Ion channels in spinal cord astrocytes in vitro. II. Biophysical and pharmacological analysis of two Na+ current types.

1. Na+ currents expressed in astrocytes cultured from spinal cord were studied by whole cell patch-clamp recording. Two subtypes of astrocytes, pancake and stellate cells, were morphologically differentiated and showed expression of Na+ channels at densities that are unusually high for glial cells (2-8 channels/microns2) and comparable to cultured neurons. 2. Na+ currents in stellate and pancake astrocytes were comparable to neuronal Na+ currents with regard to Na(+)-current activation (tau m) and inactivation (tau h) time constants, which were equally fast in both astrocyte types. However, they differed with respect to voltage dependence of activation, and current-voltage (I-V) curves were approximately 10 mV more positive in stellate cells (-11.1 +/- 5.6 mV, mean +/- SD) than in pancake cells (19.7 +/- 4.5 mV). Steady-state activation (m infinity curves) was 16 mV more negative in pancake (mean V1/2 = -48.8 mV) than in stellate cells (mean V1/2 = -32.7 mV). 3. Steady-state inactivation (h infinity curves) of Na+ currents was distinctly different in the two astrocyte types. In stellate astrocytes h infinity curves had midpoints close to -65 mV (-64.6 +/- 6.5 mV), similar to most cultured neurons. In pancake astrocytes h infinity-curves were approximately 25 mV more negative, with midpoints close to -85 mV (84.5 +/- 9.5 mV). 4. The two forms of Na+ currents were additionally distinguishable by their sensitivity to tetrodotoxin (TTX). Na+ currents in stellate astrocytes were highly TTX sensitive [half-maximal inhibition (Kd) = 5.7 nM] whereas Na+ currents in pancake astrocytes were relatively TTX resistant, requiring 100- to 1,000-fold higher concentrations for blockage (Kd = 1,007 nM). 5. Na+ currents were fit by the Hodgkin-Huxley (HH) model. In pancake astrocytes, as in squid gigant axons, Na(+)-current kinetics could be well described with an m3h model, whereas in stellate astrocytes Na+ currents were better described with higher-order power terms for activation (m). On average, best fits were obtained using an m4h model. 6. Pancake astrocytes were capable of generating action-potential (AP)-like responses under current clamp whereas stellate astrocytes were not. The h infinity curve for APs shows that membrane potentials more negative than -70 mV are required to allow these responses to occur.(ABSTRACT TRUNCATED AT 400 WORDS)     

Relationship between Na+ current expression and cell-cell coupling in astrocytes cultured from rat hippocampus

1. Cell-cell coupling between hippocampal astrocytes in culture was studied by following the intracellular spread of the low molecular weight fluorescent dye Lucifer yellow (LY). Dye coupling appeared as early as 24 h after plating, at which time approximately 20% of all astrocytes that physically contacted neighboring cells showed dye coupling. 2. The percentage of coupled cells increased with time in culture and peaked after 10 days in vitro (DIV) when approximately 50% of astrocytes showed coupling. Further time in culture, up to 20 DIV, did not increase the percentage of coupled cells. Thus, coupled and noncoupled astrocytes coexist in hippocampal cultures in approximately equal numbers. 3. Na+ currents were expressed in a subpopulation of hippocampal astrocytes and changed characteristics during in vitro development. A "neuronal type" of Na+ current, so called because of an h alpha curve that had a midpoint near -60 mV, was observed within the first 5 days post-plating. A "glial type" of Na+ current, characterized by a -25 mV shift in its h alpha curve, was only expressed after 6 days in culture. 4. Na+ current expression was restricted to hippocampal astrocytes that did not exhibit dye coupling; astrocytes that exhibited dye coupling (n = 39) did not show measurable Na+ currents. 5. The failure to see Na+ currents in coupled astrocytes cannot be explained by insufficient space-clamp since astrocytes acutely uncoupled with octanol (10 microM) did not reveal Na+ current expression. Control experiments showed that low concentrations of octanol (i.e., 10-100 microM) did not block Na+ currents; blockage of Na+ currents by octanol was only observed at high concentrations (e.g., 50-fold the concentration used for uncoupling). These observations support the idea that Na(+)-channel expression was restricted to noncoupled astrocytes. 6. The time courses for the development of cell coupling and Na+ current expression appeared to be inversely correlated and suggested a gradual increase in cell coupling in concert with a loss in Na+ current expression with time in culture.     

Ion channels in spinal cord astrocytes in vitro. I. Transient expression of high levels of Na+ and K+ channels.

1. Na+ and K+ channel expression was studied in cultured astrocytes derived from P--0 rat spinal cord using whole cell patch-clamp recording techniques. Two subtypes of astrocytes, pancake and stellate, were differentiated morphologically. Both astrocyte types showed Na+ channels and up to three forms of K+ channels at certain stages of in vitro development. 2. Both astrocyte types showed pronounced K+ currents immediately after plating. Stellate but not pancake astrocytes additionally showed tetrodotoxin (TTX)-sensitive inward Na+ currents, which displayed properties similar to neuronal Na+ currents. 3. Within 4-5 days in vitro (DIV), pancake astrocytes lost K(+)-current expression almost completely, but acquired Na+ currents in high densities (estimated channel density approximately 2-8 channels/microns2). Na+ channel expression in these astrocytes is approximately 10- to 100-fold higher than previously reported for glial cells. Concomitant with the loss of K+ channels, pancake astrocytes showed significantly depolarized membrane potentials (-28.1 +/- 15.4 mV, mean +/- SD), compared with stellate astrocytes (-62.5 +/- 11.9 mV, mean +/- SD). 4. Pancake astrocytes were capable of generating action-potential (AP)-like responses under current clamp, when clamp potential was more negative than resting potential. Both depolarizing and hyperpolarizing current injections elicited overshooting responses, provided that cells were current clamped to membrane potentials more negative than -70 mV. Anode-break spikes were evoked by large hyperpolarizations (less than -150 mV). AP-like responses in these hyperpolarized astrocytes showed a time course similar to neuronal APs under conditions of low K+ conductance. 5. In stellate astrocytes, AP-like responses were not observed, because the K+ conductance always exceeded Na+ conductance by at least a factor of 3. Thus stellate spinal cord astrocyte membranes are stabilized close to EK as previously reported for hippocampal astrocytes. 6. It is concluded that spinal cord pancake astrocytes are capable of synthesizing Na+ channels at densities that can, under some conditions, support electrogenesis. In vivo, however, AP-like responses are unlikely to occur because the cells' resting potential is too depolarized to allow current activation. Thus the absence of electrogenesis in astrocytes may be explained by two mechanisms: 1) a low Na-to-K conductance ratio, as in stellate spinal cord astrocytes and in other previously studied astrocyte preparations; or, 2) as described in detail in the companion paper, a mismatch between the h infinity curve and resting potential, which results in Na+ current inactivation in spinal cord pancake astrocytes.     

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

Ca2(+)-independent voltage-activated potassium currents were investigated during the differentiation of rat retinal ganglion cells. Whole-cell patch-clamp recordings of Ca2(+)-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 Ba2+, 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. Ca2(+)-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 Ba2(+)-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 Ca2(+)-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 Ca2(+)-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.     

Characterization of sodium current in developing rat diencephalic neurons in serum-free culture.

1. Dissociated, synchronized (G1 phase of cell cycle), and birth-dated fetal rat diencephalic neurons were grown in a serum-free defined medium. The gigaseal whole-cell voltage-clamp technique was used to measure the inward Na+ currents (INa) from morphologically identified bipolar neurons. The earliest expressed somatic INa has been characterized and compared with that present at a later date. 2. The identity of the INa was established on the basis of its reversal potential and reversible blockade by tetrodotoxin (TTX). Close agreement between the measured reversal potentials (68.5 +/- 1.3 and 38.3 +/- 2.4 mV, mean +/- SE) and calculated Nernst equilibrium potentials (64.6 and 34.7 mV) at two different bath Na+ concentrations (120 and 35 mM, respectively) suggests that the channels are highly selective for Na+. 3. The peak INa density increased from 47.7 +/- 2.9 pA/pF in younger neurons (5-6 days in culture) to 93.9 +/- 6.4 pA/pF in older neurons (12-13 days in culture). The activation voltage and the voltage for peak current were also shifted by 10 mV in the hyperpolarizing direction, from -30 and +10 mV in younger neurons to -40 and 0 mV in older neurons, respectively. However, the reversal potential did not change (69.2 +/- 2.3 and 68.5 +/- 1.3 mV in younger and older neurons, respectively). 4. In older neurons the steady-state inactivation parameters (V1/2, the voltage at which inactivation was 50% of maximum, and kh, the voltage at which there is an e-fold change in inactivation) were significantly altered. V1/2 was shifted from -41.5 +/- 2.3 to -48.8 +/- 1.8 mV, and kh was increased from 6.2 +/- 0.5 to 8.9 +/- 0.4 mV. However, the time course of activation and the rates of inactivation and recovery from inactivation were unchanged. 5. In both groups, the INa decays were best described by a sum of two exponentials. The corresponding time constants were voltage dependent. Also, the amplitudes of the two components were differentially affected by membrane potential and niflumic acid. 6. The extrapolated amplitudes of both the fast and the slow components of INa were larger in older neurons, but the ratio of the amplitudes of the two components did not change with age. The voltage dependencies of the time constants of both components were altered. 7. We conclude that INa in fetal rat diencephalic neurons grown in a defined medium with only essential nutrients undergoes in vitro changes in current density and in some, but not all, kinetic parameters.(ABSTRACT TRUNCATED AT 250 WORDS)     

Voltage-dependent Na(+) currents in mammalian retinal cone bipolar cells

Voltage-dependent Na(+) channels are usually expressed in neurons that use spikes as a means of signal coding. Retinal bipolar cells are commonly thought to be nonspiking neurons, a category of neurons in the CNS that uses graded potential for signal transmission. Here we report for the first time voltage-dependent Na(+) currents in acutely isolated mammalian retinal bipolar cells with whole cell patch-clamp recordings. Na(+) currents were observed in approximately 45% of recorded cone bipolar cells but not in rod bipolar cells. Both ON and OFF cone bipolar cells were found to express Na(+) channels. The Na(+) currents were activated at membrane potentials around -50 to -40 mV and reached their peak around -20 to 0 mV. The half-maximal activation and steady-state inactivation potentials were -24.7 and -68.0 mV, respectively. The time course of recovery from inactivation could be fitted by two time constants of 6.2 and 81 ms. The amplitude of the Na(+) currents ranged from a few to >300 pA with the current density in some cells close or comparable to that of retinal third neurons. In current-clamp recordings, Na(+)-dependent action potentials were evoked in Na(+)-current-bearing bipolar cells by current injections. These findings raise the possibility that voltage-dependent Na(+) currents may play a role in bipolar cell function.     

A novel mutation L619F in the cardiac Na+ channel SCN5A associated with long-QT syndrome (LQT3): a role for the I-II linker in inactivation gating

Congenital long QT syndrome type 3 (LQT3) is caused by mutations in the gene SCN5A encoding the alpha-subunit of the cardiac Na(+) channel (Nav1.5). Functional studies of SCN5A mutations in the linker between domains III and IV, and more recently the C-terminus, have been shown to alter inactivation gating. Here we report a novel LQT3 mutation, L619F (LF), located in the domain I-II linker. In an infant with prolonged QTc intervals, mutational analysis identified a heterozygous missense mutation (L619F) in the domain I-II linker of the cardiac Na(+) channel. Wild-type (WT) and mutant channels were studied by whole-cell patch-clamp analysis in transiently expressed HEK cells. LF channels increase maintained Na(+) current (0.79 pA/pF for LF; 0.26 pA/pF for WT) during prolonged depolarization. We found a +5.8mV shift in steady state inactivation in LF channels compared to WT (WT, V(1/2)=-64.0 mV; LF, V(1/2)=-58.2 mV). The positive shift of inactivation, without a corresponding shift in activation, increases the overlap window current in LF relative to WT (1.09 vs. 0.58 pA/pF), as measured using a positive voltage ramp protocol (-100 to +50 mV in 2s). The increase in window current, combined with an increase in non-inactivating Na(+) current, may act to prolong the AP plateau and is consistent with the disease phenotype observed in patients. Moreover, the defective inactivation imposed by the L619F mutation implies a role for the I-II linker in the Na(+) channel inactivation process.     

Developmental regulation of calcium channel-mediated currents in retinal glial (Müller) cells

Whole cell voltage-clamp recordings of freshly isolated cells were used to study changes in the currents through voltage-gated Ca(2+) channels during the postnatal development of immature radial glial cells into Müller cells of the rabbit retina. Using Ba(2+) or Ca(2+) ions as charge carriers, currents through transient low-voltage-activated (LVA) Ca(2+) channels were recorded in cells from early postnatal stages, with an activation threshold at -60 mV and a peak current at -25 mV. To increase the amplitude of currents through Ca(2+) channels, Na(+) ions were used as the main charge carriers, and currents were recorded in divalent cation-free bath solutions. Currents through transient LVA Ca(2+) channels were found in all radial glial cells from retinae between postnatal days 2 and 37. The currents activated at potentials positive to -80 mV and displayed a maximum at -40 mV. The amplitude of LVA currents increased during the first postnatal week; after postnatal day 6, the amplitude remained virtually constant. The density of LVA currents was highest at early postnatal days (days 2-5: 13 pA/pF) and decreased to a stable, moderate level within the first three postnatal weeks (3 pA/pF). A significant expression of currents through sustained, high-voltage-activated Ca(2+) channels was found after the third postnatal week in approximately 25% of the investigated cells. The early and sole expression of transient currents at high-density may suggest that LVA Ca(2+) channels are involved in early developmental processes of rabbit Müller cells.     

Characterization and developmental changes of Na+ currents of petrosal neurons with projections to the carotid body

Carotid body chemoreceptors transduce a decrease in arterial oxygen tension into an increase in spiking activity on the sinus nerve, and this response increases with postnatal age over the first week or two of life. Previous work from our laboratory has suggested a major role of axonal Na(+) channels in the initiation of afferent spiking activity. Using RT-PCR of the petrosal ganglia we identified Na(+) channel TTX-S isoforms Na(v)1.1, Na(v)1.6, and Na(v)1.7 and the TTX-resistant (TTX-R) isoforms Na(v)1.8 and Na(v)1.9 at high levels. Electrophysiologic recordings (at 3 ages: 3 days, 9 days, 18-20 days) of neurons that project to the carotid body exhibited predominantly fast-inactivating sodium currents, with a bimodal recovery from inactivation at -80 mV (fast component approximately 8 ms; slow component approximately 90 ms). Developmental age had little effect with no change in peak current density (approximately 1.4 nA/pF) and was associated with a slight, but significant increase in the speed of recovery from inactivation at -140 and -120 mV but not at other potentials. Assuming that the same Na(+) channel complement is present at the nerve terminal as at the soma, the association of a sensory modality (chemoreception) with a relatively uniform Na(+) channel profile suggests that the rapid kinetics of TTX-S channels may be essential for some aspects of chemoreceptor function beyond mediating simple axonal conduction.     

Effects of endothelin-1 on calcium and potassium currents in undiseased human ventricular myocytes

Endothelins have been reported to exert a wide range of electrophysiological effects in mammalian cardiac cells. These results are controversial and human data are not available. Our aim was to study the effects of endothelin-1 (ET-1, 8 nmol/l) on the L-type calcium current (ICa-L) and various potassium currents (rapid component of the delayed rectifier, IKr; transient outward current, Ito; and the inward rectifier K current, IK1) in isolated human ventricular cardiomyocytes. Cells were obtained from undiseased donor hearts using collagenase digestion via the segment perfusion technique. The whole-cell configuration of the patch-clamp technique was applied to measure ionic currents at 37 degrees C. ET-1 significantly decreased peak ICa-L from 10.2+/-0.6 to 6.8+/-0.8 pA/pF at +5 mV (66.7% of control, P<0.05, n=5). This reduction of peak current was accompanied by a lengthening of inactivation. The voltage dependence of steady-state activation and inactivation was not altered by ET- 1. IKr, measured as tail current amplitudes at 40 mV, decreased from 0.31+/-0.02 to 0.06+/-0.02 pA/pF (20.3% of control, P<0.05, n=4) after exposure to ET-1. ET-1 failed to change the peak amplitude of Ito, measured at +50 mV (9.3+/-4.6 and 9.0+/-4.4 pA/pF before and after ET-1, respectively), or steady-state IK1 amplitude, measured at the end of a 400-ms hyperpolarization to -100 mV (3.6+/-1.4 and 3.7+/-1.4 pA/pF, n=4). The present results indicate that in undiseased human ventricular myocytes ET-1 inhibits both ICa-L and IKr; however, the degree of suppression of the two currents is different.     

Neurotrophin regulation of sodium and calcium channels in human neuroblastoma cells

Neurotrophins, acting through tyrosine kinase family genes, are essential for neuronal differentiation. The expression of tyrosine kinase family genes is prognostic in neuroblastoma, and neurotrophins reduce proliferation and induce differentiation, indicating that neuroblastomas are regulated by neurotrophins. We tested the effects of nerve growth factor and brain-derived neurotrophic factor on Na(+) and Ca(2+) currents, using the whole-cell patch-clamp technique, in human neuroblastoma NB69 cells. Control cells exhibited a slow tetrodotoxin-resistant (IC(50)=98 nM) Na(+) current and a high-voltage-activated Ca(2+) current. Exposure to nerve growth factor (50 ng/ml) and/or brain-derived neurotrophic factor (5 ng/ml) produced the expression of a fast tetrodotoxin-sensitive (IC(50)=10 nM) Na(+) current after day 3, and suppressed the slow tetrodotoxin-resistant variety. The same type of high-voltage-activated Ca(2+) current was expressed in control and treated cells. The treatment increased the surface density of both Na(+) and Ca(2+) currents with time after plating, from 17 pA/pF at days 3-5 and 1-5 to 34 and 30 pA/pF after days 6-10, respectively. Therefore, both nerve growth factor and brain-derived neurotrophic factor, acting through different receptors of the tyrosine kinase family and also possibly the tumor necrosis factor receptor-II, were able to regulate differentiation and the expression of Na(+) and Ca(2+) channels, partially reproducing the modifications induced by diffusible astroglial factors.We show that neurotrophins induced differentiation to a neuronal phenotype and modified the expression of Na(+) and Ca(2+) currents, partially reproducing the effects of diffusible astroglial factors.     

焦亚硫酸钠对大鼠海马CA1区神经元钠电流的影响

目的：探讨SO2 及其体内衍生物（亚硫酸盐和亚硫酸氢盐）对中枢神经元钠通道的影响。 方法: 采用全细胞膜片钳技术研究了焦亚硫酸钠（SMB）对大鼠海马CA1区神经元钠电流的影响及超氧化物歧化酶(SOD)、过氧化氢酶(CAT)及谷胱甘肽过氧化物酶(GPx)相应的保护作用。 结果： ① 焦亚硫酸钠可剂量依赖性地增大全细胞钠电流，剂量为2 μmol/L和20 μmol/L时，钠电流分别增大（22.36±3.28）% 和（65.05±5.75）%（n=10）。② 10　μmol/L的焦亚硫酸钠不影响钠电流的激活过程，却非常显著地影响其失活过程，使失活曲线显著右移，作用前后的半数失活电压分别为（-82.38±0.54）mV和（-69.39±0.41）mV (n=10, P<0.01), 但失活曲线的斜率因子未见改变。③ SOD(1×106 U/L)、CAT(2×106 U/L) 及GPx (1×104 U/L) 均可使SMB（10 μmol/L）增大的钠电流部分恢复。 结论： SMB增大钠电流并抑制其失活过程，从而影响神经细胞的兴奋性，这一效应可能与硫中心或氧中心自由基的损伤作用有关。     

Steady-state contribution of the sodium pump to the resting potential of a molluscan neurone

1. The electrogenic contribution of the Na(+)-K(+) exchange pump to the membrane potential of the Anisodoris giant neurone (G cell) was examined under steady-state and Na(+) loaded conditions.2. The membrane potential was variable for the first 1-4 hr after impalement, but, in the absence of experimental manipulation, remained constant thereafter. The average membrane potential for ten cells maintained at 11-13 degrees C and measured 5-36 hr after impalement was 55.8 +/- 1.0 mV (S.E. of mean).3. Low concentrations of external ACh caused a reversible increase in membrane Na(+) conductance. Brief exposure to ACh proved a fast and reversible technique to load the cell with Na(+) ions, and transiently stimulate the electrogenic Na(+) pump.4. In ten cells maintained from 5 to 36 hr at 11-13 degrees C the reduction in membrane potential produced by inhibition of the Na(+) pump with ouabain was remarkably constant between cells and averaged + 9.7 mV.5. Cells maintained under steady-state conditions (at 11-13 degrees C) for extended periods of time were shown to be relatively insensitive to changes in temperature and to small changes in external K(+).6. It is estimated that the Na(+)-K(+) exchange pump contributes approximately - 10 mV to the steady-state resting potential of the G cell, and that two Na(+) ions are extruded for every K(+) ion transported into the cell per pump cycle.     

Voltage-activated currents through calcium channels in normal bovine lactotrophs

The properties of whole cell Ba2+ currents were studied in immunocytochemically identified, normal bovine lactotrophs using the patch clamp technique. In the current clamp mode, current-induced and spontaneous Ba2+ action potentials were recorded. These were of longer duration and showed less inactivation with stimulation frequency when compared with Na+ action potentials. Under voltage clamp, isolated Ba2+ currents had an activation threshold of about -35 mV and peak value at -15 mV to +20 mV. Inactivation of the current to a potential-dependent, non-zero steady-state level indicated the presence of one rapidly and one slowly inactivating component to the current. These two components were also distinguished by: (1) the voltage dependence of the inactivation time constant of the current, (2) the differential frequency-dependent inactivation of the peak and steady-state currents, and (3) the presence of two half-inactivation potentials for the current. Analysis of the ensemble current variance of the non-inactivating component gave a single-channel amplitude of 0.19 pA at 0 mV and a slope conductance of 3 pS. Fluctuation analysis of the voltage-activated Ba2+ current noise revealed two time constants, one which was voltage dependent and the other was independent of potential. The contribution of these two currents to Ca2+-dependent hormone secretion remains to be clarified.     

Nitric oxide is an autocrine regulator of Na(+) currents in axotomized C-type DRG neurons

In this study, we examined whether nitric oxide synthase (NOS) is upregulated in small dorsal root ganglion (DRG) neurons after axotomy and, if so, whether the upregulation of NOS modulates Na(+) currents in these cells. We identified axotomized C-type DRG neurons using a fluorescent label, hydroxystilbamine methanesulfonate and found that sciatic nerve transection upregulates NOS activity in 60% of these neurons. Fast-inactivating tetrodotoxin-sensitive (TTX-S) Na(+) ("fast") current and slowly inactivating tetrodotoxin-resistant (TTX-R) Na(+) ("slow") current were present in control noninjured neurons with current densities of 1.08 +/- 0. 09 nA/pF and 1.03 +/- 0.10 nA/pF, respectively (means +/- SE). In some control neurons, a persistent TTX-R Na(+) current was observed with current amplitude as much as approximately 50% of the TTX-S Na(+) current amplitude and 100% of the TTX-R Na(+) current amplitude. Seven to 10 days after axotomy, current density of the fast and slow Na(+) currents was reduced to 0.58 +/- 0.05 nA/pF (P < 0.01) and 0.2 +/- 0.05 nA/pF (P < 0.001), respectively. Persistent TTX-R Na(+) current was not observed in axotomized neurons. Nitric oxide (NO) produced by the upregulation of NOS can block Na(+) currents. To examine the role of NOS upregulation on the reduction of the three types of Na(+) currents in axotomized neurons, axotomized DRG neurons were incubated with 1 mM N(G)-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor. The current density of fast and slow Na(+) channels in these neurons increased to 0.82 +/- 0.08 nA/pF (P < 0.01) and 0.34 +/- 0.04 nA/pF (P < 0.05), respectively. However, we did not observe any persistent TTX-R current in axotomized neurons incubated with L-NAME. These results demonstrate that endogenous NO/NO-related species block both fast and slow Na(+) current in DRG neurons and suggest that NO functions as an autocrine regulator of Na(+) currents in injured DRG neurons.     

Isolation and kinetic analysis of inward currents in neuroblastoma cells

The suction pipette method for combined voltage clamp and intracellular dialysis was applied to isolate the two components of voltage-gated inward current across membranes of NIE-115 neuroblastoma cells. In order to analyze the kinetic behavior of the Na+ and Ca2+ channels responsible for generating these components, current through K+ channels was effectively blocked by substituting impermeant Cs+ for internal and external K+. Block was confirmed independently by examining the effects of the application of external tetraethylammonium or Cd2+; and comparing the time course of Ca2+ tail currents with the decay of current during a maintained depolarization. Na+ currents studied at 8-10 degrees C, developed as a fourth order process giving a maximum e-fold conductance change for a 3 mV depolarization, with half activation occurring at -10 mV. The instantaneous current-voltage relationship was linear. Time constants of the activation parameter (m) varied from 0.5 ms (-50 mV) to 3-4 ms (-10 to -40 mV) at 10 degrees C. Inactivation (h) was a first order process having a time constant between 4 ms (+10 to +60 mV) and 225 ms (-60 mV). Steady-state inactivation for Na+ channels attained a value of 0.5 at -50 mV. A slow inactivation process, however, also is involved in gating of Na+ channels, and has a time course at least two orders of magnitude slower than that for h. The temperature sensitivity of Na+ currents was found to be similar to that found for other preparations. Ca2+ currents were studied at 24-29 degrees C in the presence of internal ethyleneglycolbis-(aminoethylether)-tetra-acetate (EGTA) and an external Ca2+ concentration of 20 mM. Ca2+ channel activation could also be described by a fourth order process giving an e-fold conductance change for a 5-6 mV change in potential and the half activation potential of -13 mV. Internal EGTA (20 mM) did not abolish inactivation of Ca2+ currents and no recovery from inactivation caused by a prepulse could be measured as the prepulse potential approached the null potential for Ca2+ influx. Time constants of both activation and inactivation of Ca2+ channels were measured between -20 and +50 mV. Currents through K+ channels could be completely eliminated by substitution of K+ with Cs+, although a residual non-linear leakage current remained, in addition to currents through the Na+ and Ca2+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Voltage-dependent K+ currents in rat cardiac dorsal root ganglion neurons

We have assessed the expression and kinetics of voltage-gated K(+) currents in cardiac dorsal root ganglion (DRG) neurons in rats. The neurons were labelled by prior injection of a fluorescent tracer into the pericardial sack. Ninety-nine neurons were labelled: 24% small (diameter<30 microm), 66% medium-sized (diameter 30 microm>.48 microm) and 10% large (>48 microm) neurons. Current recordings were performed in small and medium-sized neurons. The kinetic and pharmacological properties of K(+) currents recorded in these two groups of neurons were identical and the results obtained from these neurons were pooled. Three types of K(+) currents were identified:a) I(As), slowly activating and slowly time-dependently inactivating current, with V(1/2) of activation -18 mV and current density at +30 mV equal to 164 pA/pF, V(1/2) of inactivation at -84 mV. b) I(Af) current, fast activating and fast time-dependently inactivating current, with V(1/2) of activation at two mV and current density at +30 mV equal to 180 pA/pF, V(1/2) of inactivation at -26 mV. At resting membrane potential I(As) was inactivated, whilst I(Af), available for activation. The I(As) currents recovered faster from inactivation than I(Af) current. 4-Aminopiridyne (4-AP) (10 mM) and tetraethylammonium (TEA) (100 mM) produced 98% and 92% reductions of I(Af) current, respectively and 27% and 66% of I(As) current, respectively. c) The I(K) current that did not inactivate over time. Its V(1/2) of activation was -11 mV and its current density equaled 67 pA/pF. This current was inhibited by 95% (100 mM) TEA, whilst 4-AP (10 mM) produced its 23% reduction. All three K(+) current components (I(As), I(Af) and I(K)) were present in every small and medium-sized cardiac DRG neuron.We suggest that at hyperpolarized membrane potentials the fast reactivating I(As) current limits the action potential firing rate of cardiac DRG neurons. At depolarised membrane potentials the I(Af) K(+) current, the reactivation of which is very slow, does not oppose the firing rate of cardiac DRG neurons.     

A voltage-dependent transient K(+) current in rat dental pulp cells

We characterized a voltage-dependent transient K(+) current in dental pulp fibroblasts on dental pulp slice preparations by using a nystatin perforated-patch recording configuration. The mean resting membrane potential of dental pulp fibroblasts was -53 mV. Depolarizing voltage steps to +60 mV from a holding potential of -80 mV evoked transient outward currents that are activated rapidly and subsequently inactivated during pulses. The activation threshold of the transient outward current was -40 mV. The reversal potential of the current closely followed the K(+) equilibrium potential, indicating that the current was selective for K(+). The steady-state inactivation of the peak outward K(+) currents described by a Boltzmann function with half-inactivation occurred at -47 mV. The K(+) current exhibited rapid activation, and the time to peak amplitude of the current was dependent on the membrane potentials. The inactivation process of the current was well fitted with a single exponential function, and the current exhibited slow inactivating kinetics (the time constants of decay ranged from 353 ms at -20 mV to 217 ms at +60 mV). The K(+) current was sensitive to intracellular Cs(+) and to extracellular 4-aminopyridine in a concentration-dependent manner, but it was not sensitive to tetraethylammonium, mast cell degranulating peptide, and dendrotoxin-I. The blood depressing substance-I failed to block the K(+) current. These results indicated that dental pulp fibroblasts expressed a slow-inactivating transient K(+) current.     

Sodium channel currents in rat hippocampal NG2 glia: characterization and contribution to resting membrane potential

We have recently reported that most of NG2 glycoprotein expressing glial cells, or NG2 glia, in rat hippocampus persistently express sodium channel currents (I_Na) during development, but little is known about its function. We report here that hippocampal NG2 glia recorded in either acute slices or freshly isolated preparations from postnatal days (P) 7–21 rats express low density I_Na (9.5–15.7 pA/pF) that is characterized by a fast activation and rapid inactivation kinetics with a tetrodotoxin (TTX) IC₅₀ value of 39.3 nM. The I_Na expression correlated with a 25 mV more depolarized resting membrane potential (RMP) as compared with non-I_Na-expressing GLAST(+) astrocytes in situ at the same age. In the presence of the sodium channel blocker TTX (0.1 μM), these depolarized RMPs were negatively shifted by an average of 19 mV and 16 mV for I_Na-expressing glia recordings from in situ and freshly isolated preparations, respectively. The I_Na expressing glia actually showed a positive RMP (+12 mV) in the absence of potassium conductance that was inhibited to 0 mV by 0.1 μM TTX. Analysis of the I_Na activation/inactivation curves yields an I_Na “window current” at −40±20 mV, implying a persistent I_Na component being active around the NG2 glia RMP of −45 mV. According to the constant-field equation analysis, this active I_Na component leads to a pNa/pK ratio of 0.14 at RMP which is threefold higher than astrocytes (0.05). These results indicate that a TTX sensitive I_Na component in NG2 glia contributes significantly to the depolarized NG2 glia RMP in the developing brain.     

Inward rectification in the inner segment of single retinal cone photoreceptors.

1. Single cone photoreceptors were dissociated from the retina of a lizard with the aid of papain. The majority of the cells lost their outer segments but had well-preserved, large synaptic pedicles. Electrical properties of the cells were studied with tight-seal electrodes in the whole cell configuration. On the average, cone inner segments had a resting potential of -55 mV, and at this potential their input resistance was 2.6 G omega and their capacitance was 8 pF. 2. Under current clamp the cones exhibited a pronounced anomalous voltage rectification in response to hyperpolarizing currents. The voltage rectification was eliminated by external Cs+. 3. The Cs(+)-sensitive current underlying voltage rectification was isolated by blocking other currents present in the cone. Co2+ blocked a voltage-dependent Ca2+ current and a Ca2(+)-dependent Cl- current, and tetraethylammonium (TEA)+ blocked a delayed-rectifier K+ current. 4. The Cs(+)-sensitive current was activated by hyperpolarization to potentials more negative than -50 mV, and its current-voltage (I-V) relationship exhibited inward rectification. 5. The inward-rectifying current was selective for K+, but not exclusively. Increasing external K+ concentration 10-fold shifted the reversal potential by 13 mV. If Na ions also permeate through the inward-rectifying channels, the ratio of permeabilities (PK+/PNa+) in normal solution is approximately 3.9. 6. The kinetics of the inward-rectifying current were described by the sum of two exponentials, the amplitudes and time constants of which were voltage dependent. 7. The voltage dependence of the inward-rectifying current was described by Boltzmann's function, with half-maximum activation at -79 mV and a steepness parameter of 7.5 mV. 8. The voltage dependence and kinetics of the inward-rectifying current suggest that it is inactive in a cone photoreceptor in the dark. However, it becomes activated in the course of large hyperpolarizations generated by bright-light illumination. This activity will modify the waveform of the photovoltage--the current will generate a depolarizing component that opposes the light-generated hyperpolarization.