Voltage clamp analysis of tetrodotoxin-sensitive and -insensitive sodium channels in rat muscle cells developing in vitro

Sodium currents in cultured rat muscle cells converted to myoballs by treatment with colchicine were recorded using a giga-ohm seal voltage clamp procedure in the whole cell configuration. The mean peak Na+ conductance of the myoballs was 90 pS/microns2 of surface membrane. Half-maximal activation of Na+ currents was observed for test pulses to -31 mV and half-maximal inactivation was observed for prepulses to -74 mV. Titration of the inhibition of Na+ currents by tetrodotoxin (TTX) yielded a biphasic inhibition curve consistent with the presence of two classes of Na+ channels differing in affinity for TTX. The TTX-sensitive channels carried 28% of the Na+ current and had an apparent KD for TTX of 13 nM at 20 degrees C. The TTX-insensitive Na+ channels had an apparent KD for TTX of 3.2 microns. Inhibition of TTX-insensitive Na+ channels by TTX was enhanced by repetitive stimulation of the myoballs at 2 Hz, whereas the inhibition of TTX-sensitive Na+ channels by TTX was not frequency dependent. We conclude that rat muscle cells developing in vitro synthesize physiologically functional, TTX-sensitive Na+ channels in the absence of innervation. These channels, which are characteristic of adult skeletal muscle, function in parallel with TTX-insensitive Na+ channels that are present in embryonic muscle.     

Expression and kinetic properties of Na(+) currents in rat cardiac dorsal root ganglion neurons

The expression and properties of voltage-gated Na(+) currents in cardiac dorsal root ganglion (DRG) neurons were assessed in this study. Cardiac DRG neurons were labelled by injecting the Fast Blue fluorescent tracer into the pericardium. Recordings were performed from 138 cells. Voltage-dependent Na(+) currents were found in 115 neurons. There were 109 neurons in which both tetrodotoxin-sensitive (TTX-S, blocked by 1 microM of TTX) and tetrodotoxin-resistant (TTX-R, insensitive to 1 microM of TTX) Na(+) currents were present. Five cells expressed TTX-R current only and one cell only the TTX-S current. The kinetic properties of Na(+) currents and action potential waveform parameters were measured in neurons with cell membrane capacitance ranging from 15 to 75 pF. The densities of TTX-R (110.0 pA/pF) and TTX-S (126.1 pA/pF) currents were not significantly different. Current threshold was significantly higher for TTX-R (-34 mV) than for TTX-S (-40.4 mV) currents. V(1/2) of activation for TTX-S current (-19.6 mV) was significantly more negative than for TTX-R current (-9.2 mV), but k factors did not differ significantly. V(1/2) and the k constant for inactivation for TTX-S currents were -35.6 and -5.7 mV, respectively. These values were significantly lower than those recorded for TTX-R current for which V(1/2) and k were -62.3 and -7.7 mV, respectively. The action potential threshold was lower, the 10-90% rise time and potential width were shorter before than after the application of TTX. Based on this we drew the conclusion that action potential recorded before adding tetrodotoxin was mainly TTX-S current dependent, while the action potential recorded after the application of toxin was TTX-R current dependent. We also found 23 cells with mean membrane capacitance ranging from 12 to 35 pF (the smallest labelled DRG cells found in this study) that did not express the Na(+) current. The function of these cells is unclear. We conclude that the overwhelming majority of cardiac dorsal root ganglion neurons in which voltage-dependent Na(+) currents were present, exhibited both TTX-S and TTX-R Na(+) currents with remarkably similar expression and kinetic properties.     

Na(v)1.5 underlies the 'third TTX-R sodium current' in rat small DRG neurons

In addition to slow-inactivating and persistent TTX-R Na(+) currents produced by Na(v)1.8 and Na(v)1.9 Na(+) channels, respectively, a third TTX-R Na(+) current with fast activation and inactivation can be recorded in 80% of small neurons of dorsal root ganglia (DRG) from E15 rats, but in only 3% of adult small DRG neurons. The half-time for activation, the time constant for inactivation, and the midpoints of activation and inactivation of the third TTX-R Na(+) currents are significantly different from those of Na(v)1.8 and Na(v)1.9 Na(+) currents. The estimated TTX K(i) (2.11+/-0.34 microM) of the third TTX-R Na(+) current is significantly lower than those of Na(v)1.8 and Na(v)1.9 Na(+) currents. The Cd(2+) sensitivity of third TTX-R Na(+) current is closer to cardiac Na(+) currents. A concentration of 1 mM Cd(2+) is required to completely block this current, which is significantly lower than the 5 mM required to block Na(v)1.8 and Na(v)1.9 currents. The third TTX-R Na(+) channel is not co-expressed with Na(v)1.8 and Na(v)1.9 Na(+) channels in DRG neurons of E18 rats, at a time when all three currents show comparable densities. The physiological and pharmacological profiles of the third TTX-R Na(+) current are similar to those of the cardiac Na(+) channel Na(v)1.5 and RT-PCR and restriction enzyme polymorphism analysis, show a parallel pattern of expression of Na(v)1.5 in DRG during development. Taken together, these results demonstrate that Na(v)1.5 is expressed in a developmentally regulated manner in DRG neurons and suggest that Na(v)1.5 Na(+) channel produces the third TTX-R current.     

Changes in ultrastructure and voltage-dependent currents at the glia limitans of the frog optic nerve following retinal ablation

The surface of the frog optic nerve consists of astrocytic processes separated by narrow extracellular clefts underlying a pial sheath of loose connective tissue. Macroscopic voltage dependent currents can be recorded from this surface using the loose patch-clamp technique. In this study the changes in ultrastructure and voltage dependent Na currents have been studied for up to 1 year following removal of the retina. During the first 1–4 weeks, many of the myelinated and unmyelinated axons of the retinal ganglion cells degenerate, and the debris is phagocytosed by macrophages and glial cells. However, some morphologically intact axons remain even 12 weeks after surgery. Finally, after 16 weeks all the axons have disappeared, leaving a nerve consisting only of glial cells, some of which contain phagosomes. At 40–52 weeks after enucleation, the nerve persists, at 20–40% of the normal diameter, consisting mostly of normal looking astrocytes. The amplitude of the voltage dependent Na currents recorded from nerves during the first 1–4 weeks after enucleation, with the pial sheath intact, decreases by about 50%. After 8 weeks, the Na current recorded from the surface is about 30% of control. At 16–52 weeks after removal of the retina, when there are no intact axons, the Na current is reduced by 90%. If, however, the pial sheath is stripped away, the Na currents recorded from the glial surface are 40–50% of control during this same 16- to 52-week period, suggesting that in the all-glia nerve, the currents are shunted by the relatively thicker pial sheath. In contrast to their normal TTX sensitivity, the voltage dependent Na currents recorded from these all glial nerves are insensitive to TTX (5 μM) but disappear when the external Na is replaced by TMA. The results suggest that in situ glial voltage dependent membrane properties depend on interactions between the glial cells and the neighboring axons. © 1993 Wiley-Liss, Inc.     

Altered sodium channel behaviour causes myotonia in dominantly inherited myotonia congenita.

The cause of increased excitability in autosomal dominant myotonia congenita (MyC) was studied in resealed greater than 3-cm long segments of muscle fibres from eight patients. Three hours after biopsy only about 50% of the fibre segments had regained a normal resting potential. This differs from our experiences with normal muscle or other disorders of myotonia (e.g. recessive generalized myotonia) where nearly all cut fibres reseal and repolarize during this time. When the depolarized MyC fibre segments were placed in a solution containing 1 microM tetrodotoxin (TTX) they repolarized to -80 to -90 mV. In fibre segments with normal resting potential, in the absence of TTX, spontaneous myotonic runs were recorded intracellularly, occasionally with double spikes. For only one of the eight patients, the Cl- conductance was reduced (50% of the total membrane conductance vs the usual 75%), for the rest of the patients the steady-state current-voltage relationship was normal. Sodium currents through single membrane channels were recorded with a patch clamp. For every patient re-openings of the Na+ channels were observed throughout 10-ms depolarizing pulses. These are very uncommon in normal muscle. At potentials positive to the resting potential, the duration of the re-openings increased, but the current amplitude was the same. It is concluded that in myotonia congenita re-openings of Na+ channels are the major cause of hyperexcitability and that Cl- conductance is normal. If it is reduced in rare cases, it may potentiate the myotonia.     

Cell-specific posttranslational events affect functional expression at the plasma membrane but not tetrodotoxin sensitivity of the rat brain IIA sodium channel alpha-subunit expressed in mammalian cells.

The rat brain IIA Na+ channel alpha-subunit was expressed and studied in mammalian cells. Cells were infected with a recombinant vaccinia virus (VV) carrying the bacteriophage T7 RNA polymerase gene and were transfected with cDNA encoding the IIA Na+ channel alpha-subunit under control of a T7 promoter. Whole-cell patch-clamp recording showed that functional IIA channels were expressed efficiently (approximately 10 channels/microns2 in approximately 60% of cells) in Chinese hamster ovary (CHO) cells and in neonatal rat ventricular myocytes but were expressed poorly in undifferentiated BC3H1 cells and failed to express in Ltk- cells. However, voltage-dependent Drosophila Shaker H4 K+ channels and Escherichia coli beta-galactosidase were expressed efficiently in all four cell types with VV vectors. Because RNA synthesis probably occurs without major differences in the cytoplasm of all infected cell types under the control of the T7 promoter and T7 polymerase, we conclude that cell type-specific expression of the Na+ channel probably reflects differences at posttranslational steps. The gating properties of the IIA Na+ currents expressed in cardiac myocytes differed from those expressed in CHO cells; most noticeably, the IIA Na+ currents displayed more rapid macroscopic inactivation when expressed in cardiac myocytes. These differences also suggest cell-specific posttranslational modifications. IIA channels were blocked by approximately 90% by 90 nM TTX when expressed either in CHO cells or in cardiac myocytes; the latter also continued to display endogenous TTX-resistant Na+ currents. Therefore, the TTX binding site of the channel is not affected by cell-specific modifications and is encoded by the primary amino acid sequence.     

Two subtypes of sodium channel with tetrodotoxin sensitivity and insensitivity detected in denervated mammalian skeletal muscle

The action potential (AP) in most nerve and muscle preparations depends upon nanomolar concentrations of the neurotoxins saxitoxin (STX) and tetrodotoxin (TTX). In some excitable tissues lacking mature innervation, a toxin-resistant AP has been described by electrophysiological results. However, multiple attempts to detect corresponding toxin-resistant Na channels with radiolabelled STX and TTX have been unsuccessful. We report here the detection of Na channels with low-affinity binding of STX and TTX, accounting for 50-60% of the Na channels in rat hindlimb muscle 4-5 days after denervation.     

Membrane defects in paramyotonia congenita (Eulenburg)

Membrane potentials, current-voltage relationships, and component conductances were determined in resting excised external intercostal muscle fibers from five patients with paramyotonia congenita. At 37 degrees C all investigated parameters were normal. At 27 degrees C the resting potentials decreased to about -40 mV, and the fibers were inexcitable. At this stage the membrane currents were much larger than in normal fibers owing to increases in the membrane conductances for Na and Cl ions. The earlier finding that in the cold the Na permeability is abnormally large was confirmed. The Cl permeability was shown to be normal even in the cold. The decrease of the resting potential and the changes in the current-voltage relationship at 27 degrees C could be prevented by the use of the Na channel blocker tetrodotoxin (TTX) or by bathing the fibers in a Na-free solution. Our previous conclusion that the Cl conductance at 27 degrees C was also increased when TTX was present was not confirmed. Exposure of a muscle bundle to 7 mmol/l potassium did not lead to excessive depolarization and paralysis.     

Differential properties of tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels in rat dorsal root ganglion neurons.

TTX-sensitive (TTX-S) and TTX-resistant (TTX-R) sodium channel currents were analyzed in acutely dissociated dorsal root ganglion (DRG) neurons isolated from 3-12-d-old and adult rats. Currents were recorded using the whole-cell patch-clamp technique. TTX-R current was more likely to be present in younger animals (3-7 d), whereas TTX-S current was more common in older animals (7-10 d), although TTX-R current was recorded from adult rat DRG neurons. The TTX-R and TTX-S currents differed in their steady-state inactivation, with 50% inactivation voltage at -40 +/- 5 mV (n = 10) for TTX-R currents and -70 +/- 4 mV (n = 10) for TTX-S currents. These current types also differed in their activation kinetics, with 50% activation values of -15 +/- 5 mV (n = 5) for TTX-R currents and -26 +/- 6 mV (n = 5) for TTX-S currents. The interactions of TTX-R and TTX-S channels with various pharmacological agents and divalent cations were studied. The Kd values for TTX-S and TTX-R currents were estimated to be 0.3 nM and 100 microM for TTX, 0.5 nM and 10 microM for saxitoxin, and 50 microM and 200 microM for lidocaine, respectively. TTX-S channels did not exhibit a marked use-dependent block by lidocaine, whereas lidocaine significantly decreased TTX-R current in a use-dependent manner at frequencies ranging from 1 to 33.3 Hz. Several external divalent cations exerted different effects on these current types.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ontogenic development of the TTX-sensitive and TTX-insensitive Na+ channels in neurons of the rat dorsal root ganglia.

Developmental changes in the sensitivity of neurons to tetrodotoxin (TTX) were studied in relation to the cell size in rat dorsal root ganglia (DRG). Na+ currents were recorded from neurons of various stages of development. Two types of Na+ channels were identified on the basis of their sensitivity to TTX. One type was insensitive to a very high concentration (0.1 mM) of TTX, while the other type was blocked by a low concentration (1 nM) of TTX. These two types of Na+ channels were observed throughout the developmental stages examined from day 17 of gestation and adulthood. Thus, both types of Na+ channels are already established at the early stage of neuronal development and appear to be retained throughout the life-span of the DRG neuron. The concentration-response relationships for the block of TTX-sensitive Na+ current by TTX did not appreciably change during development. Although two types of Na+ channels had strikingly different kinetic properties, the kinetic properties of each channel type were basically similar throughout development. The TTX-sensitive Na+ channels were mainly concentrated in cells with large cell diameters throughout developmental stages examined. These large cells appear to correspond to the 'large-light' cells. On the contrary, the TTX-insensitive Na+ channels were found in smaller diameter cells which may correspond to the 'small-dark' cells. Thus, it is concluded that there are heterogeneous categories of neurons which have Na+ channels with different physiological and pharmacological properties. Since Na+ channels play a pivotal role in the action potential generation, these heterogeneity of DRG neurons appear to be instrumental in integrating the sensory signals.     

Lidocaine stabilizes the open state of CNS voltage-dependent sodium channels

We have previously reported that the lidocaine action is different between CNS and muscle batrachotoxin-modified Na+ channels [Salazar et al., J. Gen. Physiol. 107 (1996) 743-754; Brain Res. 699 (1995) 305-314]. In this study we examined lidocaine action on CNS Na+ currents, to investigate the mechanism of lidocaine action on this channel isoform and to compare it with that proposed for muscle Na+ currents. Na+ currents were measured with the whole cell voltage clamp configuration in stably transfected cells expressing the brain alpha-subunit (type IIA) by itself (alpha-brain) or together with the brain beta(1)-subunit (alphabeta(1)-brain), or the cardiac alpha-subunit (hH1) (alpha-cardiac). Lidocaine (100 microM) produced comparable levels of Na+ current block at positive potentials and of hyperpolarizing shift of the steady-state inactivation curve in alpha-brain and alphabeta(1)-brain Na+ currents. Lidocaine accelerated the rates of activation and inactivation, produced an hyperpolarizing shift in the steady-state activation curve and increased the current magnitude at negative potentials in alpha-brain but not in alphabeta(1)-brain Na+ currents. The lidocaine action in alphabeta(1)-brain resembled that observed in alpha-cardiac Na+ currents. The lidocaine-induced increase in current magnitude at negative potentials and the hyperpolarizing shift in the steady-state activation curve of alpha-brain, are novel effects and suggest that lidocaine treatment does not always lead to current reduction/block when it interacts with Na+ channels. The data are explained by using a modified version of the model proposed by Vedantham and Cannon [J. Gen. Physiol., 113 (1999) 7-16] in which we postulate that the difference in lidocaine action between alpha-brain and alphabeta(1)-brain Na+ currents could be explained by differences in the lidocaine action on the open channel state.     

Activation of beta-adrenergic receptor induces Na-dependent inward currents in acutely dissociated motoneurons of bullfrog spinal cord

Effects of adrenergic drugs on single motoneurons acutely dissociated from the lumbar enlargement of adult bullfrogs were examined. The dissociated large cells were identified as motoneurons by retrograde labeling with a fluorescent dye. Adrenaline caused membrane depolarization with a decrease in input resistance. Under whole-cell voltage clamp conditions at a holding potential of -70 mV, adrenergic drugs induced inward currents in a dose-dependent manner. Adrenaline was more potent than noradrenaline. Under K(+)-free conditions, adrenaline (10(-6)-10(-5) M) induced inward currents which were blocked by propranolol (10(-6) M) but not by phentolamine (10(-5) M). CoCl2 (1 mM) did not affect the currents. Substitution of choline+ in the recording solution for Na+ abolished the currents, but tetrodotoxin (TTX, 10(-6) M) had no effect on them. The adrenaline-induced currents exhibited a characteristic voltage-dependency: the conductance became large at hyperpolarized membrane potential (-150 to -30 mV) and approached zero at the depolarized membrane potential (greater than -30 mV), but was never reversed up to 30 mV, suggesting that the currents are different from non-specific cation currents. Substitution of isethionate- for Cl- in the recording solution had no effect on the voltage-dependency of the adrenaline-induced currents, whereas substitution of choline+ for Na+ apparently attenuated the voltage-dependency of the currents. These results indicate that adrenaline induces Na(+)-dependent inward currents through activation of beta-adrenergic receptors in bullfrog motoneurons.     

Noninactivating,tetrodotoxin-sensitive Na+ conductance in peripheral axons

A noninactivating, persistent sodium current has been demonstrated previously in dorsal root ganglia neurons and in rat optic nerve. We report here that Na(+) channel blockade with tetrodotoxin (TTX) in isolated dorsal and ventral roots elicits membrane hyperpolarization, suggesting the presence of a persistent Na(+) current in peripheral axons. We used a modified sucrose-gap chamber to monitor resting and action potentials and observed a hyperpolarizing shift in the nerve potential of rat dorsal and ventral roots by TTX. The block of transient inward Na(+) currents was confirmed by the abolition of compound action potentials (CAPs). Moreover, depolarization of nerve roots by elevating extracellular K(+) concentrations to 40 mM eliminated CAPs but did not significantly alter TTX-induced hyperpolarizations, indicating that the persistent Na(+) currents in nerve roots are not voltage-dependent. Tetrodotoxin-sensitive persistent inward Na(+) currents are present in both dorsal and ventral root axons at rest and may contribute to axonal excitability.     

Tetrodotoxin-resistant Na+ channels in human neuroblastoma cells are encoded by new variants of Nav1.5/SCN5A

Both tetrodotoxin-sensitive (TTX-S) and TTX-resistant (TTX-R) voltage-dependent Na+ channels are expressed in the human neuroblastoma cell line NB-1, but a gene encoding the TTX-R Na+ channel has not been identified. In this study, we have cloned cDNA encoding the alpha subunit of the TTX-R Na+ channel in NB-1 cells and designated it hNbR1. The longest open reading frame of hNbR1 (accession no. AB158469) encodes 2016 amino acid residues. Sequence analysis has indicated that hNbR1 is highly homologous with human cardiac Nav1.5/SCN5A with > 99% amino acid identity. The presence of a cysteine residue (Cys373) in the pore-loop region of domain I is consistent with the supposition that hNbR1 is resistant to TTX. Analysis of the genomic sequence of SCN5A revealed a new exon encoding S3 and S4 of domain I (exon 6A). In addition, an alternative splicing variant, lacking exon 18, that encodes 54 amino acids in the intracellular loop between domains II and III was found (hNbR1-2; accession no. AB158470). Na+ currents in human embryonic kidney cells (HEK293) transfected with hNbR1 or hNbR1-2 showed electrophysiological properties similar to those for TTX-R I(Na) in NB-1 cells. The IC50 for the TTX block was approximately 8 microM in both variants. These results suggest that SCN5A has a newly identified exon for alternative splicing and is more widely expressed than previously thought.     

Na+ current kinetics are not the determinants of the action potential duration in neurons of the rat ventral tegmental area.

Na+ currents were recorded from two morphological subpopulations of neurons acutely dissociated from the rat ventral tegmental area (VTA). About 45% of 56 VTA cells examined possessed only the ordinary type of Na+ current which was blocked by a low concentration (0.2 microM) of TTX. However, the remaining 55% had the Na+ current which contained a small fraction of the TTX-insensitive component, irrespective of morphological variations and action potential durations of VTA cells. The peak amplitude of the TTX-insensitive component was less than 10% of the peak amplitude of the total Na+ current. The activation and inactivation kinetics of the TTX-insensitive component were much the same as those of the overwhelming TTX-sensitive component of the Na+ current in VTA cells but differed from those of the TTX-insensitive Na+ current reported in peripheral sensory neurons. Thus, it was concluded that the well-known different action potential durations found for subpopulations of VTA cells are not due to multiplicity of Na+ channel kinetics.     

Modification of the Na-dependent action potential in myelinated fibers of rat sciatic nerve exposed to phorbol ester

Exposure of rat sciatic nerve to the active phorbol 1,2-beta-myristate-13-acetate (b-PMA), but not to the active analogue 4-alpha-phorbol-12,13-didecanoate (a-PDD), is followed by a decrease of the compound action potential amplitude, rate of rise, and conduction velocity, and an increase of the threshold, and of the duration of the refractory period. The effect is concentration-dependent, the Kd being 250 nM. The attenuated Na-dependent action potential is tetrodotoxin (TTX)-sensitive, but after exposure to b-PMA the sensitivity to TTX is decreased from Kd = 45 nM to 400 nM. Action potential depression is larger when Ca is replaced by Mg (but not by Ba), or when Na is replaced by Li. The replacement of K by Cs, or exposure to potassium channel blockers such as 4-aminopyridine (4AP) and tetra-ethyl ammonium (TEA) has no effect. The results indicate that in the myelinated axons of rat sciatic nerve, exposure to b-PMA induces modification of Na channels.     

Enhancement of persistent sodium current by internal fluorescence in isolated hippocampal neurons

Following up on an earlier chance observation, voltage-dependent whole-cell currents were recorded from isolated hippocampal neurons filled with the fluorescent dyes Fluo-3 and Fura-red, that were intermittently excited by 488 nm laser light. In the absence of any ion channel blocking drugs, in most cells depolarizing voltage steps initially evoked only the 'Hodgkin-Huxley' type early, fast inward surge followed by sustained outward current. Over 5-20 min of intermittent electrical stimulation and laser-excited fluorescence pulses, a voltage-dependent, slowly inactivating inward current also appeared and grew, while sustained outward current diminished. When K(+) currents were blocked, a small persistent inward current was usually detectable immediately, and then it increased in amplitude. This current was blocked by tetrodotoxin (TTX) and it had current-voltage (I-V) characteristics of a persistent sodium current, I(Na,P). In cells not filled with dye but illuminated by laser, and in cells with dye but not illuminated, I(Na,P) remained small. There was a more than 12-fold difference in the maximal amplitude of I(Na, P) of fluorescent compared to non-fluorescent cells. Once induced, I(Na,P) decreased very slowly. Fluorescence increased the duration but not the amplitude of the transient Na(+) current, I(Na,T). With membrane potential clamped to a constant voltage, the laser-induced fluorescence did not evoke a membrane current. It is not certain whether fluorescence-induced I(Na,P) potentiation is related to photodynamic action.     

Ionic channels in mouse astrocytes in culture

We observed Na, K, and Cl voltage-dependent currents in a patch-clamp study of mouse brain astrocytes. In whole-cell recordings, depolarizations activated inward currents that were identified as Na currents since they were blocked by TTX, although complete block required high concentrations (greater than 1 microM). The corresponding single-channel Na currents were observed in outside-out patches. The channels were opened by a depolarizing pulse applied from a holding potential identical to the resting potential (-70 to -80 mV). Therefore, they may be considered functional Na channels. After addition of veratridine and an alpha-scorpion toxin, the decay of Na currents in whole-cell recordings was slower than observed under control conditions. At the single-channel level, the channels appeared to open in bursts. Depolarization did not increase the duration of the bursts, but inside each burst, increased the time spent in the open state. The K currents observed in the whole-cell recording mode were separated into inactivating and noninactivating currents. The inactivating current resembled the A current in its kinetics, its insensitivity to tetraethylammonium, and its sensitivity to 4-aminopyridine. At the single-channel level, at least 3 classes of K channels were observed at steady depolarized potentials. They resembled the K channels found in chromaffin cells by Marty and Neher (1985). Large conductance channels (385 pS) activated around 0 mV were identified as Cl channels.     

Expression of membrane currents in rat diencephalic neurons in serum-free culture

The whole-cell gigaseal voltage clamp technique has been used to investigate the timing of expression and type of voltage-dependent ionic currents in dissociated primary cultures of fetal rat (E17) diencephalic neurons grown in a serum-free defined medium. The expression of membrane currents varied among cells at any particular time in culture. Despite this variability, certain characteristics of the appearance of ionic currents emerge from this study. These are: (i) the earliest appearing membrane current is a voltage-dependent outward current carried by K+. In some cells, it is the classical delayed rectifier current, whereas in others it is the transient outward current (IA). (ii) The earliest appearing inward current is carried by Na+. In some cells the channels are first expressed in the neurites and then in or near the cell body. The early neuritic Na+ channels are blocked by cobalt or cadmium as well as by tetrodotoxin (TTX). In others, the early Na+ channels appear in or near the cell body and are only blocked by TTX. (iii) With additional time in culture, a majority of cells exhibit a Ca2+ current at the time of Na+ channel appearance in or near the cell body as well as a transient Ca2+-dependent outward current. The Ca2+ current is only a small fraction of the total inward current. These inward currents show the classical pharmacologic profile. The complex pattern of expression of ionic current may reflect multiple populations of neurons with different developmental sequences resulting from differences in cell age and lineage.