Properties of the interaction of the sodium channel with permeant monovalent cations

The use of sea anemone toxin, veratridine and scorpion toxin which specifically interact with the gating system of the sodium channel and maintain the channel in an open conformation has permitted a study of the mechanism of transport of monovalent cations through the selectivity filter of this channel. The initial rate of 22Na+ influx through the tetrodotoxin-sensitive Na+ channels of excitable cells is dependent upon the external concentrations of Na+ and Na+-substitutes with the following properties. (a) It is saturable at high Na+ concentrations and increases with the external Na+ concentration in a cooperative manner (nH = 1.6). (b) At low external Na+ concentrations (1 mM), it is activated and then inhibited by increasing external concentrations of monovalent cations such as Li+, guanidinium, hydrazinium, hydroxylamine and K+. The activating effect of these cations disappears at higher external Na+ concentrations (10 mM). The experimental data are consistent with a model involving at least two allosteric cation-binding sites per Na+ channel. The binding of monovalent cations to Na+ sites is characterized by a high positive homotropic cooperativity. Most of the work describes the properties of the Na+ channel in neuroblastoma cells. The mechanism has also been shown to be valid for excitable cells of other types and origins.     

Slow and incomplete inactivations of voltage-gated channels dominate encoding in synthetic neurons.

Electrically excitable channels were expressed in Chinese hamster ovary cells using a vaccinia virus vector system. In cells expressing rat brain IIA Na+ channels only, brief pulses (< 1 ms) of depolarizing current resulted in action potentials with a prolonged (0.5-3 s) depolarizing plateau; this plateau was caused by slow and incomplete Na+ channel inactivation. In cells expressing both Na+ and Drosophila Shaker H4 transient K+ channels, there were neuron-like action potentials. In cells with appropriate Na+/K+ current ratios, maintaining stimulation produced repetitive firing over a 10-fold range of frequencies but eventually led to "lock-up" of the potential at a positive value after several seconds of stimulation. The latter effect was due primarily to slow inactivation of the K+ currents. Numerical simulations of modified Hodgkin-Huxley equations describing these currents, using parameters from voltage-clamp kinetics studied in the same cells, accounted for most features of the voltage trajectories. The present study shows that insights into the mechanisms for generating action potentials and trains of action potentials in real excitable cells can be obtained from the analysis of synthetic excitable cells that express a controlled repertoire of ion channels.     

The effect of nerve growth factor on the development of sodium channels in PC12 cells

We have studied the development of the action potential Na+ channels in PC12 cells, an established line that has been useful as a model for neuronal differentiation. In continuous culture PC12 cells, although electrically inexcitable, nevertheless have a low level of Na+ channels as judged by the increase in 22Na+ uptake in the presence of veratridine and scorpion toxin. These two neurotoxins have been shown to promote activation of Na+ channels in a variety of electrically excitable cells. Following treatment with nerve growth factor (NGF), conditions which induce differentiation to an electrically excitably neuronal-cell type, the neurotoxin-activated 22Na+ uptake increases approximately 12-fold, on a per cell basis, reaching a maximum in 12-16 days. The dose-response curves for veratridine and scorpion toxin are unchanged by NGF treatment (K0.5 for veratridine, 18-14 microM; K0.5 for scorpion toxin, 120-96 nM). Na+ channels in both undifferentiated and differentiated cells are tetrodotoxin sensitive and NGF treatment has no effect on the inhibition constant (Ki, 10-12 nM). Na+ channel sites were measured directly by the specific binding of [3H]saxitoxin. In NGF-treated cells, the saxitoxin receptor density reaches 154 fmol/mg protein (Kd, 1.3 nM), a level comparable to other excitable cells. Levels in control cells were too low to measure accurately. These findings show that NGF treatment of PC12 cells leads to a substantial increase in the expression of neurotoxin-sensitive Na+ channels. Furthermore, these channels are pharmacologically similar, if not identical, to those which exist in undifferentiated cells and therefore do not appear to result from the conversion of preexisting channels.     

A Na+- and Cl- -activated K+ channel in the thick ascending limb of mouse kidney

This study investigates the presence and properties of Na+-activated K+ (K(Na)) channels in epithelial renal cells. Using real-time PCR on mouse microdissected nephron segments, we show that Slo2.2 mRNA, which encodes for the K(Na) channels of excitable cells, is expressed in the medullary and cortical thick ascending limbs of Henle's loop, but not in the other parts of the nephron. Patch-clamp analysis revealed the presence of a high conductance K+ channel in the basolateral membrane of both the medullary and cortical thick ascending limbs. This channel was highly K+ selective (P(K)/P(Na) approximately 20), its conductance ranged from 140 to 180 pS with subconductance levels, and its current/voltage relationship displayed intermediate, Na+-dependent, inward rectification. Internal Na+ and Cl- activated the channel with 50% effective concentrations (EC50) and Hill coefficients (nH) of 30 +/- 1 mM and 3.9 +/- 0.5 for internal Na+, and 35 +/- 10 mM and 1.3 +/- 0.25 for internal Cl-. Channel activity was unaltered by internal ATP (2 mM) and by internal pH, but clearly decreased when internal free Ca2+ concentration increased. This is the first demonstration of the presence in the epithelial cell membrane of a functional, Na+-activated, large-conductance K+ channel that closely resembles native K(Na) channels of excitable cells. This Slo2.2 type, Na+- and Cl--activated K+ channel is primarily located in the thick ascending limb, a major renal site of transcellular NaCl reabsorption.     

Stimulation of sodium and calcium uptake by scorpion toxin in chick embryo heart cells.

Scorpion toxins, the basic miniproteins of scorpion venom, stimulated the passive uptake of Na+ and Ca2+ in chick embryo heart cells. Half-maximum stimulation was obtained for 20-30 nM Na+ and 40-50 nM Ca2+. Scorpion toxin-activated Na+ and Ca2+ uptakes were fully inhibited by tetrodotoxin, a specific inhibitor of the action potential Na+ ionophore in excitable membranes. Half-maximum inhibition was obtained with the same concentration of tetrodotoxin (10 nM) for both Na+ and Ca2+. Scorpion toxin-stimulated Ca2+ uptake was dependent on extracellular Na+ concentration and was not inhibited by Ca2+ channel blocking drugs which are inactive on heart cell action potential. Thus, in heart cells scorpion toxin affects the passive Ca2+ transport, which is coupled to passive Na+ ionphore. Other results suggest that (1) tetrodotoxin and scorpion toxin bind to different sites of the sarcolemma and (2) binding of scorpion toxin to its specific sites may unmask latent tetrodotoxin - sensitive fast channels.     

Initiation of cultured rat hepatocyte proliferation does not involve Na+-dependent plasma membrane Ca2+ fluxes

Adult rat hepatocytes in primary culture were examined to determine if Na+-dependent transmembrane Ca2+ fluxes precede reinitiation of DNA synthesis. Studies with 45Ca2+ and atomic absorption measurements of 40Ca2+ showed that hepatocytes lack plasma membrane Na+-Ca2+ exchange activity. Under chemically defined conditions, combinations of mitogens - EGF, insulin, and glucagon - failed to induce transmembrane Ca2+ fluxes early in the prereplicative phase. In addition, a Ca2+ ionophore, A23187, was non-mitogenic. Thus, plasma membrane Na+-Ca2+ exchange is not a mitogenic signal for hepatocytes. Elevated intracellular Ca2+ levels are thought to mediate early prereplicative events required for animal cell proliferation. These conclusions stem partly from findings that A23187, a Ca2+ ionophore, stimulates transmembrane Ca2+ fluxes and proliferation in several cell systems (reviewed in Boynton et al., 1982). Sodium ion fluxes also are implicated as "initiating" mitogenic signals (Koch and Leffert, 1979). In particular, amiloride-sensitive Na+ influxes, stimulated by growth factors, may be necessary to initiate DNA synthesis in rat hepatocytes, mouse and human fibroblasts, rat liver derived cell lines, mouse sympathetic neurons, human lymphocytes, and monkey kidney epithelial cells (reviewed in Leffert, 1982). Several investigators, using cells from electrically excitable tissues (Schellenberg and Swanson, 1981; Eckert and Grosse, 1982), have reported that plasma membrane Na+-Ca2+ exchange carriers regulate intracellular Na+ and Ca2+ concentration. It is unclear if this exchange system exists in non-electrically excitable membranes, especially with regard to hepatocytes (Judah and Ahmed, 1964; van Rossum, 1970). We have here investigated the possible association of Na+ influxes with transmembrane Ca2+ movement following reinitiation of hepatocyte growth.     

Ouabain-insensitive net sodium influx in erythrocytes of normotensive and essential hypertensive humans

The Na+ content of erythrocytes is elevated in people with essential hypertension. There is conflicting evidence about its cause. The present study was designed to investigate whether the increase in content is due to a defect in a ouabain-resistant Na+ flux. Net Na+ influx was determined from the increase in Na+ content of erythrocytes during incubation in the presence of ouabain. Na+ content of erythrocytes from 24 normotensive Caucasian subjects with no known family history of hypertension was 6.9 +/- 1.3 mmol per litre of cells. It was 7.9 +/- 2.0 mmol per litre of cells in 18 subjects with essential hypertension. The difference was less and not significant when the two non-Caucasian subjects of the hypertensive group were excluded. Net Na+ influx was 1.83 mmol/h per litre of cells in the normotensive group. In eight subjects it was measured on a second occasion after an interval of several months. The coefficient of a variation of the duplicate tests was 2.4%. Net Na+ influx was significantly higher in the hypertensive group, the value was 2.18 +/- 0.15 mmol/h per litre of cells. In 11 of these subjects, Na+ influx was measured on a second occasion. The coefficient of variation was 6.2%, significantly greater than in the control group. In some of these subjects Na+ influx was within the normal range on one of the two occasions. When the groups were compared with use of the mean values from the duplicate tests, net Na+ influx was elevated in 17 of the 18 hypertensive subjects. The findings are discussed with reference to previous work and in relation to the established facilitatory effects of an increased intracellular Na+ concentration on excitable cells that influence blood pressure.     

Bursting excitable cell models by a slow Ca2+ current

Bursting in excitable cells is a phenomenon that has attracted the interest of many electrophysiologists and non-linear dynamicists. In this paper, we present two models that give rise to bursting in action potentials. The membrane of the first model contains a voltage-activated Ca2+ channel that inactivates very slowly upon depolarization and a delayed K+ channel that is activated by voltage. This model consists of three dynamic variables--the gating variable of K+ channel (n), inactivation gating variable of the Ca2+ channel (f), and membrane potential (V). The membrane of the second model contains a voltage-activated Na+ channel that inactivates rather fast upon depolarization. This model contains altogether five dynamic variables--the Na+ inactivation gating variable (h) and Ca2+ activation variable (d), in addition to the three dynamic variables in the first model. With the first model, we show how various interesting bursting patterns may arise from such a simple three dynamic variable model. We also demonstrate that a slowly inactivating voltage-dependent Ca2+ channel may play the key role in the genesis of bursting. With the second model, we show how the participation of a quickly inactivating fast inward current may lead to a neuronal type of bursting, multi-peaked oscillations, and chaos, as the rates of the gating variables change.     

Voltage-activated currents in the CRI-G1 rat insulin-secreting cell-line.

1. The whole-cell configuration of the patch-clamp recording technique was used to characterize the electrophysiological properties of CRI-G1 insulin-secreting cells. 2. Current-clamp recordings demonstrated the excitable nature of these cells. 3. Voltage-clamp recordings revealed the presence of an inward Na+ current, an inward Ca2+ current and a delayed outward K+ conductance. 4. The electrophysiological properties of CRI-G1 closely resemble those of pancreatic beta-cells, thereby rendering this cell-line as a useful alternative to freshly isolated cells for the study of pancreatic beta-cell electrophysiology and pharmacology.     

Mouse brain synaptosomal sodium channels: activation by aconitine, batrachotoxin, and veratridine, and inhibition by tetrodotoxin

Batrachotoxin, veratridine and aconitine, activators of the voltage-dependent sodium channel in excitable cell membranes, increase the rate of 22Na+ uptake by mouse brain synaptosomes. Batrachotoxin was both the most potent (K0.5, 0.49 microM) and most effective activator of specific 22Na+ uptake. Veratridine (K0.5, 34.5 microM) and aconitine (K0.5, 19.6 microM) produced maximal stimulations of 22Na+ uptake that were 73% and 46%, respectively, of that produced by batrachotoxin. Activation of 22Na+ uptake by veratridine was completely inhibited by tetrodotoxin (I50, 6 nM ), a specific blocker of nerve membrane sodium channels. These results identify appropriate conditions for measuring sodium channel-dependent 22Na+ flux in mouse brain synaptosomes. The pharmacological properties of mouse brain synaptosomal sodium channels described here are distinct from those previously described for sodium channels in rat brain synaptosomes and mouse neuroblastoma cells.     

A comparative study of the effects of tetrodotoxin and the removal of external Na+ on the resting potential: Evidence of separate pathways for the resting and excitable Na currents in squid axon

To investigate whether the Na permeability of the resting membrane is determined predominantly by the excitable Na channel, we examined the effects of tetrodotoxin (TTX) and the complete removal of external Na+ on the resting potential. In the intact squid axon bathed in K-free artificial seawater, both TTX and the removal of Na+ produced small hyperpolarizations. The effect of Na removal, however, was larger than that of TTX. In the perfused squid axon, the hyperpolarization produced by the removal of external Na+ was greatly enhanced when the internal K concentration ([K+]i) was reduced. The effect of TTX, on the other hand, was not sensitive to the [K+]i or to the membrane potential. For [K+]i = 50 mM and [K+]o = 0, the average hyperpolarization produced by TTX was 1.2 mV, while the hyperpolarization produced by Na removal was approximately 21 mV. The difference between these two effects suggests that the majority of the resting Na current passes through pathways other than the excitable Na channel.     

Na+-Ca2+ exchange in plasma membranes of crayfish striated muscle

Na+-Ca2+ exchange rates and some physico-chemical properties of the exchanger were studied in crayfish striated muscle membranes enriched in plasma membranes prepared by differential centrifugation of muscle microsomal fraction on discontinuous sucrose density gradient. The lightest subfraction with the highest Na+, K+-ATPase and Mg2+-ATPase activities also showed the highest Na+-Ca2+ exchange rates. A number of physico-chemical characteristics of the Na+-Ca2+ exchanger found in the present experiments were similar to those reported for excitable membranes of mammals, except for the temperature optimum (20 degrees C for the crayfish).     

Differential expression of sodium channel activities during the development of chick skeletal muscle cells in culture

The expression of Na+ channels during differentiation of cultured embryonic chick skeletal muscle cells was investigated using saxitoxin (STX) and batrachotoxin (BTX), which previously have been shown to interact with distinct, separate receptor sites of the voltage-sensitive Na+ channel of excitable cells. In the present study, parallel measurements of binding of [3H]-STX (STX) and of BTX-activated 22Na+ uptake (Na influx) were made in order to establish the temporal relationship of the appearance of these two Na+ channel activities during myogenesis. Na influx was clearly measurable in 2-d cells; from day 3 to day 7 the maximum Na influx approximately doubled when measured with saturating BTX concentrations potentiated by Leiurus scorpion toxin, while the apparent affinity of BTX, measured without scorpion toxin, also increased. Saturable STX binding did not appear consistently until day 3; from then until day 7 the STX binding capacity increased about threefold, whereas the equilibrium dissociation constant (KD) decreased about fourfold. Although Na influx in cells of all ages was totally inhibited by STX or tetrodotoxin (TTX) at 10 microM, lower concentrations (2-50 nM) blocked the influx in 7-d cells much more effectively than that in 3-d cells, where half the flux was resistant to STX at 20-50 nM. Similar but smaller differences characterized the block by TTX. In addition, when protein synthesis is inhibited by cycloheximide, both Na influx and STX binding activities disappear more rapidly in 3-d than in 7-d cells, which shows that these functions are less stable metabolically in the younger cells.     

Ion conductances related to development of repetitive firing in mouse retinal ganglion neurons in situ

ABSTRACT: In the retina, the ability to encode graded depolarizations into spike trains of variable frequency appears to be a specific property of retinal ganglion neurons (RGNs). To deduce the developmental changes in ion conductances underlying the transition from single to repetitive firing, patch-clamp recordings were performed in the isolated mouse retina between embryonic day 15 (E15) and postnatal day 5 (P5). Immature neurons of the E15 retina were selected according to their capacity to generate voltage-activated Na+ currents (I(Na)(v)). Identification of P5 RGNs was based on retrograde labeling, visualization of the axon, or the amplitude of I(Na)(v). At E15, half of the cells were excitable but none of them generated more than one spike. At P5, all cells were excitable and a majority discharged in tonic fashion. Ion conductances subserving maintenance of repetitive discharge were identified at P5 by exposure to low extracellular Ca2+, Cd2+, and charybdotoxin, all of which suppressed repetitive discharge. omega-Conotoxin GVIA and nifedipine had no effect. We compared passive membrane properties and a variety of voltage-activated ion channels at E15 and P5. It was found that the density of high voltage-activated (HVA) Ca2+ currents increased in parallel with the development of repetitive firing, while the density of Ni2+-sensitive low voltage-activated (LVA) Ca2+ currents decreased. Changes in density and activation kinetics of tetrodotoxin-sensitive Na+ currents paralleled changes in firing thresholds and size of action potentials, but seemed to be unrelated to maintenance of repetitive firing. Densities of A-type K+ currents and delayed rectifier currents did not change. The results suggest that HVA Ca2+ channels, and among them a toxin-resistant subtype, are specifically engaged in activation of Ca2+-sensitive K+ conductance and thereby account for frequency coding in postnatal RGNs.     

Membrane excitability expressed in human neuroblastoma cell hybrids

The voltage-sensitive Na+ channel is responsible for the action potential of membrane electrical excitability in neuronal tissue. Three methods were used to demonstrate the presence of neurotoxin-responsive Na+ channels in two hybrid cell lines resulting from the fusion of excitable human neuroblastoma cells with mouse fibroblasts. Only one of the two electrically active hybrid cell lines maintained the sensitivity of the neuroblastoma parent to tetrodotoxin (TTX). The other hybrid, although electrically active, was not responsive to TTX or scorpion venom. Comparisons of the patterns of expression of membrane excitability and of chromosome complements in these human neuroblastoma cell hybrids suggest that the phenotype of membrane excitability is composed of genetically distinct elements.     

Functions of erg K+ channels in excitable cells

Ether-à-go-go-related gene (erg) channels are voltage-dependent K+ channels mediating inward-rectifying K+ currents because of their peculiar gating kinetics. These characteristics are essential for repolarization of the cardiac action potential. Inherited and acquired malfunctioning of erg channels may lead to the long QT-syndrome. However, erg currents have also been recorded in many other excitable cells, like smooth muscle fibres of the gastrointestinal tract, neuroblastoma cells or neuroendocrine cells. In these cells erg currents contribute to the maintenance of the resting potential. Changes in the resting potential are related to cell-specific functions like increase in hormone secretion, frequency adaptation or increase in contractility.     

Perturbation of glycoprotein processing affects the neurotoxin-responsive Na+ channel in neuroblastoma cells

The activity of neurotoxin-responsive Na+ channels in mouse neuroblastoma cells, N-18, was examined after treating the cells with compounds that are reported to perturb intracellular traffic. The compounds used have been shown to either alter glycoprotein synthesis and processing, (swainsonine, castanospermine, monensin, and retinoic acid) or receptor mediated endocytosis (mevinolin, 7-ketocholesterol, and chloroquine), or both. All of these compounds inhibited the activity of the neurotoxin-responsive Na+ channel with the exception of retinoic acid which increased the activity. Na+ channel activity was measured by two methods: (a) In vivo, the efflux of 86Rb was measured by use of the cells in monolayer culture, and (b) in vitro, the flux of 86Rb was measured from artificial phospholipid vesicles containing the partially purified Na+ channel. In both cases, 86Rb flux responded to stimulating neurotoxins, veratridine and scorpion venom, and was inhibited by tetrodotoxin as characteristic of excitable membranes. One of the perturbing compounds, swainsonine, was examined in detail. Treatment of N-18 cells with 10 microM swainsonine for 24 h markedly reduced the activity of the neurotoxin-responsive Na+ channel, as shown by the neurotoxin-stimulated efflux of 86Rb in vivo. In addition, after reconstitution into phospholipid vesicles of the partially purified Na+ channel from swainsonine-treated cells, reduced 86Rb flux was observed when compared with that of nontreated cells. Furthermore, the activity was not recovered in other less purified fractions. A comparison of the glycopeptides from the treated and nontreated cells by size, charge, and lectin-binding affinities was consistent with the formation of hybrid oligosaccharides after swainsonine treatment. It is concluded that the oligosaccharide residues of the Na+ channel glycoprotein must be processed to the mature complex-type for full activity. The stimulation of channel activity by treatment with retinoic acid supported this conclusion.     

Ontogenic appearance of Na+ channels characterized as high affinity binding sites for tetrodotoxin during development of the rat nervous and skeletal muscle systems

The appearance of the voltage-dependent Na+ channel during the fetal and post-natal development of rat brain, cerebellum and skeletal muscle has been followed using a highly radiolabelled derivative of tetrodotoxin. The number of Na+ channels is low at the fetal stage and increases drastically during post-natal development. The time-course of this increase is different in brain, cerebellum and skeletal muscle. Changes in affinity of the Na+ channel for tetrodotoxin occur during brain and cerebellum development. The results are discussed in relation with the maturation of the three types of excitable tissues.     

Resting Membrane Potentials Recorded On-site in Intact Skeletal Muscles from Deep Sea Fish (Sigmops gracile) Salvaged from Depths up to 1.000 m

The effect of elevated ambient pressures in deep sea fish residing at certain bottom depths or even covering different depth levels during migration is poorly understood. Elevated pressures are known to influence membrane properties of various excitable tissues in many species. Reliable results on membrane properties require freshly isolated living cells and short decompression times. During a scientific cruise south of Japan, deep sea fish were sampled from depths up to 1.000 m by using the intelligent operative net sampling system IONESS. On-site electrophysiological recordings of resting membrane potentials were performed in freshly isolated skeletal muscles from Sigmops gracile. Experiments were conducted at various extracellular K+ concentrations to derive relative membrane ion permeabilities and estimate intracellular K+ concentrations [K+]i in the muscles studied. With increasing sampling depth, a tendency for depolarized resting membrane potentials was observed. This could be explained by an increase in relative Na+ over K+ resting membrane permeabilities. Fish samples from deeper sites also had larger [K+]i values compared with shallower sites. This study represents a first approach to perform sophisticated physiological live-cell experiments on board a fully operating ship. These data are expected to more realistically reflect the physiological state of biological preparations residing in the deep sea.