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B Attali

Publications and source records attributed to B Attali.

At least 37 records · Page 2Linked to original sources
PubMed1996

Chronic morphine administration enhances the expression of Kv1.5 and Kv1.6 voltage-gated K+ channels in rat spinal cord.

Prolonged opiate administration leads to the development of tolerance and dependence. These phenomena are accompanied by selective regulation of distant cellular proteins and mRNAs, including ionic channels. Acute opiate administration differentially affects voltage-dependent K+ currents. Whereas, opiate activation of K+ channels is well established opioid-induced inhibition of K+ conductance has also been studied. In this study, we focused on the effect of chronic morphine exposure on voltage-dependent Shaker-related Kv1.5 and Kv1.6 K+ channel gene expression and on Kv1.5 protein levels in the rat spinal cord. Several experimental approaches including in-situ hybridization, RNAse protection, reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry were employed. We found that motor neurons are highly enriched in Kv1.5 and Kv1.6 mRNA and in Kv1.5 channel protein. Moreover, we found significant increases in the amount of mRNA encoding for these two K+ channels and in Kv1.5 channel protein in the spinal cord of morphine-treated rats, compared with controls. For example, quantitative in-situ hybridization, revealed a 2.1 +/- 0.15- and 2.3 +/- 0.5-fold increase in Kv1.5 and Kv1.6 channel mRNA levels, respectively. Similar results were obtained by semiquantitative RT-PCR analyses. Kv1.5 protein level was increased by 1.9-fold in the spinal cord or morphine-treated rats. Our results suggest that Kv1.5 and Kv1.6 Shaker K+ channels play an important role in regulating motor activity that increases in mRNA and protein levels of the spinal cord K+ channels after chronic morphine exposure could be viewed as a cellular adaptation which compensates for a persistent opioid-induced inhibition of K+ channel activity. These alterations may account, in part, for the cellular events leading to opiate tolerance and dependence.

Animals↗
PubMed1996

Cytoplasmic and extracellular IsK peptides activate endogenous K+ and Cl- channels in Xenopus oocytes. Evidence for regulatory function.

IsK is a 14.5-kDa type III membrane glycoprotein which induces slowly activating K+ and Cl- currents when expressed in Xenopus oocytes and HEK 293 cells. Recently, mutagenesis experiments identified amino- and carboxyl-terminal domains of IsK as critical for induction of Cl- and K+ currents, respectively. This hypothesis was tested by examining effects of synthetic IsK hydrophilic peptides on untreated Xenopus oocytes. In agreement with IsK membrane topology, we show here that peptides derived from carboxyl and amino termini are sufficient to activate slow K+ and Cl- channels whose biophysical and pharmacological characteristics are similar to those exhibited by the native IsK protein. That data provide further evidence that IsK is a regulatory subunit of pre-existing silent channel complexes rather than a channel per se.

Amino Acid Sequence↗
PubMed1996

K+ channel antisense oligodeoxynucleotides inhibit cytokine-induced expansion of human hemopoietic progenitors.

Primitive human hemopoietic progenitor cells identified by surface membrane markers CD33-CD34+ are capable of expansion into lineage-restricted precursors following in vitro stimulation by hemopoietic regulators such as stem cell factor (SCF) and interleukin-3 (IL-3). In search of ionic currents involved in cytokine-induced progenitor cell growth and differentiation, human umbilical cord blood CD33-CD34+ cells were subjected to perforated patch-clamp recordings following overnight incubation with SCF and/or IL-3. An inward rectifying potassium channel (Kir) was found in 33% of control unstimulated cells, in 34% of cells incubated with IL-3, in 31% of cells incubated with SCF and in 75% of cells incubated with IL-3 plus SCF. Kir activity increased with elevation of extracellular potassium and was blocked by extracellular Cs+ or Ba2+ Antisense oligodeoxynucleotides directed against Kir blocked both mRNA and functional expression of Kir channels. Kir antisense also inhibited the in vitro expansion of cytokine-stimulated CD33-CD34+ cells into erythroid (BFU-E) and myeloid (GM-CFU) progenitors in 7-day suspension cultures. Extracellular Cs+ or Ba2+ induced a similar degree of inhibition (40-60%) of progenitor cell generation. These findings strongly suggest an essential role for Kir in the process of cytokine-induced primitive progenitor cell growth and differentiation.

Antigens, CD↗
PubMed1995

Increased expression of synapsin I mRNA in defined areas of the rat central nervous system following chronic morphine treatment.

Chronic opiate administration leads to a selective regulation of several cellular proteins and mRNAs. This phenomenon has been viewed as a compensatory mechanism to the opiate signaling leading to the development of opiate addiction. In this study, in situ hybridization histochemistry experiments were employed to investigate the effect of chronic morphine treatment on synapsin I gene expression. We show here for the first time that prolonged morphine exposure causes a selective increase in the mRNA levels of synapsin I in several brain regions which are considered to be important for opiate action. Quantitative analysis of the signals, obtained by hybridization of digoxigenin-labeled antisense RNA probe, revealed a 5.8- and 7-fold increase of synapsin I mRNA levels in the locus coeruleus and the amygdala of morphine-treated rats, respectively, as compared with control untreated rats. Increased expression of synapsin I mRNA was also observed in the spinal cord of morphine-treated rats (by 3.8-fold). Since opiates were shown to attenuate neurotransmitter release and reduce synapsin I phosphorylation, it is suggested that the increase in synapsin I levels would lead to the requirement of higher amounts of opiate agonists to obtain the opiate physiological effects. These results suggest that the increases in mRNA levels of synapsin I in these specific areas can be part of the molecular mechanism(s) underlying opiate tolerance and withdrawal.

Animals↗
PubMed1995

A corticosteroid-induced gene expressing an "IsK-like" K+ channel activity in Xenopus oocytes.

Screening a rat colon cDNA library for aldosterone-induced genes resulted in the molecular cloning of a cDNA whose corresponding mRNA is strongly induced in the colon by dexamethasone, aldosterone, and a low NaCl diet. A similar mRNA was detected in kidney papilla but not in brain, heart, or skeletal muscle. Xenopus laevis oocytes injected with cRNA synthesized from this clone, designated CHIF (channel-inducing factor), express a K(+)-specific channel activity. The biophysical, pharmacological, and regulatory characteristics of this channel are very similar to those reported before for IsK (minK). These include: slow (tau > 20 s) activation by membrane depolarization with a threshold potential above -50 mV, blockade by clofilium, inhibition by phorbol ester, and activation by 8-bromoadenosine 3',5'-cyclic monophosphate and high cytoplasmic Ca2+. The primary structure of this clone, however, shows no homology to IsK. Instead, CHIF exhibits > 50% similarity to two other short bitopic membrane proteins, phospholemman and the gamma subunit of Na+K(+)-ATPase. The data are consistent with the possibility that CHIF is a member of a family of transmembrane regulators capable of activating endogenous oocyte transport proteins.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1994

External blockade of the major cardiac delayed-rectifier K+ channel (Kv1.5) by polyunsaturated fatty acids.

The present work shows that arachidonic acid and some other long chain polyunsaturated fatty acids such as docosahexaenoic acid, which is abundant in fish oil, produce a direct open channel block of the major voltage-dependent K+ channel (Kv1.5) cloned in cardiac cells. The inhibitory action of these selected fatty acids is seen when they are applied extracellularly but not when they are included in the patch pipette. Fatty acids then appear to bind to an external site on the Kv1.5 channel structure. Inhibition of Kv1.5 channel activity by polyunsaturated fatty acids (acceleration of the apparent inactivation and decrease of the peak current) is similar to that produced by the class III antiarrhythmic tedisamil. Docosahexaenoic acid and arachidonic acid also inhibit the delayed-rectifier K+ channel currents in cultured mouse and rat cardiomyocytes. These results are discussed in the light of the reported fatty acids effects on cardiac function in diseased states. Since Kv1.5 is also present in the brain, the results reported here could also have a significance in terms of processes such as long-term potentiation or depression.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed1993

Multiple mRNA isoforms encoding the mouse cardiac Kv1-5 delayed rectifier K+ channel.

The mouse Kv1-5 K+ channel cDNA has been cloned from heart. This channel was highly expressed in heart and, to a lesser extent, in other tissues, including brain and thymus. Two alternatively spliced isoforms were found. The longer form encoded a 602-amino acid protein, while in the short form (Kv1-5 delta 5'), the first 200 amino acids lying upstream the transmembrane segment S1 were deleted. RNase protection experiments showed that both Kv1-5 mRNA isoforms are present in the mouse tissues examined, the longer form being predominant. The short mRNA (Kv1-5 delta 5') arose by an unusual splicing event within the exonic sequence. An additional short cDNA clone (Kv1-5 delta 3') that codes for a carboxyl-terminal truncated protein has been isolated. The gene coding sequence contained a single exon and has been mapped on human chromosome 12 (p13) and on mouse chromosome 6 (band F). Expression in Xenopus oocytes revealed that the long (Kv1-5) and the amino-terminal deleted (Kv1-5 delta 5') isoforms elicited similar K+ currents with a drastically decreased efficacy for Kv1-5 delta 5'. The carboxyl-terminal truncated Kv1-5 delta 3' clone was not functional but inhibited the expression of the long isoform.

Alternative Splicing↗
PubMed1993

The protein IsK is a dual activator of K+ and Cl- channels.

The protein IsK (M(r) 14,500) is present in epithelial cells, heart, uterus and lymphocytes and induces slowly activating K+ currents when expressed in Xenopus oocytes. The finding that mutations of its single transmembrane segment altered channel gating or selectivity has suggested that IsK is a channel-forming protein. But IsK does not exhibit the K+ channel hallmarks (a conserved K+ selective pore (H5) flanked by either six or two membrane-spanning regions). Here we report that IsK expression in Xenopus oocytes also induces a Cl- selective current very similar to the Cl- current produced by phospholemman expression and with biophysical, pharmacological and regulation characteristics very different from those of the IsK-induced K+ channel activity. IsK mutagenesis identifies amino- and carboxy-terminal domains as critical for the induction of Cl- and K+ channel activities, respectively. Our data lead to a model in which the IsK protein (now called IsK, Cl) acts as a potent activator of endogenous and otherwise silent K+ or Cl- channels.

Amino Acid Sequence↗
PubMed1993

Heterologous desensitization and reduced G protein ADP-ribosylation following exposure to alpha 2-adrenoceptor and muscarinic receptor agonists.

We investigated the acute and chronic effects of alpha 2-adrenoceptor and muscarinic receptor agonists on dihydropyridine-sensitive voltage-dependent Ca2+ channels in spinal cord-dorsal root ganglion cocultures. Clonidine and oxotremorine inhibited the voltage-dependent Ca2+ influx (42 +/- 2% and 35 +/- 6% with 100 microM, respectively). The respective antagonists, yohimbine and atropine, abolished these effects. Pertussis toxin attenuated the inhibitory effects of clonidine and oxotremorine on Ca2+ influx, demonstrating involvement of G proteins in the transduction process. Chronic treatment with clonidine or oxotremorine desensitized the Ca2+ channel response to the agonist applied as well as to the other receptor agonist (heterologous desensitization). Such treatment with clonidine or oxotremorine decreased the pertussis toxin-catalyzed ADP-ribosylation of Gi alpha and G(o) alpha subunits, an effect which could be largely reversed by the detergent Lubrol PX. Yohimbine and atropine blocked the effects of clonidine or oxotremorine on pertussis toxin-catalyzed ADP-ribosylation. Results suggest that alpha 2-adrenoceptor and muscarinic receptors couple to the dihydropyridine-sensitive voltage-dependent Ca2+ channels via pertussis toxin-sensitive G proteins. Chronic agonist treatment leads to heterologous desensitization and to a reduced capacity of Gi and G(o) to undergo pertussis toxin-catalyzed ADP-ribosylation.

Adenosine Diphosphate Ribose↗
PubMed1993

Are Xenopus oocytes unique in displaying functional IsK channel heterologous expression?

IsK is a novel membrane protein that induces a slow voltage-gated K+ current when expressed in Xenopus oocytes. Attempts were made to express this channel in several eukaryotic cells including the skeletal muscle cell line C2C12, the fibroblastic cell line CHO and the T lymphocyte cell line Jurkat. In spite of the clear demonstration of successful transfection, no IsK current could be recorded from these cells. Overexpression was also obtained using the vaccinia/T7 and baculovirus systems, again without any success in demonstrating the presence of the K+ current in the plasma membrane of infected cells. Bilayer reconstitution experiments using membranes highly enriched in IsK protein ruled out the possibility that the IsK protein could induce a K+ channel located in intracellular organelles. Collectively, these data suggest the possibility that IsK protein is part of a K+ channel complex but is only active in association with another protein endogenous to the Xenopus oocyte. Other hypotheses are also discussed.

Animals↗
PubMed1992

Effects of the level of mRNA expression on biophysical properties, sensitivity to neurotoxins, and regulation of the brain delayed-rectifier K+ channels Kv1.2.

Injection of 0.2 ng of cRNA encoding the brain Kv1.2 channel into Xenopus oocytes leads to the expression of a very slowly inactivating K+ current. Inactivation is absent in oocytes injected with 20 ng of cRNA although activation remains unchanged. Low cRNA concentrations generate a channel which is sensitive to dendrotoxin I (IC50 = 2 nM at 0.2 ng of cRNA/oocyte) and to less potent analogs of this toxin from Dendroaspis polylepis venom. A good correlation is found between blockade of the K+ current and binding of the different toxins to rat brain membranes. High cRNA concentrations generate another form of the K+ channel which is largely insensitive to dendrotoxin I (IC50 = 200 nM at 20 ng of cRNA per oocyte). At low cRNA concentrations, the expressed Kv1.2 channel is also blocked by other polypeptide toxins such as MCD peptide (IC50 = 20 nM), charybdotoxin (IC50 = 50 nM), and beta-bungarotoxin (IC50 = 50 nM), which bind to distinct and allosterically related sites on the channel protein. The pharmacologically distinct type of K+ channel expressed at high cRNA concentrations (20 ng of cRNA/oocyte) is nearly totally resistant to 100 nM MCD peptide and hardly altered by charybdotoxin and beta-bungarotoxin at concentrations as high as 1 microM. Both at low and at high cRNA concentrations, the expressed Kv1.2 channel is blocked by an increase in intracellular Ca2+ from the inositol trisphosphate sensitive pools and by the phorbol ester PMA that activates protein kinase C.

Allosteric Site↗
PubMed1992

Developmental expression of voltage-sensitive K+ channels in mouse skeletal muscle and C2C12 cells.

The developmental expression of voltage-sensitive K+ channels was analyzed by Northern blot in mouse skeletal muscle. Of nine Shaker-like genes studied, eight are expressed in this mammalian muscle. Their expression is differentially regulated during development. The mouse cell line C2C12 has been used to study expression of voltage-sensitive K+ channels during in vitro myotube differentiation. Different voltage-sensitive K+ channel messages are also expressed in these cells which display a pattern of expression depending upon the differentiation stage. The message for the very peculiar K+ channel of IsK type could only be detected by polymerase chain reaction on skeletal muscle mRNA.

Animals↗
PubMed1992

Regulation of a major cloned voltage-gated K+ channel from human T lymphocytes.

When expressed into Xenopus oocytes, HLK3 K+ channel (Kv1-3) induced a slowly inactivating voltage-dependent K+ current. We have studied the modulation of this K+ current by co-expressing a cloned 5-HT2 receptor together with HLK3 K+ channel protein. Application of 5-HT caused a long-lasting inhibition of the voltage-gated K+ current. This inhibitory modulation was mimicked by intracellular injection of inositol triphosphate or Ca2+, as well as by incubation with phorbol esters or diacylglycerol analogs. Oocytes pretreatment with staurosporine and EGTA fully prevented 5-HT inhibitory action. Elevation of cAMP and cGMP levels into oocytes did not produce any detectable effect on the current recorded in the absence or the presence of 5-HT. These data suggest that the second messengers generated by phospholipase C activation may be important modulators of HLK3 K+ channels in the immune and the central nervous systems.

Animals↗
PubMed1992

Receptor-mediated regulation of IsK, a very slowly activating, voltage-dependent K+ channel in Xenopus oocytes.

Expression of IsK in Xenopus oocytes has been obtained in 2 ways: (i) by injection of cardiac polyA+ RNA from neonatal mouse heart; (ii) by injection of a cRNA synthesized in vitro. It was observed that polyA+ RNA not only directs the expression of the IsK channel but also contains purinergic P2 and endothelin receptors. Stimulation of these receptors, that produce intracellular Ca2+ increase together with diacylglycerol production activating protein kinase C, increases IsK activity. The same type of results and the same conclusions were obtained by co-injecting cRNA's corresponding to the 5-HT2 receptor and the IsK channel into oocytes. This stimulatory effect was shown to be due to Ca2+ via a calmodulin-dependent kinase process. Conversely, activation of protein kinase C pathway alone by phorbol esters leads to inhibition of IsK activity.

Adenosine Triphosphate↗
PubMed1992

Cloning, functional expression, and regulation of two K+ channels in human T lymphocytes.

Low stringency screening of a Jurkat cDNA library with a rat brain K+ channel (RCK1) probe has resulted in the isolation of HLK3, a voltage-gated K+ channel. In Xenopus oocytes, the HLK3 clone directs the expression of a rapidly activating transient outward K+ current similar to the type n K+ current recorded in Jurkat T cells. The HLK3 gene is located on the short arm of human chromosome 1 (p13.3). Polymerase chain reaction was used to clone HIsK from Jurkat cDNA. The HIsK clone shares the same sequence with a previously described genomic clone (Murai, T., Kazikuka, A., Takumi, T., Ohkubo, H., and Nakanishi, S. (1989) Biochem. Biophys. Res. Commun. 161, 176-181). In Xenopus oocytes, it encodes a slowly activating, noninactivating K+ channel which cannot be recorded in Jurkat cells by conventional patch-clamp techniques. Transcripts of both clones are present at a similar level before and after activation of purified human T lymphocytes and Jurkat cells, reflecting a constitutive expression of K+ channel messages. This finding is in good agreement with the electrophysiological results for type n K+ current density on the same cells. HLK3 current is very sensitive to the scorpion toxin charybdotoxin (IC50 = 0.8 nM). HIsK current is totally insensitive to this toxin but is blocked by the antiarrhythmic clofilium (IC50 = 80 microM). While charybdotoxin has no effect on interleukin 2 mRNA induction, clofilium potently inhibits interleukin 2 mRNA expression upon mitogen-induced T cell activation. It is concluded that the HLK3 channel is not an important component of the T cell mitogenic response. Other targets for K+ channel blockers, such as the HIsK protein, could be involved in the activation process.

Amino Acid Sequence↗
PubMed1992

ISK, a slowly activating voltage-sensitive K+ channel. Characterization of multiple cDNAs and gene organization in the mouse.

mISK is a protein consisting of 129 amino acids with a single putative transmembrane domain. The injection of mISK cRNA into Xenopus oocytes directs the expression of a voltage-gated K+ current. A heart mRNA blot probed with mISK DNA revealed at least two transcripts. The messenger diversity of mISK was investigated by cloning and characterization of multiple cDNAs of one genomic clone, and by performing primer extension experiments. All cDNAs characterized have the same protein-coding sequence, and heterogeneity of the transcripts arises from alternative splicing, and multiple sites of transcription start and polyadenylation. ISK is encoded by a single gene in the mouse genome. The gene organization reveals the existence of an exon containing the whole protein-coding sequence and of two alternative exons corresponding to the 5' untranslated sequences. We failed to detect the presence of another exon capable of extending the protein-coding sequence. The diversity of mISK messengers is not associated with a diversity of the mISK protein.

Amino Acid Sequence↗
PubMed1992

Different types of K+ channel current are generated by different levels of a single mRNA.

A cloned human voltage-sensitive K+ channel HLK3 which is present in T-lymphocytes and in the brain was expressed in Xenopus oocytes and after permanent transfection of a human B-lymphocyte cell line (IM9). Injections of low cRNA concentrations into Xenopus oocytes led to the expression of a transient K+ current, with saturating current-voltage (I-V) relationship, which was abolished by repetitive stimulations due to a slow recovery from inactivation. This transient K+ channel current was fully inhibited by 10 nM charybdotoxin. Injection of high concentrations of the same RNA led to a non-inactivating K+ current, with linear I-V curve, which did not undergo use-dependent inactivation and was hardly sensitive to 10 nM charybdotoxin. Intermediate behaviour due to changing proportions of these two types of K+ channel expression were observed at intermediate RNA concentrations. Transient and non-inactivating K+ currents were also observed by both whole-cell and single channel patch-clamp recording from HLK3 transfected IM9 cells. The main conductance of the channel in the two different modes (inactivating and charybdotoxin-sensitive or non-inactivating and charybdotoxin-resistant) is the same (12-14 pS). Destruction of the cytoskeletal elements with cytochalasin D, colchicine or botulinum C2 toxin in oocyte experiments prevented expression of the sustained mode of the K+ channel. The results suggest that the sustained mode obtained at high RNA concentrations corresponds to channel clustering involving cytoskeletal elements. This differential functional expression of K+ channels associated with different levels of mRNA appears as a new important factor to explain the biophysical and pharmacological diversity of voltage-sensitive K+ channels.

Animals↗