The lobster nerve sodium channel: solubilization and purification of the tetrodotoxin receptor protein

Solubilization and purification of the tetrodotoxin (TTX) binding protein of the lobster walking-leg nerve Na+ channel were carried out utilizing [3H]tetrodotoxin [( 3H]tetrodotoxin) as a marker. The nerve membrane was solubilized with Lubrol-PX and the Na+ channel protein was purified with diethylaminoethyl Bio-Gel A, Bio-Gel hydroxylapatite powder and two Sepharose 6B columns. Care was taken to keep the temperature of the Na+ channel preparation as close to 1 degrees C as possible and to use solutions (pH 7.5) that contain Na channel protectors, i.e., egg phosphatidylcholine/Lubrol-PX mixture, TTX, EDTA, EGTA, phenylmethylsulfonyl fluoride, pepstatin A, iodoacetamide, antipain, phosphoramidon, soybean trypsin inhibitor, leupeptin and bacitracin. From an initial specific binding of 20.1 pmol of [3H]TTX/mg protein for the solubilized membrane, the binding increased to 1241 pmol/mg protein for the most active fraction of the last Sepharose 6B column. The [3H]TTX specific binding of the Sepharose 6B fractions correlated with a large peptide of Mr 260,000 (240-280K), although other peptides were also present in lesser amounts.     

Influence of sodium conductances on platelet activation

The effects of extracellular Na+ and tetrodotoxin on resting membrane potential, cytosolic free Ca2+ levels and aggregation of human platelets have been studied. Neither the decrease in extracellular Na+-concentration (from 140 mmol/l to 0 mmol/l) nor the addition of tetrodotoxin (10(-7) to 10(-5) mol/l) modified the platelet membrane potential. Zero extracellular Na+ concentration or the presence of tetrodotoxin in the medium inhibited platelet aggregation; however, K+-depolarized platelets showed an unchanged aggregation induced by ADP or thrombin in media with zero or low extracellular Na+ concentrations or in the presence of tetrodotoxin. Moreover, zero extracellular Na+ concentration or tetrodotoxin inhibited calcium mobilization in platelets during activation induced by thrombin. Hence, voltage-dependent activation linked to Na+ influx appears to be necessary for ADP- and thrombin-induced platelet aggregation under control conditions. Mechanisms for the role of Na+ conductances in platelet function are discussed.     

Tetrodotoxin, saxitoxin, chiriquitoxin: new perspectives on ionic channels

Chiriquitoxin is a new, natural analog of tetrodotoxin, differing only in having the -CH2OH on C-6 replaced with an unidentified group of 104 mass units. On isolated frog sartorius muscle fibers, chiriquitoxin is equipotent with tetrodotoxin in blocking the Na+ channel, as shown by their identical dose-response relations on the maximum rate of rise of the action potential. Chiriquitoxin additionally interferes with some K+ channels, as shown by a slowed repolarization of the action potential, a reduced steady-state membrane conductance in current-clamped fibers, and a reduced K+ current in point-voltage-clamped fibers. The effects of chiriquitoxin on the Na+ and K+ channels are apparently exerted by the same molecule because high concentration of tetrodotoxin can either prevent or reverse the effects of chiriquitoxin on the K+ channel. Therefore, the receptor for tetrodotoxin-chiriquitoxin is probably not located inside the Na+ channel, but is on the outside surface of the membrane close to the orifice of the Na+ channel. The results also suggest that the Na+ and K+ channels are probably not randomly distributed throughout the membrane, but occur in clusters with some definite spatial relation to each other. From the structure of tetrodotoxin and a presumed structure of chiriquitoxin, the Na+ and K+ channels are estimated to be separated from each other by not less than 5 nor much more than 15A. The receptor for saxitoxin may be different, but partially overlapping with that for tetrodotoxin-chiriquitoxin.     

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.     

Trypsin-induced masking of tetrodotoxin receptor of the sodium channels in mollusc neurones

At the early stage of trypsin treatment of mollusc neurones tetrodotoxin cannot block the Na⁺ current. In the course of further exposure of neurones to trypsin, tetrodotoxin-sensitivity is restored completely, so its temporal loss results from shielding rather than destruction of the tetrodotoxin-binding site. Pronase and papain do not affect the tetrodotoxin action on the Na⁺ current.     

Na+ channels with high and low affinity tetrodotoxin binding sites in the mammalian skeletal muscle cell. Difference in functional properties and sequential appearance during rat skeletal myogenesis

High and low affinity binding sites for tetrodotoxin have been found in rat skeletal muscle cells in vitro using a radiolabeled tetrodotoxin derivative and 22Na+ flux studies. High affinity binding sites for tetrodotoxin (KD(tetrodotoxin) = 1.6 nM) cannot be detected at the myoblast stage. They appear and increase in density as myoblasts fuse into myotubes to reach a maximum binding capacity of 50 fmol/mg of proteins. Na+ channel structures with a high affinity for tetrodotoxin cannot be activated by neurotoxins specific for the Na+ channel such as veratridine and sea anemone toxinII. They are not expressed in the action potential. Na+ channels with a low affinity for tetrodotoxin (IC50(tetrodotoxin) = 1 microM) are functional since they can be activated by veratridine and sea anemone toxinII. They are already expressed in myoblasts and their density is not modified during the fusion of myoblasts into myotubes; they remain functional throughout the differentiation process. It is suggested that neuronal factors are not required for the synthesis of structures with high affinity binding sites for tetrodotoxin in the rat muscle and that they are only involved for the maturation of these structures from a nonfunctional to a functional form.     

Synaptic effects of the synthetic pyrethroid resmethrin in rat brain in vitro

Resmethrin (30 microM) induced release of transmitters was not affected by manipulation of the Na+ current with either choline or tetrodotoxin agents which readily reversed the effects of veratridine, deltamethrin and cypermethrin. Resmethrin (I50: 2.2 microM) inhibited the ATP dependent uptake of Ca2+ but deltamethrin and cypermethrin were much less effective. Resmethrin also displaced Ca2+ from crude synaptosomal membranes. The release promoting effects of resmethrin in rat brain in vitro are better explained by its effects on Ca2+ rather than through a specific effect on the Na+ channel. In contrast, the effects of deltamethrin and cypermethrin promote transmitter release by a Na+ dependent process.     

Functional expression of the rat heart I Na+ channel isoform. Demonstration of properties characteristic of native cardiac Na+ channels

We describe the expression of functional Na+ channels in Xenopus oocytes injected with cRNA transcribed from the rat heart I cDNA clone. The expressed rat heart I Na+ currents show kinetic properties and resistance to tetrodotoxin and saxitoxin which are characteristic of native cardiac Na+ currents. The primary amino acid sequence of the rat heart I alpha-subunit is therefore sufficient for expression of tetrodotoxin resistance, and the rat heart I clone is likely to account for the tetrodotoxin-resistant phenotype of cardiac and denervated skeletal muscle.     

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.     

Pharmacological and Electrophysiological Characterization of Lithium Ion Flux Through the Action Potential Sodium Channel in Neuroblastoma × Glioma Hybrid Cells

Interaction of Li+ with the voltage-dependent Na+ channel has been analyzed in neuroblastoma X glioma hybrid cells. The cells were able to generate action potentials in media containing Li+ instead of Na+. The uptake of Li+ into the hybrid cells was investigated for the pharmacological analysis of Li+ permeation through voltage-dependent Na+ channels. Veratridine and aconitine increased the uptake of Li+ to the same degree (EC50 30 microM). This increase was blocked by tetrodotoxin (IC50 20 nM). Veratridine and aconitine did not act synergistically; however, the veratridine-stimulated influx was further enhanced by the toxin of the scorpion Leiurus quinquestriatus (EC50 0.06 micrograms/ml). This stimulation was also blocked by tetrodotoxin. Thus, the voltage-dependent Na+ channel of the hybrid cells accepts both Li+ and Na+ in a similar manner.     

The presence of Na+ channels in myometrial smooth muscle cells is revealed by specific neurotoxins

This paper shows the presence, in rat myometrial smooth muscles, of low affinity binding sites for tetrodotoxin with a K0.5 value of 2 microM. Electrophysiological experiments using both intact strips and single isolated myometrial cells in culture have shown that veratridine and sea anemone toxins reveal functional Na+ channels. The activity of these channels was blocked by tetrodotoxin (10 microM) or by removal of Na+ ions. Results presented here are the first direct demonstration of the existence in rat myometrium of Na+ channels of the tetrodotoxin-resistant type.     

The voltage-dependent Na+ channel of insect nervous system identified by receptor sites for tetrodotoxin, and scorpion and sea anemone toxins

Receptor sites for some of the most important toxins known to be specific for voltage-sensitive Na+ channel in the mammalian nervous system have been identified in a purified membrane preparation of house fly brain. Very high affinities have been found for the association of tetrodotoxin or tetrodotoxin derivatives with the insect Na+ channel (Kd = 0.03 - 0.08 nM). The gamma toxin from the Brazilian scorpion Tityus serrulatus forms a complex with the Na+ channel having a Kd of 6.1 pM. The Kd value for toxin II from the sea anemone Anemonia sulcata is 0.12 microM. These results show a high degree of conservation of the pharmacological properties of the brain Na+ channels between insects and mammals.     

Sensitivity of the resting sodium conductivity of leech Retzius nerve cells to tetrodotoxin

Simultaneously, the effect of sodium-free medium and tetrodotoxin (3 X 10(-8) M/ml) were investigated on some passive electrophysiological properties of leech Retzius nerve cells. Complete replacement of Na+ with Tris or addition of tetrodotoxin to the leech Ringer was followed by an increase of input resistance in contrast to the cell-to-cell interaction which was not affected by such a procedure. At the same time tetrodotoxin was not able to block repetitive spike activity. The data imply the existence of two types of Na+ channel in leech Retzius nerve cells.     

Use-dependent block of sodium channels in frog myelinated nerve by tetrodotoxin and saxitoxin at negative holding potentials

Na+ currents were measured in myelinated frog nerve fibres in the presence of nanomolar concentrations of tetrodotoxin (TTX) or saxitoxin (STX) in the extracellular solution. The Na+ currents declined during a train of depolarizing pulses if the fibre was held at hyperpolarizing potentials between the pulses. At a pulse frequency of 0.8 Hz, the peak Na+ currents were reduced to 70 or 60% of the initial value in 9.3 nM TTX and 3.5 nM STX solutions, respectively. A decline of Na+ currents was also observed in two-pulse experiments. The peak Na+ current during a second test pulse did not depend on the duration (0.2 to 12 ms) of the first pulse. It decreased with increasing interval between the pulses, reached a minimum and increased again. The results are interpreted with a use-dependent blockage of Na+ channels by TTX or STX at negative holding potentials. The effects were described quantitatively, assuming a fast affinity increase of toxin receptors at Na+ channels triggered by Na+ activation followed by slow toxin binding to channels and relaxation of the receptor affinity.     

Activation of the action potential Na+ ionophore of cultured neuroblastoma cells by veratridine and batrachotoxin.

The activation of the action potential Na+ ionophore by veratridine and batrachotoxin is time- and concentration-dependent and completely reversible. Batrachotoxin acts more slowly than veratridine. The concentration dependence of activation at equilibrium suggests reversible interaction of each toxin with a single class of independent sites having dissociation constants at physiologic ion concentrations of 80 plus or minus 13 muM for veratridine and 0.4 plus or minus muM for batrachotoxin. The maximum velocity of Na+ uptake at 50 mM Na+ is 128 plus or minus 12 nmol/min/mg in the presence of batrachotoxin compared to 48 plus or minus 4 nmol/min/mg in the presence of veratridine. Treatment of cells with excess veratridine in addition to batrachotoxin inhibits batrachotoxin-dependent 22-Na+ uptake. The concentration dependence of this inhibition suggests that it reflects competitive displacement of batrachotoxin from its binding site by veratridine. The activation by veratridine and batrachotoxin is inhibited in a competitive manner by divalent cations. The inhibition by divalent cations exhibits significant ion specificity with Mn-2+ greater than Co-2+ greater than Ni-2+ greater than Ca-2+ greater than Mg-2+ greater than Sr-2+. The inhibition constants (KI) for Ca-2+ are 0.84 mM for veratridine-dependent 22-Na+ uptake and 1.2 mM for batrachotoxin-dependent 22-Na+ uptake. The activation by veratridine and batrachotoxin is inhibited in a noncompetitive manner by tetrodotoxin. The apparent KD for tetrodotoxin as 11 plus or minus 1 nM in the presence of 150 mM Na+ and approximately 8.5 nM in 50 mM Na+. Divalent cations do not affect the apparent KD for tetrodotoxin. A hypothesis is presented which suggests that batrachotoxin, veratridine, and divalent cations interact with an activation site associated with the action potential Na+ ionophore, whereas tetrodotoxin interacts with a physically and functionally independent site involved in the transport of monovalent cations by the ionophore.     

Developmental regulation of a Na(+)-activated fast outward K+ current in rat myoblasts.

BACKGROUND/AIMS: Myoblasts undergoing differentiation sequentially express multiple K+ channels, and that ion channel expression varies depending on species and state of development. In this report, we reported a developmental regulation of fast activated and fast inactivated outward current in rat myoblasts. METHODS: The kinetic and pharmacological property of the outward current was investigated by using the patch-clamp technique. RESULTS: The outward current was elicited by a depolarizing step from -100 mV holding potential to +40 mV- +80 mV. The activation properties of this channel changed with days in culture. The outward current was blocked by 4-AP in a concentration dependent manner, with 0.5 mM and 2 mM 4-AP inhibiting the current by 10 +/- 3% and 56 +/- 3%, respectively. When 1 mM tetrodotoxin (TTX) was added to the bath solution or the membrane potential was depolarized to -50 mV, the fast outward current was aborted. Na+ dependent inhibition was observed when Na+ in the bath solution was replaced by Li+. In addition, replacement of K+ in the pipette solution by Cs+ almost completely eliminated the outward current. CONCLUSION: The developmentally regulated outward current recorded in rat myoblasts is a Na+ influx-dependent outward K+ current, which may contribute to myoblast membrane firing of action potential or myoblast fusion.     

Voltage-dependent conductances in Limulus ventral photoreceptors

The voltage-dependent conductances of Limulus ventral photoreceptors have been investigated using a voltage-clamp technique. Depolarization in the dark induces inward and outward currents. The inward current is reduced by removing Na+ or Ca2+ and is abolished by removing both ions. These results suggest that both Na+ and Ca2+ carry voltage-dependent inward current. Inward current is insensitive to tetrodotoxin but is blocked by external Ni2+. The outward current has a large transient component that is followed by a smaller maintained component. Intracellular tetraethylammonium preferentially reduces the maintained component, and extracellular 4-amino pyridine preferentially reduces the transient component. Neither component is strongly affected by removal of extracellular Ca2+ or by intracellular injection of EGTA. It is concluded that the photoreceptors contain at least three separate voltage-dependent conductances: 1) a conductance giving rise to inward currents; 2) a delayed rectifier giving rise to maintained outward K+ current; and 3) a rapidly inactivating K+ conductance similar to the A current of molluscan neurons.     

川芎嗪增加大鼠远端结肠阴离子分泌的基侧膜机制

本研究用短路电流技术来观察在川芎嗪作用下,电解质在大鼠远端结肠上皮细胞的转运及其细胞机制。在新鲜分离的结肠上皮的基侧膜加入川芎嗪,能产生较大的短路电流。用粘膜下神经元阻断剂——河豚毒素预作用于结肠上皮,不影响随后的川芎嗪所产生的短路电流,前列腺素合成抑制剂indomethacin预作用可使随后的川芎嗪产生的短路电流减少55．2％。在结肠上皮的顶膜加入Cl^-通道阻断剂DPC和glibenclamide,能完全阻断川芎嗪产生的短路电流。Bumetanide,基侧膜钠、钾、氯共转运体阻断剂能抑制川芎嗪引起的短路电流的85．2％,而结肠上皮细胞基侧膜的非选择性钾通道阻断剂Ba^2 能阻断90％以上的短路电流,说明基侧膜的钠、钾、氯共转运体和钾通道在川芎嗪引起的短路电流中起着重要的作用。上述结果表明,川芎嗪刺激大鼠远端结肠上皮细胞分泌Cl^-是通过上皮细胞顶膜Cl^-通道和基侧膜的钠、钾、氯共转体和K^ 通道介导的。     

Constitution and properties of axonal membranes of crustacean nerves.

The purification of axonal membranes of crustaceans was followed by measuring enrichment in [3H]tetrodotoxin binding capacity and in Na+, K+-ATPase activity. A characteristic of these membranes is their high content of lipids and their low content of protein as compared to other types of plasmatic membranes. The axonal membrane contains myosin-like, actin-like, tropomyosin-like, and tubulin-like proteins. It also contains Na+, K+-ATPase and acetylcholinesterase. The molecular weights of these two enzymes after solubilization are 280,000 and 270,000, respectively. The molecular weights of the catalytic subunits are 96,000 for ATPase and 71,000 for acetylcholinesterase. We confirmed the presence of a nicotine binding component in the axonal membrane of the lobster but we have been unable to find [3H]nicotine binding to crab axonal membranes. The binding to axonal membranes og of the sodium channel, has been studied in detail. The dissociation constant for the binding of [3H]tetrodotoxin to the axonal membrane receptor is 2.9 nM at pH 7.4. The concentration of the tetrodotoxin receptor in crustacean membranes is about 10 pmol/mg of membrane protein, 7 times less than the acetylcholinesterase, 30 times less than the Na+, K+-ATPase, and 30 times less than the nicotine binding component in the lobster membrane. A reasonable estimate indicates that approximately only one peptide chain in 1000 constitutes the tetrodotoxin binding part of the sodium channel in the axonal membrane. Veratridine, which acts selectively on the resting sodium permeability, binds to the phospholipid part of the axonal membrane. [3H]Veratridine binding to membranes parallels the electrophysiological effect. Veratridine and tetrodotoxin have different receptor sites. Although tetrodotoxin can repolarize the excitable membrane of a giant axon depolarized by veratridine, veratridine does not affect the binding of [3H]tetrodotoxin to purified axonal membranes. Similarly, tetrodotoxin does not affect the binding of [3H]veratridine to axonal membranes. Scorpion neurotoxin I, a presynaptic toxin which affects both the Na+ and the K+ channels, does not interfere with the binding of [3H]tetrodotoxin or [3H]veratridine to axonal membranes. Tetrodotoxin, veratridine, and scorpion neurotoxin I, which have in common the perturbation of the normal functioning of the sodium channel, act upon three different types of receptor sites.     

The Action of Tetrodotoxin on Electrogenic Components of Squid Giant Axons

Voltage clamp measurements on squid giant axons show that externally applied puffer fish poison, tetrodotoxin, eliminates only the initial inward current component of spike electrogenesis and does not affect the subsequent outward current. The selective effect on Na activation, which is reversible, confirms the view that the movements of Na and K during spike electrogenesis occur at structurally different sites on the membrane. Spike electrogenesis is also blocked when tetrodotoxin is injected into the axon, but the interior of the membrane appears to be somewhat less sensitive to the poison. Differences in reactivity of various electrogenic membrane components to tetrodotoxin are discussed as signifying differences in chemical structures.