Inhalational anesthetics activate two-pore-domain background K+ channels.

Volatile anesthetics produce safe, reversible unconsciousness, amnesia and analgesia via hyperpolarization of mammalian neurons. In molluscan pacemaker neurons, they activate an inhibitory synaptic K+ current (IKAn), proposed to be important in general anesthesia. Here we show that TASK and TREK-1, two recently cloned mammalian two-P-domain K+ channels similar to IKAn in biophysical properties, are activated by volatile general anesthetics. Chloroform, diethyl ether, halothane and isoflurane activated TREK-1, whereas only halothane and isoflurane activated TASK. Carboxy (C)-terminal regions were critical for anesthetic activation in both channels. Thus both TREK-1 and TASK are possibly important target sites for these agents.

A TREK-1-like potassium channel in atrial cells inhibited by beta-adrenergic stimulation and activated by volatile anesthetics.

Many members of the two-pore-domain potassium (K(+)) channel family have been detected in the mammalian heart but the endogenous correlates of these channels still have to be identified. We investigated whether I(KAA), a background K(+) current activated by negative pressure (stretch) and by arachidonic acid (AA) and sensitive to intracellular acidification, could be the native correlate of TREK-1 in adult rat atrial cells. Using the inside-out configuration of the patch-clamp technique, we found that I(KAA), like TREK-1, was outwardly rectifying in physiological K(+) conditions, with a conductance of 41 pS at +50 mV. Like TREK-1, I(KAA) was reversibly activated by clinical concentrations of volatile anesthetics (in mmol/L, chloroform 0.18, halothane 0.11, and isoflurane 0.69). In cell-attached experiments, I(KAA) was inhibited by chlorophenylthio-cAMP (500 micromol/L) and also by stimulation of beta-adrenergic receptors with isoproterenol (1 micromol/L). In addition, TREK-1 mRNAs were detected in all cardiac tissues, and the TREK-1 protein was immunolocalized in isolated atrial myocytes. Such a background potassium channel might contribute to the positive inotropic effects produced by beta-adrenergic stimulation of the heart. It might also be involved in the regulation of the atrial natriuretic peptide secretion.

Transition temperatures of the electrical activity of ion channels in the nerve membrane.

The temperature dependence of some of the electrical characteristics of neuronal membranes from Aplysia giant neurons and crustacean and cuttlefish giant axons has been analyzed. Arrhenius plots for the maximum rate of depolarization of (V+max) or repolarization (V-max) of the action potential, for the resting membrane conductance, and for the speed of propagation of the action potential, exhibited clear breaks at characteristic temperatures between 17 and 20 degrees C. Lobster giant axons and frog nodes of Ranvier were voltage-clamped at different temperatures between 5 and 30 degrees C. Arrhenius plots for relaxation times related to the opening and closing processes affecting the Na+ and K+ channels were linear. No 'transition' temperature was detected. However, clear-cut changes in (Formula: see text) Na+ and K+ currents, were consistantly observed around 18 degrees C. Values for (Formula: see text) plateaued above 18 degrees C, then decreased gradually as a function of reduced temperature. Variations in temperature between 1 and 30 degrees C did not alter the binding properties of [3H]tetrodotoxin to a purified crab axonal membrane. Pharmacological properties of the Na+ channel are sensitive to temperature. The temperature-dependent effect of veratridine has been studied and indicates a change in properties of the Na+ channel below 20 degrees C. These results support the possibility that the fluidity of membrane lipids in the ionic channel microenvironment may influence the degree to which the channel can open.

Scorpion neurotoxin. Mode of action on neuromuscular junctions and synaptosomes.

Electrophysiological analysis of the effects of scorpion toxin I, one of the neurotoxins from the venom of the scorpion Androctonus australis Hector, upon crayfish neuromuscular junctions has shown that the toxin strongly associates with the nerve terminal to stimulate release of neurotransmitters. The biochemical approach has shown that the binding of scorpion toxin I to rat brain synaptosomes is accompanied by a decrease in their capacity to accumulate gamma-aminobutyric acid. The main effect of the toxin is to stimulate neurotransmitter release. The apparent dissociation constant of the toxin-receptor complex is 0.1-0.2 muM at 22 degrees C. The rate of dissociation is so slow that complex formation seems to be quasi-irreversible. The "quasi-irreversibility" has also been observed in electrophysiological experiments with the crayfish neuromuscular junction. Tetrodotoxin prevents scorpion toxin I action if it is incubated with synaptosomes or with crayfish neuromuscular junctions before scorpion toxin I application. Tetrodotoxin does not reverse scorpion toxin action if it is added to the preparation after scorpion toxin I. Prevention of scorpion toxin action by tetrodotoxin permits measurements of binding characteristics of this toxin to synaptosomes. The dissociation constant of the tetrodotoxin-receptor complex is 2.2 nM at 22 degrees C. No cooperativity is observed in the binding. Because of its high affinity for synaptosomes (and the "quasi-irreversibility" of the binding), scorpion toxin I appears to be a potentially excellent tool for further studies of the molecular mechanism of neurotransmitter secretion.

The sodium channel in non-impulsive cells. Interaction with specific neurotoxins.

The cell line C9 used in this paper has a resting potential of --50 mV (+/- 10 mV) but is unable to generate an action potential upon electrical stimulation. The cell membrane has receptors for the selectivity filter toxin tetrodotoxin as well as for the gating system toxins, veratridine, scorpion toxin and sea anemone toxin. The Na+ channel which remains silent to electrical stimulation in the absence of toxins can be chemically activated by the gating system toxins. This has been demonstarted by electrophysiological techniques and by 22Na+ flux studies. The electrophysiological approach has shown that the sea anemone toxin is able to induce a spontaneous slow-wave activity inhibited by tetrodotoxin. 22Na+ influx analyses have shown that veratridine and the sea anemone toxin produce an important increase of the initial rate of 22Na+ influx into the C9 cell. The stimulation of 22Na+ entry by these gating system toxins is similar to that found using spiking neuroblastoma cells. Veratridine and the sea anemone toxin on one hand as well as veratridine and the scorpion toxin on the other hand are synergistic in their action to stabilize an open and highly permeable form of the sodium channel. Stimulation of 22Na+ entry into the cell through the sodium channel maintained open by the gating system neurotoxins is completely suppressed by tetrodotoxin.

Interaction of pyrethroids with the Na+ channel in mammalian neuronal cells in culture.

The interaction of a series of pyrethroids with the Na+ channel of mouse neuroblastoma cells has been followed using both an electrophysiological and a 22Na+ influx approach. By themselves, pyrethroids do not stimulate 22Na+ entry through the Na+ channel (or the stimulation they give is too small to be analyzed). However, they stimulate 22Na+ entry when used in conjunction with other toxins specific for the gating system of the channel. These include batrachotoxin, veratridine, dihydrograyanotoxin II or polypeptide toxins like sea anemone and scorpion toxins. This stimulatory effect is fully inhibited by tetrodotoxin with a dissociation constant of 1.6 nM for the tetrodotoxin-receptor complex. Half-maximum saturation of the pyrethroid receptor on the Na+ channel is observed in the micromolar range for the most active pyrethroids, Decis and RU 15525. The synergism observed between the effect of pyrethroids on 22Na+ influx on the one hand, and the effects of sea anemone toxin II, Androctonus scorpion toxin II, batrachotoxin, veratridine and dihydrograyanotoxin II on the other, indicates that the binding component for pyrethroids on the Na+ channel is distinct from the other toxin receptors. It is also distinct from the tetrodotoxin receptor. Some of the pyrethroids used in this study bind to the Na+ channel but are unable to stimulate 22Na+ entry. These inactive compounds behave as are unable to stimulate 22Na+ entry. These inactive compounds behave as antagonists of the active pyrethroids. An electrophysiological approach has shown that pyrethroids by themselves are active on the Na+ channel of mammalian neurones, and essentially confirm the conclusions made from 22Na+ flux measurements. Pyrethroids are also active on C9 cells in which Na+ channels are 'silent', that is, not activatable by electrical stimulation. Pyrethroids chemically activate the silent Na+ channel in a manner similar to that with veratridine, batrachotoxin, or polypeptide toxins, which are known to slow down the inactivation process of a functional Na+ channel.

Synthesis and properties of new photoactivable derivatives of tetrodotoxin.

The Pfitzner-Moffatt oxidation procedure has been used to prepare two new photoactivable derivatives of tetrodotoxin that have been synthesized with high specific radioactivities (17.5 Ci/mmol and 30 Ci/mmol). They specifically bind to axonal membranes with affinities of 5.2-14.2 nM. They dissociate from their membrane complex with half-lives of 10.8 and 20 min. In the dark, these compounds give a reversible block of the sodium channels. After ultraviolet irradiation, they induce an irreversible blockade of the nerve channels.

Novel mutations in KvLQT1 that affect Iks activation through interactions with Isk.

OBJECTIVES: We report the functional expression of four KCNQ1 mutations affecting arginine residues and resulting in Romano-Ward (RW) and the Jervell and Lange-Nielsen (JLN) congenital long QT syndromes. RESULTS: The R539W and R190Q mutations were found in typical RW families with an autosomal dominant transmission. The R243H mutation was found in a compound heterozygous JLN patient who presents with deafness and cardiac symptoms. The fourth mutation, R533W, was a new case of recessive form of the RW syndrome since homozygous carriers experienced syncopes but showed no deafness, whereas the heterozygous carriers were asymptomatic. The R190Q mutation failed to produce functional homomeric channels. The R243H, R533W and R539W mutations induced a positive voltage shift of the channel activation but only when co-expressed with IsK, pointing out the critical role of these positively charged residues in the modulation of the gating properties of KvLQT1 by IsK. The positive shift induced by R533W was merely 15%. This small effect was compatible with the recessive character of the RW phenotype transmission. The average QTc was significantly longer (P < 0.01) in patients carrying mutations inducing a total loss of channel function and those patients were also prone to cardiac adverse symptoms (whether syncopes or sudden death) to a greater extent (62 vs. 21%, P < 0.001). CONCLUSIONS: Novel mutations are described that induce a voltage shift of the channel activation only in the presence of IsK. They appear associated with a milder cardiac phenotype.

Induction of normal ultrastructure by CGRP treatment in dysgenic myotubes.

The calcitonin gene-related peptide (CGRP) restores an apparent normal ultrastructure in mdg/mdg muscle cells in vitro, including a normal triadic organization which is known to be essential for excitation-contraction (E-C) coupling. However, neither slow L-type Ca2+ channel activity nor E-C coupling, which are absent in mdg/mdg muscle, were re-established. These observations suggest a potential role of CGRP (and also of cAMP as the intracellular messenger) in the morphological development of the muscle fiber.

Molecular cloning of a murine N-type calcium channel alpha 1 subunit. Evidence for isoforms, brain distribution, and chromosomal localization.

A cDNA encoding a N-type Ca2+ channel has been cloned from the murine neuroblastoma cell line N1A103. The open reading frame encodes a protein of 2,289 amino acids (257 kDa). Analysis of different clones provided evidence for the existence of distinct isoforms of N-type channels. High levels of mRNA were found in the pyramidal cell layers CA1, CA2 and CA3 of the hippocampus, in the dentate gyrus, in the cortex layers 2 and 4, in the subiculum and the habenula. The N-type Ca2+ channel gene has been localized on the chromosome 2, band A.

Dominant negative chimeras provide evidence for homo and heteromultimeric assembly of inward rectifier K+ channel proteins via their N-terminal end.

Chimeras have been constructed using three different fragments (N-terminal, central and C-terminal) of IRK3, a constitutive inward rectifier K+ channel subunit, and GIRK2, a G-protein activated inward rectifier K+ channel subunit and have been coinjected into Xenopus oocytes together with IRK3 or IRK1 (another constitutive inward rectifier) cRNA. Both IRK1 and IRK3 expression was inhibited by coinjection with chimeras containing a N-terminal fragment of IRK3 suggesting that subunits of K+ channels in the IRK family form a functional multimeric assembly where the N-terminal end has an important role. In situ hybridization shows that IRK1 and IRK3 are coexpressed in the same areas of the brain and probably in the same cells. Taken together both the localization and the oocyte expression results suggest that not only homomultimeric IRK1 or homomultimeric IRK3 assemblies take place but that heteromultimeric IRK1/IRK3 assemblies are also formed.

The apamin-sensitive Ca2+-dependent K+ channel molecular properties, differentiation and endogenous ligands in mammalian brain.

Apamin is a bee venom neurotoxin of 10 amino acids containing two disulphide bridges. Current-clamp and voltage-clamp experiments have shown that apamin externally applied blocks specifically at low concentration (0.1 microM) the Ca2+-dependent slow K+ conductance which mediates the long-lasting after-hyperpolarization in neuroblastoma cells and rat muscle cells in culture. The apamin-sensitive Ca2+-dependent slow K+ conductance is voltage-dependent and tetraethylamonium-insensitive. It is distinct from the high conductance Ca+-dependent K+ channel revealed by patch-clamp experiments. Biochemical characterization of the apamin receptor in rat striated muscle, neuroblastoma cells, rat synaptosomes, smooth muscles and hepatocytes was carried out with the use of a radiolabelled monoiodo-apamin derivative (125I-apamin) of high specific radioactivity (2000 Ci/mmol). The dissociation constant of the apamin-receptor complex is between 15 and 60 pM for all tissue preparations. The density of binding sites is very low: between 1 and 40 fmol/mg of protein. Radiation-inactivation analysis indicates a molecular mass for the apamin receptor of 250 000 Da whereas affinity labelling with 125I-apamin results in covalent labelling of a single polypeptide chain with a molecular mass of about 30 000 Da. Autoradiography of 125I-apamin binding sites reveals the presence of Ca2+-activated K+ channels in many regions of the brain. There is an all-or-none control of the expression of the apamin-sensitive Ca2+-dependent K+ channel by innervation in mammalian skeletal muscle. There exists an endogenous equivalent of apamin in rat brain.

Uptake of fatty acids by cultured cardiac cells from chick embryo: evidence for a facilitation process without energy dependence.

Cultured heart cells from chick embryo accumulate fatty acids up to 50 fold at a steady-state level under defined conditions [ref.1]. Studies of fatty acid uptake as a function of different cellular parameters (intracellular ATP and pH, membrane potential and electrochemical gradients for monovalent and divalent cations) show the lack of effect of these factors. The rate of uptake is temperature-dependent. The maximum velocity V is affected with no change in the Km value of the saturable component; the activation energies were found to be 35.5 kJ.mol-1 for palmitate and 42 kJ.mol-1 for oleate. The results are in favour of a facilitation process which leads to an accumulation of fatty acids without energy dependence. The accumulation of fatty acids could be due to their association to intracellular membraneous and/or cytosolic components.

Excitation-contraction uncoupling in the developing skeletal muscle of the muscular dysgenesis mouse embryo.

The muscular dysgenesis recessive autosomal mutation is characterized by a total lack of muscular contraction and a myofibrillar non-organization. Many abnormalities involved in the excitation-contraction coupling are found in mdg/mdg myotubes: 1) the internal structural organization of the membrane coupling between the sarcoplasmic reticulum (SR) and the transverse (T)-tubule forming the triadic association is defective: the triad number is decreased in the muscle and there are a lack of periodic densities between the SR and T-tubule apposed membranes. 2) the voltage-dependent Ca2+ channel contents, identified by binding with the specific blocker PN 200-110, are decreased. The two fast (30 ms) and slow (100 ms) Ca2+ currents present in normal myotubes are absent in mdg/mdg myotubes in vitro. 3) the Ca2+-dependent K+ conductance triggering an action potential followed by a long lasting after hyperpolarization (ahp) is absent in mdg/mdg myotubes. This indicates a lack of the free intracellular Ca2+ increased by the action potential. These results suggest that: 1) the lack of differentiated triadic junctions is directly correlated with very low amounts of voltage-dependent Ca2+ channels; 2) the low amount of Ca2+ channels results directly in decreased Ca2+ currents; 3) the decreased Ca2+ currents are the consequence of the low intracellular Ca2+ concentration which is not sufficient to trigger a contraction. However, the addition of normal motoneurones to mdg/mdg myotubes in culture induces, few days later, an increase in Ca2+ currents.

The bee venom peptide tertiapin underlines the role of I(KACh) in acetylcholine-induced atrioventricular blocks.

Acetylcholine (ACh) is an important neuromodulator of cardiac function that is released upon stimulation of the vagus nerve. Despite numerous reports on activation of I(KACh) by acetylcholine in cardiomyocytes, it has yet to be demonstrated what role this channel plays in cardiac conduction. We studied the effect of tertiapin, a bee venom peptide blocking I(KACh), to evaluate the role of I(KACh) in Langendorff preparations challenged with ACh. ACh (0.5 microM) reproducibly and reversibly induced complete atrioventricular (AV) blocks in retroperfused guinea-pig isolated hearts (n=12). Tertiapin (10 to 300 nM) dose-dependently and reversibly prevented the AV conduction decrements and the complete blocks in unpaced hearts (n=8, P<0.01). Tertiapin dose-dependently blunted the ACh-induced negative chronotropic response from an ACh-induced decrease in heart rate of 39+/-16% in control conditions to 3+/-3% after 300 nM tertiapin (P=0.01). These effects were not accompanied by any significant change in QT intervals. Tertiapin blocked I(KACh) with an IC(50) of 30+/-4 nM with no significant effect on the major currents classically associated with cardiac repolarisation process (I(Kr), I(Ks), I(to1), I:(sus), I(K1) or I(KATP)) or AV conduction (I(Na) and I(Ca(L))). In summary, tertiapin prevents dose-dependently ACh-induced AV blocks in mammalian hearts by inhibiting I(KACh).

Biochemical studies of the structure, mechanism and differentiation of the voltage-sensitive sodium channel.

This paper describes how neurotoxins specific of the Na(+) channel are used to study its function, its structure and its differentiation in a variety of excitable and non-impulsive cells.

Identification in mammalian brain of an endogenous substance with Na+ channel blocking activities similar to those of tetrodotoxin.

A substance with Na+ channel blocking activities has been isolated from pig brain after extraction and purification on sulfopropyl-Sephadex C-25, reversed-phase and carboxymethyl Synchropak high pressure liquid chromatography columns. The peptidic material i) displaces [3H]ethylenediamine tetrodotoxin ([3H]en-TTX) from its binding sites on rat brain membranes, (ii) it blocks 22Na+ influx induced by veratridine and sea anemone toxin on neuroblastoma and embryonic chick heart cells in culture, (iii) it specifically decreases the height of the action potential generated in frog sciatic nerve, and (iv) it blocks the fast Na+ current in voltage-clamped neuroblastoma cells. These properties are similar to those of tetrodotoxin while the endogenous factor is a peptide that is destroyed by proteases. These results suggest the presence in pig brain of a potent Na+ channel modulation activity.

Cells with neuronal properties in permanent cultures of quail embryo neuroretinas infected with Rous sarcoma virus.

Neuroretina cells from 7-day quail embryos infected 'in vitro' with the mutant ts NY-68 of Rous sarcoma virus, were established into permanent cultures. An initial stage of cellular proliferation was followed by a period of minimal multiplication. After recovery from this crisis, cell proliferation resumed. About 30% of the cells had binding sites for tetanus toxin and the monoclonal antibody A2B5 which seem to be specific for neurons, and an ultrastructural study suggested the presence of neurons and Müller (astroglial) cells. The specific activity of glutamic acid decarboxylase, the enzyme responsible for the synthesis of the neurotransmitter gamma-aminobutyric acid was high (10-30 nmol CO2/h/mg of protein) and electrophysiology showed that some cells had 'active' membranes. After about 18 months in culture, approximately 20% of the cells were able to respond to electrical stimulation by producing action potentials which were inhibited by 10(-7) M tetrodotoxin. These electrophysiological properties are stable: they have been repeatedly found at regular intervals throughout a 20 months period. Furthermore, a clone in which all tested cells are excitable, has been derived from the mass culture. Quail embryo neuroretina cells with typical neuronal properties can thus be established into permanent cultures after infection with Rous sarcoma virus.

Characterization and partial purification from pheochromocytoma cells of an endogenous equivalent of scyllatoxin, a scorpion toxin which blocks small conductance Ca(2+)-activated K+ channels.

This work describes the partial purification of a heat-stable peptide which has the same properties as the scorpion toxin, scyllatoxin, a specific blocker of one class of Ca(2+)-activated K+ channels: (i) it competes with [125I]apamin for binding to the same site, (ii) like apamin and scyllatoxin, it blocks the after-potential hyperpolarization in skeletal muscle cells in culture, (iii) like apamin and scyllatoxin, it contracts guinea-pig taenia coli relaxed by epinephrine, (iv) it cross-reacts with antibodies raised against scyllatoxin but not with antibodies raised against apamin.

Effects of two chemically related new Ca2+ channel antagonists, SR33557 (fantofarone) and SR33805, on the L-type cardiac channel.

Fantofarone (SR33557) is a substituted indolizine and SR33805 is a substituted indole. These drugs have been shown to specifically bind to the alpha 1 subunit of the L-type Ca2+ channel at the same site, distinct from those of the classical 1,4-dihydropyridine, phenylalkylamine or benzothiazepine Ca2+ antagonists, but in negative allosteric interaction with them. The present work shows that fantofarone and SR33805 block L-type but not T-type Ca2+ channels in mouse cardiac cells in primary culture. This block is voltage-dependent. Fantofarone and SR33805 are potent Ca2+ channel blockers in depolarized conditions (i.e. at a holding potential of -40 mV) with an EC50 = 1.4 and 4.1 nM, respectively. In polarized conditions (i.e. at a holding potential of -80 mV), SR33805 is a better Ca2+ channel blocker (EC50 = 33 nM) than fantofarone (EC50 = 0.15 microM). Therefore differences in their chemical structures make the blocking action of fantofarone more sensitive to voltage than that of SR33805.