Ion currents and membrane domains in the cleaving Xenopus egg

We used an extracellular vibrating probe to measure ion currents through the cleaving Xenopus laevis egg. Measurements indicate sharp membrane heterogeneities. Current leaves the first cleavage furrow after new, unpigmented membrane is inserted. This outward current may be carried by K+ efflux. No direct involvement of the Na+,K+-ATPase in the generation of this outward current is detected at first cleavage. Inward current enters the old, pigmented membrane; however, it does not enter uniformly. The inward current is largest at the old membrane bordering the new membrane. This suggests a heterogeneous ion channel distribution within the old membrane. Experiments suggest that the inward current may be carried by Na+ influx, Ca2+ influx, and Cl- efflux. No steady currents were detected during grey crescent formation, the surface contraction waves preceding cleavage, or with groove formation at the beginning of cleavage.     

Natural H Currents Traverse Growing Roots and Root Hairs of Barley (Hordeum vulgare L.)

With the aid of an extracellular vibrating electrode, natural electric fields were detected and measured in the medium near growing roots and root hairs of barley seedlings. An exploration of these fields indicates that both the root as a whole, as well as individual root hairs, drive large steady currents through themselves. Current consistently enters both the main elongation zone of the root as well as the growing tips of elongating root hairs; it leaves the surface of the root beneath the root hairs. These currents enter with a density of about 2 microamperes per square centimeter, leave with a density of about 0.5 to 1 microampere per square centimeter, and total about 30 nanoamperes.     

Oöplasmic segregation and secretion in the Pelvetia egg is accompanied by a membrane-generated electrical current

Using an ultrasensitive extracellular vibrating electrode, I have studied the membrane-generated electrical currents around the egg of the brown alga, Pelvetia, between fertilization and germination. During this period, the egg chooses an elongation axis and moves wall-precursor vesicles to the prospective growth region where they are secreted. This results in visible oöplasmic segregation which appears under the light microscope as a 1- to 2-μm-thick clear band at the cortex of the growth region. A steady electrical current enters a small region of the membrane and leaves the remainder of the egg's surface as early as 30 min after fertilization. This early spatial current pattern is unstable and shifts position, often with more than one inward current region. However, current enters mainly on the side where germination will occur and is usually largest at the prospective cortical clearing region. The average measured early current density is 0.06 μA/cm² at 50 μm from the egg's surface, implying a surface current density of between 0.2 and 1 μA/cm² due to the extrapolation uncertainty. At germination the current increases about twofold, resulting in a total transcellular current on the order of 100 pA. Unilateral growth-orienting light reversal stimulates inward current on the new dark side, and subsequent morphological polarity reversal is preceded by electrical polarity reversal. The steady current tends to increase when the external Ca²⁺ concentration is increased or the external Na⁺ concentration is decreased, suggesting that the current is carried in part by Ca²⁺. This current will generate a transcellular electrical field which may be the force driving the observed oöplasmic segregation.     

Electrical currents associated with rhythmic contractions of the blastoderm of the medaka, Oryzias latipes

1. We used a vibrating probe to measure extracellular electrical currents near the surface of dechorionated Oryzias latipes eggs as contraction waves moved slowly across the blastoderm. 2. Although we found no detectable current outside dechorionated embryos, we recorded large current pulses near the edge of wounds made in the surface of the blastoderm. 3. The maximum net inward current--or in some cases, the least net outward current--correlated temporally with the contraction of cells near the edge of the wound. 4. The current pulses were superimposed on steady currents of variable magnitude and polarity. 5. We discuss possible mechanisms for the initiation and propagation of the contraction wave.     

Interfering with calcium release suppresses I gamma, the "hump" component of intramembranous charge movement in skeletal muscle.

Four manifestations of excitation-contraction (E-C) coupling were derived from measurements in cut skeletal muscle fibers of the frog, voltage clamped in a Vaseline-gap chamber: intramembranous charge movement currents, myoplasmic [Ca2+] transients, flux of calcium release from the sarcoplasmic reticulum (SR), and the intrinsic optical transparency change that accompanies calcium release. In attempts to suppress Ca release by direct effects on the SR, three interventions were applied: (a) a conditioning pulse that causes calcium release and inhibits release in subsequent pulses by Ca-dependent inactivation; (b) a series of brief, large pulses, separated by long intervals (greater than 700 ms), which deplete Ca2+ in the SR; and (c) intracellular application of the release channel blocker ruthenium red. All these reduced calcium release flux. None was expected to affect directly the voltage sensor of the T-tubule; however, all of them reduced or eliminated a component of charge movement current with the following characteristics: (a) delayed onset, peaking 10-20 ms into the pulse; (b) current reversal during the pulse, with an inward phase after the outward peak; and (c) OFF transient of smaller magnitude than the ON, of variable polarity, and sometimes biphasic. When the total charge movement current had a visible hump, the positive phase of the current eliminated by the interventions agreed with the hump in timing and size. The component of charge movement current blocked by the interventions was greater and had a greater inward phase in slack fibers with high [EGTA] inside than in stretched fibers with no EGTA. Its amplitude at -40 mV was on average 0.26 A/F (SEM 0.03) in slack fibers. The waveform of release flux determined from the Ca transients measured simultaneously with the membrane currents had, as described previously (Melzer, W., E. Ríos, and M. F. Schneider. 1984. Biophysical Journal. 45:637-641), an early peak followed by a descent to a steady level during the pulse. The time at which this peak occurred was highly correlated with the time to peak of the current suppressed, occurring on average 6.9 ms later (SEM 0.73 ms). The current suppressed by the above interventions in all cases had a time course similar to the time derivative of the release flux; specifically, the peak of the time derivative of release flux preceded the peak of the current suppressed by 0.7 ms (SEM 0.6 ms). The magnitude of the current blocked was highly correlated with the inhibitory effect of the interventions on Ca2+ release flux.(ABSTRACT TRUNCATED AT 400 WORDS)     

Large electrical currents traverse growing pollen tubes

Using a newly developed vibrating electrode, we have explored the electric fields around lily pollen germinating in vitro. From these field measurements, we infer that each weeted pollen drives a steady current of a few hundred picoamperes through itself. Considered as a flow of positive ions, this current enters an ungerminated grain's prospective growth site and leaves it opposite end. After a grain germinates and forms a tube, this current enters most of the growing tube and leaves the whole grain. The current densities over both of these extended surface regions are relatively uniform, and the boundary zone, near the tube's base, is relatively narrow. This current continues as long as the tube grows, and even continues when elongation, as well as cytoplasmic streaming, are blocked by 1 mug/ml of cytochalasin B. After a otherwise indistinguishable minority of tubes have grown to lengths of a millimeter or more, their current comes to include an endless train of discrete and characteristic current pulses as well as a steady component. These pulses are about 30s long, never overlap, recur every 60-100s, and seem to enter a region more restricted to be growing tip than the steady current's sink. In most ways, the current through growing lily pollen resembles that known to flow through focoid eggs.     

Development of a steady electric current in neonatal rat lumbrical muscle

We have used a vibrating probe and intracellular recording techniques to study the development of a steady electric current generated by rat lumbrical muscle. In adult animals, previous work has revealed a steady outward current generated at the end plate region. In the present study, we show that at birth muscles generate a steady inward, not outward current. The inward current declines with age, disappearing about 5 days after birth. At about the same time, the steady outward current appears, and reaches adult amplitude by 2-3 weeks after birth. The two currents are generated by completely different mechanisms. The inward current is blocked by alpha-bungarotoxin and apparently results from activation of acetylcholine-gated channels at the end plate. The outward current, on the other hand, is not affected by alpha-bungarotoxin but is blocked by agents which interfere with chloride movements across the membrane, as in the adult.     

The pulse current pattern generated by developing fucoid eggs

Using a newly developed extracellular vibrating electrode, we have made the first study of the spatial distribution of the growth currents around a single developing egg. This pattern was studied during the current pulses wihic traverse two-celled Pelvetia embryos. These pulses can be stimulated to occur with a periodicity of 70 min by mild acidification of the dea water medium. Current enters only at the growing rhizoid's tip while leaving both the base of the rhizoid cell and the whole outer membrane of the thallus cell. The field in front of the rhizoid cell falls off as the inverse cube of the distance from the rhizoid cell's center in the manner of a dipole field. The total inward and outward currents are equal, agreeing with theory. The current density at the rhizoid cell's base is twice that at the top of the thallus cell and this probably represents a change in the outer membrane's properties. There are no significant differences in the durrent density over the thallus cell. These results suggest a model in which the pulse current leaks in through newly opened channels in the growing tip and leaks out elsewhere due to the resultant fall in the membrane potential.     

A damped oscillation in the intramembranous charge movement and calcium release flux of frog skeletal muscle fibers

Asymmetric membrane currents and calcium transients were recorded simultaneously from cut segments of frog skeletal muscle fibers voltage clamped in a double Vaseline-gap chamber in the presence of high concentration of EGTA intracellularly. An inward phase of asymmetric currents following the hump component was observed in all fibers during the depolarization pulse to selected voltages (congruent to -45 mV). The average value of the peak inward current was 0.1 A/F (SEM = 0.01, n = 18), and the time at which it occurred was 34 ms (SEM = 1.8, n = 18). A second delayed outward phase of asymmetric current was observed after the inward phase, in those experiments in which hump component and inward phase were large. It peaked at more variable time (between 60 and 130 ms) with amplitude 0.02 A/F (SEM = 0.003, n = 11). The transmembrane voltage during a pulse, measured with a glass microelectrode, reached its steady value in less than 10 ms and showed no oscillations. The potential was steady at the time when the delayed component of asymmetric current occurred. ON and OFF charge transfers were equal for all pulse durations. The inward phase moved 1.4 nC/microF charge (SEM = 0.8, n = 6), or about one third of the final value of charge mobilized by these small pulses, and the second outward phase moved 0.7 nC/microF (SEM = 0.8, n = 6), bringing back about half of the charge moved during the inward phase. When repolarization intersected the peak of the inward phase, the OFF charge transfer was independent of the repolarization voltage in the range -60 to -90 mV. When both pre- and post-pulse voltages were changed between -120 mV and -60 mV, the equality of ON and OFF transfers of charge persisted, although they changed from 113 to 81% of their value at -90 mV. The three delayed phases in asymmetric current were also observed in experiments in which the extracellular solution contained Cd2+, La3+ and no Ca2+. Large increases in intracellular [Cl-] were imposed, and had no major effect on the delayed components of the asymmetric current. The Ca2+ transients measured optically and the calculated Ca2+ release fluxes had three phases whenever a visible outward phase followed the inward phase in the asymmetric current. Several interventions intended to interfere with Ca release, reduced or eliminated the three delayed phases of the asymmetric current.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Bioelectric aspects of the hemipteran telotrophic ovariole (Dysdercus intermedius)

Summary Two systems of steady extra-cellular currents were found along the surface of the telotrophicDysdercus ovarioles by means of a vibrating probe. The first covers the subgerminal tropharium and all the previtellogenic follicles. The current leaves the 3 or 4 small follicles of early euplasmic growth stages laterally and enters the syncytial tropharium. We presume that a similar intracellular current flows between the trophoplasm and the ooplasm which are interconnected by narrow nurse strands. Preliminary intracellular measurements indicate a potential gradient within this continuous cytoplasm, the ooplasm being electropositive to that of the tropharium. This current system fits into a model of polarized intracytoplasmic transport by electrophoresis. It is possible to explain the well known directed and selective flow of RNA from the tropharium via the nurse strands into the oocytes by means of such a model. The second current system occurs around every one of the 2 to 8 vitellogenic follicles. The pattern is completely different from that described for the first system. In the vitellogenic stages the current enters the follicle laterally all along the now much extended surface. It is balanced by a strong peak current which leaves the interfollicular region. As data on intracellular currents are not yet avialable, it is only a matter of speculation whether the circuit is closed through the ooplasm or only by a tangential loop through the follicle epithelium. The possible significance of this second current system for vitellogenin accumulation and uptake by the vitellogenic oocytes is also uncertain as yet.Supported by the Deutsche Forschungsgemeinschaft (Schwerpunkt Differenzierung)     

Local ion currents controlling the localized cytoplasmic movement associated with feeding initiation ofNoctiluca

Summary We used an extracellular vibrating probe to investigate local transmembrane ion currents that occur just before and during localized cytoplasmic movement associated with feeding initiation in the marine dinoflagellateNoctiluca, Our results indicates that the currents flow only through a specialized cellular region, the sulcus, suggesting a heterogeneous distribution of an ion channel in the cell membrane. A current enters into the middle of the sulcus where the cytostome exists and leaves from both ends of the sulcus. The mean inward and outward current densities were approx. + 11 and — 1 A·cm^–2, respectively. The cytoplasm began to stream toward the cytostome in association with the currents and then aggregated around it. Removal of Ca²⁺, Na⁺, or Mg²⁺ ions from the external medium diminished the inward current. Ca²⁺ ions were proved to carry only 5% of the inward current. The Ca²⁺ current appears to be enough to raise Ca²⁺ concentration in a localized region of the cytoplasm, causing the cytostome-directed cytoplasmic movement. Rest of the current seems to be carried by Na⁺ ions. Most of the outward current was inhibited by an ion pump inhibitor, but the current-carrying ion species could not be identified.     

Transmembrane ion currents in pulmonary artery smooth muscle

Transmembrane ionic currents were investigated in the rabbit pulmonary artery smooth muscle under voltage clamp conditions with the use of the double sucrose gap method. With depolarizing pulses, there developed a fast inactivated outward current that was followed by a steady-state outward current. Tetraethylammonium (TEA) partly suppressed the outward current, and the fast inward current that preceded the fast outward one could be seen in these conditions. Appearance of the fast inward current in TEA-containing solution suggests the overlapping of the fast inward and outward currents. It appears that the resultant transmembrane current has an outward direction since in normal conditions the permeability of the fast potassium channels exceeds that of calcium channels. Conditioning hyperpolarization increased and depolarization decreased the fast outward current indicating that at the resting membrane potential a part of the potassium channels is inactivated and this inactivation is removed by hyperpolarization.     

Ionic currents in two strains of rat anterior pituitary tumor cells

The ionic conductance mechanisms underlying action potential behavior in GH3 and GH4/C1 rat pituitary tumor cell lines were identified and characterized using a patch electrode voltage-clamp technique. Voltage-dependent sodium, calcium, and potassium currents and calcium-activated potassium currents were present in the GH3 cells. GH4/C1 cells possess much less sodium current, less voltage-dependent potassium current, and comparable amounts of calcium current. Voltage-dependent inward sodium current activated and inactivated rapidly and was blocked by tetrodotoxin. A slower-activating voltage-dependent inward calcium current was blocked by cobalt, manganese, nickel, zinc, or cadmium. Barium was substituted for calcium as the inward current carrier. Calcium tail currents decay with two exponential components. The rate constant for the slower component is voltage dependent, while the faster rate constant is independent of voltage. An analysis of tail current envelopes under conditions of controlled ionic gradients suggests that much of the apparent decline of calcium currents arises from an opposing outward current of low cationic selectivity. Voltage-dependent outward potassium current activated rapidly and inactivated slowly. A second outward current, the calcium-activated potassium current, activated slowly and did not appear to reach steady state with 185-ms voltage pulses. This slowly activating outward current is sensitive to external cobalt and cadmium and to the internal concentration of calcium. Tetraethylammonium and 4-aminopyridine block the majority of these outward currents. Our studies reveal a variety of macroscopic ionic currents that could play a role in the initiation and short-term maintenance of hormone secretion, but suggest that sodium channels probably do not make a major contribution.     

Electric Currents around Growing Trichoderma Hyphae, before and after Photoinduction of Conidiation

Electric currents were measured around Trichoderma harzianum (Rifai) hyphae using an extracellular vibrating electrode. A steady current enters growing hyphal tips and along the side of the apical millimeter. In addition, outward currents were detected at about one-ninth of the locations tested, 60 to 150 minutes after illumination but not in dark controls. This sporadic, localized outward current pattern might be an early biophysical response to blue light.     

Effects of Internal Divalent Cations on Voltage-Clamped Squid Axons

We have studied the effects of internally applied divalent cations on the ionic currents of voltage-clamped squid giant axons. Internal concentrations of calcium up to 10 mM have little, if any, effect on the time-course, voltage dependence, or magnitude of the ionic currents. This is inconsistent with the notion that an increase in the internal calcium concentration produced by an inward calcium movement with the action potential triggers sodium inactivation or potassium activation. Low internal zinc concentrations (~1 mM) selectively and reversibly slow the kinetics of the potassium current and reduce peak sodium current by about 40% with little effect on the voltage dependence of the ionic currents. Higher concentrations (~10 mM) produce a considerable (ca. 90%) nonspecific reversible reduction of the ionic currents. Large hyperpolarizing conditioning pulses reduce the zinc effect. Internal zinc also reversibly depolarizes the axon by 20–30 mV. The effects of internal cobalt, cadmium, and nickel are qualitatively similar to those of zinc: only calcium among the cations tested is without effect.     

Two modes of gating during late Na+ channel currents in frog sartorius muscle

Na+ currents were measured during 0.4-s depolarizing pulses using the cell-attached variation of the patch-clamp technique. Patches on Cs-dialyzed segments of sartorius muscle of Rana pipiens contained an estimated 25-500 Na+ channels. Three distinct types of current were observed after the pulse onset: a large initial surge of inward current that decayed within 10 ms (early currents), a steady "drizzle" of isolated, brief, inward unitary currents (background currents), and occasional "cloudbursts" of tens to hundreds of sequential unitary inward currents (bursts). Average late currents (background plus bursts) were 0.12% of peak early current amplitude at -20 mV. 85% of the late currents were carried by bursting channels. The unit current amplitude was the same for all three types of current, with a conductance of 10.5 pS and a reversal potential of +74 mV. The magnitudes of the three current components were correlated from patch to patch, and all were eliminated by slow inactivation. We conclude that all three components were due to Na+ channel activity. The mean open time of the background currents was approximately 0.25 ms, and the channels averaged 1.2 openings for each event. Neither the open time nor the number of openings of background currents was strongly sensitive to membrane potential. We estimated that background openings occurred at a rate of 0.25 Hz for each channel. Bursts occurred once each 2,000 pulses for each channel (assuming identical channels). The open time during bursts increased with depolarization to 1-2 ms at -20 mV, whereas the closed time decreased to less than 20 ms. The fractional open time during bursts was fitted with m infinity 3 using standard Na+ channel models. We conclude that background currents are caused by a return of normal Na+ channels from inactivation, while bursts are instances where the channel's inactivation gate spontaneously loses its function for prolonged periods.     

Spatial and temporal transcellular current patterns during oogenesis.

We have used the two-dimensional vibrating probe to examine spatial and temporal patterns in the transcellular current flow around telotrophic ovarioles of the insect Rhodnius prolixus. We demonstrate a dynamic pattern of currents which correlates with various stages of vitellogenesis. Asymmetries exist in the radial current pattern around intact ovarioles, particularly around the terminal follicle, and may correlate with early developmental axes. The extra-cellular current pattern is largely reflected by a similar, though weaker pattern of currents over the germ cell membranes, indicating that both germ cell and somatic cell membranes are involved in current generation. Current enters previtellogenic oocytes and leaves oocytes entering vitellogenesis. We speculate that current reversal and loss of trophic cord contact may represent an electrophysiological feedback control mechanism during oogenesis.     

Synchronization of Na/K pump molecules by a train of squared pulses

We experimentally studied the Na/K pump currents evoked by a train of squared pulses whose pulse-duration is about the time course of Na-extrusion at physiological conditions. The magnitude of the measured pump current can be as much as three-fold of that induced by the traditional single pulse measurement. The increase in the pump current is directly dependent on the number of pre-pulses. The larger the number of the pre-pulses is, the higher the current magnitude can be obtained. At a particular number of pre-pulses, the pump current becomes saturated. These results suggest that a large number of pre-pulses may synchronize the pump molecules to work at the same pace. As a result, the pump molecules may extrude Na ions at the same time corresponding to the stimulation pulses, and pump in K ions at the same time during the pulse intervals. Therefore, the measured pump current is three-fold of that measured by a single pulse where the outward and inward pump currents are canceled each other.     

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.