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Expression of "fast", TTX-sensitive sodium and high-threshold calcium channels in the membrane of Xenopus oocytes following mRNA injection from the rat brain has been detected using two microelectrode voltage clamp technique. Barium current through expressed calcium channels was blocked by 200 mumol/l Cd2+ and was insensitive to D-600 (20 mumol/l) and nitrendipine (50 mumol/l). Expressed barium current was inhibited within 20-40 min by omega-conotoxin, a peptide neurotoxin known to block high-threshold calcium channels of the neuronal membrane, in 1 mumol/l concentration. A steady-state inactivation curve for this current could be fitted by the Boltzmann relation with V1/2 = -50 mV and k = 14 mV. Voltage-dependent and pharmacological properties of calcium channels which appeared in the oocyte membrane following mRNA injection from the mammalian brain resembled most of all those of high-threshold inactivating (HTI- or N-type) calcium channels of neurons in spite they did not demonstrate prominent time-dependent inactivation. Evidences in favour of expressed calcium channels heterogeneity were not obtained.

Parathyroid hormone (PTH) has been studied for its effect on the voltage-sensitive calcium channels in voltage-clamp experiments on intracellularly perfused snail neurons. A two-stage action of the hormone on the calcium current (Ica) was shown. An initial stage was a short-term one and consisted in an increase of ICa by 7-10%. The second stage developed slowly for 60-70 min, ICa increased twice. The exogenous cAMP and cGMP did not produce such an effect as PTH did. It is suggested that the phosphoinositide pathway mediates the PTH effect.

The action of cAMP (100 microM) and serotonin (5-HT, 1-10 microM) on the calcium current (Ica) in intracellularly perfused Helix pomatia neurons was studied with voltage clamp method. Three types of 5-HT-induced changes in the calcium current were observed in different cells: reversible blockade, increase and no changes in the current amplitude. Intracellular introduction of exogenous cAMP (100 microM) affected Ica only in cells with the stimulatory effect of 5-HT; cAMP-induced increase in the current amplitude was not additive to that elicited by 5-HT while both of these effects were similarly potentiated by cyclic nucleotide-phosphodiesterase inhibitor. The data presented show that the stimulatory action of 5-HT on the potential-activated calcium current is mediated by an increase in intracellular cAMP. Existence of two types of calcium channels differing in their dependence on cAMP metabolism is suggested in the snail neurons. The presence of the cAMP-dependent calcium channels seems to correlate with the existence of the definite type of 5-HT receptors in the cell membrane. A new approach to the investigation of isolated neurons is suggested: their functional identification.

A newly developed method of internal dialysis was applied together with the voltage clamp method to the isolated neurons of molluscs Helix pomatia and Limnea stagnalis. Effect of Ca on the Ca inward and K delayed outward currents was investigated. Removal of Ca from the external medium was equivalent to 25-35 mV hyperpolarization of the membrane. Internal Ca in concentrations up to 3.5-10-(7)M caused insignificant depression of K outward current. 5.7-10-(8)M of internal Ca completely blocked Ca inward current. Ca-chelating agents applied either internally or externally caused an appearance of Na inward current transferred through the Ca channels.

The intracellular perfusion technique rendered isolated neurons a convenient object for studying the Ca channels securing inflow of these ions across the membrane into the cell in excitation. Extensive research showed the Ca channels to pass the cations in the sequence Ba greater than Sr greater than Ca greater than Mg and bind these ions with the aid of a binding compound within the channel. Other cation (Co, Ni, Mn, Cd) which are too closely bound with this compound become competitive blocking agents for the channels. In absence of the bivalent cations in the extracellular space the Ca channels loose their selectivity and allow monovalent cations to pass, the reason for this transformation beeing a disruption between the bound Ca ions and the specific regulating compound within the Ca channels. The channels can exist in two states: conducting and nonconducting. The transition from one state to another is accompanied by an intramembrane transfer of charges ("the gate current"). The statistic kinetics of this transition can be described by means of a modified Hodgkin-Huxley equation. In prolonged depolarization the Ca channels become inactivated due to recurrent blocking effect of the Ca ions penetrating in the cell. Some types of the Ca channels, however, reveal a potential-dependent inactivation analogous to that of the sodium or potassium channels.

Kinetic and voltage-dependent characteristics of deactivation of calcium inward currents with the removal of membrane depolarization were studied in the somatic membrane of rat dorsal root ganglion neurons by intracellular dialysis technique. The "tail" of low-threshold calcium current could be described reliably by one exponent with time constant tau 1-1.2-1.8 ms at repolarization to --90 mV. The "tail" of the high-threshold calcium current represented a sum of several exponents; the time constant of the main component tau h was in the range of 250-380 microseconds. tau 1 and tau h remained practically unchanged for repolarization potentials in the subthreshold region; however, they increased if it was in the range of potentials at which the corresponding component of the calcium current started to activate. A dependence of tau 1 and tau h on the duration of depolarizing shift was observed. The results obtained are discussed in the framework of a three-level kinetic model of calcium channels.

The time-dependent decrease of calcium inward currents (inactivation) at long (about 1s) depolarizing membrane potential shifts was studied on isolated dialysed neurons of the mollusc Helix pomatia. It is shown that the time course of this decrease can be approximated by two exponents with corresponding time constants 20-70 and 250-350 ms. The fast component of the decay disappeared completely after pH increase of the intracellular solution to 8.5. The kinetics of the slow component did not change. A conclusion is made that the fast decay of the recorded current does not represent variations in the calcium current but is due to parallel activation of a nonspecific outward current whereas the slow decay represents true inactivation of the calcium current. It is shown that the inactivation velocity of this current is related to the maximal value of the latter but not to the level of membrane depolarization, and depends on conditions of accumulation of calcium ions at the inner surface of the cell membrane.

Calcium inward and potassium outward currents were studied on internally dialysed isolated neurons of the snail Helix pomatia. Different sensitivity of the corresponding channels to changes in external pH was found. This difference was used for separation of their activation regions on the potential axis so that the characteristics of the inward and outward currents could be studied with minimal overlap. It is shown that the outward current channels possess a definite permeability to Tris ions (PTris :PK=0.05). This explains the impossibility to switch off this current by substituting Tris for internal potassium. The channels for the inward calcium current inactivate slowly with a first order kinetic; their instantaneous current-voltage characteristic reveals considerable Goldman-type rectification. The selectivity of the calcium channels to other bivallent cations is Ba:Sr:Ca:Mg=2.8:2.6:1.0:0.2.

Single calcium channels of cultured dorsal root ganglion cells from mouse embryos were studied using patch clamp method in its cell-attached configuration. Two types of activity of unitary calcium channels were found. The first one which arose at membrane potentials near--50-40 mV was characterized by unitary current amplitude of 0.37 +/- 0.04 pA with 40 mmol/l Ca2+ in the pipette solution, mean open time of 0.6 ms and intraburst mean shut time of 1.2 ms. It displayed voltage- and time-dependent inactivation. The corresponding values for the second one which required much more positive depolarization to be activated (approximately 0 mV) and did not express noticeable inactivation were: 0.53 +/- 0.04 pA, 0.8 ms and 0.8 ms. It is concluded that the recorded types of unitary activity are associated respectively with low- and high-threshold calcium currents which have been found earlier while studying whole cell currents.

The surface charge density (sigma'0 and the constant of Ca2+ binding with the charged surface groups of the outer side of the membrane (KCa) are calculated on the basis of experimentally determined values of the current-voltage characteristic shifts for calcium currents in the membrane of rat spinal ganglia neurons. The obtained values are as follows: sigma'0 = 0.15 +/- 0.05 e/nm2 and KCa = 70 +/- 10 1/ml. The energy profile of the calcium channel in the membrane of investigated neurons was calculated for Ca2+, Ba2+ Cd2+ Mn2+ Co2+ ions and verapamil using a three-barrier model. It is shown that the value of calcium current is determined mainly by the potential hole depth which corresponds to the outer binding centre of the calcium channel. On the basis of the data obtained a conclusion is made that the outer binding centre contains only one carboxylic group.

The action of forskolin, adenylate cyclase activator, on the calcium inward current was studied on intracellularly perfused nonidentified snail neurons. Extracellular application of 5-20 mumol/l of forskolin was shown to cause about 20% increase of the calcium current amplitude and this confirms the previous suggestion that the stimulation of the cellular synthesis of cAMP leads to enhancement of the calcium current through the cell membrane.

The effect of varying intracellular 3.5-cAMP level on steady-state and voltage-dependent transmembrane ionic currents has been studied on both vertebrate and invertebrate nerve cells. A change in 3.5-cAMP level was achieved either by its direct introduction into the cell or by stimulation or inhibition of the activity of different enzymes of the cyclase system. Intracellular cAMP increase was found to activate a steady-state transmembrane current whose early component is related mainly to the increase of sodium and calcium conductance of the membrane, and a late one to increase of potassium conductance (possibly, activated by Ca2+ influx). The decrease of intracellular cAMP concentration (by intracellular dialysis) results in reduction of the potential-activated inward calcium current, whereas its increase restores the current. Restoration of calcium current can be achieved by activation of cellular adenylate cyclase, inhibition of phosphodiesterase or by direct intracellular introduction of the catalytic subunit of cAMP-dependent protein kinase. The evidence is presented that the observed regulatory effects are mediated through cAMP-dependent phosphorylation of proteins of corresponding ionic channels. The increase of intracellular Ca2+ level closely cooperates with this regulatory metabolic system by activation of a number of its enzymatic processes.

A change in the relationship between high-threshold calcium channels and intracellular metabolism of cyclic nucleotides during postnatal development was found in experiments on rat dorsal root ganglion neurons. In the first age group (5-9 postnatal days) intracellular administration of cAMP-ATP-Mg2+ complex has resulted in restoration of the maximal amplitude of high threshold calcium current for 70% of neurons, whereas in the second (45 days) and third (90 days) age groups this effect was observed only in 26% and 10% of neurons, respectively. Kinetic and voltage-dependent characteristics of high-threshold calcium current in these three age groups were identical. The effect of introduction of cAMP-ATP-Mg2+ complex was different for neurons with different combination of inward currents. Neurons which possessed only two types of inward currents--"fast" sodium and high-threshold calcium ones, show no effect. Conventional effect of the intracellular cAMP injection occurred always in neurons which had exhibited a "slow" (TTX-resistant) sodium inward current together with the two main inward currents.

The nature of a significant deviation of the reversal potential for the slow inward current component from calcium equilibrium potential was investigated in the snail neuron somatic membrane. It is shown that this deviation may be due to a development of a non-specific outward current during large depolarizing shifts of the clamped membrane potential. The real equilibrium potential for calcium ions in nerve cells may be of about +200 mV.

The outward currents remaining after addition of 20-50 mM tetraethylammonium (TEA) to the extracellular solution were studied on perfused isolated neurons from Helix pomatia. A potassium-carried noninactivating outward current with potential-dependence and kinetics different from those of TEA-sensitive potassium currents was found. This TEA-resistant current includes a component depending on the presence of the inward calcium current. It could be abolished by replacing extracellular calcium by magnesium ions, by blocking the calcium channels with extracellular cadmium ions and their distruction by intracellular fluorid ions. An increase in the level of intracellular free carcium (by perfusing the cell with solutions containing Ca-EGTA buffer) potentiated the TEA-resistant component of the outward current and the removal of free calcium by EGTA decreased it. A conclusion is made that the somatic membrane contains outward current channels which can be activated only when calcium ions are bound to its inner surface.

Correlations between densities of different types of inward currents in the somatic membrane of dorsal root ganglion neurons were studied in three age groups of rats (5-9 days, 45 days and 90 days postnatally). A linear dependence between the densities of high-threshold calcium and slow sodium currents was found. No correlation was observed between the densities of different inward currents in neurons with low-threshold calcium inward current. An inverse dependence was observed between the densities of transmembrane currents in cells having only two types of channels ("fast" sodium and high-threshold calcium ones). Neurons exhibiting slow TTX-resistant sodium and/or low-threshold calcium channels did not show inverse dependence between the densities of "fast" sodium and high-threshold calcium currents.

Membrane currents induced by the intake of sodium ions during tetanic stimulation or steady depolarization under voltage clamp conditions were studied on giant neurons of the mollusc Helix pomatia. It is shown that the induced current may be separated into two components: the fast one having a reversal potential close to the EK level, and the slow one slightly dependent on the membrane potential in the region --50 divided by --90 mV Strophanthin K and replacement of external sodium with lithium or calcium ions eleminated for slow component and reduced the fast one. A conclusion is made that the slow component is produced by the activation of the electrogenic sodium pump, and the fast one-- by an increase in potassium permeability probably coupled to intensive activity of this pump.

Distribution of different types of ionic channels carrying inward currents was studied in the somatic membrane of rat dorsal root ganglion neurons within three age groups: 5-9 days, 45 days and 90 postnatally. The number of neurons whose membrane contained simultaneously four types of inward current channels ("fast" tetrodotoxin-sensitive and "slow" tetrodotoxin-resistant sodium low- and high-threshold calcium one's) was progressively reduced in successive groups. The first group contained 14.5%, the second 5% and the third group 1% of such neurons. These changes were due to disappearance of "slow" sodium and low-threshold calcium channels from the membrane; the number of neurons whose somatic membrane contained only two types of inward current channels ("fast" sodium and high-threshold calcium) has increased, respectively.