Two types of calcium-dependent channels of potassium outward current in the somatic membrane of Helix pomatia neurons

Characteristics of delayed potassium outward current were investigated during voltage clamp experiments on nonidentified intracellularly perfused neurons isolated from the snailHelix pomatia. A calcium-dependent potassium curent displaying special properties was shown to exist, apart from the voltage-operated potassium currents dependent on intracellular calcium ions . This type of current increases with a rise in the extracellular concentration of calcium ions , is not blocked by intracellular application of 10 mM EGTA and 77 mM fluoride, and may be suppressed by adding 1.5 mM cobalt ions to the extracellular fluid. This current, unlike , only takes a few milliseconds to peak, after which it fades to a steady level, comparable with that of .A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 19, No. 2, pp. 185–191, March–April, 1987.     

Mechanisms of up-regulation of single calcium channels by serotonin in Helix pomatia neurons

Action of serotonin (5-HT) on single Ca(2+) channel activity was studied in identified neurons of snail Helix pomatia. Only one type of Ca(2+) channels of 5 pS unitary conductance was determined under patch-clamp cell-attached mode. Kinetic analysis have shown a monotonically declining distribution of channel open times (OT) with mean time constant of 0.2 ms. The distribution of channel closed times (CT) could be fitted by double-exponential curve with time constants 1 and 12 ms. We established that 5-HT acts on Ca(2+) channel activity indirectly via cytoplasm. 5-HT prolonged the OT (up to 0.3 ms) and shortened the CT proportionally for both constants to 0.4 and 6 ms correspondingly. A conclusion is made that enhancement of Ca(2+) macro-current by 5-HT is determined by kinetic changes, increase of the number of active channels, and increase of the probability of OT. At the same time the transmitter did not affect the unitary channel conductance.     

Mechanisms of membrane potential oscillation in bursting neurons on the snail,Helix pomatia

• 1.1. The mechanism of generation of membrane potential (MP) oscillations was studied in identified bursting neurons from the snail Helix pomatia.
• 2.2. Long-lasting stimulation of an identified peptidergic interneuron produced a persistent bursting activity in a non-active burster.
• 3.3. External application of calcium channel blockers (1 mM Cd²⁺ or 5 mM La²⁺) resulted in a transient increase in the slow-wave amplitude and subsequent prevention of pacemaker activity generation in bursting neurons. Application of these blockers together with endogenous neuropeptide initiating bursting activity generation, increased MP wave amplitude without prevention of bursting activity generation.
• 4.4. Replacement of all NaCl in normal Ringer's solution with isoosmotic CaCl₂, glucose or Tris-HCl produced a reversible block of bursting activity generation. Stationary current-voltage relation (CVR) of bursting neuron membrane has a region of negative resistance (NRR) and does not intersect the potential axis in threshold region for action potential (AP) generation in normal Ringer's solution. In Na-free solution stationary CVR is linear and intersects the potential axis near — 52 mV.
• 5.5. Novel potential- and time-dependent outward (E_rev = − 58 mV) current, I_B, activated by hyperpolarization was found in the bursting neuron membrane. Having achieved a maximal value, this current decayed with a time constant of about 1 sec. Hyperpolarization inactivated maximal conductance, g_B, responsible for I_B, and depolarization abolished inactivation of g_B.
• 6.6. Short-lasting (0.01 sec) hyperpolarization of the bursting neuron membrane by inward current pulse induced the development of prolonged hyperpolarization wave lasting up to 10 sec.
• 7.7. These results suggest that: (a) persistent bursting activity of RPal neuron in the snail Helix pomatia is not endogenous but is due to a constant activation of peptidergic synaptic inputs of these neurons; (b) Ca²⁺ ions do not play a pivotal role in the ionic mechanism of MP oscillations but play a determining role in the process of secretion of a peptide initiating bursting activity by the interneuron presynaptic terminal; (c) depolarizing phase of the MP wave is due to specific properties of stationary CVR and hyperpolarization phase is due to regenerative properties of hyperpolarization-activated outward current I_B. The minimal mathematical version of MP oscillations based on the experimental data is presented.

     

Selectivity in biomineralization of barium and strontium

The desmid green alga Closterium moniliferum belongs to a small number of organisms that form barite (BaSO₄) or celestite (SrSO₄) biominerals. The ability to sequester Sr in the presence of an excess of Ca is of considerable interest for the remediation of ⁹⁰Sr from the environment and nuclear waste. While most cells dynamically regulate the concentration of the second messenger Ca²⁺ in the cytosol and various organelles, transport proteins rarely discriminate strongly between Ca, Sr, and Ba. Herein, we investigate how these ions are trafficked in C. moniliferum and how precipitation of (Ba,Sr)SO₄ crystals occurs in the terminal vacuoles. Towards this goal, we simultaneously visualize intracellular dynamics of multiple elements using X-ray fluorescence microscopy (XFM) of cryo-fixed/freeze-dried samples. We correlate the resulting elemental maps with ultrastructural information gleaned from freeze-fracture cryo-SEM of frozen-hydrated cells and use micro X-ray absorption near edge structure (micro-XANES) to determine sulfur speciation. We find that the kinetics of Sr uptake and efflux depend on external Ca concentrations, and Sr, Ba, and Ca show similar intracellular localization. A highly ion-selective cross-membrane transport step is not evident. Based on elevated levels of sulfate detected in the terminal vacuoles, we propose a “sulfate trap” model, where the presence of dissolved barium leads to preferential precipitation of (Ba,Sr)SO₄ due to its low solubility relative to SrSO₄ and CaSO₄. Engineering the sulfate concentration in the vacuole may thus be the most direct way to increase the Sr sequestered per cell, an important consideration in using desmids for phytoremediation of ⁹⁰Sr.     

Parvalbumin-immunoreactive neurons in the brain of Helix pomatia

Parvalbumin-immunoreactive material was detected in the central nervous system of the snail, Helix pomatia. Each ganglion investigated contained parvalbumin-immunoreactive neurons. The molecular weight of Helix parvalbumin-immunoreactive material as determined by Western blots is about 40 kilodaltons. ⁴⁵Ca²⁺ overlays showed that this protein binds Ca²⁺. In contrast to vertebrates, in Helix neurons parvalbuminlike material was not colocalized with the neurotransmitter gamma-aminobutyric acid (GABA).     

The effect of oxytocin on potential-dependent calcium current in snail neurons, Helix pomatia.

1. The effect of external application of oxytocin on inward calcium current in dialyzed snail neurons has been investigated under clamp conditions. 2. External application of oxytocin in a dose-dependent manner (Kd 0.9 microM) inhibits inward calcium current in dialyzed neurons of the snail, Helix pomatia. 3. Inhibition of calcium current developed with the time constant of about 2 min. The degree of restoration of calcium current after oxytocin washout depends on duration of oxytocin action. 4. It has been suggested that inhibition of calcium current by oxytocin occurs in two stages, the initial one is more fast and reversible and the second one--more slow and irreversible. The participation of soluble second messengers in the inhibitory effect of oxytocin on calcium current is discussed.     

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.     

Multiplicity of pacemaker zones in Helix pomatia neurons

Intracellular microelectrode recordings from neurons ofHelix pomatia revealed several local zones of action potential generation both on the soma and on some of the branches of the neurons. Under certain conditions the activity of individual loci of the neuron membrane was synchronized to produce a normal action potential. It is suggested that the somatic membrane of neurons is heterogeneous in structure and consists of separate loci of an electrically excitable membrane, incorporating active and latent pacemaker zones. Neurons ofH. pomatia are characterized by two types of action potential with different triggering mechanisms: one (synaptic) type is generated under the influence of the EPSP, the other (pacemaker) arises through activation of endogenous factors for the neuron (pacemaker potentials). The interaction between synaptic and pacemaker potentials during integrative activity of the neuron is discussed.M. V. Lomonosov Moscow State University. Translated from Neirofiziologiya, Vol. 5, No. 1, pp. 88–94, January–February, 1973.     

Effects of microiontophoretically injected AMP and cAMP on calcium current in dialyzed Helix pomatia neurons

The effects of injecting cells with adenosine monophosphate (AMP) and cyclic adenosine monophosphate (cAMP) on calcium current were investigated during intracellular dialysis ofHelix pomatia neurons. Microiontophoretically injected AMP was found to lead to reinstatement of calcium current following dialysis-induced wash-out, as well as considerable stabilization of this current with the extracellular medium at normal pH. Current-voltage relationship of the current would then undergo a 10 mV shift towards depolarization values. Perfusing the cell with a solution containing 10 mM AMP then produced a qualitatively identical effect. Injecting the neuron iontophoretically with cAMP led to a decline in the amplitude of calcium current under the same conditions. Neither raising the pH of the intracellular solution to 8.1 nor adding 4-aminopyridine in order to depress the hydrogen ion current produced a qualitative alteration in the effects of injecting AMP and cAMP on calcium current.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 20, No. 6, pp. 769–776, November–December, 1988.     

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.     

Two calcium currents in the somatic membrane of mollusc neurons

Two new types of calcium channels were discovered during research in ionic currents in the somatic membrane ofHelix pomatia neurons, using an intracellular perfusion technique. Apart from the principal calcium current described in the literature with a holding potential of about –110 mV, an additional calcium current was observed activated at depolarizations of –40 to –80 mV and was not reduced when the cell was perfused with solutions containing fluoride anions. The kinetics of this current were well described in the context of the Hodgkin and Huxley model with a time constant of activation of 6–8 msec and of inactivation of 300–600 msec. It increased in amplitude as the Ca⁺⁺ rose in the cellular environment but was reduced by extracellular addition of the Ca⁺⁺ antagonists Co⁺⁺, Ni⁺⁺, and Cd⁺⁺, and the organic blockers nifedipine and verapamil. The association constants of these substances with corresponding channels determined from the maximum of the current-voltage relationship were 2 (Ca⁺⁺), 3 (Co⁺⁺), 0.06 (nifedipine), and 0.2 mM (verapamil). The properties detected in this component of calcium conductance are compared with those of calcium channels in other excitatory formations and its possible functional role is discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 17, No. 5, pp. 627–633, September–October, 1985.     

Sites of action potential generation in Helix pomatia neurons

The site of action potential generation in unipolar snail neurons was identified by stimulating neurons isolated together with the initial portion of the process from the neuropile. Stimulation consisted of a sinusoidal from electrical current passed along the soma-axonal axis in saline solution. No low threshold sites of action potential generation were found in 80% of test neurons using this technique. Spontaneous activity was determined by the operation of one dominant site on the neuronal process. Antidromic activation of the soma by axonal action potentials (even with simultaneous hyperpolarization of the soma) induced somatic potentials more successfully than direct somatic depolarization by the current flowing through the solution.Institute of Chemical Physics, Academy of Sciences of the USSR, Moscow. Institute of Higher Nervous Activity and Neurophysiology, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 20, No. 1, pp. 90–98, January–February, 1988.     

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.     

Dose-inward current relationships for current-carrying calcium and strontium ions in isolated Limnea stagnalis neurons

The dose-inward current relationships for current-carrying Ca⁺⁺ and Sr⁺⁺ ions were studied by means of intracellular dialysis and voltage clamp methods in isolated neurons ofLimnea stagnalis. These relationships rose to a ceiling for these ions. The data are explained in terms of an activation theory using a double-barrier model of the calcium channels. Relative values of velocity constantsv _X for passage of ion X²⁺ across the second energy barrier and of the constant K_X, defining the binding effectiveness at the entrance to the channel were determined:vCa:vSr = 1:2; K_Ca = 1400 M^–1; K_Sr = 270 M^–1.Research Institute of Physiology and Pathology of the Cardiovascular System, Medical Institute, Ministry of Higher Middle and Specialized Education of the Lithuanian SSR, Kaunas; Institute of Biophysics, Academy of Sciences of the USSR, Pushchino-on-Oka. Translated from Neirofiziologiya, Vol. 11, No. 4, pp. 362–366, July–August, 1979.     

Reduction of calcium currents in identified neurons of Helix pomatia: intracellular injection of D890

The actions of intracellularly applied D890 on membrane currents of the identified neurons B1, B2 and B3 of Helix pomatia were investigated. The TTX-resistant component of the inward current, the inward currents in Na+-free sucrose solution and in Ca2+-free Ba2+ solution were reduced. In Ca2+-free Co2+ solution the inward current was not affected. The late outward currents were strongly reduced. In solutions containing 20 mmol/l NiCl2 the remaining parts of these currents were blocked only to a lesser extent. The early outward current remained unchanged. It is concluded that intracellularly applied D890 mainly exerts its effects on the calcium current.