Functional properties of rat brain sodium channels lacking the beta 1 or beta 2 subunit

The sodium channel purified from rat brain is a heterotrimeric complex of alpha (Mr 260,000), beta 1 (Mr 36,000), and beta 2 (Mr 33,000) subunits. alpha and beta 2 are attached by disulfide bonds. Removal of beta 1 subunits by incubation in 1.0 M MgCl2 followed by reconstitution into phospholipid vesicles yielded a preparation of alpha beta 2 which did not bind [3H]saxitoxin, mediate veratridine-activated 22Na+ influx, or bind the 125I-labeled alpha-scorpion toxin from Leiurus quinquestriatus (LqTx). In contrast, removal of beta 2 subunits by reduction of disulfide bonds with 1.5 mM dithiothreitol followed by reconstitution into phospholipid vesicles yielded a preparation of alpha beta 1 that retained full sodium channel function. Alpha beta 1 bound [3H]saxitoxin with a KD of 4.1 nM at 36 degrees C. It mediated veratridine-activated 22Na+ influx at a comparable initial rate as intact sodium channels with a K0.5 for veratridine of 46 microM. Tetracaine and tetrodotoxin blocked 22Na+ influx. Like intact sodium channels, alpha beta 1 bound 125I-LqTx in a voltage-dependent manner with a KD of approximately 6 nM at a membrane potential of -60 mV and was specifically covalently labeled by azidonitrobenzoyl 125I-LqTx. When incorporated into planar phospholipid bilayers, alpha beta 1 formed batrachotoxin-activated sodium channels of 24 pS whose voltage-dependent activation was characterized by V50 = -110 mV and an apparent gating charge of 3.3 +/- 0.3. These results indicate that beta 2 subunits are not required for the function of purified and reconstituted sodium channels while a complex of alpha and beta 1 subunits is both necessary and sufficient for channel function in the purified state.     

未成熟钠通道调控大鼠胚胎神经干细胞分化

钠通道在各类神经元上高表达,参与细胞多种生理功能的调节,是神经元实现功能活动的基本单位．未成熟神经元上钠/钙通道所诱发和自发的电位活动对后期的发育成熟至关重要．然而,发育中的钠通道是否参与神经干细胞(neural stem cells, NSCs)分化的调控尚不清楚．本研究证明,未成熟的钠通道参与NSCs分化调控．Western印迹结果显示,在分化第1,3,5,7 d的NSCs上钠通道和胞外信号调节激酶（ERK）的蛋白表达与分化时间正相关.免疫组化结果发现,与对照组比较,加入电压门控钠通道阻断剂TTX可明显下调NeuN、GFAP和Gal-c在NSCs中的表达（P＜0.05）,提示钠通道参与NSCs分化的调控.当采用veratridine激动钠通道后,激光共聚焦检测到细胞内Ca2+浓度明显升高,免疫组化和Western印迹结果显示细胞内Ca2+浓度明显升高,p-ERK表达量明显上调;相反,TTX可明显阻断Veratridine所引起的细胞内Ca2+浓度上调,并使p-ERK峰值明显降低和延后（P＜0.05）．研究结果表明,未成熟钠通道可通过激活ERK信号途径促进NSCs的分化．钠通道的这种作用可能是由钙离子介导的,其详尽机制有待进一步研究．     

Reconstitution of the voltage-sensitive sodium channel of rat brain from solubilized components

The voltage-sensitive sodium channel of rat brain synaptosomes was solubilized with sodium cholate. The solubilized sodium channel migrated on a sucrose density gradient with an apparent S20,w of approximately 12 S, retained [3H]saxitoxin ([3H]STX) binding activity that was labile at 36 degrees C but no longer bound 125I-labeled scorpion toxin (125I-ScTX). Following reconstitution into phosphatidylcholine vesicles, the channel regained 125I-ScTX binding and thermal stability of [3H]STX binding. Approximately 50% of the [3H]STX binding activity and 58% of 125I-ScTX binding activity were recovered after reconstitution. The reconstituted sodium channel bound STX and ScTX with KD values of 5 and 10 nM, respectively. Under depolarized conditions, veratridine enhanced the binding of 125I-ScTX with a K0.5 of 20 microM. These KD and K0.5 values are similar to those of the native synaptosome sodium channel. 125I-ScTX binding to the reconstituted sodium channel, as with the native channel, was voltage dependent. The KD for 125I-ScTX increased with depolarization. This voltage dependence was used to demonstrate that the reconstituted channel transports Na+. Activation of sodium channels by veratridine under conditions expected to cause hyperpolarization of the reconstituted vesicles increased 125I-ScTX binding 3-fold. This increased binding was blocked by STX with K0.5 = 5 nM. These data indicate that reconstituted sodium channels can transport Na+ and hyperpolarize the reconstituted vesicles. Thus, incorporation of solubilized synaptosomal sodium channels into phosphatidylcholine vesicles results in recovery of toxin binding and action at each of the three neurotoxin receptor sites and restoration of Na+ transport by the reconstituted channels.     

Excessive release of [3H] noradrenaline by veratridine and ischemia in spinal cord

In this study, the properties of ischemic condition-induced and veratridine-evoked [3H]noradrenaline ([3H]NA) release from rat spinal cord slices were compared. It was expected that ischemia mimicked by oxygen and glucose deprivation results in the impairment of Na+/K+ -ATPase with a consequent elevation of the intracellular Na+ -level which reverses the NA carrier and promotes excessive NA release, and veratridine, by the activation of Na+ channels, releases NA both carrier-mediated and Ca2+ -dependent, i.e. vesicular manner. In our experiments, veratridine (1-100 microM) dose-dependently increased the resting [3H]NA release, and its effect was only partially blocked by low temperature or the lack of external calcium, whereas the sodium channel inhibitor tetrodotoxin (TTX, 1 microM) completely prevented it, indicating that veratridine induces NA release via axonal depolarization and reversing the transporters by eliciting Na+ -influx. In contrast to TTX, the local anesthetic lidocaine (100 microM) only partially blocked the veratridine-induced [3H]NA release due to its inhibitory action on K+ channels. The ischemia-induced [3H]NA release was abolished at 12 degrees C, a temperature known to block only the transporter-mediated release of transmitters. However, lidocaine was also partially effective to reverse the action of ischemia on the NA release, indicating that lidocaine is not a useful compound in the treatment of spinal cord-injured patients against the excessive excytotoxic NA release.     

Developmental Appearance of Sodium Channel Subtypes in Rat Skeletal Muscle Cultures

22Na influx was measured in the established muscle cell line L-6 and in primary rat skeletal muscle cultures following activation of sodium channels by veratridine and sea anemone toxin II. Inhibition of the activated channels by tetrodotoxin (TTX) was analyzed with computer-assisted fits to one- or two-site binding models. In L-6 cultures, two inhibitable sodium channel populations were resolved at all ages in culture: a TTX-sensitive (K = 0.6-5.0 X 10(-8) M) and an insensitive population (Ki = 3.3-4.9 X 10(-6) M). In primary rat muscle cultures, the sensitivity of the toxin-stimulated channels to TTX changed with time in culture. In 4-day-old cultures, a single sodium channel population was detected using TTX (Ki = 2.4 X 10(-7)M). A single population was also found in 6-day-old cultures (Ki = 5.3 X 10(-7) M). By day 7 in culture, the inhibition of 22Na influx by TTX could be resolved into two components with high- and low-affinity sites for the toxin (Ki = 1.3 X 10(-9) M and 9.6 X 10(-7) M). We conclude that a single, toxin-activated sodium channel population with low affinity for TTX exists at early stages, whereas a second, high-affinity population evolves with time in primary rat muscle cultures. The expression of a high-affinity site apparently does not require ongoing neuronal involvement and may reflect an intrinsic property of the muscle cells.     

Internalization of Voltage-Dependent Sodium Channels in Fetal Rat Brain Neurons: A Study of the Regulation of Endocytosis

Abstract: In fetal rat brain neurons, activation of voltage-dependent Na⁺ channels induced their own internalization, probably triggered by an increase in intracellular Na⁺ level. To investigate the role of phosphorylation in internalization, neurons were exposed to either activators or inhibitors of cyclic AMP- and cyclic GMP-dependent protein kinases, protein kinase C, and tyrosine kinase. None of the tested compounds mimicked or inhibited the effect of Na⁺ channel activation. An increase in intracellular Ca²⁺ concentration induced either by thapsigargin, a Ca²⁺-ATPase blocker, or by A23187, a Ca²⁺ ionophore, was unable to provoke Na⁺ channel internalization. However, Ca²⁺ seems to be necessary because both neurotoxin- and amphotericin B-induced Na⁺ channel internalizations were partially inhibited by BAPTA-AM. The selective inhibitor of Ca²⁺/calmodulin-dependent protein kinase II, KN-62, caused a dose-dependent inhibition of neurotoxin-induced internalization due to a blockade of channel activity but did not prevent amphotericin B-induced internalization. The rate of increase in Na⁺ channel density at the neuronal cell surface was similar before and after channel internalization, suggesting that recycling of internalized Na⁺ channels back to the cell surface was almost negligible. Pretreatment of the cells with an acidotropic agent such as chloroquine prevented Na⁺ channel internalization, indicating that an acidic endosomal/lysosomal compartment is involved in Na⁺ channel internalization in neurons.     

Regulation of the (Na+ + K+)-ATPase in cultured chick skeletal muscle. Modulation of expression by the demand for ion transport

The levels of (Na+ + K+)-ATPase expression during muscle development and in response to modulation of demand for ion transport were studied in chick skeletal muscle cells in culture. The number of (Na+ + K+)-ATPase molecules on the myogenic cell surface, quantified with 125I-labeled monoclonal antibodies, increased 20-fold during muscle differentiation, with a substantial increase in (Na+ + K+)-ATPase molecules/unit area of membrane. The demand for sodium ion transport by the (Na+ + K+)-ATPase was modulated by activating voltage-sensitive sodium channels with veratridine or exposing cultures to low [K+]o (0.5 mM). Exposure to veratridine (10 microM) resulted in a 60-100% increase in cell surface and a smaller increase in intracellular (Na+ + K+)-ATPase over a 24-36-h period. Neither high [K+]o (50 mM) nor Ca2+ ionophore A23187 (1 microM) produced any such change, suggesting that neither membrane depolarization nor elevated cytosolic calcium was mediating the effect of veratridine. Veratridine stimulated up-regulation was specific for the (Na+ + K+)-ATPase, blocked by tetrodotoxin, and completely reversible. The kinetics of the reversal (down-regulation) process were much faster (t1/2 = 3 h) than those of up-regulation (t1/2 = 18 h). Up-regulation of the (Na+ + K+)-ATPase by veratridine occurred by a combination of two mechanisms: the first an early phase involving a stimulated biosynthesis of the (Na+ + K+)-ATPase and a later phase in which the biosynthetic rate returned to approximately control levels while the degradation rate slowed (t1/2 control = 31 h, t1/2 veratridine = 64 h).     

Dihydropiridines Mechanism of Action in Striatal Isolated Nerve Endings: Comparison with ω-Agatoxin IVA

The relative contribution of Ca2+ and Na+ channels to the mechanism underlying the action of the dihydropiridines (DHPs), nimodipine, nitrendipine and nifedipine was investigated in rat striatum synaptosomes. The rise in internal Ca2+ (Ca(i), as determined with fura-2) induced by high K+ was unchanged by the DHPs, which like tetrodotoxin (TTX) inhibited both the rise in internal Na+ (Na(i), as determined with the Na+ selective indicator dye, SBFI) and the rise in Ca(i) induced by veratridine. Nimodipine and nitrendipine were much more potent than nifedipine. Oppositely to TTX and to the DHPs, the P/Q type Ca2+ channel blocker, omega-agatoxin IVA did not inhibit the rise in Ca(i) induced by veratridine, but inhibited the rise in Ca(i) induced by high K+. Veratridine-evoked release of dopamine, GABA, Glu, and Asp (detected by HPLC) was inhibited by nimodipine, nitrendipine, and TTX, while high K+-evoked release was unchanged by the DHPs or TTX. It is concluded that the reduction in presynaptic Na+ channel permeability might contribute to the cerebral effects of DHPs.     

Biochemical properties and subcellular distribution of an N-type calcium channel alpha 1 subunit.

A site-directed anti-peptide antibody, CNB-1, that recognizes the alpha 1 subunit of rat brain class B calcium channels (rbB) immunoprecipitated 43% of the N-type calcium channels labeled by [125I]omega-conotoxin. CNB-1 recognized proteins of 240 and 210 kd, suggesting the presence of two size forms of this alpha 1 subunit. Calcium channels recognized by CNB-1 were localized predominantly in dendrites; both dendritic shafts and punctate synaptic structures upon the dendrites were labeled. The large terminals of the mossy fibers of the dentate gyrus granule neurons were heavily labeled, suggesting that the punctate labeling pattern represents calcium channels in nerve terminals. The pattern of immunostaining was cell specific. The cell bodies of some pyramidal cells in layers II, III, and V of the dorsal cortex, Purkinje cells, and scattered cell bodies elsewhere in the brain were also labeled at a low level. The results define complementary distributions of N- and L-type calcium channels in dendrites, nerve terminals, and cell bodies of most central neurons and support distinct functional roles in calcium-dependent electrical activity, intracellular calcium regulation, and neurotransmitter release for these two channel types.     

Activation of the Voltage-Sensitive Sodium Channel by a β-Scorpion Toxin in Rat Brain Nerve-Ending Particles

Neurotoxins purified from scorpion venoms previously had been divided into two classes according to their binding properties in rat brain synaptosomes. However, the pharmacological action of beta-scorpion toxin (beta-ScTx) on this preparation has not yet been described. In this report we show that a beta-ScTx induced an increase in 22Na+ uptake through synaptosomal voltage-sensitive sodium channels since this stimulation was abolished by tetrodotoxin (TTX). The increase was smaller than with veratridine and no synergy was observed between beta-ScTx and veratridine, as is the case for alpha-scorpion toxin (alpha-ScTx) and veratridine. The effects of alpha- and beta-ScTx were additive and the concentration-effect curves for each type of toxin were not modified by the other, suggesting that these two types of toxins act through distinct and noninteracting receptor sites. This was confirmed by the absence of mutual modification of the equilibrium and kinetic binding properties. beta-ScTx was shown to inhibit the uptake and to stimulate the release of [3H]gamma-aminobutyric acid. These effects were blocked by TTX, and no synergy was observed with veratridine. It was concluded that all these effects are mediated by the activation of voltage-sensitive sodium channels induced by the binding of beta-ScTx to a receptor site (site 4) distinct from those for other neurotoxins acting on sodium channels.     

Minimal sodium channel pore consisting of S5-P-S6 segments preserves intracellular pharmacology

We studied the properties of a sodium channel comprised only of S5-P-S6 region of the rat sodium channel alpha-subunit Nav1.4 (micro1pore). Results obtained in HEK cell lines permanently transfected with the sodium channel alpha-subunit or with the micro1pore were compared with data of the native HEK cells. Sodium channel blockers, tetrodotoxin and tetracaine, protect cells transfected with the complete sodium channel against death produced by incubation with veratridine. Veratridine-induced cell death in cell lines expressing the micro1pore construct is antagonised by tetracaine, but not by tetrodotoxin. Whole-cell conductance also increases in the presence of veratridine in micro1pore transfected cells and tetracaine inhibits these currents. Our pharmacological and electrophysiological data suggest that micro1pore keeps binding sites for veratridine and tetracaine, but not for TTX, and reconstitutes the permeation pathway for Na+ ions.     

Reconstitution of neurotoxin-stimulated sodium transport by the voltage-sensitive sodium channel purified from rat brain

Incorporation of the saxitoxin receptor of the sodium channel solubilized with Triton X-100 and purified 250-fold from rat brain into phosphatidylcholine vesicles is described. Fifty to 80% of the saxitoxin receptor sites are recovered in the reconstituted vesicles (KD = 3 nM). Unlike the detergent-solubilized saxitoxin receptor, the reconstituted saxitoxin binding activity is stable to incubation at 36 degrees C. Approximately 75% of the reconstituted saxitoxin receptor sites are externally oriented and 25% are inside-out. The initial rate of 22Na+ uptake into reconstituted vesicles is increased up to 3- to 4-fold by veratridine with a K0.5 of 11 microM. Seventy per cent of this increase is blocked by external tetrodotoxin (TTX) with a Ki of 10 nM. All of the veratridine-stimulated 22Na+ uptake is blocked when TTX is present on both sides of the vesicle membrane, or when tetracaine is added to the external medium. The apparent binding constants for veratridine, saxitoxin, and TTX are essentially identical to those in intact rat brain synaptosomes. The results demonstrate reconstitution of sodium transport, as well as neurotoxin binding and action, from substantially purified sodium channel preparations.     

Internalization of insulin receptors in the isolated rat adipose cell. Demonstration of the vectorial disposition of receptor subunits

The internalization of the insulin receptor in the isolated rat adipose cell and the spatial orientation of the alpha (Mr = 135,000) and beta (Mr = 95,000) subunits of the receptor in the plasma membrane have been examined. The receptor subunits were labeled by lactoperoxidase/Na125I iodination, a technique which side-specifically labels membrane proteins in intact cells and impermeable membrane vesicles. Internalization was induced by incubating cells for 30 min at 37 degrees C in the presence of saturating insulin. Plasma, high density microsomal (endoplasmic reticulum-enriched), and low density microsomal (Golgi-enriched) membrane fractions were prepared by differential ultracentrifugation. Receptor subunit iodination was analyzed by immunoprecipitation with anti-receptor antibodies, sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and autoradiography. When intact cells were surface-labeled and incubated in the absence of insulin, the alpha and beta receptor subunits were clearly observed in the plasma membrane fraction and their quantities in the microsomal membrane fractions paralleled plasma membrane contamination. Following receptor internalization, however, both subunits were decreased in the plasma membrane fraction by 20-30% and concomitantly and stoichiometrically increased in the high and low density microsomal membrane fractions, without alterations in either their apparent molecular size or proportion. In contrast, when the isolated particulate membrane fractions were directly iodinated, both subunits were labeled in the plasma membrane fraction whereas only the beta subunit was prominently labeled in the two microsomal membrane fractions. Iodination of the subcellular fractions following their solubilization in Triton X-100 again clearly labeled both subunits in all three membrane fractions in identical proportions. These results suggest that 1) insulin receptor internalization comprises the translocation of both major receptor subunits from the plasma membrane into at least two different intracellular membrane compartments associated, respectively, with the endoplasmic reticulum and Golgi-enriched membrane fractions, 2) this translocation occurs without receptor loss or alterations in receptor subunit structure, and 3) the alpha receptor subunit is primarily, if not exclusively, exposed on the extracellular surface of the plasma membrane while the beta receptor subunit traverses the membrane, and this vectorial disposition is inverted during internalization.     

NMDA receptor dependent and independent components of veratridine toxicity in cultured cerebellar neurons are prevented by nanomolar concentrations of terfenadine

Summary. Exposure of cultured neurons to nanomolar concentrations of terfenadine prevented the NMDA receptor-mediated early appearance (30 min.) of toxicity signs induced by the voltage sensitive sodium channel activator veratridine. Terfenadine also provided an histamine-insensitive protection against delayed neurotoxicity by veratridine (24 h), occurring independently of NMDA receptor activation, while not protecting from excitotoxicity following direct exposure of neurons to glutamate. Terfenadine reduced tetrodotoxin-sensitive inward currents, and reduced intracellular cGMP formation following veratridine exposure. Our data suggest that nanomolar concentrations of TEF may reduce excitatory aminoacid release following neuronal depolarization via a presynaptic mechanism involving voltage sensitive sodium channels, and therefore may be considered as a prototype for therapeutic drugs in the treatment of diseases that involve excitatory aminoacid neurotransmission. Received August 31, 1999 Accepted September 20, 1999     

Identification of an intracellular domain of the sodium channel having multiple cAMP-dependent phosphorylation sites

Cyclic AMP-dependent protein kinase catalyzes the incorporation of 3-4 mol of phosphate into the alpha subunit of rat brain sodium channels in vitro or in situ. Digestion of phosphorylated sodium channels with CNBr yielded three major phosphorylated fragments of 25, 31, and 33 kDa. These fragments were specifically immunoprecipitated with site-directed antisera establishing their location within an intracellular loop between the first and second homologous domains containing residues 448 to 630 of sodium channel RI or residues 450-639 of sodium channel RII. Five of the seven major tryptic phosphopeptides generated from intact sodium channel alpha subunits were contained in each of the 25-, 31-, and 33-kDa CNBr fragments, indicating that most cAMP-dependent phosphorylation sites are in this domain. Since CNBr digestion of sodium channels which had been metabolically labeled with 32P in intact neurons yielded the same phosphorylated fragments, the phosphorylated region we have identified is the major location of phosphorylation in situ. Only serine residues were phosphorylated by cAMP-dependent protein kinase in vitro, while approximately 16% of the phosphorylation in intact neurons was on threonine residues that must lie outside the domain we have identified. Since this domain is phosphorylated in intact neurons, our results show that it is located on the intracellular side of the plasma membrane. These results are considered with respect to models for the transmembrane orientation of the alpha subunit.     

Genetic basis of tetrodotoxin resistance in pufferfishes

Tetrodotoxin (TTX) is a highly potent neurotoxin that selectively binds to the outer vestibule of voltage-gated sodium channels. Pufferfishes accumulate extremely high concentrations of TTX without any adverse effect. A nonaromatic amino acid (Asn) residue present in domain I of the pufferfish, Takifugu pardalis, Na v1.4 channel has been implicated in the TTX resistance of pufferfishes . However, the effect of this residue on TTX sensitivity has not been investigated, and it is not known if this residue is conserved in all pufferfishes. We have investigated the genetic basis of TTX resistance in pufferfishes by comparing the sodium channels from two pufferfishes (Takifugu rubripes [fugu] and Tetraodon nigroviridis) and the TTX-sensitive zebrafish. Although all three fishes contain duplicate copies of Na v1.4 channels (Na v1.4a and Na v1.4b), several substitutions were found in the TTX binding outer vestibule of the two pufferfish channels. Electrophysiological studies showed that the nonaromatic residue (Asn in fugu and Cys in Tetraodon) in domain I of Na v1.4a channels confers TTX resistance. The Glu-to-Asp mutation in domain II of Tetraodon channel Na v1.4b is similar to that in the saxitoxin- and TTX-resistant Na+ channels of softshell clams . Besides helping to deter predators, TTX resistance enables pufferfishes to selectively feed on TTX-bearing organisms.     

Functional characterization of sodium channel blockers by membrane potential measurements in cerebellar neurons: prediction of compound preference for the open/inactivated state

Voltage-gated sodium channel (VGSC) blockers are widely used in the therapy, but most currently available blockers have suboptimal profile. However, discovery of new drug candidates has been hampered by the lack of appropriate in vitro assays. We established a fluorometric, plate reader-based membrane potential assay for testing the inhibitory potency of various VGSC blocking drugs, using primary cultures of cerebellar neurons, and veratridine, as activator of VGSCs. Since inhibition was strongly dependent on the depolarizing effect of veratridine, the EC(80) value of veratridine was determined on each experimental day, and this concentration was used for drug testing. This strict control on agonist effect seems to improve the reliability of the dose-inhibition measurements with antagonists. Veratridine responses could be completely inhibited by tetrodotoxin (TTX, IC(50)=17 nM), consistent with the exclusive expression of TTX-sensitive VGSCs. A variety of compounds known to block sodium channels inhibited veratridine-induced membrane depolarization concentration-dependently. Furthermore, inhibitory potencies of drugs strongly depended on whether their administration preceded or followed veratridine application. Potency of lamotrigine, carbamazepine, phenytoin and lidocaine was approximately 10-fold higher when applied after a steady-state depolarization had been achieved by a supramaximal veratridine dose, compared with those from a different protocol, where cells were preincubated with the antagonists prior to veratridine application. On the contrary, there was only relatively small difference between the IC(50) values of GBR 12909 obtained from the two different protocols (0.51 microM versus 1.23 microM). In contrast with most sodium channel blockers, this compound lacks binding preference to inactivated channels. We suggest that comparison of the results obtained with a particular blocker in the pretreatment and post-treatment schedules may be suitable for drawing conclusions regarding the state-dependency of its action. Thus, relevant information can be obtained about the potential therapeutic utility of different drugs by applying non-electrophysiological methods.     

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.     

Hydrophobic properties of the beta 1 and beta 2 subunits of the rat brain sodium channel

Voltage-sensitive sodium channels purified from rat brain in functional form consist of a stoichiometric complex of three glycoprotein subunits, alpha of 260 kDa, beta 1 of 36 kDa, and beta 2 of 33 kDa. The alpha and beta 2 subunits are linked by disulfide bonds. The hydrophobic properties of these three subunits were examined by covalent labeling with the photoreactive hydrophobic probe 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine [( 125I]TID) which labels transmembrane segments in integral membrane proteins. All three subunits of the sodium channel were labeled by [125I]TID when the purified protein was solubilized in mixed micelles of Triton X-100 and phosphatidylcholine (4:1). The half-time for photolabeling was approximately 7 min consistent with the half-time of 9 min for photolysis of TID under our conditions. Comparable amounts of TID per mg of protein were incorporated into each subunit. Purified sodium channels reconstituted in phosphatidylcholine vesicles were also labeled by TID with comparable incorporation per mg of protein into all three subunits. The efficiency of photolabeling of the three subunits was reduced from 39 to 44% by a 2-fold expansion of the hydrophobic phase of the reaction mixture but was unaffected by a 2-fold expansion of the aqueous phase, confirming that the photolabeling reaction took place in the lipid phase of the vesicle bilayer. The hydrophobic properties of the sodium channel subunits were examined further using phase separation in the nonionic detergent Triton X-114. Under conditions in which beta 1 is dissociated from alpha, the beta 1 subunit was preferentially extracted into the Triton X-114 phase, and the disulfide-linked alpha beta 2 complex was retained in the aqueous phase. When the disulfide bonds between the alpha and beta 2 subunits were reduced with dithioerythritol, the beta 2 subunit was also preferentially extracted into the Triton X-100 phase leaving the free alpha subunit in the aqueous phase. A preparative method for isolation of the beta 1 and beta 2 subunits was developed based on this technique. Considered together, the results of our hydrophobic labeling and phase separation experiments indicate that the alpha, beta 1, and beta 2 subunits all have substantial hydrophobic domains that may interact with the hydrocarbon phase of phospholipid bilayer membranes. Since the alpha subunit is known to be a transmembrane protein with many potential membrane-spanning segments, we conclude that the beta 1 and beta 2 subunits are likely to also be integral membrane proteins with one or more membrane-spanning segments.(ABSTRACT TRUNCATED AT 400 WORDS)