Biomedical subjects
Publications and source records attributed to F Lesage.
The recent cloning of numerous voltage-activated K+ channels provides new information concerning the architecture of K+ channel proteins. The combination of molecular genetic and biophysical methods gives us a new insight into the molecular mechanisms of K+ channel pharmacology.
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.
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.
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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.
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.
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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.
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.
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.
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.
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.
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.
For several years, the authors have been using a columellar transalar incision through the medial crus along the nasal septum, reaching the vault and continued laterally by a transalar cartilaginous incision. The nasal tip is thus opened like a car-hood, leaving the alar cartilage attached to the skin and exposing the triangular cartilage and the nasal septum. This approach allows septoplasty to be performed with excellent exposure. The tip is easily revised backwards allowing anomalies to be viewed directly. The advantages of this external approach compared with the classical external Rethi's approach are multiple: simplicity and rapidity of the technique, excellent access to triangular cartilages and the triangular-septum junction; possibility of always going from an endonasal approach to an external one and excellent access to the tip. Moreover, there is no long term swelling of the tip. The indications of this external approach are asymmetry of the tip, excessive tip projection, saddle nose deformities and secondary rhinoplasties. No complication has been observed in a series of 32 patients with a follow-up of 6 to 48 months. There is no disturbance of the tip position and the skin scar is imperceptible.
Neonatal mouse cardiac poly(A)+ mRNA microinjection into Xenopus oocytes directed the expression of a delayed rectifier K+ current. A cDNA encoding this channel, called mIsK, was cloned from a neonatal mouse heart cDNA library whose properties were studied after expression of the complementary RNA in Xenopus oocytes. Among the different known K+ channel blockers, only the class III antiarrhythmic clofilium inhibited mIsK in the 10-100 microM range. The channel was completely insensitive to other antiarrhythmics such as quinine, quinidine, sotalol or amiodarone. mIsK was enhanced by increasing intracellular Ca2+ and by microinjected Ca(2+)-calmodulin dependent protein kinase II. These stimulations were reversed by the calmodulin antagonist W7. Conversely, the phorbol ester PMA, the diacylglycerol analog OAG and microinjected purified protein kinase C inhibited mIsK. This inhibitory effect could be prevented by the protein kinase C inhibitor staurosporine. These results were consistent with the presence of consensus sequences for kinase II and kinase C in the mIsK structure. Cultured newborn mouse ventricular cardiac cells exhibited a delayed rectifier K+ current which had biophysical properties similar to those of cloned mIsK and which was inhibited by clofilium and protein kinase C activators. In situ hybridization experiments revealed that mIsK mRNA was homogeneously distributed in the cardiac tissue. Neonatal mouse heart expressed the most mIsK mRNA compared with various other rat and mouse tissues. Since this K+ channel generates a current which appears to be involved in the control of both the action potential duration and the beating rate, these results suggest an important role for the mIsK channel in cardiac cell physiology and cardiac pathology.
The authors review the notes of 40 patients who underwent surgery for large sacral pressure sore over the last 10 years. In most cases a gluteus maximus flap with skin graft has been employed to cover sacral sore, as originally described by Ger in 1971. The results of this series are then compared with those published by other authors using different techniques, with particular attention to gluteus maximus musculo-cutaneous island flaps and purely cutaneous flaps. All procedures carry a small mortality rate because of the advanced age and frail conditions of most patients. In our series 2 patients died in the postoperative period; in all the others we eventually achieved a sound healing of the sacral sore. We conclude that the gluteus maximus rotation flap is a safe and effective for the treatment of this condition. Its only major drawback is represented by the prolonged period required for the epithelisation by secondary intention of some fairly frequent areas of failure on the skin graft. The importance of a multidisciplinary team approach in the management of these patients cannot be overemphasised.
Combined allergological and anaesthetic consultations have been started in the last few years in eight French Teaching Hospitals so as to explore peranaesthetic anaphylactoid shocks. A survey was carried out in these centers in order to collect patients investigated with the same protocol, for the assessment of the incidence of anaphylaxis in France, as well as the involved drugs. Investigations were always carried out at least 6 to 8 weeks after the accident. The tests used to diagnose IgE-dependent anaphylaxis were skin tests (prick and intradermal tests, carried out in all eight centers), the radioimmunological assay of specific anti-quaternary ammonium IgE, together with an inhibition test with thiopentone and propofol (six centers), leukocyte histamine release (five centers) and human basophil degranulation tests (three centers) for those drugs for which no specific antibody assay exists. The collected data involved 1,240 patients, investigated within the last four years. Anaphylaxis was diagnosed in 821 patients (66.2%). Muscle relaxants were responsible in 668 cases (80% of cases of anaphylaxis). Suxamethonium was the main cause (54.3% of shocks due to muscle relaxants), followed by vecuronium (15.3%). General anaesthetics (hypnotics and benzodiazepines) were responsible for 9.2% of all cases of anaphylaxis opioids for 2.6%. There were only three cases of shock due to local anaesthetic agents. Latex and ethylene oxide are becoming increasingly involved. It would therefore seem mandatory to carry out after any anaphylactoid accident an assessment with sensitive and specific tests for anaphylaxis. Diagnosing anaphylaxis means that the involved drug should be used never again in that patient. Because muscle relaxants are by far the most involved drugs, anaesthetists should use them only when really required.
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