Changes in the ionic mechanisms of the electro-excitability of the somatic membrane of rat sensory neurons during ontogenesis. Density ratios of inward currents

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.     

Changes in the ionic mechanisms of the electro-excitability of the somatic membrane of rat sensory neurons during ontogenesis. Relation between calcium channels and intracellular metabolism

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 potential dependence of calcium current deactivation via the somatic membrane of sensory neurons in the mouse

Potential dependence of calcium inward current deactivation kinetics was studied in the somatic membrane of mouse dorsal root ganglion neurons by intracellular dialysis technique. The decay of the high-threshold calcium current upon repolarization was reasonably described by single-exponential process with the time constant tau less than or equal to 130 microseconds (V = = -80 mV), when the intracellular solution contained tris-PO4, and by two-exponential process (tau congruent to 0.1 and tau = 0.8 divided by ms, V = -80 mV), when the intracellular solution contained Cs-aspartate and EGTA. Both time constants were strongly voltage dependent. The amplitude of the fast component of the tail current had sigmoidal voltage dependence, and the slow component had V-shaped voltage dependence. The low-threshold calcium current deactivation occurs more slowly with high voltage dependent kinetics (tau = 1.1 divided by 1.2 ms, V = -160 mV). A dependence of low-threshold current deactivation time constant on the type of penetrating cation was observed. A kinetic model of calcium current deactivation was proposed considering three types of calcium channels presented in the somatic membrane of the neurons studied.     

Voltage-gated influx ion channels expressed in Xenopus oocytes after the administration of brain mRNA]

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.     

Deactivation of calcium currents in the soma of spinal ganglion neurons after elimination of the depolarizing shift in the membrane potential

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.     

Electroregulated sodium channels of the somatic membrane of sympathetic neurons

Electrically-operated sodium channels in the somatic membrane of isolated neurons from the rat superior cervical ganglion have been studied by means of intracellular dialysis technique under voltage clamp conditions. It was shown that in this preparation sodium currents can be carried by two independent systems of sodium channels. The mathematical analysis of voltage-dependent TTX-sensitive fast sodium currents was performed by the Hodgkin-Huxley formalism; their kinetic properties were compared with those described in other objects. TTX-sensitive sodium channels in the somatic membrane of sympathetic neurons were found to be highly selective for Na+ ions. Kinetic and voltage-dependent characteristics of slow TTX-resistant sodium current were also described. This component of the sodium current was observed only in a few neurons (not more than 2%).     

Effect of replacing calcium ions with barium ions in studies of the inward currents of mammalian neurons

The effect of replacement of Ca2+ ions by Ba2+ ions in the external artificial solution on a high-threshold calcium current of the somatic membrane of rat dorsal root ganglion neurons was studied by intracellular dialysis technique and voltage clamp method. The conductance of the corresponding channels for Ba2+ ions assessed by the increase in the maximal current amplitude was shown to increase about twice. The decrease of the maximal current amplitude in the course of dialysis associated with the washout of the intracellular content slowed down considerably, probably, due to the removal of the blocking effect of intracellular Ca2+ on the calcium channels. The link of high-threshold calcium channels with cyclic nucleotide metabolism was not disturbed after the replacement of Ca2+ by Ba2+. The data obtained are discussed within the framework of existing ideas about the functioning of calcium channels in excitable membranes.     

Serotonin- and proton-induced and modified ionic currents in frog sensory neurons

Using the whole-cell patch-clamp technique, the effects of serotonin (5-HT) and increased acidity to produce membrane currents and to modify high threshold voltage-dependent calcium currents were studied in isolated dorsal root ganglion (DRG) cells of the frog maintained in short-term culture. DRG cells were classified by morphology into two types: (1) cells with a large number of dark rusty brown granules, and (2) cells devoid of these granules or with few scattered pale granules. Fast application of 5-HT (10–30 μM) induced a rapidly desensitizing inward current with a reversal potential at about 0 mV in 38 of 50 granule-containing neurons (76%) which was never observed (0/35) in “clear” neurons. This current was blocked by 10 nM (+)-tubocurarine. In addition, a small noninactivating outward current was also observed in most DRG neurons during 5-HT superfusion. A sudden decrease of pH from 7.4 to 6 or 5.8 induced a fast inactivating inward current of 100–300 pA in 74% of the “clear” neurons and only 24% of the granule-containing neurons. Small noninactivating membrane currents induced by lowering pH were observed in all neurons. Both 5-HT and increased extracellular H⁺ reduced the magnitude of high threshold calcium currents in all DRG neurons. It is suggested that the 5-HT receptors are expressed on a morphologically distinct population of neurons while the cells with channels responsible for the fast inactivating proton-induced current cannot be related to any distinct morphological cell type. © 1995 Wiley-Liss, Inc.     

The role of altered sodium currents in action potential abnormalities of cultured dorsal root ganglion neurons from trisomy 21 (down syndrome) human fetuses

Trisomy 21 (Down syndrome) results in abnormalities in electrical membrane properties of cultured human fetal dorsal root ganglion (DRG) neurons. Action potentials have faster rates of depolarization and repolarization, with decreased spike duration, compared to diploid neurons. In order to analyze the faster depolarization rate observed in trisomic neurons, we examined sodium currents of cultured human fetal DRG neurons from trisomy 21 and control subjects, using the whole-cell patch-clamp technique. The neurons were replated in culture to reduce dendritic spines. Two components of the sodium current were identified: (1) a fast, tetrodotoxin (TTX)-sensitive current; and (2) a slow, TTX-resistant component. The inactivation curves of both current types in trisomic neurons showed a shift of approximately 10 mV towards more depolarized potentials compared to control neurons. Thus, whereas essetially all of the fast sodium channels were inactivated at normal resting potentials in control neurons, approximately 10% of these channels were available for activation in trisomy 21 cells. Furthermore, the fast current showed accelerated activation kinetics in trisomic neurons. The slow sodium current of trisomic neurons showed slower deactivation kinetics than control cells. No differences were observed between trisomic and control neurons in the maximal conductance or current densities of either fast or slow current components. These data indicate that the greater rate of depolarization in trisomy 21 neurons at resting potentials is primarily due to activation of residual fast sodium channels that also have a faster time course of activation.     

Voltage-dependent calcium currents in Purkinje cells from rat cerebellar vermis.

Whole-cell patch clamp recording was used to characterize calcium currents in Purkinje cells dissociated from the cerebellar vermis of 1-3-week postnatal rats. A subset of Purkinje cells had a low-threshold, transient current similar to the T-type current in peripheral neurons. All Purkinje cells had a high-threshold, slowly inactivating current. Only a small component of the high-threshold current was sensitive to dihydropyridine (DHP) antagonists or to the dihydropyridine agonist BAY K8644. omega-Conotoxin had very little effect on the high-threshold current. The results suggest that these Purkinje cells have at least three types of calcium channels: T-type channels (present in only a fraction of cells), DHP-sensitive L-type channels (contributing a small fraction of the high-threshold current), and a predominant type of high-threshold channel that is pharmacologically distinct from L-type and N-type channels characterized in peripheral neurons.     

Separation of the sodium and potassium channels in the surface membrane of mollusk nerve cells]

Characteristics of transmembrane ionic currents under controlled changes in ionic composition of extra-and intracellular medium were studied by means of intracellular dialysis and voltage clamp in isolated neurons from the molluscs Helix pomatia and Limnea stagnalis. The outward potassium currents were eliminated by replacement of intracellular potassium by Tris and the pure inward current could be measured. Replacement of the Ringer solution by NA-free or Ca-free solutions in the extracellular medium made it possible to separate the inward current into additive components, one of which is carried by sodium ions, and the other, by calcium ions. The sodium and calcium inward currents are shown to have different kinetics and potential dependence: taumNa = 1+/-0.5 ms, taumCa = = 3+/-1 ms, tauhNa = 8+/-2 ms, tauhCa = 115+/-10 ms when Vm = 0, GNa = 0.5 when Vm==-21+/-2 mV, GCa = 0.5 when Vm=-8+/-2 mV. Both currents were not altered by tetrodoxin (TTX), however calcium current is specifically blocked by externally applied calcium ions (2 X 10(-3) M), verapamil, D = 600 as well as by fluoride while introduced inside a cell. These data prove the existence of separate systems of sodium and calcium ion-conducting channels in the somatic membrane.     

Effect of ethanol on subthreshold currents of Aplysia pacemaker neurons

The subthreshold currents in bursting pacemaker neurons of the Aplysia abdominal ganglion were individually studied with the voltage clamp technique for sensitivity to 4% ethanol. The most prevalent effect of ethanol on unclamped bursting neurons was a hyperpolarization. This was shown to be due to a decrease of a voltage independent inward leakage current. Direct measurement of the Na-dependent slow inward current showed that this current was eliminated by 4% ethanol. Direct measurement of the Ca-dependent slow inward current showed that this current was substantially reduced by 4% ethanol. Injection of EGTA into cell bodies did not eliminate the ethanol-induced block of the slow inward calcium current. Thus, ethanol cannot be reducing the Ca-dependent slow inward current solely by an increase of internal calcium concentration. The effect of ethanol on voltage dependent outward current was measured by blockage of all inward current. The peak outward current was increased by ethanol. The rate of inactivation of this outward current was also increased. Calcium activated potassium current (IK(Ca)) is particularly complicated in its response to ethanol because it is dependent on both Ca and voltage for its activation. The level of IK(Ca) elicited in response to constant Ca injection was increased by ethanol treatment. The level of this current as activated by voltage clamp pulses was either increased or decreased depending on the neuron type. Ca2+ activated potassium conductance increased e-fold for a 26 mV depolarization in membrane holding potential. Ethanol decreased this voltage dependence to e-fold for a 55 mV change in potential. This result was interpreted to mean that ethanol shifted an effective Ca2+ binding site of these channels from about halfway through the membrane field to one quarter of the way across. The same theoretical approach allowed the further conclusion that ethanol caused an increased internal free calcium concentration probably by decreasing calcium binding by intracellular buffers.     

Calcium channels in the somatic membrane of the rat dorsal root ganglion neurons, effect of cAMP

Isolated rat dorsal root ganglion neurons have been perfused with potassium-free solutions containing cAMP, ATP and Mg2+ ions. In these conditions stable inward calcium currents can be recorded in the somatic membrane of all investigated cells. The kinetics of these currents can be approximated by a modified Hodgkin-Huxley equation using a square power of the m-variable; its inactivation is extremely slow. The corresponding channels pass Ba2+ ions about twice more effective than Ca2+.     

Effects of in vitro lead exposure on voltage-sensitive calcium channels differ among cell types in central neurons of Lymnaea stagnalis

The effects of acute in vitro lead exposure on slowly inactivating voltage-sensitive calcium channels in central neurons of the freshwater pond snail Lymnaea stagnalis were studied under voltage clamp. Three physiologically distinct cell types were used: two subsets of the B cell cluster (Bpos and Bneg) and the pedal giant neuron (RPeD1). In Bpos neurons, 5 nM free Pb2+ irreversibly inhibited current flow through calcium channels by 38 +/- 10%. In Bneg neurons, 5 nM free Pb2+ slightly inhibited inward currents (12 +/- 6%) and may have shifted their voltage dependence to more depolarized voltages. The inhibition and voltage shift were irreversible. In RPeD1 neurons, Pb2+ caused a small, statistically insignificant inhibition of inward current (5 nM free Pb2+; 18 +/- 19%; 30 nM free Pb2+: 31 +/- 23%). The effects of Pb2+ were fully reversible. These data indicate that (1) voltage-sensitive calcium channels in Lymnaea neurons are inhibited by nanomolar concentrations of free Pb2+; (2) there are multiple types of calcium channels in Lymnaea neurons; and (3) the effects of in vitro lead exposure differ qualitatively among channel types.     

2 selective filters in the calcium channel of the somatic membrane of mollusk neurons

The modification of inward currents in the somatic membrane of mollusc neurons produced by EDTA and other CA-chelating agents was investigated. The results obtained indicate the presence of two selective filters in the calcium channel of this membrane. The first one is located near the outer mouth of the calcium channel. It binds divalent cations in the following sequence--pKCa: pKSr: pKBa: pKMg=6.6 : 5.5 : 4.8 : 4.2. This outer filter controls channel selectivity according to the magnitude of cation charge and presumably contains several carboxylic groups. The second selective filter is located inside the channel and controls its permeability for cations with the same charge. It is assumed that the structure of the inner selective filter resembles very much the structure postulated by Hille for the selective filter of sodium channel and that it contains only one carboxylic group. The investigation of the effect of Ca2+ and Cd2+ ions on sodium currents of the same membrane has shown that the fast sodium current is not blocked by these ions and the observed decrease of its amplitude is connected with the change of the membrane surface potential and corresponding change of near-membrane concentration of carrier ions. On the basis of these experiments, it is suggested that the selectivity filter of sodium channel of this membrane does not contain a carboxylic group.     

Potential-dependent ion currents in the membrane of the striated muscle of the lamprey

Membrane ionic currents were recorded in thin striated muscle bundles of lamprey suction apparatus by means of double sucrose gap method. In response to depolarization fast inward Na+ and delayed outward K+ currents appeared with steady-state characteristics similar to that in frog muscle membrane. The only difference consisted in lower steepness of the inactivation curve for K+ current. This probably suggests a greater density of slow potassium channels. The presence of two fractions in potassium current is suggested from changes both in reversal potential and in speed of the current deactivation during long lasting depolarizing pulses. No functioning voltage-dependent calcium channels were detected in the lamprey muscle membrane.     

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

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.     

Mercury modulation of GABA-activated chloride channels and non-specific cation channels in rat dorsal root ganglion neurons.

The effects of mercuric chloride and methylmercury chloride on the rat dorsal root ganglion neurons in primary culture were studied by the whole-cell patch clamp technique. gamma-Aminobutyric acid-induced chloride currents were augmented by mercuric chloride in a potent and efficacious manner; at concentrations of 1 and 10 microM, the current amplitude was increased to 130% and 200% of the control. Methylmercury even at 100 microM did not augment but rather decreased the GABA-induced chloride current. Both mercuric chloride and methylmercury generated slow inward currents by themselves. These currents are not mediated by the GABA-activated chloride channels or by voltage-activated sodium, potassium or calcium channels, and are likely to be due to non-specific cation channels.     

Subcellular distribution of L-type Ca2+ channels responsible for plateau potentials in motoneurons from the lumbar spinal cord of the turtle

L-type calcium channels mediate the persistent inward current underlying plateau potentials in spinal motoneurons. Electrophysiological analysis shows that plateau potentials are generated by a persistent inward current mediated by low threshold L-type calcium channels located in the dendrites. As motoneurons express L-type calcium channels of the CaV1.2 and CaV1.3 subtypes, we have investigated the subcellular distribution of these channels using antibody labelling. The plateau generating a persistent inward current is modulated by the activation of metabotropic receptors. For this reason, we also examined the relationship between CaV1.2 and CaV1.3 subunits in motoneurons and presynaptic terminals labelled with antibodies against synapsin 1a. Motoneurons in the spinal cord of the adult turtle were identified as large neurons, immunopositive for choline acetyltransferase, located in the ventral horn. In these neurons, CaV1.2 subunits were present in the cell bodies and axons. Patches of CaV1.3 subunits were seen in association with the cell membrane of the somata and both the proximal and distal dendrites. Double labelling with an antibody against synapsin 1a showed that CaV1.3 subunits, but not CaV1.2 subunits, were always located at synaptic sites. The distribution of CaV1.2 and CaV1.3 strongly suggests that the persistent inward current underlying plateau potentials in spinal motoneurons is mediated by CaV1.3 and not by CaV1.2. Our findings also show that CaV1.3 may be located in the somatic and dendritic membrane adjacent to particular presynaptic terminals.     

Features of the kinetic and stationary characteristics of aconitine-modified sodium channels

Kinetic and steady-state characteristics of aconitine-modified sodium channels were studied in the Ranvier node membrane. Aconitine-modified sodium channels are shown to be inactivated only partially. The voltage dependence of the fraction of noninactivated channels (h infinity) may be described by a three-state model of the channel with closed, open and inactivated states. A reasonable agreement with the data was obtained when parameters of the inactivated state were supposed to be not changed after aconitine modification of the channels. The membrane repolarization to -70 divided by -110 mV, after long (10 ms) depolarizing shift induces firstly fast current decay ("tail") and then its rather slow increase to a steady-state level. Kinetics of this current requires two or more open states to be postulated.