Biomedical subjects

M Lazdunski

Publications and source records attributed to M Lazdunski.

At least 37 records · Page 2Linked to original sources

PubMed2001

Cloning and cDNA sequence analysis of Lys(49) and Asp(49) basic phospholipase A(2) myotoxin isoforms from Bothrops asper.

Snake venom myotoxic phospholipases A(2) contribute to much of the tissue damage observed during envenomation by Bothrops asper, the major cause of snake bites in Central America. Several myotoxic PLA(2)s have been identified, but their mechanism of myotoxicity is still unclear. To aid in the molecular characterization of these venom toxins, the complete open reading frames encoding two Lys(49) and one Asp(49) basic PLA(2) myotoxins from the Central American snake B. asper (terciopelo) were obtained by cDNA cloning from venom gland poly-adenylated RNA. The amino acid sequence deduced from the myotoxins II and III open reading frames corresponded in each case to one of the reported amino acid sequence isoforms. The sequence of a new myotoxin IV-like sequence (MT-IVa) contains conservative Val-->Leu(18) and Ala-->Val(23) substitutions when compared with the reported N-terminus of the native myotoxin IV, suggesting minor isoform variations among specimens of a single species. Sequence alignment studies indicated significant (>75% sequence identity) identities with other crotalid venom Lys(49) PLA(2)s, particularly bothropstoxin I/Ia isoforms of B. jararacussu and myotoxin II of B. asper.

Amino Acid Sequence↗

PubMed2000

Cloning and recombinant expression of a structurally novel human secreted phospholipase A2.

Mammals contain a diverse set of secreted phospholipases A(2) (sPLA(2)s) that liberate arachidonic acid from phospholipids for the production of eicosanoids and exert a variety of physiological and pathological effects. We report the cloning, recombinant expression, and kinetic properties of a novel human sPLA(2) that defines a new structural class of sPLA(2)s called group XII. The human group XII (hGXII) cDNA contains a putative signal peptide of 22 residues followed by a mature protein of 167 amino acids that displays homology to all known sPLA(2)s only over a short stretch of amino acids in the active site region. Northern blot and reverse transcription-polymerase chain reaction analyses show that the tissue distribution of hGXII is distinct from the other human sPLA(2)s with strong expression in heart, skeletal muscle, kidney, and pancreas and weaker expression in brain, liver, small intestine, lung, placenta, ovaries, testis, and prostate. Catalytically active hGXII was produced in Escherichia coli and shown to be Ca(2+)-dependent despite the fact that it is predicted to have an unusual Ca(2+)-binding loop. Similar to the previously characterized mouse group IIE sPLA(2)s, the specific activity of hGXII is low in comparison to that of other mammalian sPLA(2), suggesting that hGXII could have novel functions that are independent of its phospholipase A(2) activity.

Amino Acid Sequence↗

PubMed2000

Cloning and recombinant expression of human group IIF-secreted phospholipase A(2).

Mammalian-secreted phospholipases A(2) (sPLA(2)) form a diverse family of at least nine enzymes that hydrolyze phospholipids to release free fatty acids and lysophospholipids. We report here the cloning and characterization of human group IIF sPLA(2) (hGIIF sPLA(2)). The full-length cDNA codes for a signal peptide of 20 amino acid followed by a mature protein of 148 amino acids containing all of the structural features of catalytically active group II sPLA(2)s. hGIIF sPLA(2) gene is located on chromosome 1 and lies within a sPLA(2) gene cluster of about 300 kbp that also contains the genes for group IIA, IIC, IID, IIE, and V sPLA(2)s. In adult tissues, hGIIF is highly expressed in placenta, testis, thymus, liver, and kidney. Finally, recombinant expression of hGIIF sPLA(2) in Escherichia coli shows that the enzyme is Ca(2+)-dependent, maximally active at pH 7-8, and hydrolyzes phosphatidylglycerol versus phosphatidylcholine with a 15-fold preference.

Amino Acid Sequence↗

PubMed2000

KCNE2 confers background current characteristics to the cardiac KCNQ1 potassium channel.

Mutations in HERG and KCNQ1 (or KVLQT1) genes cause the life-threatening Long QT syndrome. These genes encode K(+) channel pore-forming subunits that associate with ancillary subunits from the KCNE family to underlie the two components, I(Kr) and I(Ks), of the human cardiac delayed rectifier current I(K). The KCNE family comprises at least three members. KCNE1 (IsK or MinK) recapitulates I(Ks) when associated with KCNQ1, whereas it augments the amplitude of an I(Kr)-like current when co-expressed with HERG. KCNE3 markedly changes KCNQ1 as well as HERG current properties. So far, KCNE2 (MirP1) has only been shown to modulate HERG current. Here we demonstrate the interaction of KCNE2 with the KCNQ1 subunit, which results in a drastic change of KCNQ1 current amplitude and gating properties. Furthermore, KCNE2 mutations also reveal their specific functional consequences on KCNQ1 currents. KCNQ1 and HERG appear to share unique interactions with KCNE1, 2 and 3 subunits. With the exception of KCNE3, mutations in all these partner subunits have been found to lead to an increased propensity for cardiac arrhythmias.

Animals↗

PubMed2000

Human TREK2, a 2P domain mechano-sensitive K+ channel with multiple regulations by polyunsaturated fatty acids, lysophospholipids, and Gs, Gi, and Gq protein-coupled receptors.

Mechano-sensitive and fatty acid-activated K(+) belong to the structural class of K(+) channel with two pore domains. Here, we report the isolation and the characterization of a novel member of this family. This channel, called TREK2, is closely related to TREK1 (78% of homology). Its gene is located on chromosome 14q31. TREK2 is abundantly expressed in pancreas and kidney and to a lower level in brain, testis, colon, and small intestine. In the central nervous system, TREK2 has a widespread distribution with the highest levels of expression in cerebellum, occipital lobe, putamen, and thalamus. In transfected cells, TREK2 produces rapidly activating and non-inactivating outward rectifier K(+) currents. The single-channel conductance is 100 picosiemens at +40 mV in 150 mm K(+). The currents can be strongly stimulated by polyunsaturated fatty acid such as arachidonic, docosahexaenoic, and linoleic acids and by lysophosphatidylcholine. The channel is also activated by acidification of the intracellular medium. TREK2 is blocked by application of intracellular cAMP. As with TREK1, TREK2 is activated by the volatile general anesthetics chloroform, halothane, and isoflurane and by the neuroprotective agent riluzole. TREK2 can be positively or negatively regulated by a variety of neurotransmitter receptors. Stimulation of the G(s)-coupled receptor 5HT4sR or the G(q)-coupled receptor mGluR1 inhibits channel activity, whereas activation of the G(i)-coupled receptor mGluR2 increases TREK2 currents. These multiple types of regulations suggest that TREK2 plays an important role as a target of neurotransmitter action.

Amino Acid Sequence↗

PubMed2000

TWIK-2, an inactivating 2P domain K+ channel.

We cloned human and rat TWIK-2 and expressed this novel 2P domain K(+) channel in transiently transfected COS cells. TWIK-2 is highly expressed in the gastrointestinal tract, the vasculature, and the immune system. Rat TWIK-2 currents are about 15 times larger than human TWIK-2 currents, but both exhibit outward rectification in a physiological K(+) gradient and mild inward rectification in symmetrical K(+) conditions. TWIK-2 currents are inactivating at depolarized potentials, and the kinetic of inactivation is highly temperature-sensitive. TWIK-2 shows an extremely low conductance, which prevents the visualization of discrete single channel events. The inactivation and rectification are intrinsic properties of TWIK-2 channels. In a physiological K(+) gradient, TWIK-2 is half inhibited by 0.1 mm Ba(2+), quinine, and quinidine. Finally, cysteine 53 in the M1P1 external loop is required for functional expression of TWIK-2 but is not critical for subunit self-assembly. TWIK-2 is the first reported 2P domain K(+) channel that inactivates. The base-line, transient, and delayed activities of TWIK-2 suggest that this novel 2P domain K(+) channel may play an important functional role in cell electrogenesis.

Amino Acid Sequence↗

PubMed2000

M-type KCNQ2-KCNQ3 potassium channels are modulated by the KCNE2 subunit.

KCNQ2 and KCNQ3 subunits belong to the six transmembrane domain K+ channel family and loss of function mutations are associated with benign familial neonatal convulsions. KCNE2 (MirP1) is a single transmembrane domain subunit first described to be a modulator of the HERG potassium channel in the heart. Here, we show that KCNE2 is present in brain, in areas which also express KCNQ2 and KCNQ3 channels. We demonstrate that KCNE2 associates with KCNQ2 and/or KCNQ3 subunits. In transiently transfected COS cells, KCNE2 expression produces an acceleration of deactivation kinetics of KCNQ2 and of the KCNQ2-KCNQ3 complex. Effects of two previously identified arrhythmogenic mutations of KCNE2 have also been analyzed.

Amino Acid Sequence↗

PubMed2000

Isolation of a tarantula toxin specific for a class of proton-gated Na+ channels.

Acid sensing is associated with nociception, taste transduction, and perception of extracellular pH fluctuations in the brain. Acid sensing is carried out by the simplest class of ligand-gated channels, the family of H(+)-gated Na(+) channels. These channels have recently been cloned and belong to the acid-sensitive ion channel (ASIC) family. Toxins from animal venoms have been essential for studies of voltage-sensitive and ligand-gated ion channels. This paper describes a novel 40-amino acid toxin from tarantula venom, which potently blocks (IC(50) = 0.9 nm) a particular subclass of ASIC channels that are highly expressed in both central nervous system neurons and sensory neurons from dorsal root ganglia. This channel type has properties identical to those described for the homomultimeric assembly of ASIC1a. Homomultimeric assemblies of other members of the ASIC family and heteromultimeric assemblies of ASIC1a with other ASIC subunits are insensitive to the toxin. The new toxin is the first high affinity and highly selective pharmacological agent for this novel class of ionic channels. It will be important for future studies of their physiological and physio-pathological roles.

Acid Sensing Ion Channels↗

PubMed2000

TREK-1 is a heat-activated background K(+) channel.

Peripheral and central thermoreceptors are involved in sensing ambient and body temperature, respectively. Specialized cold and warm receptors are present in dorsal root ganglion sensory fibres as well as in the anterior/preoptic hypothalamus. The two-pore domain mechano-gated K(+) channel TREK-1 is highly expressed within these areas. Moreover, TREK-1 is opened gradually and reversibly by heat. A 10 degrees C rise enhances TREK-1 current amplitude by approximately 7-fold. Prostaglandin E2 and cAMP, which are strong sensitizers of peripheral and central thermoreceptors, reverse the thermal opening of TREK-1 via protein kinase A-mediated phosphorylation of Ser333. Expression of TREK-1 in peripheral sensory neurons as well as in central hypothalamic neurons makes this K(+) channel an ideal candidate as a physiological thermoreceptor.

Animals↗

PubMed2000

Polyunsaturated fatty acids are potent neuroprotectors.

Results reported in this work suggest a potential therapeutic value of polyunsaturated fatty acids for cerebral pathologies as previously proposed by others for cardiac diseases. We show that the polyunsaturated fatty acid linolenic acid prevents neuronal death in an animal model of transient global ischemia even when administered after the insult. Linolenic acid also protects animals treated with kainate against seizures and hippocampal lesions. The same effects have been observed in an in vitro model of seizure-like activity using glutamatergic neurons and they have been shown to be associated with blockade of glutamatergic transmission by low concentrations of distinct polyunsaturated fatty acids. Our data suggest that the opening of background K(+) channels, like TREK-1 and TRAAK, which are activated by arachidonic acid and other polyunsaturated fatty acids such as docosahexaenoic acid and linolenic acid, is a significant factor in this neuroprotective effect. These channels are abundant in the brain where they are located both pre- and post-synaptically, and are insensitive to saturated fatty acids, which offer no neuroprotection.

Animals↗

PubMed2000

Cloning and expression of human TRAAK, a polyunsaturated fatty acids-activated and mechano-sensitive K(+) channel.

The two P domain hTRAAK K(+) channel has been cloned from human brain. hTRAAK cDNA encodes a 393 amino acid polypeptide with 88% of homology with its mouse counterpart. The hTRAAK gene has been mapped to chromosome 11q13 and the study of its organization indicates that the hTRAAK open reading frame is contained in six exons. hTRAAK is expressed abundantly in brain and placenta. In COS cells, hTRAAK currents are K(+)-selective, instantaneous and non-inactivating. These currents are insensitive to the classical K(+) channels blockers 4-aminopyridine, tetraethylammonium, barium and quinidine, but are strongly stimulated by application of arachidonic acid as well as other polyunsaturated fatty acids. hTRAAK can also be activated by a stretch of the membrane.

Amino Acid Sequence↗

PubMed2000

Molecular cloning, functional expression and chromosomal localization of an amiloride-sensitive Na(+) channel from human small intestine.

Amiloride-sensitive Na(+) channels belonging to the recently discovered NaC/DEG family of genes have been found in several human tissues including epithelia and central and peripheral neurons. We describe here the molecular cloning of a cDNA encoding a novel human amiloride-sensitive Na(+) channel subunit that is principally expressed in the small intestine and has been called hINaC (human intestine Na(+) channel). This protein is similar to the recently identified rodent channel BLINaC and is relatively close to the acid sensing ion channels (ASICs) (79 and 29% amino acid identity, respectively). ASICs are activated by extracellular protons and probably participate in sensory neurons to nociception linked to tissue acidosis. hINaC is not activated by lowering the external pH but gain-of-function mutations can be introduced and reveal when expressed in Xenopus oocytes, an important Na(+) channel activity which is blocked by amiloride (IC(50)=0.5 microM). These results suggest the existence of a still unknown physiological activator for hINaC (e.g. an extracellular ligand). The presence of this new amiloride-sensitive Na(+) channel in human small intestine probably has interesting physiological as well as physiopathological implications that remain to be clarified. The large activation of this channel by point mutations may be associated with a degenerin-like behavior as previously observed for channels expressed in nematode mechanosensitive neurons. The hINaC gene has been mapped on the 4q31.3-q32 region of the human genome.

Acid Sensing Ion Channels↗

PubMed2000

Lysophospholipids open the two-pore domain mechano-gated K(+) channels TREK-1 and TRAAK.

The two-pore (2P) domain K(+) channels TREK-1 and TRAAK are opened by membrane stretch as well as arachidonic acid (AA) (Patel, A. J., Honoré, E., Maingret, F., Lesage, F., Fink, M., Duprat, F., and Lazdunski, M. (1998) EMBO J. 17, 4283-4290; Maingret, F., Patel, A. J., Lesage, F., Lazdunski, M., and Honoré, E. (1999) J. Biol. Chem. 274, 26691-26696; Maingret, F., Fosset, M., Lesage, F., Lazdunski, M. , and Honoré, E. (1999) J. Biol. Chem. 274, 1381-1387. We demonstrate that lysophospholipids (LPs) and platelet-activating factor also produce large specific and reversible activations of TREK-1 and TRAAK. LPs activation is a function of the size of the polar head and length of the acyl chain but is independent of the charge of the molecule. Bath application of lysophosphatidylcholine (LPC) immediately opens TREK-1 and TRAAK in the cell-attached patch configuration. In excised patches, LPC activation is lost, whereas AA still produces maximal opening. The carboxyl-terminal region of TREK-1, but not the amino terminus and the extracellular loop M1P1, is critically required for LPC activation. LPC activation is indirect and may possibly involve a cytosolic factor, whereas AA directly interacts with either the channel proteins or the bilayer and mimics stretch. Opening of TREK-1 and TRAAK by fatty acids and LPs may be an important switch in the regulation of synaptic function and may also play a protective role during ischemia and inflammation.

Amiloride↗

PubMed2000

Axonal transport of TREK and TRAAK potassium channels in rat sciatic nerves.

The recent cloning, functional expression and brain localization of two new potassium channels, TREK and TRAAK, led us to examine whether both channels are present in peripheral nerves and can move along axons by means of axonal transport mechanisms. Using specific antibodies directed against TREK and TRAAK peptides, we found that immunoreactivity for both potassium channels accumulates above and below a ligature in rat sciatic nerves. The process was rapid and bidirectional suggesting that the channels are associated with vesicles. This represents the first report on the axonal transport of potassium channels.

Animals↗

PubMed2000

Novel human secreted phospholipase A(2) with homology to the group III bee venom enzyme.

Venom and mammalian secreted phospholipases A(2) (sPLA(2)s) have been associated with numerous physiological, pathological, and toxic processes. So far, structurally related group I and II sPLA(2)s have been found in vertebrates such as mammals and snakes, whereas group III sPLA(2)s have mainly been found in venom from invertebrates such as bees and scorpions. Here we report the cloning and expression of a cDNA coding for a human group III (hGIII) sPLA(2). The full-length cDNA codes for a signal peptide of 19 residues followed by a protein of 490 amino acids made up of a central sPLA(2) domain (141 residues) flanked by large N- and C-terminal regions (130 and 219 residues, respectively). The sPLA(2) domain is 31% identical to bee venom sPLA(2) and displays all of the features of group III sPLA(2)s including 10 cysteines. The hGIII sPLA(2) gene consists of at least 7 exons and maps to chromosome 22q. By Northern blot analysis, a 4.4-kilobase hGIII transcript was found in kidney, heart, liver, and skeletal muscle. Transfection of hGIII sPLA(2) cDNA in COS cells led to accumulation of sPLA(2) activity in the culture medium, indicating that the cDNA codes for a secreted enzyme. Using small unilamellar vesicles as substrate, hGIII sPLA(2) was found to be a Ca(2+)-dependent enzyme showing an 11-fold preference for phosphatidylglycerol over phosphatidylcholine and optimal activity at pH 8.

Amino Acid Sequence↗

PubMed2000

Two phospholipase A2 inhibitors from the plasma of Cerrophidion (Bothrops) godmani which selectively inhibit two different group-II phospholipase A2 myotoxins from its own venom: isolation, molecular cloning and biological properties.

Myotoxic phospholipases A(2) (PLA(2)s; group II) account for most of the muscle-tissue damage that results from envenomation by viperid snakes. In the venom of the Godman's viper (Cerrophidion godmani, formerly Bothrops godmani), an enzymically active PLA(2) (myotoxin I) and an inactive, Lys-49 variant (myotoxin II) induce extensive muscle damage and oedema. In this study, two distinct myotoxin inhibitor proteins of C. godmani, CgMIP-I and CgMIP-II, were purified directly from blood plasma by selective binding to affinity columns containing either myotoxin I or myotoxin II, respectively. Both proteins are glycosylated, acidic (pI=4) and composed of 20-25-kDa subunits that form oligomers of 110 kDa (CgMIP-I) or 180 kDa (CgMIP-II). In inhibition studies, CgMIP-I specifically neutralized the PLA(2) and the myotoxic, oedema-forming and cytolytic activities of myotoxins I, whereas CgMIP-II selectively inhibited the toxic properties of myotoxin II. N-terminal amino acid sequence analysis and sequencing of cDNAs encoding the two inhibitors revealed that CgMIP-I is similar to gamma-type inhibitors, which share a pattern of cysteine residues present in the Ly-6 superfamily of proteins, whereas CgMIP-II shares sequence identity with alpha-type inhibitors that contain carbohydrate-recognition-like domains, also found in C-type lectins and mammalian PLA(2) receptors. N-terminal sequencing of myotoxin I revealed a different primary structure from myotoxin II [De Sousa, Morhy, Arni, Ward, Díaz and Gutiérrez (1998) Biochim. Biophys. Acta 1384, 204-208], which provides insight into the nature of such pharmacological specificity.

Amino Acid Sequence↗

PubMed2000

K(ATP) channel openers, adenosine agonists and epileptic preconditioning are stress signals inducing hippocampal neuroprotection.

Many models of induced ischemic and epileptic tolerance have now been described in the brain. Although detailed mechanisms underlying such protections still remain largely unknown, induction of heat shock proteins is amongst the endogenous responses believed to play an important role in cellular defense mechanisms. This study reveals that the development of epileptic tolerance also coincides with the induction of the 70,000 mol. wt heat shock protein expression within the time window of protection. Adenosine agonists or ATP-sensitive potassium channel openers have also been shown to exert strong neuroprotective effects when injected shortly prior to a severe ischemic or epileptic insult. The present work shows that adenosine receptor activation and ATP-sensitive potassium channel opening induce 70,000 mol. wt heat shock protein expression in the rat hippocampus and are able to mimic neuroprotection driven by preconditioning. R-phenylisopropyladenosine, a purine agonist, or (-)cromakalim, an ATP-sensitive potassium channel opener, was administered three days prior to a lethal ischemic or epileptic episode to mimic preconditioning. Neurodegeneration was assessed using Cresyl Violet staining and cellular DNA fragmentation visualized by the terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling method. 70, 000 mol. wt heat shock protein expression was analysed by western blotting and immunohistochemistry. The results show a long-lasting neuroprotection induced by activation of adenosine receptors or ATP-sensitive K(+) channels as early as three days prior to induction of a severe ischemic or epileptic challenge. This protective effect is associated with enhanced 70,000 mol. wt heat shock protein expression also occurring three days following administration of R-phenylisopropyladenosine or (-)cromakalim. These findings support the idea that preconditioning doses of R-phenylisopropyladenosine and (-)cromakalim act as mild cellular stresses inducing neuroprotection in a manner similar to a mild kainate treatment prior to a lethal ischemic or severe epileptic insult three days later. They also suggest that a delayed 70,000 mol. wt heat shock protein expression induced by excitatory neuronal stresses such as short ischemia, mild kainic acid treatment or activation of adenosine receptors and ATP-sensitive potassium channels is predictive of neuronal survival against a subsequent lethal injury.

Adenosine↗

PubMed2000

Immunolocalization of the arachidonic acid and mechanosensitive baseline traak potassium channel in the nervous system.

TRAAK is the sole member of the emerging class of 2P domain K+ channels to be exclusively expressed in neuronal cells. TRAAK produces baseline K+ currents which are strongly stimulated by arachidonic acid and by mechanical stretch, and which are insensitive to the classical K+ channel blockers tetraethylammonium, Ba2+, and Cs+. This report describes the immunolocalization of TRAAK in brain, spinal cord, and retina of the adult mouse. The most striking finding is the widespread distribution of the TRAAK immunoreactivity, with a prominent staining of the cerebellar cortex, neocortex, hippocampus, dentate gyrus, subiculum, the dorsal hippocampal commissure, thalamus, caudate-putamen, olfactory bulb, and several nuclei in the brainstem. Virtually all neurons express TRAAK, and the highest immunoreactivity was seen in soma, and to a lesser degree in axons and/or dendrites in most areas in brain and spinal cord. In the retina, the TRAAK protein is concentrated to the soma of ganglion cells and to the dendrites of all other neurons. Taken together, these results show a wide distribution of TRAAK, a mechanosensitive and arachidonic acid-stimulated neuron-specific baseline K+ channel, in brain, spinal cord and retina.

Animals↗