extracellular calcium concentration, (b) it was blocked by the addition of the divalent cations cadmium or nickel, and reduced in magnitude by the addition of manganese or cobalt, and (c) barium was able to replace calcium as an inward current carrier. The threshold potential for inward calcium current was around -20 mV in 10mM extracellular calcium and about -35 mV in 2 mM calcium. Currents were net inward over part of their time course for potentials up to at least +30 mV. At temperatures of 20-26 degrees C, the peak inward current (at approximately 0 mV) was 139 +/- 14 microA/cm2 (mean +/- SD), increasing to 226 +/- 28 microA/cm2 at temperatures of 27-37 degrees C. The late outward current exhibited considerable fiber-to-fiber variability. In some fibers it was primarily a time-independent, nonlinear leakage current. In other fibers it was primarily a time-independent, nonlinear leakage current. In other fibers it appeared to be the sum of both leak and a slowly activated outward current. The rate of activation of inward calcium current was strongly temperature dependent. For example, in a representative fiber, the time-to-peak inward current for a +10-mV test pulse decreased from approximately 250 ms at 20 degrees C to 100 ms at 30 degrees C. At 37 degrees C, the time-to-peak current was typically approximately 25 ms. The earliest phase of activation was difficult to quantify because the ionic current was partially obscured by nonlinear charge movement. Nonetheless, at physiological temperatures, the rate of calcium channel activation in rat skeletal muscle is about five times faster than activation of calcium channels in frog muscle. This pathway may be an important source of calcium entry in mammalian muscle.     

Measurement of electric current evoked by substrate transport via bi-directional H+/oligopeptide transporter over-expressed in HeLa cells: electrogenic efflux and existence of a newly observed channel-like state

In the present study, we measured an electric current induced by substrate transport in a HeLa cell over-expressing a human intestinal di/tri-peptide transporter using the whole-cell patch-clamp technique. Gly-Sar, a typical substrate, induced an inward current associated with its uptake, which showed concentration-dependency following Michaelis-Menten-type kinetics with an apparent K(0.5) of 1.3mM as well as voltage-dependency. An outward current accompanying the efflux of Gly-Sar was also observed after washing out the cell. This outward current was voltage-dependent and was reduced by the inward proton gradient. In the case of hydrophobic dipeptides such as Gly-Phe and Gly-Leu, a distinctive current was observed: after washing out the cells, no outward current was observed, but rather, an 'inward leak' current was sustained in spite of the absence of transportable substrate. This leaky current was abolished by the perfusion of Gly-Sar and subsequent washing. It is considered that the hydrophobic substrate sticks within the substrate-binding site and causes the newly observed state, or the 'inward leak' current.     

Prevention of Pseudomonas aeruginosa adhesion by electric currents

The process of controlling bacterial adhesion using an electric current deserves attention because of its ease of automation and environmentally friendly nature. This study investigated the role of electric currents (negative, positive, alternating) for preventing adhesion of Pseudomonas aeruginosa and achieving bacterial inactivation. Indium tin oxide (ITO) film was used as a working electrode to observe adhesion and inactivation under electric polarization. Electric current types were classified into negative, positive, and alternating current. The working electrode acted as a cathode or anode by applying a negative or positive current, and an alternating current indicates that the negative current was combined sequentially with the positive current. The numbers of adhered cells were compared under a flow condition, and the in situ behavior of the bacterial cells and the extent of their inactivation were also investigated using time-lapse recording and live/dead staining, respectively. The application of a negative current prevented bacterial adhesion significantly (～81% at 15.0 μA cm(-2)). The positive current did not significantly inhibit adhesion (<20% at 15.0 μA cm(-2)), compared to the nonpolarized case. The alternating current had a similar effect as the negative current on preventing bacterial adhesion, but it also exhibited bactericidal effects, making it the most suitable method for bacterial adhesion control.     

Influence of chloride, potassium, and tetraethylammonium on the early outward current of sheep cardiac Purkinje fibers

In voltage clamp studies of cardiac Purkinje fibers, a large early outward current is consistently observed during depolarizations to voltages more positive than -20 mV. After the outward peak of the current, the total membrane current declines slowly. Dudel et al. (1967. Pfluegers Arch. Eur. J. Physiol. 294:197--212) reduced the extracellular chloride concentration and found that the outward peak and the decline of the current were abolished. They concluded that the total membrane current at these voltages was largely determined by a time- and voltage-dependent change in the membrane chloride conductance. We reinvestigated the chloride sensitivity of this current, taking care to minimize possible sources of error. When the extracellular chloride concentration was reduced to 8.6% of control, the principal effect was a 20% decrease in the peak amplitude of the outward current. This implies that the membrane chloride conductance is not the major determinant of the total current at these voltages. The reversal potential of current tails obtained after a short conditioning depolarization was not changed by alterations in the extracellular chloride or potassium concentrations. We suspect that the tail currents contain both inward and outward components, and that the apparent reversal potential of the net tail current largely reflects the kinetics of the outward component, so that this experiment does not rule out potassium as a possible charge carrier. The possibility that potassium carries much of the early outward current was further investigated using tetraethylammonium, which blocks potassium currents in nerve and skeletal muscle. This drug substantially reduced the early outward current, which suggests that much of the early outward current is carried by potassium ions.     

The Drosophila Shaker gene codes for a distinctive K+ current in a subset of neurons

A transient K+ current coded by the Shaker gene was identified in muscle and expressed in Xenopus oocytes by injecting cRNA transcribed from a cloned cDNA. The Shaker current has not previously been identified in neurons. Mutational analysis now reveals that in neurons, Shaker is required for expression of a very rapidly inactivating K+ current with a depolarized steady-state inactivation curve. Together, these properties distinguish the Shaker-coded current from similar fast transient K+ currents coded by other genes. The Sh5 mutation further enhanced the depolarization of the Shaker current steady-state inactivation curve. Deletion of the Shaker gene completely removes the transient K+ current from a small percentage of neurons (15%) in a mixed population, and removes a portion of the whole-cell current in about 35% of neurons. The remaining 50% of neurons were apparently unaffected by deletion of the Shaker gene. The unique combination of rapid inactivation and depolarized steady-state inactivation of the Shaker current may reflect a unique functional role for this current in the nervous system such as the rapid repolarization of action potentials.     

Barium currents in developing skeletal muscle cells of normal and mutant mice foetuses with 'muscular dysgenesis'.

The ontogenesis of Ca channel activities was studied in the developing myotubes of normal mice and mutant mice foetuses with 'Muscular Dysgenesis'. The ionic current through Ca channels was measured with Ba2+ as charge carrier using the whole cell clamp technique. All dissociated myotubes from foetuses (14th to 18th day of gestation) showed two distinct inward Ba currents: a low threshold, transient current (T-type) and a high threshold sustained current. In normal myotubes, T-type current density increased from the 14th day to the 16th day of gestation. After day 16, T-type current density decreased gradually until birth. Similar changes in T-type current density were observed in developing dysgenic myotubes where the current density was about 40% of that measured in normal myotubes throughout the prenatal period studied. The high threshold sustained current (L-type current) density increased gradually with age in normal myotubes while absent in dysgenic muscle. The latter, regardless of age, showed a high threshold current (Idys) which is distinct from the L-type current. Idys density did not change during the prenatal myogenesis period studied.     

Inactivation of calcium current in the somatic membrane of snail neurons

The decline of calcium inward currents evoked by a long-lasting membrane depolarization was studied on isolated snail neurons internally perfused with a K+-free solution. Two exponential components superimposed on a steady inward current could be distinguished, a slow decline with a time constant of several hundreds of milliseconds, observed at all the testing potentials used, and a fast one with a time constant of several dozens of milliseconds, which appeared at depolarizations to about -10 mV and above. When the calcium current was blocked by extracellular Cd2+ or verapamil, an outward current could be recorded at the same depolarizations. Subtraction of the latter current from the total current, recorded prior to the blockage, largely reduced the fast component of the decline of the total current. An increase in pHi from 7.3 to 8.1 led to the elimination of both the outward current and the fast component of the calcium current decline. The slow component remained practically unchanged, with its rate depending upon the current amplitude. It was slowed following intracellular administration of EDTA, and after equimolar substitution of Ba2+ for Ca2+. It is concluded that the fast component of the calcium inward current decline is mainly due to the superposition of the outward current produced by low selective channels. Only the slow component represents an actual decline of the inward current through calcium channels; it is due to ion accumulation at the inner surface of the cell membrane.     

Prevention of Pseudomonas aeruginosa adhesion by electric currents

The process of controlling bacterial adhesion using an electric current deserves attention because of its ease of automation and environmentally friendly nature. This study investigated the role of electric currents (negative, positive, alternating) for preventing adhesion of Pseudomonas aeruginosa and achieving bacterial inactivation. Indium tin oxide (ITO) film was used as a working electrode to observe adhesion and inactivation under electric polarization. Electric current types were classified into negative, positive, and alternating current. The working electrode acted as a cathode or anode by applying a negative or positive current, and an alternating current indicates that the negative current was combined sequentially with the positive current. The numbers of adhered cells were compared under a flow condition, and the in situ behavior of the bacterial cells and the extent of their inactivation were also investigated using time-lapse recording and live/dead staining, respectively. The application of a negative current prevented bacterial adhesion significantly (～81% at 15.0 μA cm^?2). The positive current did not significantly inhibit adhesion (<20% at 15.0 μA cm^?2), compared to the nonpolarized case. The alternating current had a similar effect as the negative current on preventing bacterial adhesion, but it also exhibited bactericidal effects, making it the most suitable method for bacterial adhesion control.     

Calcium-dependent potassium current in barnacle photoreceptor

When barnacle lateral eye photoreceptors are depolarized to membrane potentials of 0 to +50 mV in the dark, the plot of outward current through the cell membrane against time has two distinct maxima. The first maximum occurs 5-10 ms after the depolarization began. The current then decays to a minimum at approximately 500 ms after the onset of depolarization, and then increases to a second maximum 4-6 s after the depolarization began. If depolarization is maintained, the current again decays to reach a steady value approximately 1 min after depolarization began. The increase in current to the maximum at 4-6s from the minimum at approximately 500 ms is termed the "late current." It is maximum for depolarizations to around +25 mV and is reduced in amplitude at more positive potentials. It is not observed when the membrane is depolarized to potentials more positive than +60 mV. The late current is inhibited by external cobaltous ion and external tetraethylammonium ion, and shows a requirement for external calcium ion. When the calcium-sequestering agent EGTA is injected, the late current is abolished. Illumination of a cell under voltage clamp reduces the amplitude of the late current recorded subsequently in the dark. On the basis of the voltage dependence and pharmacology of the late current, it is proposed that the current is a calcium-dependent potassium current.     

Energy of Plasma Flows Accelerated in a Current Sheet as a Function of the Current Flowing through the Sheet

The energy of directed plasma flows accelerated in a current sheet is studied experimentally as a function of the current flowing through the sheet. It is found that the plasma flow energy rapidly increases with increasing current amplitude, nearly according to a power law with an exponent of 1.8–1.9. Using relations for the neutral current sheet and the concept of plasma acceleration under action of the Ampère forces, the energy of directed plasma flows is estimated as a function of the total current flowing through the sheet. It is shown that, as the current rises, the ion energy grows due to an increase in both the Ampère forces and the width of the current sheet within which plasma is accelerated.