Potassium Current Density Research Articles

Developmental changes in the density of ionic currents in antennal-lobe neurons of the sphinx moth, Manduca sexta.

Early in metamorphic adult development, action potentials elicited from Manduca sexta antennal lobe neurons are small in amplitude, long in duration, and calcium dependent. As development proceeds, the action potential waveform becomes larger in amplitude, shorter in duration, and increasingly sodium dependent. Whole cell voltage-clamp analysis of Manduca antennal-lobe neurons in vitro has been used to identify voltage-activated currents that contribute to developmental changes in the electrical excitability of these cells. Proximal Branching neurons [putative projection (output) neurons] and Rick Rack neurons (putative local antennal-lobe interneurons) are examined in detail early (pupal stage 5) and late (pupal stage 14) in adult metamorphosis. In both cell types, four voltage-gated and two calcium-dependent ionic currents have been identified. Cell-type-specific changes in the density of sodium, calcium, and potassium currents correlate temporally with changes in cell excitability and spike waveform. Developmental changes in ionic current profiles are accompanied also by the emergence of cell-type-specific response characteristics in the cells. Together with the accompanying paper, this study provides an important foundation for examining the impact of developmental changes in electrical excitability on the growth, electrical properties and connectivity of neurons in central olfactory pathways of the moth.

Depressed transient outward potassium current density in catecholamine-depleted rat ventricular myocytes.

The effect of catecholamine depletion (induced by prior treatment with reserpine) was studied in Wistar rat ventricular myocytes using whole cell voltage-clamp methods. Two calcium-independent outward currents, the transient outward potassium current (I(to)) and the sustained outward potassium current (I(sus)), were measured. Reserpine treatment decreased tissue norepinephrine content by 97%. Action potential duration in the isolated perfused heart was significantly increased in reserpine-treated hearts. In isolated ventricular myocytes, I(to) density was decreased by 49% in reserpine-treated rats. This treatment had no effect on I(sus). The I(to) steady-state inactivation-voltage relationship and recovery from inactivation remained unchanged, whereas the conductance-voltage activation curve for reserpine-treated rats was significantly shifted (6.7 mV) toward negative potentials. The incubation of myocytes with 10 microM norepinephrine for 7-10 h restored I(to), an effect that was abolished by the presence of actinomycin D. Norepinephrine (0.5 microM) had no effect on I(to). However, in the presence of both 0.5 microM norepinephrine and neuropeptide Y (0.1 microM), I(to) density was restored to its control value. These results suggest that the sympathetic nervous system is involved in I(to) regulation. Sympathetic norepinephrine depletion decreased the number of functional channels via an effect on the alpha-adrenergic cascade and norepinephrine is able to restore expression of I(to) channels.

Voltage-dependent ion channels in CAD cells: A catecholaminergic neuronal line that exhibits inducible differentiation.

Cell lines derived from tumors engineered in the CNS offer promise as models of specific neuronal cell types. CAD cells are an unusual subclone of a murine cell line derived from tyrosine hydroxylase (TH) driven tumorigenesis, which undergoes reversible morphological differentiation on serum deprivation. Using single-cell electrophysiology we have examined the properties of ion channels expressed in CAD cells. Despite relatively low resting potentials, CAD cells can be induced to fire robust action potentials when mildly artificially hyperpolarized. Correspondingly, voltage-dependent sodium and potassium currents were elicited under voltage clamp. Sodium currents are TTX sensitive and exhibit conventional activation and inactivation properties. The potassium currents reflected two pharmacologically distinguishable populations of delayed rectifier type channels while no transient A-type channels were observed. Using barium as a charge carrier, we observed an inactivating current that was completely blocked by nimodipine and thus associated with L-type calcium channels. On differentiation, three changes in functional channel expression occurred; a 4-fold decrease in sodium current density, a 1.5-fold increase in potassium current density, and the induction of a small noninactivating barium current component. The neuronal morphology, excitability properties, and changes in channel function with differentiation make CAD cells an attractive model for study of catecholaminergic neurons.

Restoration of cardiac transient outward potassium current by norepinephrine in diabetic rats.

In cardiac ventricle, the density of the transient outward potassium current, Ito, is clearly related to sympathetic nervous system integrity. This sympathetic regulation of Ito expression may be greatly significant to the genesis of cardiac complications of several diseases such us diabetes mellitus. Autonomic neuropathy, including cardiac neuropathy, is a complication of chronic diabetes. The objective of the present study was to identify the possible role of cardiac sympathetic neuropathy in the reduction of Ito current density in diabetic ventricular myocardium. Thus, we employed the patch-clamp technique to test whether Ito can be restored in diabetic myocytes incubated with norepinephrine. We also measured, using HPLC, the catecholamine content of the stellate ganglion, which is responsible for cardiac sympathetic innervation, in normal and diabetic animals. The main result of the present study was to show that a 24-h incubation of diabetic cells with norepinephrine restores Ito density to control values. The restoration of Ito current density by norepinephrine suggests that the diabetes-induced reduction of Ito is at least partially attributable to a reduced trophic effect of norepinephrine on the expression of Ito.

Benzalkonium Chloride and Gentamicin Cause a Leak in Corneal Epithelial Cell Membrane

The purposes of this study are to characterize the pathophysiological effects of benzalkonium chloride and gentamicin on corneal epithelial cells and to determine the concentration dependent effect of these agents on membrane currents of these cells. Rabbit corneal epithelial cells were isolated and subdivided into small, medium and large cells according to their cell capacitance. Using whole cell clamp technique, potassium current of corneal epithelial cells was recorded. Transmembrane current was measured again after bathing in benzalkonium chloride 1, 3, 10, 30 and 100μ m ml−1for 3min. The effect of gentamicin was tested at concentrations of 0.4, 1.0, 2.0, 4.0 and 10mg ml−1. Synergistic effect of gentamicin of the above mentioned concentrations in the presence of benzalkonium chloride 1μ g ml−1was also measured. We found that small corneal epithelial cells had the highest depolarization-gated, outward potassium current density and large cells had the lowest current density, while medium cells had a current density in between. Benzalkonium chloride induced a concentration dependent increase in the leak current with increasing concentration from 1 to 100μ g ml−1. The increase of leak current in medium and large cells was less than that in the small cells. Gentamicin also caused a concentration dependent increase in leak current density from 0.4 to 10.0mg ml−1. The increase in leak current density was statistically significant when the concentration was 2.0mg ml−1or higher in small corneal epithelial cells and 1.0mg ml−1or higher in the medium and large cells. Benzalkonium chloride 1.0μ g ml−1augmented the effect of gentamicin on epithelial cell membrane. The extent of enhancement was more prominent in larger than smaller cells. Using whole-cell clamp technique, we were able to determine the threshold concentration of gentamicin and benzalkonium chloride on the integrity of corneal epithelial cell membrane. The toxic action of both agents is mediated by an increase in leak current. We propose that the whole-cell clamp technique is a sensitive and useful tool in determining cytotoxic effects of various agents.

Developmental changes in voltage-activated potassium currents of rat retinal ganglion cells

Ca 2+-independent voltage-activated potassium currents were investigated during the differentiation of rat retinal ganglion cells. Whole-cell patch-clamp recordings of Ca 2+-independent voltage-activated potassium currents and their individual current components, i.e. a sustained, tetraethylammonium-sensitive current, a transient, 4-aminopyridine-sensitive current, and a slowly decaying current that was blocked by Ba 2+, revealed distinct ontogenetic modifications in current densities and in activation and inactivation parameters. All three current types were expressed simultaneously at embryonic day 17/18 and were present in all retinal ganglion cells thereafter without showing any significant changes until the end of the first postnatal week. Ca 2+-independent voltage-activated potassium current densities then increased strongly from postnatal day 8 onwards. Tetraethylammonium-sensitive current density increased about eightfold from 74 pA/pF in embryonic stages to 586 pA/pF in adult cells, whereas the transient potassium currents blocked by 4-aminopyridine increased only about 2.5-fold from 174 pA/pF to 442 pA/pF. The Ba 2+-sensitive current increased simultaneously from 35 pA/pF to 332 pA/pF. The much higher increase in the sustained current components during retinal ganglion cell differentiation accounted for the changes in decay kinetics of Ca 2+-independent voltage-activated potassium current observed in later postnatal stages. Alterations in current densities were paralleled by pronounced changes in current kinetics. From postnatal day 8 onwards, activation of Ca 2+-independent voltage-activated potassium current was right-shifted for about 10 mV owing to a shift in tetraethylammonium-sensitive current-activation, whereas activation of other K + components remained unaltered. Tetraethylammonium-sensitive current steady-state inactivation was incomplete at all developmental stages. About 50% of the tetraethylammonium-sensitive current elicited by a depolarization to +36 mV did not inactivate after prepulse potentials positive to −10 mV. In contrast, transient potassium current blocked by 4-aminopyridine almost fully inactivated during embryonic stages, whereas in adult retinal ganglion cells about 40% of this current component did not inactivate after prepulse potentials positive to −20 mV. Parallel investigation of the resting membrane potential during retinal ganglion cells differentiation showed an exponential increase from −3 mV at embryonic day 15/16 when no voltage-activated ion currents were expressed to a final value of −58 mV at postnatal day 8. These results show that fundamental potassium current modifications occur relatively late in retinal ganglion cell development and only after the resting potential is at its final value.

Regional differences in action potential characteristics and membrane currents of guinea-pig left ventricular myocytes.

Regional differences in action potential characteristics and membrane currents were investigated in subendocardial, midmyocardial and subepicardial myocytes isolated from the left ventricular free wall of guinea-pig hearts. Action potential duration (APD) was dependent on the region of origin of the myocytes (P < 0.01, ANOVA). Mean action potential duration at 90 % repolarization (APD90) was 237 +/- 8 ms in subendocardial (n = 30 myocytes), 251 +/- 7 ms in midmyocardial (n = 30) and 204 +/- 7 ms in subepicardial myocytes (n = 36). L-type calcium current (ICa) density and background potassium current (IK1) density were similar in the three regions studied. Delayed rectifier current (IK) was measured as deactivating tail current, elicited on repolarization back to -45 mV after 2 s step depolarizations to test potentials ranging from -10 to +80 mV. Mean IK density (after a step to +80 mV) was larger in subepicardial myocytes (1.59 +/- 0.16 pA pF-1, n = 16) than in either subendocardial (1.16 +/- 0.12 pA pF-1, n = 17) or midmyocardial (1. 13 +/- 0.11 pA pF-1, n = 21) myocytes (P < 0.05, ANOVA). The La3+-insensitive current (IKs) elicited on repolarization back to -45 mV after a 250 ms step depolarization to +60 mV was similar in the three regions studied. The La3+-sensitive tail current, (IKr) was greater in subepicardial (0.50 +/- 0.04 pA pF-1, n = 11) than in subendocardial (0.25 +/- 0.05 pA pF-1, n = 9) or in midmyocardial myocytes (0.38 +/- 0.05 pA pF-1, n = 11, P < 0.05, ANOVA). The contribution of a Na+ background current to regional differences in APD was assessed by application of 0.1 microM tetrodotoxin (TTX). TTX-induced shortening of APD90 was greater in subendocardial myocytes (35.7 +/- 7.1 %, n = 11) than in midmyocardial (15.7 +/- 3. 8 %, n = 10) and subepicardial (20.2 +/- 4.3 %, n = 11) myocytes (P < 0.05, ANOVA). Regional differences in action potential characteristics between subendocardial, midmyocardial, and subepicardial myocytes isolated from guinea-pig left ventricle are attributable, at least in part, to differences in IK and Na+-dependent currents.

Modifications of potassium currents at the nerve growth factor-induced differentiation of pheochromocytoma cells

Potassium currents were studied in non-differentiated and neuron type-differentiated (induction by nerve growth factor) cells of P12 pheochromocytoma. In the differentiated cells, an increase in the density of non-inactivating potassium current was observed. Fast and slow inactivating potassium currents underwent no changes.

Female gender as a risk factor for drug-induced cardiac arrhythmias: evaluation of clinical and experimental evidence.

One of the most pronounced gender-based differences in response to drugs is women's far greater risk of developing the life-threatening ventricular arrhythmia called torsades de pointes (TdP). A review of the literature and databases of the Food and Drug Administration reveals that a much higher percentage of women than men develop TdP arrhythmias after taking a variety of drugs, such as antihistamines (terfenadine, astemizole), antibiotics (erythromycin), antimalarials (halofantrine), antiarrhythmics (quinidine, d-sotalol), and miscellaneous other drugs. All of these drugs have in common the ability to block potassium currents, thereby prolonging cardiac repolarization and the QT interval on the ECG. The available experimental data support the hypothesis that gender differences in specific cardiac ion current densities are responsible, at least in part, for the greater susceptibility of females for developing TdP arrhythmias. In isolated perfused rabbit hearts (Langendorff technique), female rabbit hearts display greater baseline and drug-induced (quinidine and d-sotalol) changes in QT intervals than male hearts, and at least two different repolarizing potassium current densities (IKr and IKl) are found to be significantly lower in ventricular cardiomyocytes from female rabbits compared with those from males. Thus, it appears that as in humans, clear gender differences exist in the electrophysiologic characteristics governing cardiac repolarization in rabbits. This model and perhaps others should be examined as predictors of functional and pharmacologic differences between men and women. Understanding the potential mechanisms responsible for the greater risk of drug-induced arrhythmias in women could lead to screening methods for identification of individuals at risk for drug-induced arrhythmias or to the development of drugs with reduced risk of inducing arrhythmia.

Action Potential Prolongation and Potassium Currents in Left-Ventricular Myocytes Isolated from Hypertrophied Rabbit Hearts

We have studied the action potential characteristics and potassium currents in single left ventricular myocytes isolated from control and hypertrophied rabbit hearts. Left-ventricular hypertrophy (LVH) was induced following perinephritis-induced hypertension. Control animals underwent a sham operation. Animals were killed at 10 weeks post-operation. Left-ventricular myocytes were isolated by an enzyme dissociation technique. Action potential duration (APD) at 50 and 90% repolarisation was prolonged in myocytes obtained from hypertrophied compared to control hearts over the range of stimulation frequencies (0.1–1.5 Hz). This prolongation in APD was more pronounced in epicardial compared to endocardial myocytes. Steady-state ionic current, measured at the end of voltage clamp steps of 3-s duration, stepping at intervals of 10 mV, from a holding potential of −40 mV, was similar in control and hypertrophied myocytes. However, when normalised for capacitative cell surface area, steady-state current was significantly smaller in hypertrophied myocytes over the voltage range −40 to −60 mV and at potentials greater than +10 mV. Inward rectifier potassium current (IKl), identified as the barium chloride (0.1 mm)-sensitive current, contributed to the steady state current at negative potentials. Normalised IKlwas significantly smaller in hypertrophied compared to control myocytes at potentials negative to −60 mV. Peak transient outward potassium current (Ito) density was reduced in hypertrophied compared to control myocytes at 0 and +10 mV, from a holding potential −80 mV (12.9±2.3v24.9±3.9μA cm2, at +10 mV,P<0.05). Steady-state inactivation of Itowas similar in control and hypertrophied myocytes. In conclusion, LVH induced by perinephritis hypertension in the rabbit is associated with a prolongation in APD. Reductions in IKl, sustained outward current and Itomay contribute to the prolongation in APD.

Tumor necrosis factor enhancement of transient outward potassium currents in cultured rat cortical neurons.

The effect of recombinant human tumor necrosis factor-alpha (TNF) on voltage-gated membrane currents of cultured neurons derived from embryonic rat cerebral cortex was studied using the whole-cell patch-clamp technique. Treatment of neurons with TNF resulted in an increase in outward potassium current density, dependent upon the concentration of TNF and the incubation time, without affecting other membrane currents such as barium and N-methyl-D-aspartate (NMDA). Long exposures (12-48 hr) to TNF (10-100 ng/ml) increased transient outward potassium current (A-current) density without affecting the parameters of activation and inactivation of the current. Prolonged exposures to TNF diminished its increasing effect on the A-current. Since the increase of A-current density induced by TNF is inhibited by both the anti-TNF receptor antibody and cycloheximide treatment, the effect of TNF might be mediated through receptors and by de novo synthesis of the channel protein itself and/or modulating proteins associated with the channel activities. Results indicate that phosphatidylcholine-specific phospholipase C and protein kinase C, but not ceramide, are involved in the signal transduction. In toxicological experiments, TNF had no neurotoxicity. Moreover, a 12 hr pretreatment of TNF protected neurons against NMDA-induced neurotoxicity. This protective effect of TNF was cancelled by 4-aminopyridine, an A-current blocker, suggesting that the increase of A-current densities induced by TNF contributes to the neuroprotection.

Tumor necrosis factor enhancement of transient outward potassium currents in cultured rat cortical neurons

The effect of recombinant human tumor necrosis factor-α (TNF) on voltage-gated membrane currents of cultured neurons derived from embryonic rat cerebral cortex was studied using the whole-cell patch-clamp technique. Treatment of neurons with TNF resulted in an increase in outward potassium current density, dependent upon the concentration of TNF and the incubation time, without affecting other membrane currents such as barium and N-methyl-D-aspartate (NMDA). Long exposures (12–48 hr) to TNF (10–100 ng/ml) increased transient outward potassium current (A-current) density without affecting the parameters of activation and inactivation of the current. Prolonged exposures to TNF diminished its increasing effect on the A-current. Since the increase of A-current density induced by TNF is inhibited by both the anti-TNF receptor antibody and cycloheximide treatment, the effect of TNF might be mediated through receptors and by de novo synthesis of the channel protein itself and/or modulating proteins associated with the channel activities. Results indicate that phosphatidylcholine-specific phospholipase C and protein kinase C, but not ceramide, are involved in the signal transduction. In toxicological experiments, TNF had no neurotoxicity. Moreover, a 12 hr pretreatment of TNF protected neurons against NMDA-induced neurotoxicity. This protective effect of TNF was canceled by 4-aminopyridine, an A-current blocker, suggesting that the increase of A-current densities induced by TNF contributes to the neuroprotection. J. Neurosci. Res. 50:990–999, 1997. © 1997 Wiley-Liss, Inc.

Alterations in Ito1, IKr and Ik1 density in the BIO TO-2 strain of syrian myopathic hamsters.

The BIO TO-2 strain of cardiomyopathic hamster provides a model of dilated low output heart failure. The goal of the study was to determine whether changes in potassium currents occur in this model of heart failure. The densities of Ito1, IKr and IK1 in 8-month-old myopathic hamsters were not significantly different from their age-matched controls. The half-maximum activation voltage (V1/2) of IKr and Ito1, as well as the voltage-dependence of Ito1 inactivation were also similar in both groups at 8 months. Ito1 inactivation exhibited a double exponential time-course; the slow component (tau2), but not the rapid component (tau1), was larger in the myopathic animals. The densities of Ito1, IKr and IK1 were not significantly different in the 8- and 10-month-old control animals. However, the densities of Ito1, IKr and IK1 were all significantly lower in the 10-month-old myopathic hamsters relative to the 10-month-old controls. The V1/2 for IKr and Ito1 activation was the same in myopathic and control animals. tau2, but not tau1, of Ito1 inactivation was again larger in the myopathic animals. The voltage-dependence of Ito1 inactivation was shifted slightly, but significantly, positive in the myopathic animals. Lastly, a sustained outwardly rectifying current that activated upon depolarization was found to be larger in the myopathic animals at both 8 and 10 months of age. In conclusion, many of the alterations in potassium current densities in the 10-month-old cardiomyopathic hamsters are qualitatively similar to the changes observed in the failing human heart.

Postnatal maturation of rat hypothalamoneurohypophysial neurons: evidence for a developmental decrease in calcium entry during action potentials.

Action potentials and voltage-gated currents were studied in acutely dissociated neurosecretory cells from the rat supraoptic nucleus during the first three postnatal weeks (PW1-PW3), a period corresponding to the final establishment of neuroendocrine relationships. Action potential duration (at half maximum) decreased from 2.7 to 1.8 ms; this was attributable to a decrease in decay time. Application of cadmium (250 microM) reduced the decay time by 43% at PW1 and 21% at PW3, indicating that the contribution of calcium currents to action potentials decreased during postnatal development. The density of high-voltage-activated calcium currents increased from 4.4 to 10.1 pA/pF at postnatal days 1-5 and 11-14, respectively. The conductance density of sustained potassium current, measured at +20 mV, increased from 0.35 (PW1) to 0.53 (PW3) nS/pF. The time to half-maximal amplitude did not change. Conductance density and time- and voltage-dependent inactivation of the transient potassium current were stable from birth. At PW1, the density and time constant of decay (measured at 0 mV) were 0.29 nS/pF (n = 12) and 17.9 ms (n = 10), respectively. Voltage-dependent properties and density (1.1 nS/pF) of the sodium current did not change postnatally. During PW1, fitting the mean activation data with a Boltzmann function gave a half-activation potential of -25 mV. A double Boltzman equation was necessary to adequately fit the inactivation data, suggesting the presence of two populations of sodium channels. One population accounted for approximately 14% of the channels, with a half-inactivation potential of -86 mV; the remaining population showed a half-inactivation potential of -51 mV. A mathematical model, based on Hodgkin-Huxley equations, was used to assess the respective contributions of individual currents to the action potential. When the densities of calcium and sustained potassium currents were changed from immature to mature values, the decay time of the action potentials generated with the model decreased from 2.85 to 1.95 ms. A similar reduction was obtained when only the density of the potassium current was increased. Integration of the calcium currents generated during mature and immature action potentials demonstrated a significant decrease in calcium entry during development. We conclude that the developmental reduction of the action potential duration 1) is a consequence of the developmentally regulated increase in a sustained potassium current and 2) leads to a reduction of the participation of calcium currents in the action potential, resulting in a decreased amount of calcium entering the cell during each action potential.

The properties and distribution of inward rectifier potassium currents in pig coronary arterial smooth muscle.

1. Whole-cell potassium currents were studied in single smooth muscle cells enzymatically isolated from pig coronary arteries. 2. In cells isolated from small diameter branches of the left anterior descending coronary artery (LAD), an inward rectifier potassium current (IK(IR)) was identified, which was inhibited by extracellular barium ions, suggesting the presence of inward rectifier potassium (KIR) channels. 3. The conductance for IK(IR) measured in 6, 12, 60 and 140 mM extracellular potassium was a function of membrane potential and the extracellular potassium concentration. 4. On hyperpolarization, IK(IR) activated along an exponential time course with a time constant that was voltage dependent. 5. Inward rectifier current was compared in cells isolated from coronary vessels taken from different points along the vascular tree. Current density was greater in cells isolated from small diameter coronary arteries; at -140 mV it was -20.5 +/- 4.4 pA pF-1 (n = 23) in 4th order branches of the LAD, but -0.8 +/- 0.2 pA pF-1 (n = 11) in the LAD itself. 6. In contrast to IK(IR), there was little effect of arterial diameter on the density of voltage-dependent potassium current; densities at +30 mV were 12.8 +/- 1.3 pA pF-1 (n = 19) in 4th order branches and 17.4 +/- 3.1 pA pF-1 (n = 11) in the LAD. 7. We conclude that KIR channels are present in pig coronary arteries, and that they are expressed at a higher density in small diameter arteries. The presence of an enhanced IK(IR) may have functional consequences for the regulation of cell membrane potential and tone in small coronary arteries.

?2-Interferon- and oligoadenylate-induced morphological differentiation and modulation of the ion channels in neuroblastoma cells

The experiments were carried out on the IMR-32 human neuroblastoma and NIE-115 murine neuroblastoma cultured cells. Peculiarities of the ion channel expression and their correlation with the main morphological parameters characterizing neuronal differentiation were studied under conditions of incubation of the cells with α2-interferon and 2′,5′-oligoadenylate, 2–5A. Twenty-four hours after addition of 1000 U act./ml interferon to the culture medium, a 56% increase in the mean projective surface of the IMR-32 cells was observed, and after a nine-day-long exposition this increase was 132%, as compared with the control. Mean total length of the cellular protrusions nearly doubled after nine-day-long incubation. Morphological and electrophysiological properties of the N1E-115 murine neuroblastoma cells showed practically no changes after incubation with human α2-interferon. Cultivation of the IMR-32 human neuroblastoma cells in the presence of 2–5A evoked insignificant changes in their morphological parameters. By contrast, the mean total length of neurites of the N1E-115 neuroblastoma protrusion-supplied cells increased more than a factor of five after eight-day-long incubation with 2–5A in a 1.0 µM concentration, and at a 0.01 µM 2–5A concentration this increase was about fourfold. At the same time, the projective surface exhibited no significant changes either in the neurite-supplied or the neurite-free cellular subpopulations. Twenty-four hours after the incubation with human α2-interferon had been begun, the density of sodium current in the IMR-32 human neuroblastoma cells increased by 250% compared with the control. A similar effect was observed after the addition of 2–5A to the medium: the density of sodium current approximately doubled. Cultivation of the neurite-supplied N1E-115 murine neuroblastoma cells was followed by a gradual increase in the density of fast sodium current both in control and 2–5A-influenced samples, but in the latter case this increase was significantly faster. In the neurite-free cells, the density of sodium current was 27% higher after their 24-h-long incubation with 2–5A, as compared with the control values (11.05±0.9 and 8.7±0.9 pA/pF, respectively). Longer incubation resulted in a sharp decrease in the density of potassium current. The results of our study are in agreement with the data about species-related individuality of α2-interferon and different intensity of its effects on the cells passing different stages of cellular differentiation.

Sodium and potassium current in neonatal rat carotid body cells following chronic in vivo hypoxia

Chronic hypoxic acclimatization modifies ventillatory reflexes arising from carotid body stimulation. To explore this, the effects of in vivo chronic hypoxia on membrane currents were quantified in chemoreceptive carotid body glomus cells. Pregnant rats were maintained in either normoxia (NORM: inspired oxygen tension 141 mmHg), or hypoxia (CHX: inspired oxygen tension 80 mmHg) from day 3 of gestation, to day 5–10 postpartum. Whole cell patch clamp recordings were then made from the mechanically and enzymatically dissociated carotid body glomus cells of the rat pups (NORM: 41 cells, CHX: 36 cells) and comparisons of means ± S.E.M. were made with unpaired t-tests. Glomus cells were bright under phase contrast illumination, formed clusters, were histochemically positive for catecholamines and possessed voltage-potassium currents that were depressed by acute hypoxia. Acclimatization to chronic hypoxia did not affect rat pup whole body mass (CHX: 12.0 ± 0.7 g vs. NORM: 11.0 ± 0.2 g), but it significantly increased blood hematocrit (CHX: 48.7 ± 0.9% vs. NORM: 37.8 ± 0.5%%, P < 0.05). Sodium current was not uniformly present in glomus cells from either group, but sodium current was observed in a greater proportion of glomus cells isolated from the chronically hypoxic pups (CHX: 72% vs. NORM: 46%, P < 0.05). The mean peak tetrodotoxin-sensitive sodium current evoked by −70 to + 10 mV depolarizations was greater after hypoxic acclimatization (CHX: −100 ± 25 vs. NORM: −38 ± 15 pA, P < 0.05), but the sodium current density (pA/pF) was unchanged. In contrast, the mean peak voltage potassium current (pA) evoked by −70 mV to 0 mV depolarizations was unchanged by acclimatization, but the potassium current density (pA/pF) was reduced ( P < 0.05). Unchanged sodium current density coupled with decreased potassium current density may make glomus cells more excitable during exposure to chronic in vivo hypoxia.

Electrical activity and calcium influx regulate ion channel development in embryonic Xenopus skeletal muscle

The development of electrical excitability involves complex coordinated changes in ion channel activity. Part of this coordination appears to be due to the fact that the expression of some channels is dependent on electrical activity mediated by other channel types. For example, we have previously shown that normal potassium current development in embryonic skeletal muscle cells of the frog Xenopus laevis is dependent on sodium channel activity. To examine the interrelationships between the development of different ionic currents, we have made a detailed study of electrical development in cultured Xenopus myocytes using whole-cell patch-clamp recording. The initial expression of potassium, sodium, and calcium currents is followed by a brief period during which the densities of potassium currents decrease, while at the same time sodium and calcium current densities continue to increase, which may increase electrical excitability during this time. The normal developmental increase in both potassium and sodium currents is inhibited by the sodium channel blocker tetrodotoxin, suggesting that electrical activity normally stimulates the expression of both these currents. These effects of electrical activity appear to be mediated via activation of voltage-gated calcium channels. We suggest that the developmental acquisition of sodium and calcium channels by these cells, possibly coupled with a transient decrease in potassium current density, lead to an increase in electrical excitability and calcium entry, and that this calcium entry provides a critical developmental cue controlling the subsequent development of mature electrical properties.

Voltage-gated currents and firing properties of embryonic Drosophila neurons grown in a chemically defined medium.

This study reports the composition of a chemically defined medium (DDM1) that supports the survival and differentiation of neurons in dissociated cell cultures prepared from midgastrula stage Drosophila embryos. Cells with neuronal morphology that stain with a neural-specific marker are clearly differentiated by 1 day in vitro and can be maintained in culture for up to 2 weeks. Although the whole cell capacitance measurements from neurons grown in DDM1 were 5- to 10-fold larger than those of neurons grown in a conventional serum-supplemented medium, the potassium current densities were similar in the two growth conditions. A small but significant increase in the sodium current density was observed in the neurons grown in DDM1 compared with those in serum-supplemented medium. The majority of neurons grown in DDM1 fired either single or trains of action potentials in response to injection of depolarizing current. Contributing to the observed heterogeneity in the firing properties between individual neurons grown in DDM1 was heterogeneity in the levels of expression and gating properties of voltage-dependent sodium, calcium, and potassium currents. The ability of embryonic Drosophila neurons to differentiate in a chemically defined medium and the fact that they are amenable to both voltage-clamp and current-clamp analysis makes this system well suited to studies aimed at understanding the mechanisms regulating expression of ion channels involved in electrical excitability.

Reduction of calcium-independent transient outward potassium current density in DOCA salt hypertrophied rat ventricular myocytes

Saline-drinking, left-nephrectomized rats made hypertensive by deoxycorticosterone acetate (DOCA) pellet implantation at the time of surgery develop a cardiac hypertrophy, which becomes maximal after 6-7 weeks. The hypertrophy results in a marked increase in the amplitude and duration of both the early and the late component of the ventricular action potential plateau recorded in the isolated perfused rat heart. The 4-aminopyridine(4-AP)-sensitive calcium-independent transient outward potassium current was markedly depressed in hypertrophied ventricular myocytes resulting in a highly significant decrease in current density (from 19.9 +/- 3.5 to 6.4 +/- 3.1 pA/pF at +60 mV). Activation/voltage and steady-state inactivation/voltage relationships were moderately although non-significantly shifted towards negative potentials. The steady-state outward current measured at the end of 1-s depolarizing pulses was not significantly changed in hypertrophied myocytes. 4-AP induced a smaller increase in plateau amplitude and duration in hypertrophied rather than in control hearts, a point that is well explained by the depression of the transient outward current resulting from hypertrophy. We also demonstrated that a complete recovery of both cell capacitance and transient outward current amplitude occurs in myocytes from saline-drinking rats studied 13 weeks after DOCA pellet implantation, showing that hypertrophy regresses as a result of pellet elimination. Several mechanisms can be involved in the observed phenomena, including the possibility that the expression of potassium channels responsible for the transient outward current is not enhanced by hypertrophy in contrast with what occurs in the case of calcium channels.(ABSTRACT TRUNCATED AT 250 WORDS)