Action of calcium on the somatic membrane of mollusk giant neurons

Early membrane currents of the isolated neuron soma of the mollusksHelix pomatia,Limnaea stagnalis, andPlanorbis corneus in normal and sodium-free solutions differing in their calcium ion concentration were investigated by the voltage clamp method. The early inward current was shown to continue when the sodium ions in the external solution were replaced by an equivalent number of calcium ions and to be increased with an increase in the concentration of those ions in all neurons of these mollusks investigated. A change in the calcium concentration in the external solution shifted the inactivation curves and also the curves of conductance for the inward current along the potential axis. It is concluded that a system of calcium channels exists in the somatic membrane of neurons in these species of mollusks.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 5, No. 6, pp. 621–627, November–December, 1973.     

Effect of the alkaloid lappaconitine on ionic currents through the somatic membrane ofHelix pomatia neurons

The effect of the alkaloid lappaconitine on passive ion transport through the somatic membrane of identified neurons of the snailHelix pomatia was studied under voltage clamp conditions. In a concentration of 4 mM lappaconitine has a reversible blocking action on the calcium channels of the excitable membrane. To study the effect of the alkaloid on inward sodium currents a solution in which calcium ions were replaced by the equivalent number of magnesium ions was used. Lappaconitine has no appreciable effect on the inward sodium current.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Institute of Chemistry of Plant Substances, Academy of Sciences of the Uzbek SSR, Tashkent. Translated from Neirofiziologiya, Vol. 11, No. 5, pp. 469–474, September–October, 1979.     

Investigations of the ionic mechanisms of bursting activity in Helix pomatia neurons

Steady-state current-voltage characteristics of the membrane and ionic currents arising during changes in membrane potential in bursting neurons ofHelix pomatia were studied by the voltage clamp method. The steady-state current-voltage characteristics of the membrane were shown to have a nonlinear region. Replacement of sodium ions by Tris-HC1 ions in the external solution completely abolishes this nonlinearity. Hyperpolarization of the membrane under voltage clamp conditions leads to the development of an outward current which reaches a maximum and then is inactivated. This current has a reversal potential in the region of the potassium equilibrium potential. Depolarization of the membrane to the threshold value for excitation of uncontrollable regions of the axon hillock causes the appearance of a slow inward current. After reaching a maximum, the inward current falls to zero. A model of generation of waves in a bursting neuron is suggested.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 10, No. 2, pp. 193–202, March–April, 1978.     

Electrically operated sodium channels in the somatic membrane of sympathetic neurons

Electrically operated sodium channels in the somatic membrane of isolated neurons from the rat superior cervical ganglion were investigated using an intracellular dialysis technique and voltage clamping. It was found that sodium currents can be conveyed along two independent systems of sodium channels in these neurons. A mathematical analysis was made of voltage-dependent tetrodotoxin-sensitive fast sodium currents within the framework of the Hodgkin-Huxley model and their kinetic properties were compared with those described in other subjects. It was also shown that the tetrodotoxin-sensitive sodium channels in the somatic membrane of sympathetic neurons have a high affinity for sodium ions. The kinetic and voltage-dependent characteristics of slow tetrodotoxin-sensitive inward sodium current are described. It is also noted that this component of the sodium current was observed in only a limited number of neurons (not more than 2%).A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 18, No. 1, pp. 108–117, January–February, 1986.     

Separation of sodium and calcium channels in the surface membrane of molluscan nerve cells

By intracellular dialysis of isolated neurons of the mollusksHelix pomatia andLimnaea stagnalis and by a voltage clamp technique the characteristics of transmembrane ionic currents were studied during controlled changes in the ionic composition of the extracellular and intracellular medium. By replacing the intracellular potassium ions by Tris ions, functional blocking of the outward potassium currents was achieved and the inward current distinguished in a pure form. Replacement of Ringer's solution in the extracellular medium with sodium-free or calcium-free solution enabled the inward current to be separated into two additive components, one carried by sodium ions, the other by calcium ions. Sodium and calcium inward currents were found to have different kinetics and different potential-dependence: _mNa=1±0.5 msec, _mCa=3±1 msec, _hNa=8±2 msec, _hCa=115±10 msec (V_m=0), G_Na=0.5 (V_m=–21±2 mV), G_Ca=0.5 (V_m=–8±2 mV). Both currents remained unchanged by tetrodotoxin, but the calcium current was specifically blocked by cadmium ions (2·10^–3 M), verapamil, and D=600, and also by fluorine ions if injected intracellularly. All these results are regarded as evidence that the soma membrane of the neurons tested possesses separate systems of sodium and calcium ion-conducting channels. Quantitative differences are observed in the relative importance of the systems of sodium and calcium channels in different species of mollusks.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 8, No. 2, pp. 183–191, March–April, 1976.     

Mechanisms of generation of long action potentials in barium solutions

Under voltage clamp conditions ionic currents of neurons of the molluskHelix were studied in solutions containing barium ions. Replacement of the calcium ions in the normal external solution by barium ions led to displacement of the potassium conductivity versus membrane potential curve along the voltage axis toward more positive potentials and also to a decrease in the limiting value of the potassium conductance of the membrane. In sodium- and calcium-free solutions containing barium ions two fractions of the inward current are recorded: quickly (I) and slowly (II) inactivated. The rates of activation of these fractions are comparable. Barium ions are regarded as carriers of both fractions of the inward current. It is postulated that both fractions of the barium current are carried along the calcium channels of the membrane.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 9, No. 4, pp. 408–414, July–August, 1977.     

Changes in the ionic mechanisms of the electrical excitability of rat sensory neuronal somatic membrane during ontogenesis. Distribution of ionic channels carrying inward currents

The distribution of different types of ionic channels carrying inward currents was investigated in the somatic membranes of spinal ganglion neurons in rats belonging to three different age groups: at 5–9 days, 45 days, and 3 months. A decrease was found in the number of neuronal membranes operating all four types of inward current channels simultaneously: "fast" (tetrodotoxin-sensitive), "slow" (tetrodotoxin-resistant) sodium currents and low- and high-threshold calcium currents. There were 14.5% of such neurons in the first age group, 5% in the second, and 1% on the third. It was found that this change was related to the disappearance of "slow" (tetrodotoxin-resistant) sodium and high-threshold calcium channels from the membrane. The number of neuronal somatic membranes with only two types of inward current channels ("fast" sodium and high-threshold calcium channels) increased proportionately.A. A. Bogomolets Institute of Technology, Academy of Sciences of the Ukrainian SSR, Kiev Translated from Neirofiziologiya, Vol. 18, No. 6, pp. 813–820, November–December, 1986.     

Changes in the ionic mechanisms of electrical excitability in the somatic membrane of rat dorsal root ganglion neurons during ontogenesis. Correlations between inward current densities

Correlations between densities of various types of inward currents in the somatic membrane of dorsal root ganglion neurons were studied in three different rat age groups: 5–9 days, 45 days, and 90 days. A linear relationship was found in neurons with "slow" tetrodotoxin-sensitive sodium current between the densities of high-threshold calcium current and "slow" sodium current (Bravias-Pearson's correlation coefficient: r=0.84 and 0.70 for n₁=16 and n₂=28, respectively). No such correlation was observed in neurons with low-threshold calcium inward current. Cells with only two types of channel — "fast" sodium and high-threshold calcium — present in their somatic membrane manifested an inverse correlation (r=–0.48, where n₄=95) between the densities of transmembrane currents passing through these channels. No inverse relationship was observed in the density of "fast" sodium and high-threshold calcium currents in neurons with tetradotoxinresistant "slow" sodium and/or low threshold calcium channels.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 18, No. 6, pp. 820–827, November–December, 1986.     

Changes in the ionic mechanisms of electrical excitability in the somatic membrane of rat dorsal root ganglion neurons during ontogenesis. Relationship between calcium channels and intracellular metabolism

It was found during experiments on rat sensory neurons that the relationship between high-threshold calcium channels and the system of intracellular cyclic nucleotide metabolism declined in the course of postnatal ontogenesis. Intracellullar administration of the cAMP-ATP-Mg²⁺ complex led to restoration after dialysis-induced decline in peak amplitude of high-threshold calcium currents in 70% of cells belonging to the first age group of 5–9-day-old animals, as against 26% of those examined in the 2nd (45-day-old) and only 10% of all those investigated in the third (90-day-old) group. Kinetics and voltage-dependence of high-threshold calcium current in the neuronal soma were identical in rats of all three age groups. The effect of recovery in calcium conductivity produced by intracellular application of the cAMP-ATP-Mg²⁺ complex was different in neurons with different inward current combinations. This recovery did not occur in cells with "fast" sodium and high-threshold calcium currents only. Conventional effects of intracellular cAMP application were seen in neurons mainfesting a "slow" TTX-resistant sodium inward current together with the two main inward currents.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vo.. 18, No. 6, pp. 827–832, November–December, 1986.     

Ionic mechanisms of the transmembrane current evoked by injection of cyclic amp into identified Helix pomatia neurons

Ionic mechanisms of the transmembrane current evoked by injection of cyclic AMP into identified neurons ofHelix pomatia were investigated by the voltage clamp method. Injection of cyclic AMP into neurons RPa3, LPa2, LPa3, and LPl1 was shown to cause the development of a two-component transmembrane (cyclic AMP) current. The current-voltage characteristic curve of the early component is linear in the region from –40 to –90 mV; the reversal potential of the early component, determined by extrapolation, lies between –5 and +20 mV; the current-voltage characteristic curve of the late component also is linear and has a reversal potential between –55 and –60 mV. A decrease in the sodium concentration in the external medium from 100 to 25 mM led to a decrease in amplitude of the cyclic AMP current and to a shift of the reversal potential for the early component by 30–32 mV toward hyperpolarization. It is suggested that the early component of the cyclic AMP current in neurons RPa3, LPa2, LPa3, and LPl1 is associated with an increase in permeability of the neuron membrane chiefly for sodium ions, whereas the late component is correspondingly connected with permeability for potassium ions. Injection of cyclic AMP also caused the appearance of a transmembrane inward current in neuron LPa8, but it was independent of the holding potential and was unaccompanied by any change in membrane permeability. It is suggested that this current may be due to a change in the activity of the electrogenic ion pump.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 12, No. 5, pp. 526–532, September–October, 1980.     

Effects of allapinine on sodium currents in neurons isolated from the rat trigeminal ganglion and cardiomyocytes

The effects were investigated of allapinine, diterpene alkaloid on ionic currents in voltage-clamped trigeminal neurons and cardiomyocytes isolated from rats. Allapinine application was found to exert an inhibitory effect on inward tetrodotoxin-sensitive sodium currents without changing their voltage dependence. Potential differences between the mechanisms of antiarrhythmic action of diterpene alkaloids and standard antiarrhythmic substances are examined.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Institute of Plant Chemistry, Academy of Sciences of the Uzbek SSR, Tashkent. Translated from Neirofiziologiya, Vol. 22, No. 2, pp. 201–206, March–April, 1990.     

Hydrogen currents through aconitine-modified sodium channels in the nerve fiber membrane

Ionic currents through aconitine-modified sodium channels of the Ranvier node membrane were measured by a voltage clamp method in an external medium free from sodium ions. A shift of pH of the solution below 4.6 led to the appearance of inward ionic currents, whose kinetics and activation region were characteristic of aconitine-modified sodium channels at low pH. These currents were blocked by the local anesthetic benzocaine in a concentration of 2 mM. Experiments with variation of the concentration of Ca⁺⁺, Tris⁺, TEA⁺, and choline⁺ in acid sodium-free solutions showed that these cations make no appreciable contribution to the inward current. It is concluded that the inward currents observed under these conditions are carried by H⁺ (or H₃O⁺) through aconitine-modified sodium channels. From the shifts of reversal potentials of the ionic currents the relative permeability (P_H/P_Na) for H⁺ was determined: 1059 ± 88. The results agree with the view that the aconitine-modified sodium channel is a relatively wide water pore, and that movement of H⁺ through it is limited by its binding with an acid group.Institute of Cytology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 14, No. 5, pp. 508–516, September–October, 1982.     

Conductances contributing to the action potential of Sternopygus electrocytes

In Sternopygus macrurus, electrocyte action potential duration determines the electric organ discharge pulse duration. Since the electric organ discharge is a sexually-dimorphic behavior under the control of steroid hormones, and because electrocyte action potential durations can range from 3–14 ms, the electrocytes provide a unique opportunity to study how sex steroids regulate membrane excitability. In this study, the voltage-sensitive ionic currents of electrocytes were identified under current- and voltage-clamp as a prelude to further studies on their regulation by sex steroid hormones.Bath application of TTX completely abolished the spike and eliminated an inward current under voltage clamp, indicating that the action potential is due primarily to a sodium current. Calcium-free saline had no effect on spike waveform or voltage-clamp currents, indicating that neither calcium nor calcium-dependent currents contribute to the action potential. Application of potassium channel blocking agents, such as tetraethylammonium and cesium ions, caused changes in the spike which, together with voltage-clamp results, indicate the presence of two potassium currents: an inward rectifier and a classical delayed rectifier. In addition, these cells have a large, presumably voltage-insensitive, chloride current. Differences in one or more of these currents could be responsible for the range of action potential durations found in these cells and for the steroid-mediated changes in spike duration.Abbreviations EOD electric organ discharge - VC voltage clamp - CC current clamp - AP action potential - VI/IV voltage-current/current-voltage     

L-glutamate activated membrane conductivity in spinal cord neurons during early chick embryogenesis

Transient and steady-state components of L-glutamate-activated membrane currents were investigated using intracellular perfusion, voltage clamp, and concentration clamp techniques in spinal cord neurons of 6–11 day chick embryos. Hill's coefficient was found to equal 1 for transient and 2 for steady-state components. It was shown that the L-glutamate-activated receptors are present, which appear in the membrane of spinal neurons at the early stages of development.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 19, No. 2, pp. 251–258, March–April, 1987.     

Postsynaptic mechanisms initiating bursting activity in theHelix pomatia RPal neuron under the influence of an interneuron

Postsynaptic mechanisms of the connection between the interneuron in the visceral ganglion initiating bursting activity in RPal and B7 neurons and these neurons themselves were investigated in the snail (Helix pomatia). Using voltage clamping at the membrane of these cells, stimulation of the interneuron gave rise to a slow inward current with a 2 sec latency; it rose in amplitude as stimulation increased in duration. Reducing the temperature from 25 to 5°C diminished the rise and decay rate of this current with a temperature coefficient of about 10. The current-voltage relationship of the slow inward current was nonlinear, with a maximum of –65 mV. Reducing the concentration of sodium ions in the extracellular fluid increased the amplitude of the current. While hyperpolarization of the burster neuron membrane produced a burst of inward current prior to stimulation, this same hyperpolarization induced a pulse of outward current at the peak of the slow inward current. Stimulating the interneuron is thus thought to activate at least two types of ionic channel in the cell body of the burster neurons: a steady sodium and a voltage- and time-dependent channel for outward current. This process could well be mediated by a biochemical cytoplasmic chain reaction.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 19, No. 1, pp. 28–36, January–February, 1987.     

Long-lasting inward current in snail neurons in barium solutions in voltage-clamp conditions

Summary The inward membrane current was recorded under voltage clamp from nonbursting neurons of the snailHelix pomatia in Na-free solutions containing Ba ions but no other divalent cations. The inward current was separated into two components: (i) an early fast inactivating component and (ii) a smaller long-lasting component. Both components were dependent on the external Ba concentration. It is concluded that both components of the inward current are carried by Ba ions. The activation of the early fast inactivating component of the inward current occurred at more positive membrane potential than that of the long-lasting component. The shape of the inactivation curve for the peak value of the inward current was similar to that for the long-lasting component. The potentials of half-inactivation for the peak value of the inward current and for its long-lasting component were –28 and –22 mV, respectively. The blocking effect of Co⁺⁺ on the early fast inactivating component was substantially greater. In some neurons after treatment with 15mm Co⁺⁺ only the long-lasting component was recorded. The activation kinetics of the long-lasting component of the inward current were analyzed using the Hodgkin-Huxley equations. The results could be explained by assuming that two components of the inward current in Na–Ca-free solution with Ba ions flowed through the two different channels. The significance of the long-lasting inward current for the normal spike generation is discussed.     

Hydrogen ion current through sodium channels in myelinated nerve fiber membrane

Ionic currents through the frog Ranvier node membrane were measured by the voltage clamp method on the membrane of a single myelinated frog's nerve fiber under conditions when Na⁺ in the external solution was replaced by nonpenetrating cations. When pH fell below 4.0, small (under 0.1 nA) inward currents were found and on the basis of various features (kinetics, region of activation, and blocking by the local anesthetic benzocaine — 1.0 mM) were identified as currents through sodium channels. The results of control experiments with variation of the concentrations of cations in the external solution led to the conclusion that the H⁺ (or H₃O⁺) ion is the main carrier of the measured inward current. According to the results of measurement of the reversal potential of these currents, the relative permeability of sodium channels for hydrogen ions (P_H/P_Na) averages 205 ± 14. The results are discussed in terms of a model of the water pore with saturation. It is concluded that the energy barriers for H⁺ in the sodium channel are low. It was also shown that the velocity of passage of protons through the channel is limited by binding with an acid group.Institute of Cytology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 14, No. 5, pp. 499–507, September–October, 1982.     

Action of neurotoxin from the sea anemoneHomostichanthus duerdemi on inward sodium current in mammalian neurons

The action of purified toxin from the sea anemoneHomostichanthus duerdemi (HTX-1) on the inward sodium current was studied in experiments on isolated neurons from rat spinal ganglia and neuroblastoma cells of clone N-18F1, by an intracellular perfusion and voltage clamp method. HTX-1 was found to delay inactivation of the tetrodotoxin-(TTX-)sensitive inward sodium current and to make it incomplete, but virtually without affecting its activation. The relationship between the fraction of sodium channels modified by the toxin and the HTX-1 concentration is described by a Langmuir isotherm with association constant of (1.1 ± 0.1)·10^–7 M (holding potential –100 mV). Under the influence of the toxin the peak inward sodium current was increased by about 80%. Binding of HTX-1 with TTX-sensitive sodium channels is distinguished by strong potential-dependence: at a holding membrane potential of 0 mV the binding constant was an order of magnitude less than at a potential of –100 mV. In the case of brief action of HTX-1 on the nerve cell membrane (under 5 min) the effect of the toxin was completely reversible, but if the time of action of HTX-1 exceeded 30 min, subsequent washing with normal solution for 90 min did not abolish the effect completely.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Pacific Institute of Bioorganic Chemistry, Far Eastern Scientific Center, Academy of Sciences of the USSR, Vladivostok. Translated from Neirofiziologiya, Vol. 14, No. 4, pp. 402–409, July–August, 1982.     

Interaction between sea anemone toxin BgTX8 and ionic channels of neurons isolated from mammals

The action of the toxin BgTX₈ separated from the sea actiniaBunodosoma granolifera on transient tetrodotoxin-sensitive sodium and outward potassium currents of units isolated from rat sensory ganglia was investigated using techniques of voltage clamping at the membrane and intracellular perfusion. It was found that BgTX₈ decelerates the inactivation kinetics but has little effect on activation kinetics of sodium current. At the same time, a 5–10% increase in the amplitude of inward current was often observed at holding potentials of about –100 to –120 mV at the membrane. The dissociation constant of the receptor-toxin equals 4×10^–6 M and is adequately described by Langmuir's isotherm. It was also established that intracellular perfusion of neurons with anemone toxin-containing solution leads to a reduction in the amplitude of sodium current and decelerates its inactivation process. Suppression of outward potassium current was also noted.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Institute of Brain Research, Academy of Sciences, Havana, Cuba. Translated from Neirofiziologiya, Vol. 20, No. 1, pp. 32–37, January–February, 1988.