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.     

Action potentials of the mollusk giant neuron with changes in extracellular concentrations of sodium and calcium ions

The action potential (AP) of the giant neuron of the molluskPlanorbis corneus exhibits an increased sensitivity of the spike overshoot to external sodium concentration in solutions containing a significantly lowered concentration of calcium. These results suggest that during the AP both sodium and calcium ions may act as carriers of the inward-directed current. During repeated responses the role of calcium ions in AP generation increases while that of sodium decreases. A delay in repolarization can occasionally be observed at the beginning of the falling phase of the AP. This delay is considered to be a result of a decrease in efficiency of the repolarizing action of the outward potassium current due to competition from a current entering the cell at the time of the falling phase. Results suggest that the carrier of this inward current is calcium.A. A. Bogomolets' Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 1, No. 1, pp. 109–117, July–August, 1969.     

Separation of potassium and calcium channels in the nerve cell soma membranes

Investigation of isolated neurons ofHelix pomatia during intracellular dialysis revealed differences in the sensitivity of the channels for the outward potassium and inward calcium currents to changes in pH of the external medium. As a result of this difference, considerable separation of the regions of activation of the currents was obtained along the potential axis in solutions with low pH and the characteristics of the inward and outward currents could be studied during their minimal application. Channels for the outward current were shown to have some permeability for tris ions (P_Tris:P_K=0.05), which is the reason why it is impossible to block this current completely by replacing the intracellular potassium by Tris. Channels for the inward calcium current are characterized by slow inactivation, with a first-order kinetics; their momentary voltage-current characteristic curve reveals significant Goldman's rectification. The selectivity of the calcium channels for other bivalent cations is: Ba:Sr:Ca:Mg=2.8:2.6:1.0:0.2.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 10, No. 6, pp. 645–653, November–December, 1978.     

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.     

Investigation of the TEA-resistant outward current in the somatic membrane of perfused nerve cells

Outward currents remaining after addition of 20–50 mM of tetraethylammonium (TEA) ions to the extracellular or intracellular solution, were investigated in perfused isolatedHelix neurons. After this addition, the inactivated inward current carried by potassium ions, the potential-dependent and kinetic characteristics of which differ from those of potassium outward currents suppressed by TEA, is preserved in the membrane. A component dependent on the inward calcium current was found in this TEA-resistant outward current; it was abolished by replacement of the extra-cellular calcium ions by magnesium ions, by blocking of the calcium channels by extracellular cadmium ions, and by their destruction by intracellular fluoride ions. Increasing the intracellular concentration of free calcium ions by perfusing the cell with solutions containing calcium-EGTA buffer potentiated the TEA-resistant component of the outward current, whereas removal of these ions with EGTA weakened it. It is concluded that a system of outward current channels whose activation depends on the presence of calcium ions near the inner surface of the membrane is present in the somatic membrane. It is suggested that to keep these channels capable of being activated, calcium ions must bind with the structures forming their internal opening.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 11, No. 5, pp. 460–468, September–October, 1979.     

Action of calcium ions on channels of inward and outward currents in the molluscan neuron membrane

Changes in the characteristics of activity of sodium, calcium, and potassium channels in the surface membrane during variation of the calcium ion concentration in the extracellular and intracellular medium were investigated by the voltage clamp method during intracellular dialysis of isolated neurons of the mollusksLimnea stagnalis andHelix pomatia. Besides their direct role in passage of the current through the membrane, calcium ions were shown to have two actions, differing in their mechanism, on the functional properties of this membrane. The first was caused by the electrostatic action of calcium ions on the outer surface of the membrane and was manifested as a shift of the potential-dependent characteristics of the ion transport channels along the potential axis; the second is determined by closer interaction of calcium ions with the specific structures of the channels. During the action of calcium-chelating agents EGTA and EDTA on the inner side of the membrane the conductivity of the potassium channels is substantially reduced. With an increase in the intracellular free calcium concentration the conductivity is partially restored. The action of EGTA and EDTA on the outer side of the membrane causes a substantial decrease in the ion selectivity of the calcium channels and changes the kinetics of the portal mechanism. These changes are easily abolished by rinsing off the chelating agents or by returning calcium ions to the external medium. A specific blocking action of an increase in the intracellular free calcium concentration on conductivity of the calcium channels was found.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 9, No. 1, pp. 69–77, January–February, 1977.     

Single potassium channel of anomalous (inward) rectification in mollusk neurons

Currents passing through individual potassium channels with anomalous (inward) rectification were recorded at the neuronal membrane ofPlanorbarius corneus using the patch clamp technique. These currents could be detected, whether in "right side out" or "inside out" configurations in the presence of 50 mM potassium ions or one of the potassium channel blockers: tetraethylammonium (TEA), barium, or cesium (2–20 mM) on the external side of the membrane. Inward currents were observed in individual channels at potentials more negative than level of potassium equilibrium potential (E_k); conductance of these measured 81±12 pS (n=11). At more positive potentials than E_k, conductance fell to zero. Potassium channels with anomalous (inward) rectification inPlanorbarius corneus resemble equivalent channels in other cells in their kinetics: time scale of the open state may be described by a single exponential function. This would imply that the ionic channel has a single open state. Time scale of the closed state was biexponential, thus indicating the possible existence of two kinetically different nonconducting states of the potassium channel with anomalous (inward) rectification at the neuronal membrane ofPlanorbarius corneus.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 21, No. 1, pp. 31–38, January–February, 1989.     

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.     

Blocking of Ca-dependent outward current in molluscan neuron somatic membrane by raised intracellular pH

The action of a raised intracellular pH (pH_i) on transmembrane ionic currents was investigated on isolated unidentified neurons ofHelix pomatia under intracellular dialysis and membrane voltage clamping conditions. With a rise in pH_i from 7.3 to 9.0 and in the simultaneous presence of an inward calcium current, the outward potassium current was considerably reduced and the current-voltage characteristic curve was shifted toward more positive membrane potential values. The inward calcium current was practically unchanged in this case. If, however, the calcium current was inhibited by the action of cadmium ions, no decrease in the outward current was observed, only a shift of the I_K(V) curve toward more positive values of membrane potential. It is suggested that an increase in pH_i selectively blocks the Ca-dependent component of the outward potassium current.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 14, No. 4, pp. 426–430, July–August, 1982.     

Effects of strontium and barium ions on calcium bindings and transport in nerve cells

Absorption of strontium and barium ions by intracellular organelles after loading the cell with these cations together with their effects on Ca release from the intracellular stores were investigated in neurons isolated fromHelix pomatia using fura-2, a Ca-sensitive fluorescent probe. It was found that strontium ions can successively replace intracellular calcium ions in this response, whereas barium ions are not absorbed by the cell; they block calcium channels of the intracellular stores as well as at the surface membrane.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 21, No. 6, pp. 820–825, November–December, 1989.     

Effect of calcium ions on outward currents in the somatic membrane of mollusk giant neurons

Potassium currents through the somatic membrane of giant neurons ofHelix pomatia in normal (10 mM Ca) Ringer's solution and low-calcium (1 mM Ca) solution were studied by the voltage clamp method. With a decrease in the Ca concentration to 1 mM peak potassium conductance versus membrane, potential curves and inactivation curves were shifted along the voltage axis in the negative direction by about 10 mV. Inactivation of the delayed potassium current was slowed in low Ca solution. The effect of a decrease in external calcium concentration on volt-ampere and inactivation characteristics increased with a rise in external pH. These effects of a low Ca concentration on potassium mechanisms of the giant neuron somatic membrane can be attributed to changes in the negative surface potential in the region of the potassium channels.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Institute of Biology, Hungarian Academy of Sciences, Tihany. Translated from Neirofiziologiya, Vol. 8, No. 4, pp. 400–409, July–August, 1976.     

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.     

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.     

Action potential-like responses due to the inward rectifying potassium channel

Summary This paper describes experiments carried out in the absence of sodium and calcium in the external solution. Frog atrial trabeculae were stimulated in current clamp with the double sucrose gap technique. The voltage responses looked like slow action potentials with a clear threshold. These responses were not suppressed in the presence of EGTA, in the presence of sodium or calcium channel blockers, or when sulfate ions replaced chloride. Guinea pig isolated ventricular myocytes were studied in whole cell clamp mode with a pathch pipette. Under current clamp, they displayed also voltage responses with a threshold. These responses were resistant to cadmium (5mm), and were suppressed by barium (0.5mm). A negative slope conductance is required to take into account these results. The membrane current in current clamp can be estimated by plotting the response in the phase plane. This analysis shows that on both types of preparations, the current responsible for the negative slope is not time dependent. This current is suppressed by barium. It can be concluded that it is the outward current flowing through the inward rectifying potassium channels. To confirm this hypothesis, data obtained in voltage clamp on the same preparations were introduced into a computer model to predict the response in current clamp. The results were in agreement with the experiments. Similar responses could be recorded and analyzed on skeletal muscle in isotonic potassium solution. These results show that the inward rectifier can induce by itself properties looking like excitability on different preparations. The physiological significance of this effect in normal conditions is discussed. The voltage responses described in this paper look similar to the slow action potentials on heart, which are sensitive to modifications of the calcium channels, but also of the potassium channels. Some implications in cardiac pharmacology are discussed.     

Effects of extracellular potassium ions on outward potassium current dependent on extracellular calcium ions

Changes in outward potassium current occurring in response to changes in the concentration of potassium ions in the extracellular medium were investigated in unidentified neurons isolated fromHelix pomatia using an intracellular perfusion technique. It was found that introducing potassium ions (5–10 mM) into the extracellular solution produces a reversible increase in the component of outward potassium current which is dependent on extracellular calcium ions. Increased amplitude of this component occurs as a result of attenuated inactivation of the current under the action of extracellular potassium.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 19, No. 3, pp. 351–356, May–June, 1987.     

Blocking action of calmidazolium and chlorpromazine on outward potassium current dependent on extra-cellular calcium ions

The effects of the calmodulin antagonists, calmidazolium (R 24571) and chlorpromazine on delayed outward potassium current at the somatic membrane were investigated in non-identified intracellularly perfused neurons isolated fromHelix pomatia. Voltage was clamped at the membrane. Extracellular application of these substances produced effective depression of the outward current. This effect even occurred at test substance concentrations of 10^–9–10^–8 M. Block-ade of delayed outward current was produced mainly as a result of suppressing the potassium current component dependent on intracellular potassium ions (I_k(Ca/in)). The possibility that the receptor for intracellular calcium responsible for modulating this current may be of a calmodulin-like nature is discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 19, No. 3, pp. 356–361, May–June, 1987.     

Inactivation of calcium currents in the molluscan neuron somatic membrane

The time course of weakening of inward calcium currents (inactivation) during prolonged (of the order of 1 sec) depolarizing shifts of membrane potential was studied in isolated dialyzed neurons of snailHelix pomatia. This decay of the current recorded in this way can be approximated by two exponential functions with time constants of 20–70 and 250–350 msec, respectively. With an increase in pH of the intracellular solution to 8.5 the fast component of the decay disappeared completely; the kinetics of the slow component in this case was very slightly retarded. It is concluded that the fast component of decay of the recorded current does not reflect a change in the calcium current but is due to parallel activation of the nonspecific outward current; the slow component, however, is true in activation of the calcium current. The rate of inactivation of this current was shown to be determined by its maximal value and not by the level of the depolarizing potential shift and it depends on the conditions of accumulation of calcium ions near the inner surface of the membrane.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 14, No. 5, pp. 525–531, September–October, 1982.     

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.     

Effect of sodium-free solutions on ionic currents of snail giant neurons

The ionic currents of the snail giant neurons were investigated by the voltage clamp method. The effect of sodium-free solutions on the inward and outward currents was studied. It is shown that the current entering the cells is created mainly by sodium ions. When a preparation is immersed into a solution not containing sodium ions, most neurons (tentatively neurons of type "a") "lose" the inward currents. In other neurons (tentatively of type "b") this process lasts 40 min and more. A number of peculiarities of type "b" neurons were noted. The response of the excitable membrane to conditioning polarization was also investigated. The data obtained permit the conclusion that 85–90% of the sodium-transfer system is activated in the case of a voltage clamp from the level of the resting potential.A. A. Bogomolets Institute of Physiology, Kiev. Translated from Neirofiziologiya, Vol. 2, No. 3, pp. 314–320, May–June, 1970.     

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.