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.     

Na(+)-current expression in rat hippocampal astrocytes in vitro: alterations during development

1. With the use of whole-cell patch-clamp recording. Na(+)-current expression was studied in hippocampal astrocytes in vitro, individually identified by filling with Lucifer yellow (LY) and staining for glial fibrillary acidic protein (GFAP) and vimentin. 2. The proportion of astrocytes that express Na+ currents in rat hippocampal cultures changes during development in vitro and decreases from approximately 75% at day 1 to approximately 30% after 10 days in culture. 3. The sodium currents expressed in astrocytes can be differentiated into two types on the basis of kinetics. At early times in culture the time course of Na+ currents is fast in both onset and decay with an average decay time constant of 1.27 ms, whereas after 6 days Na+ currents become comparatively slow and decayed with an average time constant of 1.86 ms. 4. As with the time-course of Na+ currents, the two age groups of astrocytes (i.e., days 1-5 and day 6 and older) differ with respect to their steady-state inactivation characteristics. Early after plating and up to day 5, the midpoint of the steady-state inactivation curve is close to -60 mV, as also observed in hippocampal neurons of various ages; in contrast, after 6 days in culture the curve is shifted by approximately 25 mV toward more hyperpolarized potentials with a midpoint close to -85 mV. 5. In contrast to h infinity-curves, current-voltage (I-V) curves of Na(+)-current activation were identical in all astrocytes studied and did not change with time in culture. 6. In astrocytes expressing Na+ currents, current densities (average of 35 pA/pF on day 1) decreased throughout the first 5 days and were almost abolished around days 4 and 5 in culture. Beginning on day 6, however, current densities increased again and maintained a steady level (average of 14 pA/pF) for the duration of the time period in culture (20 days). This biphasic time course closely correlates with the time course of changes in Na(+)-current kinetics and steady-state inactivation. 7. These data suggest that Na+ currents in cultured hippocampal astrocytes show characteristic changes with increasing time in culture. During the first 4-5 days in culture, hippocampal astrocytes display Na+ currents with properties similar to those of hippocampal neurons. Our data further suggest that Na+ currents with distinctive, "glial-type" characteristics are only expressed in hippocampal astrocytes after 6 days in culture.     

Blocking and modifying actions of octanol on Na channels in frog myelinated nerve

The actions of externally applied n-octanol on Na channels in myelinated frog nerve fibres were studied under voltage clamp conditions. Upon octanol application peak Na inward currents declined in two phases: 90% of the reduction occurred in less than 2 min but a steady-state was reached only after 15 min. During washout the currents came to a stable level within 10 min. The reduction of Na inward currents by octanol was dependent on the amplitude and duration of prepotentials. At the resting potential (VH = 0 mV) 0.4 mM octanol reduced peak Na inward currents at V = 60 mV by 50%. After a prepulse of -60 mV and 50 ms duration Na currents decreased only by 20%. At a hyperpolarizing holding potential of VH = -28 mV 0.7 mM octanol reduced peak inward Na currents to one half. Octanol depressed Na currents at all potentials by approximately the same factor. The Na reversal potential VNa remained unchanged. 0.7 mM external octanol shifted the Na activation curve m infinity (V) by 5 mV to more positive and the inactivation curve h infinity (V) by 14 mV to more negative potentials. The midpoint slopes of both curves were reduced. The time constants of Na activation and inactivation at small depolarizations were decreased. The conductance gamma of a single Na channel and the number No of conducting Na channels per node were determined from nonstationary Na current fluctuations. 0.7 mM octanol increased gamma by a factor of 1.6 and reduced No by a factor of 0.34. It is concluded that octanol blocks some Na channels and modifies the remaining unblocked channels.     

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.     

Electrophysiological and pharmacological properties of the human brain type IIA Na+ channel expressed in a stable mammalian cell line

The human brain voltage-gated Na+ channel type IIA alpha subunit was cloned and stably expressed in Chinese hamster ovary cells and its biophysical and pharmacological properties were studied using whole-cell voltage-clamp. Fast, transient inward currents of up to -8,000 pA were elicited by membrane depolarization of the recombinant cells. Channels activated at -50 mV and reached maximal activation at -10 mV to 0 mV. The reversal potential was 62 +/- 2 mV which is close to the Na+ equilibrium potential. The half-maximal activation and inactivation voltages were -24 +/- 2 mV and -63 +/- 1 mV, respectively. Currents were reversibly blocked by tetrodotoxin with a half-maximal inhibition of 13 nM. The effects of four commonly used anti-convulsant drugs were examined for the first time on the cloned human type IIA channel. Lamotrigine and phenytoin produced concentration- and voltage-dependent inhibition of the type IIA currents, whereas, sodium valproate and gabapentin (up to 1 mM) had no effect. These results indicate that recombinant human type IIA Na+ channels conduct tetrodotoxin-sensitive Na+ currents with similar properties to those observed in recombinant rat brain type IIA and native rat brain Na+ channels. This stable cell line should provide a useful tool for more detailed characterization of therapeutic modulators of human Na+ channels.     

Ionic currents in crustacean neurosecretory cells.

1. The patterns of electrical activity and membrane characteristics of a population of neurosecretory-cell somata in the X-organ of the crayfish were investigated with microelectrodes and whole-cell, voltage-clamp techniques. Some neurons (56%) were silent but could be excited by intracellular current injection: other cells showed spontaneous tonic activity (35%), and some had spontaneous bursting activity (9%). The spiking activity was abolished by tetrodotoxin (TTX) exposure and by severing the axon near the cell body. After axotomy, only a small, slow, regenerative depolarization remained that could be blocked by Cd2+. 2. Under voltage clamp the steady-state I-V curve in low [Ca2+]i (9 X 10(-9) M) showed a slope conductance of 16.7 +/- 3.9 (SD) nS (n = 10) at -50 mV and zero current potential of -50.1 +/- 7.7 mV. In current-clamp mode these neurons were either silent or fired tonically. With high [Ca2+]i (1.7 X 10(-6) M) both the slope conductance and inward and outward currents were reduced. In some neurons high [Ca2+]i reveals a negative slope resistance in the range of -46 to -41 mV. It could be supressed by removing [Na+]o, but it was TTX insensitive. These are the neurons that under current clamp showed bursting activity. 3. The main inward current in cell somata was a Ca2+ current of 2 +/- 0.6 nA (n = 18), activated at -40 mV and peaking at 20 mV. It showed relaxation with prolonged pulses. No Na(+)-dependent, TTX-sensitive inward currents were recorded with short (100-ms) pulses in axotomized neurons. 4. Two outward currents could be distinguished.(ABSTRACT TRUNCATED AT 250 WORDS)     

Slow depolarizing afterpotentials in neocortical neurons are sodium and calcium dependent.

Depolarizing afterpotentials (DAPs) were studied in intracellular recordings from neocortical slices bathed in tetrodotoxin (TTX) (1 microM) and tetraethylammonium chloride (TEA) (24 mM), to block voltage-dependent Na+ currents and most K+ currents. The DAP was Ca(2+)-dependent, in that its magnitude varied as a function of the duration of the preceding Ca2+ plateau. It had an apparent reversal potential of between -40 and -5 mV. The DAP was blocked when choline replaced all extracellular Na+; there was a hyperpolarizing shift in apparent reversal potential when extracellular Na+ was lowered. The DAP was blocked by amiloride (1 mM), which also decreased the preceding Ca2+ plateau. The data are consistent with the hypothesis that the DAP is due to electrogenic Na+/Ca2+ exchange.     

Evidence for two types of sodium conductance in axons perfused with sodium fluoride solution

1. Voltage clamp experiments were carried out on squid giant axons internally perfused with 300 mM-NaF + sucrose. K-free artificial sea-water, -0.3 to 3.5 degrees C, was used externally.2. Membrane currents were corrected for capacitative and leakage components, and the resulting Na current was converted to Na conductance, g(Na). An attempt was made to fit changes in g(Na) according to the Hodgkin-Huxley model, namely [Formula: see text].According to the model g(Na) is a constant, m(infinity) and h(infinity) are steady-state values which depend only on voltage, tau(m) (-1) and tau(h) (-1) are rate constants which also are functions only of voltage.3. Stepwise depolarizations from the holding potential (-67 to -83 mV) to a potential which varied from -10 to +63 mV resulted in an exponential decline of h from its initial level to a final, non-zero level. If the test depolarization was preceded by a positive prepulse (duration, 19-105 msec; voltage, -6 to 94 mV) the rate constant for h, tau(h) (-1), was increased roughly threefold with practically no change in the final level.4. The steady-state level of h was studied by using prepulses of varying amplitude followed by a test depolarization. In one such experiment a value of 0.34 was obtained for a 105 msec prepulse to -49 mV. The same value for the steady level of h was obtained from analysing a record taken at +52 mV. If the potential was switched from -49 to +52 mV there was a transient increase in g(Na) although h(infinity) had the same value at these two potentials.5. Recovery from depolarization was studied by repolarizing the fibre for varying lengths of time, then applying a test depolarization. If the first depolarization was strongly positive (for example, 70 mV), so that the steady level of h was large (0.39), the currents associated with the test pulse could not be fitted on the basis of an exponential increase in h during the recovery period. Rather, the results suggested that on repolarization h rapidly decreased initially, then slowly increased.6. These results can be explained by assuming that h is given by the sum of two components, h(1) and h(2). Changes are represented kinetically by h(1) right harpoon over left harpoon x right harpoon over left harpoon h(2), where x signifies the inactive state. The distribution is shifted to the left at negative potentials and to the right for positive ones. The resulting Na conductance is comprised of two types: the first type, g(Na)m(3)h(1), is similar to the Hodgkin-Huxley system and underlines the usual transient increase in g(Na) associated with depolarization; the second type, g(Na)m(3)h(2), is maintained with depolarization and gives rise to a steady level of g(Na).     

Voltage-dependent currents in isolated vestibular afferent calyx terminals

Na(+) currents were studied by whole cell patch clamp of chalice-shaped afferent terminals attached to type I hair cells isolated from the gerbil semicircular canal and utricle. Outward K(+) currents were blocked with intracellular Cs(+) or with extracellularly applied 20 microM linopirdine and 2.5 mM 4-aminopyridine (4-AP). With K(+) currents blocked, inward currents activated and inactivated rapidly, had a maximum mean peak amplitude of 0.92 +/- 0.60 (SD) nA (n = 24), and activated positive to -60 mV from holding potentials of -70 mV and more negative. The transient inward currents were blocked almost completely by 100 nM TTX, confirming their identity as Na(+) currents. Half-inactivation of Na(+) currents occurred at -82.6 +/- 0.9 mV, with a slope factor of 9.2 +/- 0.8 (n = 7) at room temperature. In current clamp, large overshooting action potential-like events were observed only after prior hyperpolarizing current injections. However, spontaneous currents consistent with quantal release from the hair cell were observed at holding potentials close to the zero-current potential. This is the first report of ionic conductances in calyx terminals postsynaptic to type I hair cells in the mammalian vestibular system.     

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)     

Expression of a tetrodotoxin-sensitive Na+ current in cultured human retinal pigment epithelial cells.

We observed a tetrodotoxin (TTX)-sensitive Na+ current in cultured fetal and adult cells of the human retinal pigment epithelium (RPE), but not in any freshly isolated fetal (n = 54) or adult (n = 47) cells, using the whole-cell version of the patch-clamp technique. A similar current was found in cultured, but not in freshly isolated, adult monkey RPE cells. The rapid activation and inactivation of this current resembled that of the voltage-dependent Na+ current of excitable cells. The voltage dependence of inactivation followed a Boltzmann function with half-maximal inactivation at -52.1 +/- 4.8 mV (n = 9), thus classifying this current as 'neuronal' in type. Recovery from inactivation followed a single exponential function with a time constant of 12.0 +/- 1.4 ms (n = 5) at -100 mV. The current was very sensitive to the Na+ channel blocker TTX, with a half-inhibition concentration of 1.87 +/- 0.37 nM (n = 5). Of special interest are the findings that current density was high when cells were rapidly proliferating and had lost their melanin pigment, and that the density declined after the cells reached confluence and repigmented. This pattern of current expression was consistently found in cells cultured with three different protocols, including a serum-free medium, indicating that serum was not necessary for its expression. We hypothesize that expression of this Na+ current in culture is regulated by an intrinsic programme related to cell differentiation. It may represent a tendency of proliferating RPE cells to dedifferentiate towards a more embryonic and neuroepithelial phenotype. Similar expression of Na+ current might occur in vivo when RPE cells proliferate, as in wounding.     

Hyperpolarization-activated currents regulate excitability in stellate cells of the mammalian ventral cochlear nucleus

The differing biophysical properties of neurons the axons of which form the different pathways from the ventral cochlear nucleus (VCN) determine what acoustic information they can convey. T stellate cells, excitatory neurons the axons of which project locally and to the inferior colliculus, and D stellate cells, inhibitory neurons the axons of which project to the ipsi- and contralateral cochlear nuclei, fire tonically when they are depolarized, and, unlike other cell types in the VCN, their firing rates are sensitive to small changes in resting currents. In both types of neurons, the hyperpolarization-activated current (I(h)) reversed at -40 mV, was activated at voltages negative to -60 mV, and half-activated at approximately -88 mV; maximum hyperpolarization-activated conductances (g(h max)) were 19.1 +/- 2.3 nS in T and 30.3 +/- 2.6 nS in D stellate cells (means +/- SE). Activation and deactivation were slower in T than in D stellate cells. In both types of stellate cells, 50 microM 4(N-ethyl-N-phenylamino)1,2-dimethyl-6-(methylamino) pyridinium chloride (ZD7288) and 2 mM Cs(+) blocked a 6- to 10-fold greater conductance than the voltage-dependent g(h) determined from Boltzmann analyses at -62 mV. The voltage-insensitive, ZD7288-sensitive conductance was proportional to g(h max) and g(input). 8-Br-cAMP shifted the voltage dependence of I(h) in the depolarizing direction, increased the rate of activation, and slowed its deactivation in both T and D stellate cells. Reduction in temperature did not change the voltage dependence but reduced the maximal g(h) with a Q(10) of 1.3 and slowed the kinetics with a Q(10) of 3.3.     

Block of sodium channels by tyramine and its analogue (N-feruloyl tyramine) in frog ventricular myocytes.

Pharmacological effects of tyramine and its analogue, N-feruloyl tyramine (NFT), on sodium and calcium currents in frog ventricular myocytes were examined using the whole-cell voltage-clamp technique. To improve the temporal and spatial control of the membrane potential, sodium currents (INa) were recorded in 45.5 mM [Na+]o at 10 degrees C. Both tyramine and NFT (1-100 microM) induced a concentration-dependent decrease in INa evoked from a holding potential of -80 mV without affecting a change in either the time to peak or the time constant for the falling phase of INa. Similarly the reversal potential for INa remained unchanged at a value close to that predicted from the Nernst equation. The finding that both tyramine and NFT decreased INa when activated maximally, from a holding potential of -120 mV, indicates that the amplitude of INa can be reduced independently of a change in the kinetics of the current. In addition, tyramine (100 microM) shifted the membrane potential for half maximal inactivation (Vh) of the steady-state inactivation (h infinity)-curve from -74 to -84 mV without affecting its slope. In contrast, NFT failed to affect the h infinity-curve. The calcium current (ICa) recorded in the presence of 0.3 microM TTX was not affected by either 100 microM tyramine or NFT. We concluded that tyramine directly blocks Na channel by shifting h infinity-curve and by suppressing maximum Na channel conductance, while NFT suppresses only maximum Na channel conductance.     

The effect of temperature on Na currents in rat myelinated nerve fibres

Voltage clamp experiments were performed in single myelinated nerve fibres of the rat and the effect of temperature on Na currents was investigated between 0 degrees C and 40 degrees C. The amplitude of the peak Na current changed with a Q10 = 1.1 between 40 degrees and 20 degrees C and with a Q10 = 1.3 between 20 degrees and 10 degrees C. Below 10 degrees C the peak Na current changed with a Q10 = 1.9. The temperature coefficient for time-to-peak (tp), the measure for Na activation, and tau h1 and tau h2, the time constants for Na inactivation changed throughout the temperature range. Q10 for all of these kinetic parameters increased from 1.8-2.1 between 40 degrees and 20 degrees C to 2.6-2.7 between 20 degrees and 10 degrees C. Below 10 degrees C Q10 increased to 3.7 for tau h1 and tp, and to 2.9 for tau h2. When the series resistance artifacts were minimized by addition of 6 nM TTX, the Q10's at T less than 10 degrees C were 2.9-3.0. When the temperature was decreased from 20 degrees to 0 degrees C, both the curve relating Na permeability to potential, PNa(V), and the steady state Na inactivation curve, h infinity (V), were reversibly shifted towards more negative potentials by 6 mV and 11 mV, respectively. When the temperature was increased from 20 degrees to 37 degrees C no shifts occurred. The Hodgkin-Huxley rate constants alpha h(V) and beta h(V) were calculated from h infinity (V) and tau h (or tau h1) at 20 degrees and 4 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrophysiological characterization of rat type II pneumocytes in situ

Optimal aeration of the lungs is dependent on an alveolar fluid clearance, a process that is governed by Na+ and Cl- transport. However, the specific contribution of various ion channels in different alveolar cell types under basal or stimulated conditions is not exactly known. We established a novel functional model of rat lung slices suitable for nystatin-perforated whole-cell patch-clamp experiments. Lung slices retained a majority of live cells for up to 72 hours. Type II pneumocytes in situ had a mean capacitance of 8.8 +/- 2.5 pF and a resting membrane potential of -4.4 +/- 1.9 mV. Bath replacement of Na+ with NMDG+ decreased inward whole-cell currents by 70%, 21% and 52% of which were sensitive to 10 microM and 1 mM of amiloride, respectively. Exposure of slices to 0.5 microM dexamethasone for 1 hour did not affect ion currents, while chronic exposure (0.5 microM, 24-72 h) induced an increase in both total Na+-entry currents and amiloride-sensitive currents. Under acute exposure to 100 microM cpt-cAMP, Type II cells in situ rapidly hyperpolarized by 25-30 mV, due to activation of whole-cell Cl- currents sensitive to 0.1 mM of 5-Nitro-2-(3-phenylpropylamino)benzoic acid. In addition, in the presence of cpt-cAMP, total sodium currents and currents sensitive to 10 microM amiloride increased by 32% and 70%, respectively. Thus, in Type II pneumocytes in situ: (1) amiloride-sensitive sodium channels contribute to only half of total Na+-entry and are stimulated by chronic exposure to glucocorticoids; (2) acute increase in cellular cAMP content simultaneously stimulates the entry of Cl- and Na+ ions.     

Excitable properties and voltage-sensitive ion conductances of horizontal cells isolated from catfish (Ictalurus punctatus) retina

External horizontal cells were enzymatically dissociated from intact catfish (Ictalurus punctatus) retina and pipetted onto a small chamber attached to the stage of an inverted phase-contrast microscope. Individual horizontal cells were recognized by their large size and restricted dendritic arborization. Low-resistance (3-12 M omega) patch-type electrodes were used to record intracellular potentials and to pass current across the cell membrane under either current or voltage-clamp conditions. The average resting potential of isolated horizontal cells was -67 V + 6.9 mV (mean +/- SD, n = 40). At the resting potential, the cell membrane appears to be mainly permeable to K. A depolarizing current step evoked an action potential in the cell. The maximum rate of rise of the action potential (dV/dt) in normal physiological solution was 6.5 +/- 1.8 V/s (means +/- SD, n = 24) and was reduced to 1.2 +/- 0.39 V/s (means +/- SD, n = 9) in 1-10 micron tetrodotoxin (TTX) and 3.2 +/- 1.4 V/s (means +/- SD, n = 6) in Ca-free solution. The maximum dV/dt was reduced in 10 mM extracellular K concentration [K]o to about half of that seen in standard saline, and values in 30 or 80 mM [K]o were similar to that measured in TTX. Following an action potential, the membrane potential reached a plateau potential of + 17.4 +/- 8.1 mV (means +/- SD, n = 17) and remained depolarized for variable periods of time lasting from less than a second to a few minutes. When the plateau potential was long lasting, the cell repolarized slowly and upon reaching zero rapidly repolarized to the original resting potential. The duration of the plateau potential decreased or was absent in saline containing one of the following calcium channel antagonists: La, Cd, Co, or Ni. The voltage-clamp technique was used to identify the membrane currents responsible for the membrane potential changes seen under current clamp. Experiments were carried out using either a single or two individual electrodes. Fast and steady-state inward currents were recorded from isolated horizontal cells in the voltage range between -20 and +20 mV. These currents were a result of increased membrane conductance to both Na and Ca ions. The Na channels are inactivated at depolarized potentials and are TTX sensitive. Ca channels are partially inactivated at depolarized potentials. The Ca conductance is decreased by Cd, Co, Ni, and La. Ba can substitute for Ca in the channel.(ABSTRACT TRUNCATED AT 400 WORDS)     

Kinetic analysis of acetylcholine-induced current in isolated frog sympathetic ganglion cells

1. Kinetic properties of activation and inactivation phases of the ACh-gated nicotinic current were investigated in isolated frog sympathetic ganglion cells using "concentration-clamp" technique under voltage-clamp conditions. This technique combines internal perfusion with a rapid change of the external solution within a few milliseconds. 2. The dose-response curve for the peak current induced by ACh showed a sigmoidal increase, in which the apparent dissociation constant Kd and Hill coefficient were 2.6 x 10(-5) M and 2.0, respectively. 3. The current-voltage relationship of ACh-induced currents were linear at potentials more negative than the reversal potential (EACh) of -5.5 +/- 1.3 mV (mean +/- SE) but showed a slight inward rectification at positive potentials over +20 mV. Since this current could be generated predominantly by an increase of Na+ and K+ conductances, the value of EACh was close to the theoretical potential, -1.3 mV, for the total amount of both Na+ and K+ or Cs+ in the extracellular and intracellular solutions. 4. There was no difference between the dose-response curves of ACh- and nicotine-induced currents. The ACh-induced current was suppressed in a competitive manner by the nicotinic antagonists, d-tubocurarine and hexamethonium, but muscarine did not induce any response, indicating that the ACh-gated current might be mediated by the nicotinic ACh receptor-ionophore complex. 5. There was a latent period of the order of milliseconds in the ACh receptor activation phase before attaining exponential increase of activation process.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

Measurement of the conductance of the sodium channel from current fluctuations at the node of Ranvier.

Single myelinated nerve fibres of Rana esculenta were investigated under voltage clamp conditions at 13 degrees C. Fluctuations of steady-state membrane current were measured during the last 152 msec of 190-225 msec pulses depolarizing the membrane by 8-48 mV. Noise power spectral densities were calculated in the frequency range of 6-6-6757 Hz. 2. External application of 150 nM tetrodotoxin (TTX) and/or 10 mM tetraethylammonium (TEA) ion reduced the current fluctuations. The difference of current noise spectra measured in the presence and absence of TTX (TEA) was not changed by the presence of TEA (TTX) during both measurements, and was taken as the spectrum of the Na (K) current fluctuations. 3. Residual current noise during application of both TTX and TEA was, except for some excess noise at the low and high frequency ends of the spectrum, similar to the noise measured from a passive nerve model and could be understood in terms of Nyquist noise of the known resistances and the amplifier noise. 4. Na current fluctuation spectra were interpreted as the sum N/f+SNa(f) where SNa(F) represents the spectrum expected for a set of equal, independent Na channels with only two conductance states (open or closed) which follow Hodgkin-Huxley kinetics. With values of hinfinity, tauh and minfinity measured from macroscopic Na currents, the measured spectra were fitted well by optimizing N, SNa(0) and taum. Values of taum obtained by this method were in fair agreement with values found from macroscopic currents. 5. The 1/f component of Na current noise was roughly proportional to the square of the steady-state Na current, I2. The mean value of N/I2 was (1-1 +/- 0-3) X 10(-4). 6. The current carried by a single Na channel was calculated from fitted spectra and steady-state Na currents measured simultaneously with the current fluctuations. The single channel conductance gamma normalized to zero absolute membrane potential was calculated. The average gamma from twelve measurements at depolarizations of 8-40 mV was 7-9 +/- 0-9 pS (S.E. of mean). The apparent value of gamma was smallest with small depolarizations. Variations of the assumed kinetic properties of the model did not drastically affect the single channel conductance. 7. External application of 0-1 mM-Ni ion lengthened taum in the macroscopic currents and in the fluctuation spectra and enhanced both the steady-state Na current and the current fluctuations. In Ni-treated nodes gamma was smaller than in normal nodes.     

Oscillatory membrane potential activity in the soma of a primary afferent neuron.

In the present report, we provide evidence that mesencephalic trigeminal (Mes-V) sensory neurons, a peculiar type of primary afferent cell with its cell body located within the CNS, may operate in different functional modes depending on the degree of their membrane polarization. Using intracellular recording techniques in the slice preparation of the adult rat brain stem, we demonstrate that when these neurons are depolarized, they exhibit sustained, high-frequency, amplitude-modulated membrane potential oscillations. Under these conditions, the cells discharge high-frequency trains of spikes. Oscillations occur at membrane potential levels more depolarized than -53 +/- 2.3 mV (mean +/- SD). The amplitude of these oscillations increases with increasing levels of membrane depolarization. The peak-to-peak amplitude of these waves is approximately 3 mV when the cells are depolarized to levels near threshold for repetitive firing. The frequency of oscillations is similar in different neurons (108.9 +/- 15.5 Hz) and was not modified, in any individual neuron, by changes in the membrane potential level. These oscillations are abolished by hyperpolarization and by TTX, whereas blockers of voltage-dependent K+ currents slow the frequency of oscillations but do not abolish the activity. These data indicate that the oscillations are generated by the activation of inward Na+ current/s and shaped by voltage-dependent K+ outward currents. The oscillatory activity is not modified by perfusion with low-calcium, high-magnesium, or cobalt-containing solutions nor is it modified in the presence of cadmium or Apamin. These results indicate that a calcium-dependent K+ current does not play a significant role in this activity. We postulate that the membrane oscillatory activity in Mes-V neurons is synchronized in adjoining electrotonically coupled cells and that this activity may be modulated in the behaving animal by synaptic influences.