Biomedical subjects

M Lazdunski

Publications and source records attributed to M Lazdunski.

At least 91 records · Page 5Linked to original sources

PubMed1998

dGNaC1, a gonad-specific amiloride-sensitive Na+ channel.

Amiloride-sensitive sodium channels have been implicated in reproductive and early developmental processes of several species. These include the fast block of polyspermy in Xenopus oocytes that follows the sperm binding to the egg or blastocoel expansion in mammalian embryo. We have now identified a gene called dGNaC1 that is specifically expressed in the gonads and early embryo in Drosophila melanogaster. The corresponding protein belongs to the superfamily of cationic channels blocked by amiloride that includes Caenorhabditis elegans degenerins, the Helix aspersa FMRF-amide ionotropic receptor (FaNaC), the mammalian epithelial Na+ channel (ENaC), and acid-sensing ionic channels (ASIC, DRASIC, and MDEG). Expression of dGNaC1 in Xenopus oocytes generates a constitutive current that does not discriminate between Na+ and Li+, but is selective for Na+ over K+. This current is blocked by amiloride (IC50 = 24 microM), benzamil (IC50 = 2 microM), and ethylisopropyl amiloride (IC50 = 49 microM). These properties are clearly different from those obtained after expression of the previously cloned members of this family, including ENaC and the human alphaENaC-like subunit, deltaNaC. Interestingly, the pharmacology of dGNaC1 is not very different from that found for the Na+ channel characterized in rabbit preimplantation embryos. We postulate that this channel may participate in gametogenesis and early embryonic development in Drosophila.

Amiloride↗

PubMed1998

The Phe-Met-Arg-Phe-amide-activated sodium channel is a tetramer.

The Helix aspersa Phe-Met-Arg-Phe-amide (FMRFamide)-gated sodium channel is formed by homomultimerization of several FMRFamide-activated Na+ channel (FaNaCh) proteins. FaNaCh is homologous to the subunits that compose the amiloride-sensitive epithelial sodium channel, to Caenorhabditis elegans degenerins, and to acid-sensing ionic channels. FaNaCh properties were analyzed in stably transfected human embryonic kidney cells (HEK-293). The channel was functional with an EC50 for FMRFamide of 1 microM and an IC50 (25 degreesC) for amiloride of 6.5 microM as assessed by 22Na+ uptake measurements. The channel activity was associated with the presence of a protein at the cell surface with an apparent molecular mass of 82 kDa. The 82-kDa form was derived from an incompletely glycosylated form of 74 kDa found in the endoplasmic reticulum. Formation of covalent bonds between subunits of the same complex were observed either after formation of intersubunit disulfide bonds following cell homogenization and solubilization with Triton X-100 or after use of bifunctional cross-linkers. This resulted in the formation of covalent multimers that contained up to four subunits. Hydrodynamic properties of the solubilized FaNaCh complex also indicated a maximal stoichiometry of four subunits per complex. It is likely that epithelial Na+ channels, acid-sensing ionic channels, degenerins, and the other proteins belonging to the same ion channel superfamily also associate within tetrameric complexes.

Animals↗

PubMed1998

Sea anemone peptides with a specific blocking activity against the fast inactivating potassium channel Kv3.4.

Sea anemone venom is known to contain toxins that are active on voltage-sensitive Na+ channels, as well as on delayed rectifier K+ channels belonging to the Kv1 family. This report describes the properties of a new set of peptides from Anemonia sulcata that act as blockers of a specific member of the Kv3 potassium channel family. These toxins, blood depressing substance (BDS)-I and BDS-II, are 43 amino acids long and differ at only two positions. They share no sequence homologies with other K+ channel toxins from sea anemones, such as AsKS, AsKC, ShK, or BgK. In COS-transfected cells, the Kv3.4 current was inhibited in a reversible manner by BDS-I, with an IC50 value of 47 nM. This inhibition is specific because BDS-I failed to block other K+ channels in the Kv1, Kv2, Kv3, and Kv4 subfamilies. Inward rectifier K+ channels are also insensitive to BDS-I. BDS-I and BDS-II share the same binding site on brain synaptic membranes, with K0.5 values of 12 and 19 nM, respectively. We observed that BDS-I and BDS-II have some sequence homologies with other sea anemone Na+ channels toxins, such as AsI, AsII, and AxI. However, they had a weak effect on tetrodotoxin-sensitive Na+ channels in neuroblastoma cells and no effect on Na+ channels in cardiac and skeletal muscle cells. BDS-I and BDS-II are the first specific blockers identified so far for the rapidly inactivating Kv3.4 channel.

Amino Acid Sequence↗

PubMed1998

A structural homologue of colipase in black mamba venom revealed by NMR floating disulphide bridge analysis.

The solution structure of mamba intestinal toxin 1 (MIT1), isolated from Dendroaspis polylepis polylepis venom, has been determined. This molecule is a cysteine-rich polypeptide exhibiting no recognised family membership. Resistance to MIT1 to classical specific endoproteases produced contradictory NMR and biochemical information concerning disulphide-bridge topology. We have used distance restraints allowing ambiguous partners between S atoms in combination with NMR-derived structural information, to correctly determine the disulphide-bridge topology. The resultant solution structure of MIT1, determined to a resolution of 0.5 A, reveals an unexpectedly similar global fold with respect to colipase, a protein involved in fatty acid digestion. Colipase exhibits an analogous resistance to endoprotease activity, indicating for the first time the possible topological origins of this biochemical property. The biochemical and structural homology permitted us to propose a mechanically related digestive function for MIT1 and provides novel information concerning snake venom protein evolution.

Animals↗

PubMed1998

H(+)-gated cation channels: neuronal acid sensors in the NaC/DEG family of ion channels.

Novel members of the amiloride-sensitive Na+ channel/ degenerin family of ion channels were discovered recently. With the cloning of four mammalian H(+)-gated cation channel subunits, the first members of a novel class of ligand-gated cation channels were identified. H(+)-gated cation channel subunits are expressed in the central and peripheral nervous system. In sensory neurones, they are thought to be involved in the perception of pain that accompanies tissue acidosis.

Animals↗

PubMed1998

IKs, a slow and intriguing cardiac K+ channel and its associated long QT diseases.

Shaping of cardiac action potentials depends on a finely tuned orchestra of ion channels. Among them, K(+) channels probably form the most diverse family. They are responsible for inwardly rectifying (I(K1), I(KAch), I(KATP)), transient (I(to)), and sustained outward rectifying (I(Kur), I(Kr), I(Ks)) K(+) currents. The properties of these cardiac K(+) channels have recently been extensively reviewed. This article focuses on recent progress made toward understanding the molecular structure of the particular channel responsible for the slow outward K(+) current I(Ks) and its implication in the delayed ventricular repolarization that characterizes the congenital long QT syndrome.

Journal Article↗

PubMed1998

Expression of TWIK-1, a novel weakly inward rectifying potassium channel in rat kidney.

Several K+ conductances have been identified in the kidney, with specific properties and localization in distinct cell types and membrane domains. On the other hand, several K+ channels have been characterized at the molecular level. By immunolocalization, we show that a new inward rectifying K+ channel, TWIK-1, is specifically expressed in distinct tubular segments and cell types of the rat kidney. In the proximal tubule, TWIK-1 prevails in the initial portions (convoluted part), where it is restricted to the apical (brush-border) membrane. In the collecting duct, immunofluorescence was intracellular or confined to the apical membrane and restricted to intercalated cells, i.e., in cells lacking aquaporin-2, as shown by double immunofluorescence. TWIK was also expressed in medullary and cortical parts of the thick limb of the loop of Henle, identified with an anti-Tamm-Horsfall protein antibody (double immunofluorescence). The intensity of TWIK-1 immunolabeling was unchanged in rats fed a low-Na+ or a low-K+ diet. Because TWIK-1 shares common properties with the low-conductance apical K+ channel of the collecting duct, we propose that it could play a role in K+ secretion, complementary to ROMK, another recently characterized K+ channel located in principal cells of the cortical collecting duct and in the loop of Henle.

Animals↗

PubMed1998

Genetic analysis of the beta subunit of the epithelial Na+ channel in essential hypertension.

Mutations of the last exon of the beta subunit of the amiloride-sensitive epithelial Na+ channel (betaENaC) can lead to Liddle's syndrome, a rare monogenic form of hypertension. The objective of this study was to test whether more subtle changes of betaENaC could be implicated in essential hypertension. After determination of the betaENaC coding gene organization (12 exons spanning 23.5 kb), a systematic screening of the last exon of the gene was performed in 525 subjects (475 whites, 50 Afro-Caribbeans), all probands of hypertensive families. This search was extended to the remaining 11 exons in a subset of 101 probands with low-renin hypertension. Seven amino acid changes were detected: G589S, T594M, R597H, R624C, E632G (last exon), G442V, and V434M (exon 8). These genetic variants were more frequent in subjects of African origin (44%) than in whites (1%). The functional properties of the variants were analyzed in Xenopus oocytes by two independent techniques, ie, electrophysiology and 22Na+ uptake. Small but not significant differences were observed between the variants and wild type. The clinical evaluation of the family bearing the G589S variant, which provided the highest relative ENaC activity, did not show a cosegregation between the mutation and hypertension. The present study illustrates the difficulty in establishing a relation of causality between a susceptibility gene and hypertension. Furthermore, it does not favor a substantial role of the betaENaC gene in essential hypertension.

Adult↗

PubMed1998

HERG and KvLQT1/IsK, the cardiac K+ channels involved in long QT syndromes, are targets for calcium channel blockers.

We examined the effects of the calcium channel blockers nitrendipine, diltiazem, verapamil, bepridil, and mibefradil on the cloned HERG and KvLQT1/IsK K+ channels. These channels generate the rapid and slow components of the cardiac delayed rectifier K+ current, and mutations can affect them, which leads to long QT syndromes. When expressed in transfected COS cells, HERG is blocked in a concentration-dependent manner by bepridil (EC50 = 0.55 microM), verapamil (EC50 = 0.83 microM), and mibefradil (EC50 = 1.43 microM), whereas nitrendipine and diltiazem have negligible effects. Steady state activation and inactivation parameters are shifted to more negative values in the presence of the blockers. Similarly, KvLQT1/IsK is inhibited by bepridil (EC50 = 10.0 microM) and mibefradil (EC50 = 11.8 microM), while being insensitive to nitrendipine, diltiazem, or verapamil. These results demonstrate that both cloned K+ channels HERG and KvLQT1/IsK, which represent together the cardiac delayed rectifier K+ current, are sensitive targets to calcium channel blockers. This work may help in understanding the mechanisms of action of verapamil in certain ventricular tachycardia, as well as some of the deleterious adverse cardiac events associated with bepridil.

Animals↗

PubMed1997

Extracellular ATP and UTP control the generation of reactive oxygen intermediates in human macrophages through the opening of a charybdotoxin-sensitive Ca2+-dependent K+ channel.

Human monocyte-derived macrophages possess a NADPH oxidase that catalyzes superoxide formation upon phagocytosis. Extracellular ATP per se does not activate NADPH oxidase but potentiates superoxide generation triggered by opsonized zymosan. UTP can substitute for ATP with the same efficiency, suggesting that ATP mediates its effects specifically through P2U receptors. Extracellular UTP stimulates a rapid increase in cytoplasmic Ca2+ concentration in monocytic cells, which results from a release of intracellular Ca2+ stores. Moreover, UTP-induced calcium increase is sufficient to activate a charybdotoxin-sensitive Ca2+-dependent outward K+ channel (K(Ca)). The activity of this channel develops between 0.1 and 1.0 microM free cytoplasmic Ca2+ concentration; it is half-blocked by 10 nM charybdotoxin but insensitive to iberiotoxin. Under asymmetrical K+ conditions, this K(Ca) channel does not depend on membrane potential and is characterized by a linear single-current voltage relationship in the voltage range of -100 to +50 mV, giving a unitary conductance of 10 pico-Siemens. Interestingly, ATP/UTP-induced oxygen radicals release was inhibited by charybdotoxin in the same range of concentration as the UTP-induced K(Ca) channel. Furthermore, we show that ATP or UTP fail to enhance oxygen radicals production before K(Ca) channel is expressed (3 days). The electrogenic nature of the NADPH oxidase, i.e., its level of activation, being dependent on the plasmic membrane potential, might provide the causal link between the reactive oxygen intermediates generation and the opening of the K(Ca) channel.

Adenosine Triphosphate↗

PubMed1997

A modulatory subunit of acid sensing ion channels in brain and dorsal root ganglion cells.

MDEG1 is a cation channel expressed in brain that belongs to the degenerin/epithelial Na+ channel superfamily. It is activated by the same mutations which cause neurodegeneration in Caenorhabditis elegans if present in the degenerins DEG-1, MEC-4, and MEC-10. MDEG1 shares 67% sequence identity with the recently cloned proton-gated cation channel ASIC (acid sensing ion channel), a new member of the family which is present in brain and in sensory neurons. We have now identified MDEG1 as a proton-gated channel with properties different from those of ASIC. MDEG1 requires more acidic pH values for activation and has slower inactivation kinetics. In addition, we have cloned from mouse and rat brain a splice variant form of the MDEG1 channel which differs in the first 236 amino acids, including the first transmembrane region. This new membrane protein, which has been called MDEG2, is expressed in both brain and sensory neurons. MDEG2 is activated neither by mutations that bring neurodegeneration once introduced in C. elegans degenerins nor by low pH. However, it can associate both with MDEG1 and another recently cloned H+-activated channel DRASIC to form heteropolymers which display different kinetics, pH dependences, and ion selectivities. Of particular interest is the subunit combination specific for sensory neurons, MDEG2/DRASIC. In response to a drop in pH, it gives rise to a biphasic current with a sustained current which discriminates poorly between Na+ and K+, like the native H+-gated current recorded in dorsal root ganglion cells. This sustained current is thought to be required for the tonic sensation of pain caused by acids.

Acid Sensing Ion Channels↗

PubMed1997

Kv2.1/Kv9.3, a novel ATP-dependent delayed-rectifier K+ channel in oxygen-sensitive pulmonary artery myocytes.

The molecular structure of oxygen-sensitive delayed-rectifier K+ channels which are involved in hypoxic pulmonary artery (PA) vasoconstriction has yet to be elucidated. To address this problem, we identified the Shab K+ channel Kv2.1 and a novel Shab-like subunit Kv9.3, in rat PA myocytes. Kv9.3 encodes an electrically silent subunit which associates with Kv2.1 and modulates its biophysical properties. The Kv2.1/9.3 heteromultimer, unlike Kv2.1, opens in the voltage range of the resting membrane potential of PA myocytes. Moreover, we demonstrate that the activity of Kv2.1/Kv9.3 is tightly controlled by internal ATP and is reversibly inhibited by hypoxia. In conclusion, we propose that metabolic regulation of the Kv2.1/Kv9.3 heteromultimer may play an important role in hypoxic PA vasoconstriction and in the possible development of PA hypertension.

Adenosine Triphosphate↗

PubMed1997

The acid-sensitive ionic channel subunit ASIC and the mammalian degenerin MDEG form a heteromultimeric H+-gated Na+ channel with novel properties.

Proton-gated cation channels are acid sensors that are present in both sensory neurons and in neurons of the central nervous system. One of these acid-sensing ion channels (ASIC) has been recently cloned. This paper shows that ASIC and the mammalian degenerin MDEG, which are colocalized in the same brain regions, can directly associate with each other. Immunoprecipitation of MDEG causes coprecipitation of ASIC. Moreover, coexpression of ASIC and MDEG subunits in Xenopus oocytes generates an amiloride-sensitive H+-gated Na+ channel with novel properties (different kinetics, ionic selectivity, and pH sensitivity). In addition, coexpression of MDEG with mutants of the ASIC subunit can create constitutively active channels that become completely nonselective for Na+ versus K+ and H+-gated channels that have a drastically altered pH sensitivity compared with MDEG. These data clearly show that ASIC and MDEG can form heteromultimeric assemblies with novel properties. Heteromultimeric assembly is probably used for creating a diversity of H+-gated cation channels acting as neuronal acid sensors in different pH ranges.

Acid Sensing Ion Channels↗

PubMed1997

New modulatory alpha subunits for mammalian Shab K+ channels.

Two novel K+ channel alpha subunits, named Kv9.1 and Kv9.2, have been cloned. The Kv9.2 gene is situated in the 8q22 region of the chromosome. mRNAs for these two subunits are highly and selectively expressed in the nervous system. High levels of expressions are found in the olfactory bulb, cerebral cortex, hippocampal formation, habenula, basolateral amygdaloid nuclei, and cerebellum. Interestingly Kv9.1 and Kv9.2 colocalized with Kv2.1 and/or Kv2.2 alpha subunits in several regions of the brain. Neither Kv9.1 nor Kv9.2 have K+ channel activity by themselves, but both modulate the activity of Kv2.1 and Kv2.2 channels by changing kinetics and levels of expression and by shifting the half-inactivation potential to more polarized values. This report also analyzes the changes in electrophysiological properties of Kv2 subunits induced by Kv5.1 and Kv6.1, two other modulatory subunits. Each modulatory subunit has its own specific properties of regulation of the functional Kv2 subunits, and they can lead to extensive inhibitions, to large changes in kinetics, and/or to large shifts in the voltage dependencies of the inactivation process. The increasing number of modulatory subunits for Kv2.1 and Kv2.2 provides an amazingly new capacity of functional diversity.

Amino Acid Sequence↗

PubMed1997

TASK, a human background K+ channel to sense external pH variations near physiological pH.

TASK is a new member of the recently recognized TWIK K+ channel family. This 395 amino acid polypeptide has four transmembrane segments and two P domains. In adult human, TASK transcripts are found in pancreas<placenta<brain<lung, prostate<heart, kidney<uterus, small intestine and colon. Electrophysiological properties of TASK were determined after expression in Xenopus oocytes and COS cells. TASK currents are K+-selective, instantaneous and non-inactivating. They show an outward rectification when external [K+] is low ([K+]out = 2 mM) which is not observed for high [K+]out (98 mM). The rectification can be approximated by the Goldman-Hodgkin-Katz current equation that predicts a curvature of the current-voltage plot in asymmetric K+ conditions. This strongly suggests that TASK lacks intrinsic voltage sensitivity. The absence of activation and inactivation kinetics as well as voltage independence are characteristic of conductances referred to as leak or background conductances. For this reason, TASK is designated as a background K+ channel. TASK is very sensitive to variations of extracellular pH in a narrow physiological range; as much as 90% of the maximum current is recorded at pH 7.7 and only 10% at pH 6.7. This property is probably essential for its physiological function, and suggests that small pH variations may serve a communication role in the nervous system.

Amino Acid Sequence↗

PubMed1997

Properties of KvLQT1 K+ channel mutations in Romano-Ward and Jervell and Lange-Nielsen inherited cardiac arrhythmias.

Mutations in the delayed rectifier K+ channel subunit KvLQT1 have been identified as responsible for both Romano-Ward (RW) and Jervell and Lange-Nielsen (JLN) inherited long QT syndromes. We report the molecular cloning of a human KvLQT1 isoform that is expressed in several human tissues including heart. Expression studies revealed that the association of KvLQT1 with another subunit, IsK, reconstitutes a channel responsible for the IKs current involved in ventricular myocyte repolarization. Six RW and two JLN mutated KvLQT1 subunits were produced and co-expressed with IsK in COS cells. All the mutants, except R555C, fail to produce functional homomeric channels and reduce the K+ current when co-expressed with the wild-type subunit. Thus, in both syndromes, the main effect of the mutations is a dominant-negative suppression of KvLQT1 function. The JLN mutations have a smaller dominant-negative effect, in agreement with the fact that the disease is recessive. The R555C subunit forms a functional channel when expressed with IsK, but with altered gating properties. The voltage dependence of the activation is strongly shifted to more positive values, and deactivation kinetics are accelerated. This finding indicates the functional importance of a small positively charged cytoplasmic region of the KvLQT structure where two RW and one JLN mutations have been found to take place.

Amino Acid Sequence↗

PubMed1997

Molecular cloning of a non-inactivating proton-gated Na+ channel specific for sensory neurons.

We have cloned and expressed a novel proton-gated Na+ channel subunit that is specific for sensory neurons. In COS cells, it forms a Na+ channel that responds to a drop of the extracellular pH with both a rapidly inactivating and a sustained Na+ current. This biphasic kinetic closely resembles that of the H+-gated current described in sensory neurons of dorsal root ganglia (1). Both the abundance of this novel H+-gated Na+ channel subunit in sensory neurons and the kinetics of the channel suggest that it is part of the channel complex responsible for the sustained H+-activated cation current in sensory neurons that is thought to be important for the prolonged perception of pain that accompanies tissue acidosis (1, 2).

Acid Sensing Ion Channels↗

PubMed1997

Behaviors and neurodegeneration induced by two blockers of K+ channels, the mast cell degranulating peptide and Dendrotoxin I.

Both the Mast Cell Degranulating (MCD) peptide and Dendrotoxin I (DTX(I)), two blockers of fast activation and slowly inactivating K+ channels, induced epileptiform seizures and brain damage after intracerebroventricular injection (200 pmol) in Sprague-Dawley rats. A considerable variation in the response of the rats was observed for each toxin. The neurodegeneration included the hippocampal formation, subiculum, septum, amygdala, and the cerebellum for both toxins, and the neocortex and anterior thalamic nuclei exclusively for DTX(I) treatment.

Animals↗