Activity-induced internalization and rapid degradation of sodium channels in cultured fetal neurons.

A regulatory mechanism for neuronal excitability consists in controlling sodium channel density at the plasma membrane. In cultured fetal neurons, activation of sodium channels by neurotoxins, e.g., veratridine and alpha-scorpion toxin (alpha-ScTx) that enhance the channel open state probability induced a rapid down-regulation of surface channels. Evidence that the initial step of activity-induced sodium channel down-regulation is mediated by internalization was provided by using 125I-alpha-ScTx as both a channel probe and activator. After its binding to surface channels, the distribution of 125I-alpha-ScTx into five subcellular compartments was quantitatively analyzed by EM autoradiography. 125I-alpha-ScTx was found to accumulate in tubulovesicular endosomes and disappear from the cell surface in a time-dependent manner. This specific distribution was prevented by addition of tetrodotoxin (TTX), a channel blocker. By using a photoreactive derivative to covalently label sodium channels at the surface of cultured neurons, we further demonstrated that they are degraded after veratridine-induced internalization. A time-dependent decrease in the amount of labeled sodium channel alpha subunit was observed after veratridine treatment. After 120 min of incubation, half of the alpha subunits were cleaved. This degradation was prevented totally by TTX addition and was accompanied by the appearance of an increasing amount of a 90-kD major proteolytic fragment that was already detected after 45-60 min of veratridine treatment. Exposure of the photoaffinity-labeled cells to amphotericin B, a sodium ionophore, gave similar results. In this case, degradation was prevented when Na+ ions were substituted by choline ions and not blocked by TTX. After veratridine- or amphotericin B-induced internalization of sodium channels, breakdown of the labeled alpha subunit was inhibited by leupeptin, while internalization was almost unaffected. Thus, cultured fetal neurons are capable of adjusting sodium channel density by an activity-dependent endocytotic process that is triggered by Na+ influx.

Inositol phosphate regulation of voltage-dependent calcium channels in cerebellar granule neurons.

The effects of intracellularly applied inositol phosphates on voltage-dependent calcium channel currents were assessed in rat cerebellar neurons using the whole-cell recording configuration of the patch-clamp technique. Intraneuronal perfusion of 10 microM inositol 1,4,5-trisphosphate (IP3) increased the amplitude of currents elicited by depolarization from a holding potential of -40 mV. IP3 did not modify current activation, but shifted the steady-state inactivation curve toward more positive values. The dose-response curve indicated an EC50 of 0.5 microM for IP3. Inositol 1,3,4,5-tetrakisphosphate (IP4), but not inositol 4,5,-bisphosphate, mimicked the effect of IP3. The effect of IP3 persisted in the presence of 100 micrograms/ml heparin and did not depend on intracellular calcium mobilization, as similar responses were not produced by 10 mM caffeine or by intrapipette calcium buffering at pCa 6 instead of pCa 7.7. Preincubation with omega-conotoxin led to a 55% inhibition of barium current; however, inhibition was reversed by IP3, which reestablished the control current amplitude. These results imply that IP3 and IP4 can elicit calcium entry by modifying both the gating characteristics and the pharmacological properties of voltage-dependent calcium channels.

Sodium channel internalization in developing neurons.

Neurotoxin-induced activation of voltage-dependent Na+ channels provoked rapid (t1/2 = 15-20 min) channel down-regulation in cultured rat brain neurons, resulting in a 50%-70% decrease in [3H]saxitoxin and 125I-alpha-scorpion toxin binding capacities as well as a decrease in Na+ peak current. Experiments using 125I-alpha-scorpion toxin as both a Na+ channel activator and a surface channel probe showed that a fraction of the bound toxin was internalized, since it was not releasable by acidic washing. Internalization was inhibited by tetrodotoxin, abolished in Na(+)-free medium, and induced by amphotericin B, a Na+ ionophore. Moreover, down-regulation occurred only in immature neuronal tissue, either cultured fetal neurons or postnatal hippocampal slices, but was absent in adult brain. These observations indicate that Na+ channel internalization is triggered by Na+ influx into neurons and may be involved in the control of electrical activity during development.

Electrophysiological studies on embryonic heart cells in culture. Scorpion toxin as a tool to reveal latent fast sodium channel.

Trypsin-dispersed heart cells were obtained from 11-day-old chick embryos. After culture as unstirred suspensions in dimethylsulfoxide-containing medium, spherical aggregates of cells beating spontaneously and apparently synchronously for months were obtained. Two kinds of cell were characterized by electrophysiological recordings: (1) cells with a slow rate of depolarizing phase showing tetrodotoxin-resistant action potential and blocked by D 600 ('slow' cells); (2) cells with high value of rising phase which was strongly decreased by tetrodotoxin and in which D 600 provoked uncoupling of excitation-contraction ('fast' cells). Toxin II from Androctonus australis scorpion venom increased the duration of action potential, which was ascribed to a slowing down of Na+ current inactivation and enhance the maximum rate of depolarization, especially in slow cells. Effects were antagonized by tetrodotoxin in both fast and slow cells. Washing experiments confirmed the results of previous studies, namely that tetrodotoxin and scorpion toxin bind to different receptors. It is concluded that slow cells with tetrodotoxin-resistant action potential contain latent fast Na+ channels that are revealed (activated) by toxin binding to the membrane.

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.

Polypeptide components of the apamin receptor associated with a calcium activated potassium channel.

Photoaffinity labeling of rat brain membranes with [125I]ANPAA-apamin incorporated radioactivity into polypeptides of 86 and 59 kDa and occasionally a more weakly labeled component of 45 kDa. These polypeptides were immunoprecipitated with anti-apamin antibodies and treated with glycosidases. Neither the 86 nor the 59 kDa polypeptide appeared to be N-glycosylated. Partial proteolytic mapping of affinity labeled polypeptides with chymotrypsin or V8 protease generated an identical pattern. These results suggest that the 59 and 45 kDa components are not additional subunits of an oligomeric protein but result from cleavage of the 86 kDa polypeptide.

Activation of voltage-dependent sodium channels in cultured cerebellar poffule cells induces neurotoxicity that is not mediated by glutamate release.

Exposure of rat cerebellar granule cell cultures to neurotoxins that specifically enhance the open state probability of voltage-dependent Na+ channels, resulted in neuronal death as estimated by a cell viability assay based on fluorescent staining and 51Cr-uptake. Toxicity was detected within 1 h after addition of 100 microM veratridine and was complete within 10-18 h; it was dose-dependent and was found to be completely abolished by tetrodotoxin, an Na+ channel blocker. When veratridine was replaced by an alpha-scorpion toxin, similar observations were done. In contrast, when cultured neurons prepared ffom the cerebral hemisphere of fetal rat brain were exposed to either veratridine or alpha-scorpion toxin for 18 h or even for a longer time of incubation, no neuronal death was observed. DNA fragmentation analysis showed that the toxicity was not mediated by apoptosis. Neuronal death was neither prevented by glutamate receptor antagonists, nor by depletion of endogenous glutamate, nor by voltage sensitive calcium channel antagonists such as omega-Conotoxin-GVIA (N-type channels), omega-Agatoxin-IVA (P-type channels), nimodipine and nitrendipine (L-type channels). Our study indicates that prolonged opening of Na+ channels induced neuronal death of cerebellar granule cells which is not mediated by glutamate and reveals novel neurotoxic mechanism in addition to the well-established excitatory amino acid receptor pathway.

Thyrotropin-induced plasma membrane protein modifications in porcine thyroid cells.

Highly purified plasma membranes were obtained from isolated porcine thyroid cells maintained in conditions of culture in the presence of thyrotropin (stimulated cells) or in their absence (non-stimulated cells). Analyses of both types of membranes by high-resolution sodium dodecylsulfate-polyacrylamide slab gel electrophoresis showed reproducible quantitative differences in protein bands of apparent molecular weight 38,000, 36,000 and inconstantly 96,000. Phosphorylation of membranes by [gamma-32P]ATP was 2-3 times higher in membranes from thyrotropin-stimulated than in membranes from non-stimulated cells. About 20 32P-labeled bands were detected by slab gel electrophoresis in denaturing conditions, among which the catalytic subunit of Na+, K+ ATPase was characterized. In addition, plasma membranes from thyrotropin-stimulated cells contained a firmly bound [14C]glucosamine-containing glycoprotein probably related to an aggregation-promoting factor. 125I-labeled thyroglobulin and components of unknown nature were associated with plasma membranes from thyrotropin-stimulated cells. Whether they participate in the structure and function(s) of the plasma membrane or represent contaminants of the preparation is not clear at the present time.

Down-regulation of voltage-dependent sodium channels coincides with a low expression of alphabeta1 subunit complexes.

The association between the beta1 subunit and the alpha subunit of the sodium channel from rat brain was studied in hippocampus during postnatal development and in cultures of fetal rat forebrain neurons and cerebellar granule cells, using an anti-beta1 antipeptide antibody to specifically immunoprecipitate alphabeta1 complexes labeled with [3H]saxitoxin. In the hippocampus, the increase in beta1 RNA expression during development was accompanied by an increase in immunoprecipitated alphabeta1 complexes. Most of the alphabeta1 complexes were constituted during the first 3 postnatal weeks, with the steepest rise between postnatal days 5 and 12. In cultured fetal neurons, the amount of beta1 RNA and of alphabeta1 complexes was approximately 3-4% of that found in the adult, whereas it reached 60-70% in cultured cerebellar granule cells. We had previously described a neurotoxin-induced internalization of sodium channels which occurred in immature neurons but not in adult tissue. Internalization decreased during development in neurotoxin-treated hippocampal slices, and resistance of plasma membrane sodium channels to internalization followed the same time course than the appearance of alphabeta1 complexes. Similarly, neurotoxin activation resulted in sodium channel internalization in fetal neurons, while cerebellar granule cells, which express high levels of beta1 RNA and of alphabeta1 complexes, did not internalize their [3H]saxitoxin receptors in that same conditions. These data suggested that the association of the beta1 subunit with the alpha subunit could provide a suitable marker for the stabilization and anchoring of sodium channels in discrete membrane domains which occur during neuronal development.

Channel activators reduce the expression of sodium channel alpha-subunit mRNA in developing neurons.

The expression of rat brain sodium channel alpha-subunit (Na+I, Na+II and Na+III) and beta 1-subunit mRNAs was examined in rat fetal brain neurons in culture. A combined technique of reverse transcription and polymerase chain reaction (RT-PCR) was used. Two different PCR primer sets were designed to obtain simultaneous amplification of the three alpha-subunit mRNAs. All three molecules were detected in fetal neurons but the expression pattern (Na+III > Na+II > > Na+I) was different than that observed in adult tissue (Na+II > Na+I > Na+III). Expression of the beta 1-subunit mRNA was detected using a specific PCR primer set. Doublet bands were amplified, from fetal cells and adult brain mRNA. To get further insight into the molecular mechanism that underlie activity dependent plasticity of sodium channels, we studied the effect on the expression of sodium channel subunits mRNA of a 60 h incubation of cells in the presence of a scorpion neurotoxin that blocks channel inactivation. An overall decrease in the expression of all three alpha-subunit mRNAs was observed whereas the beta 1-subunit mRNA was unaffected by the same treatment. When cells were incubated with the scorpion neurotoxin together with tetrodotoxin, to block Na+ influx through channels, the decrease in mRNA expression was not observed. Finally, a 60 h continuous depolarization of cells induced by application of a high concentration KC1 solution did not mimic the effect of the scorpion toxin. These observations suggest that a persistent activation of the sodium channels is able to down-regulate mRNA expression for alpha-subunits but not for the beta 1-subunit.

Multiple pathways regulate the expression of genes encoding sodium channel subunits in developing neurons.

In primary cultures of fetal neurons, activation of sodium channels with either alpha-scorpion toxin or veratridine caused a rapid and persistent decrease of mRNAs encoding beta2 and different sodium channel alpha mRNAs. In contrast, beta1 subunit mRNA was up-regulated by sodium channel activation. This phenomenon was calcium-independent. The effects of activating toxins on mRNAs of different sodium channel subunits were mimicked by membrane depolarization. An important aspect of this study was the demonstration that cAMP also caused rapid reduction of alphaI, alphaII and alphaIII mRNA levels whereas beta1 subunit mRNA was up regulated and beta2 subunit mRNA was not affected. Sodium channel activation by veratridine was shown to increase cAMP immunoreactivity in cultured neurons, but alphaII mRNA down-regulation induced by activating toxins was not reversed by protein kinase A antagonists, indicating that this phenomenon is not protein kinase A dependent. The effects of cAMP and membrane depolarisation were antagonized by the PKA inhibitor H89. These results are indicative of the existence of multiple and independent regulatory pathways modulating the expression of sodium channel genes in the developing central nervous system.

Detection and photoaffinity labeling of the Ca2+-activated K+ channel-associated apamin receptor in cultured astrocytes from rat brain.

Apamin, an 18-amino acid bee venom peptide, is a specific blocker of a class of Ca2+ activated K+ channels. Mono 125I-iodoapamin was used to detect the K+ channel-associated receptor site in cultured astrocytes from rat brain. Specific high-affinity binding to intact glial cells with a Kd of about 90 pM at 1 degree C and pH 7.5 was demonstrated by equilibrium and kinetic methods. The average receptor capacity was 3 fmol/mg cell protein which is 2 to 3-fold lower than in primary cultured neurons. Binding was stimulated by K+ ions, but to a lesser extent than with neuronal receptors. Photoaffinity labeling of receptor/ion channel components using an arylazide derivative of 125I-monoiodoapamin revealed the presence of the 86- and 33-kDa polypeptides, previously detected in neurones. However a 59-kDa peptide which is present in synaptic membrane preparations from adult rat brain, but not in cultured neurons, was also clearly labeled in intact astrocytes. This indicates that the 59-kDa polypeptide is not a proteolytic fragment of the 86-kDa chain but an associated subunit which is only accessible to photolabeling in certain apamin receptor preparations. Apamin-sensitive Ca2+-activated K+ channels in astrocytes may be one of the pathways by which glial cells redistribute K+ in the central nervous system (CNS).

Quantitative analysis of fetal rat brain neurons developing in primary cultures. I. Stereological study of the neuronal differentiation.

An ultrastructural stereological analysis was performed to analyze the morphological differentiation of primary cultures of fetal rat brain neurons, growing for two weeks in a serum-free medium. The number of neurons and of gliofibrillary acidic protein (GFAP)-positive glial cells was estimated by light microscopy counting in the culture wells. These cultures provided a quasi-pure neuronal population, since the number of GFAP-positive glial cells was found to be 1% (day 7) and 2% (day 14) respectively of the total number of cultured cells. Cell counts and the stereological measurements were related to the surface area of the culture well. The neuronal differentiation was characterized by an increase in the plasma membrane surface area (x9) and volume (x8) of neurites, contrasting with the decrease in the perikarya surface area and volume. These primary stereological data were combined with the number of neurons to obtain parameters characterizing an average neuron. The increase in membrane surface area of an average neuron was found to be a linear function of time, 29 micron 2 and 445 micron 2 of new membrane being added per day of culture to perikarya and neurites respectively. The number of chemical synapses was also counted and compared to the changes in the plasma membrane surface area. After 7 days in vitro they increased in number more rapidly than the increase in the plasma membrane surface area of neurons.

Ultrastructural localization of voltage-sensitive sodium channels using [125I]alpha scorpion toxin.

The distribution of alpha scorpion toxin (alpha-ScTx) receptors was examined in differentiated mouse neuroblastoma cell cultures (N IE 115 clone) by electron microscope autoradiography using [125I]alpha-ScTx. This neurotoxin binds specifically to voltage-sensitive sodium channels, slowing down the inactivation of the sodium permeability. Quantitative analysis demonstrated that only plasma membranes were labelled. The alpha-ScTx receptors seemed to be randomly dispersed on both cell bodies and cell processes. Microvilli protruding from the cell bodies carried more sodium channels than other parts of the membrane. The specific binding site density for alpha-ScTx varied from 4 (cell body membrane) to 13 (cell process membrane) per square micrometer.

Early appearance of cells bearing Na+ channels in developing mouse brain. A quantitative analysis using light microscopic autoradiography.

125I-alpha-Scorpion toxin (alpha-ScTx) binds to a component of the voltage-sensitive Na+ channel. We have previously shown that receptor capacity on dissociated mouse brain cells increases between days 12 and 19 of fetal life as does the expression of neurotoxin-sensitive 22Na+ influx. In the present study we have investigated the distribution of Na+ channels at the cellular level. Quantitative analysis by light-microscopic autoradiography was carried out on dissociated brain cells labeled with 125I-alpha-ScTx at 13, 15 and 18 fetal days. We have shown that at day 13 a large population of cells (39% of total) is alpha-ScTx-labeled, providing direct confirmation for a wide-spread presence of Na+ channels at an early stage of mouse brain development. The subsequent increase in receptor number with age is due both to an increase in alpha-ScTx-labeled cells (to 53% and 97% at days 15 and 18, respectively) and to an increase in the receptor density on these cells (10.9, 12.7 and 34.5 silver grains/1000 microns2 of cell surface for the 3 stages studied).

High-affinity binding of alpha-scorpion toxin: a neuronal property.

alpha-Scorpion toxin binding to its receptor--one component of the voltage-sensitive sodium channel--was studied in an attempt to define its phenotypic specificity. To this end we investigated the ability of neuronal, glial myogenic and fibroblastic cell lines to bind alpha-toxin II, purified from venom of the scorpion Androctonus australis Hector. A single class of saturable high-affinity (Kd congruent to 1 nM) binding sites, was present only in cell lines exhibiting some of the characteristics of normal neuronal cells, such as the N18, NIE-115, NS20, BN10-10, NG108-15 and T28 cell lines. NIA-103, which is an electrically non-excitable neuronal cell, gave negative results. In glial (G26-20, TR6B, C6) myogenic (T984) or fibroblastic (L) cell lines, we were unable to detect high-affinity binding sites for alpha-scorpion toxin. Primary cultures of rat skeletal muscle cells were also negative. Thus specific binding in the nanomolar range seems to be selectively associated with the neuronal phenotype. alpha-Scorpion toxin binding was tested before and after induction of neurites: in N18, NIE-115, NS20 cell lines, the differentiation brought on an increase in the number of binding sites but had little effect on the dissociation constant; in the hybrids NG108-15 and T28 high affinity saturable binding sites were detectable after but not prior to morphological differentiation.

Ultrastructural visualization of Na+-channel associated [125I]alpha-scorpion toxin binding sites on fetal mouse nerve cells in culture.

Purified neurotoxin II from the scorpion Androctonus australis Hector (alpha-ScTx) has previously been shown to bind specifically to the voltage-sensitive Na+ channels of excitable cells. Recent studies, using high specific activity 125I-labeled alpha-ScTx, demonstrated specific binding to neuronal cells derived from fetal mouse brains. In the present study, 125I-labeled alpha-ScTx was used to localize the voltage-sensitive Na+ channels in cultured fetal mouse brain cells. By quantitative electron microscope autoradiography we demonstrate that specific alpha-ScTx binding sites are selectively located at the plasma membrane. Estimates of their density revealed that neurites at 13 days in vitro carry at least 6 X more specific alpha-ScTx sites than cell body membrane.

Tyrosine kinase inhibitors and cycloheximide inhibit Li+ protection of cerebellar granule neurons switched to non-depolarizing medium.

Recently, it has been shown that Li+ robustly enhances the survival of cerebellar granule neurons acutely switched to non-depolarizing medium after maturing in vitro, a condition which elicits massive apoptotic death in this cell type. Tyrosine protein phosphorylation is known to underlie the activity of a number of trophic factors. This prompted us to investigate whether specific tyrosine kinase inhibitors could modulate the Li+ protection of cultured granule neurons switched to non-depolarizing medium. Genistein and herbimycin A dose dependently abolished the Li+ effect. Furthermore, this effect was substantially prevented by the translational inhibitor cycloheximide, suggesting that it requires de novo protein synthesis. Overall, these results suggest that Li+ protection of cerebellar granule neurons switched to non-depolarizing medium involves tyrosine kinases and transcriptional activation.

Subtypes of voltage-sensitive calcium channels in cultured rat brain neurons.

Subtypes of voltage-sensitive calcium channels have been investigated in cultured rat brain neurons using two classes of specific probes, dihydropyridine compounds and omega-conotoxin. Membranes prepared from cultured neurons contain specific binding sites for [3H]PN200-110, a dihydropyridine antagonist, and for 125I-omega-conotoxin with a stoichiometry of about 1:1. A depolarization induced 45Ca2+ influx into intact brain neurons was partially inhibited by a dihydropyridine antagonist, nifedipine and stimulated by a dihydropyridine agonist, Bay K8644. This dihydropyridine sensitive 45Ca2+ flux was insensitive to omega-conotoxin at concentrations which saturate the specific toxin binding sites indicating that in cultured brain neurons, dihydropyridine-sensitive calcium channels are not sensitive to omega-conotoxin.

Omega-conotoxin sensitive calcium channels in cerebellar granule cells are not coupled to [3H]glutamate release.

We have studied the biochemical and functional aspects of omega-conotoxin GVIA (omega-CgTx)-sensitive calcium channels in cerebellar granule cells in vitro. 125I-omega-Conotoxin GVIA (125I-omega-CgTx) binding sites were detected in intact cultured cerebellar granule cells and binding parameters were measured (Bmax: 134 fmol/mg protein; kinetic association constant kappa: 3.10(6) M-1.s-1). [3H]Glutamate release was assessed under different release paradigms (namely release triggered by calcium, voltage, and sodium channel agonists) and different times (15 s and 2 min). However, in all cases, [3H]glutamate release was found to be completely insensitive to omega-CgTx. Conversely, voltage-dependent release was inhibited in a dose-dependent fashion by cadmium chloride, with total inhibition at 10(-4) M. These results indicate that N-type calcium channels are not involved in glutamate secretion from granule neurons.