Remodelage des canaux potassiques dépendants de l'ATP lors de l'hypertrophie et de l'insuffisance cardiaque

RESUME :Introduction. Les maladies cardiovasculaires représentent la première cause de mortalité dans les pays développés et l'insuffisance cardiaque (IC) est la plus fréquente. Suite à un infarctus, le coeur des patients subit un remodelage ventriculaire pouvant évoluer vers un état d'IC. L'IC se définit comme un état dans lequel le coeur n'est plus capable d'approvisionner suffisamment les organes et cet état s'accompagne souvent de troubles du rythme cardiaque. Le remodelage ventriculaire touche de nombreux gènes codant à la fois pour les voies métaboliques et pour des canaux ioniques favorisant ainsi l'apparition des arythmies responsables de la mort subite des patients atteints d'IC. Comprendre ce passage entre remodelage et IC est crucial afin de pouvoir un jour prévenir l'IC et les complications médicales qui l'accompagnent. Nous nous sommes intéressés aux canaux potassiques dépendants de l'ATP (KATP) car ces canaux ont la capacité de coupler le métabolisme de la cellule à son activité électrique. En effet, les canaux KATP s'ouvrent quand la charge énergétique (rapport ATP/ ADP) de la cellule chute. Dans les cardiomyocytes, l'ouverture des KATP induit une hyperpolarisation de la membrane cellulaire ce qui diminue indirectement la surcharge calcique et de ce fait préserve la cellule. Les canaux KATp sont formés de 4 sous-unités Kir6.x (Kir6.1 ou Kir6.2) formant le pore du canal associées à 4 sous-unités régulatrices SUR. Les propriétés électrophysiologiques ainsi que la sensibilité pharmacologique des canaux KATP dépendent de leur composition et seuls les canaux KATP formés par la sous-unité Kirô.l sont activés par le diazoxyde.Méthodes et résultats. Nous avons d'abord montré dans un modèle in vivo d'IC chez le rat adulte que les sous-unités Kir6.1 et SUR sont surexprimées dans ces conditions pathologiques. Par ailleurs, les cardiomyocytes issus des coeurs infarcis deviennent sensibles au diazoxyde reflétant la surexpression de Kir6.1. Les potentiels d'action qui sont prolongés dans l'IC et qui sont à l'origine d'arythmies majeures sont normalisés par l'ouverture des canaux KATp induite par le diazoxyde. Ainsi, l'ouverture pharmacologique des canaux KATp contribuerait à la cardio-protection. Dans une seconde partie, nous avons déterminé quels étaient les facteurs de transcription responsables de ce changement d'expression des sous-unités formant les KATP. Dans notre modèle, nous avons pu montrer que la surexpression de Kirô.l est due aux facteurs de transcription Fox03 et FoxF2 qui est aussi responsable de la surexpression des sous-unités SUR. Dans la dernière partie de ce travail, nous avons mis au point un modèle d'IC in vitro en cultivant les cardiomyocytes de rats adultes en présence d'angiotensine II (Angll) ou de TNFa. Ce modèle expérimental nous a non seulement permis de mettre en relation l'importance de L'AnglI et du TNFa sur le remodelage des canaux KATP mais aussi de développer un modèle in vitro présentant les mêmes caractéristiques que le modèle in vivo concernant le remodelage des KATP lors de l'IC. Ce dernier modèle expérimental ouvre des perspectives afin de mieux caractériser les voies de signalisation impliquées dans le remodelage des canaux KATp lors de l'IC.Conclusion. Les canaux KATp subissent un remodelage lors de l'IC et les résultats obtenus montrent le potentiel cardio-protecteur de ces canaux.ABSTRACT :Background and aim. Cardiovascular disease is the leading cause of death in developed countries and heart failure (HF) is the most common. Following myocardial infarction, the heart of the patient undergoes ventricular remodeling which may evolve toward a state of HF. HF is defined as a state in which heart is unable to supply enough blood to organs and this state is often accompanied by cardiac arrhythmias. Ventricular remodeling involves many genes coding for both metabolic enzymes and ion channels. Changes in ion channel expression can promote arrhythmias responsible for sudden death in patients with HF. A better understanding of the transition between remodeling and HF is crucial in order to prevent the complications associated to HF We were interested in ATP-dependent potassium channels (KATp) because they couple cell metabolism to electrical activity of the cell. Indeed, KATP channels open when the energy charge (ratio of ATP / ADP) of the cell collapses. In cardiomyocytes, the opening of KATP channels induces hyper- polanzation of the cell membrane which reduces calcium overload and thereby protects the cell. KATp channels are composed by 4 Kir6.x subumts (Kir6.1 or Kir6.2) forming the pore channel associated with 4 regulatory subunits SUR. The electrophysiological properties as well as pharmacological sensitivity of KATp channels depend on their composition and only KATP channels formed by Kir6.1 subunit are activated by diazoxide.Methods and results. Firstly, using an in vivo model of HF in adult rats, we showed that Kir6.1 and SUR subunits are overexpressed in HF. In addition, cardiomyocytes from post-infarction hearts became sensitive to diazoxide reflecting the overexpression of the Kir6.1 subunit. The opening of KATP by diazoxide tended to reduce the action potential duration (APD) which is extended in HF. This increase in APD is known to be a major source of arrhythmias during HF. Therefore, the opening of KATP channels by diazoxide would be cardio-protective. Secondly, we wanted to determine which transcription factors were responsible for this KATP remodeling. In our model of HF, we showed that overexpression of Kir6.1 is due to the transcription factors Fox03 and FOXF2 which is also responsible for SUR subunits overexpression. Thirdly, we developed an in vitro model of HF by cultivation of adult rat cardiomyocytes in the presence of angiotensin II (Angll) or TNFa. This model is very interesting not only because it underlines the importance of Angll and TNFa in KATp remodeling but also because this in vitro model presents the same KATP remodeling as the in vivo model of HF. These findings show that our in vitro model of HF opens up many possibilities to investigate more precisely the signaling pathways involved in remodeling of the KATP channels in HF.Conclusion. KATP channels undergo remodeling during HF and our results show the cardio¬protective potential of KATP channels in this disease.

Relation entre CaMKII et les dynamiques calciques endothéliales : impact de l'hypertension arterielle.

L’endothélium vasculaire joue un rôle prépondérant dans la régulation du tonus vasculaire en générant l’oxyde nitrique (NO), la prostacycline (PGI2) et les facteurs hyperpolarisants dérivés de l’endothélium (EDHF) comme puissants vasodilatateurs. Ces mécanismes requièrent le calcium (Ca2+) à divers niveaux, démontrant l’importance des dynamiques calciques endothéliales. Une perturbation de l’homéostasie calcique est observée dans une dysfonction endothéliale liée à l’hypertension artérielle. Il est impératif d’approfondir nos connaissances sur les signalisations calciques endothéliales impliquées dans le contrôle du tonus vasculaire. Des études récentes ont montré qu’une variation locale de la concentration en Ca2+ libre intracellulaire ([Ca2+]i) est suffisante pour générer une réponse physiologique importante. Les pulsars calciques sont caractérisés par une augmentation de [Ca2+]i spontanée et transitoire spécifiquement localisée au niveau des projections myoendothéliales (PMEs). Ces PMEs sont des sites de communication privilégiés entre les cellules endothéliales (CEs) et les cellules musculaires lisses vasculaires (CMLVs). Les pulsars calciques sont impliqués dans le mécanisme de l’EDHF via l’activation des canaux potassiques Ca2+-dépendant de moyenne conductance (KCa3.1 ou IKCa). Les travaux de cette thèse visent à améliorer nos connaissances sur les signalisations calciques locales en caractérisant une nouvelle voie de signalisation pouvant être impliquée dans la régulation du tonus vasculaire en condition physiopathologique. Outre les canaux KCa3.1 peu d’informations sont disponibles sur les cibles sensibles aux pulsars calciques. Une première étude a permis d’identifier la protéine kinase II dépendante du complexe Ca2+/calmoduline (CaMKII) sous ses isoformes α, β et δ dans les CEs d’artères natives de souris comme une cible pouvant être modulée par les pulsars calciques. Des études en immunofluorescence ont permis d’observer la localisation particulière de CaMKII endothéliale dans les PMEs, les sites des pulsars calciques. Une stimulation spécifique des pulsars calciques par la phényléphrine (PE) engendre un recrutement de CaMKII dans les PMEs. Sachant que CaMKII active l’oxyde nitrique synthase endothéliale (NOS3), nous avons évalué l’impact d’une stimulation des pulsars calciques sur la production de NO en présence d’un inhibiteur de CaMKII, le KN-93. Nous avons démontré que la production de NO est en partie dépendante de l’activation de CaMKII par les pulsars calciques. En utilisant un modèle d’hypertension induite par l’infusion chronique de PE, nous avons permis de mettre en évidence une perturbation dans la relation entre les pulsars calciques et CaMKII. Dans une seconde étude nous avons établi deux modèles (normo- et hypertendus) d’infusion chronique à l’angiotensine II (AngII) afin évaluer l’impact des ROS et de l’hypertension sur la voie de signalisation pulsars/CaMKII/NO. Nos résultats ont montré une augmentation des pulsars calciques accompagnée d’un recrutement de CaMKII dans les PMEs. Une stimulation aigue à l’AngII suggère que les ROS modulent les dynamiques calciques et que l’AngII stimule la production de NO. Cette étude propose que ces voies de signalisations impliquent les récepteurs de type 1 et 2 à l’AngII (AT1 et AT2). L’étude des pulsars calciques dépend fortement de la structure native des artères qui permet de conserver la formation des PMEs. La dernière étude présentée dans cette thèse a permis d’établir une relation entre les PMEs et les pulsars calciques dans trois lits vasculaires distincts (artères mésentériques, pulmonaires et coronariennes). Nos résultats ont montré que les paramètres cinétiques des pulsars calciques sont fortement conservés entre les différents lits vasculaires. Toutefois, la fréquence globale ainsi que le nombre de sites actifs des pulsars calciques diffèrent avec une proportion plus élevée dans les artères mésentériques et coronariennes comparativement aux artères pulmonaires. Ces résultats corrèlent avec le nombre plus élevé de PMEs retrouvé dans les artères mésentériques et coronariennes. Ces travaux suggèrent que les pulsars calciques sont fondamentaux pour les artères de résistance. Les études de cette thèse ont mené à l’identification d’une nouvelle voie de signalisation impliquant les pulsars calciques et CaMKII endothéliale dans la stimulation de la production de NO. Cette nouvelle voie de signalisation pourrait être impliquée dans la régulation du tonus vasculaire en condition physiopathologique. Les pulsars calciques semblent être fortement conservés entre les différentes artères de résistances et ce malgré la disparité dans les PMEs, suggérant un rôle prépondérant dans la fonction vasculaire. Ces travaux ouvrent une avenue pour le développement de potentielles cibles thérapeutiques pouvant contrer la dysfonction endothéliale associée à l’hypertension artérielle.

Eag 1, Eag 2 and Kcnn3 gene brain expression of isolated reared rats

The Eag1 and Eag2, voltage-dependent potassium channels, and the small-conductance calcium-activated potassium channel (Kcnn3) are highly expressed in limbic regions of the brain, where their function is still unknown. Eag1 co-localizes with tyrosine hydroxilase enzyme in the substantia nigra and ventral tegmental area. Kcnn3 deficiency leads to enhanced serotonergic and dopaminergic neurotransmission accompanied by distinct alterations in emotional behaviors. As exposure to stress is able to change the expression and function of several ion channels, suggesting that they might be involved in the consequences of stress, we aimed at investigating Eag 1, Eag2 and Kcnn3 mRNA expression in the brains of rats submitted to isolation rearing. As the long-lasting alterations in emotional and behavioral regulation after stress have been related to changes in serotonergic neurotransmission, expressions of serotonin Htr1a and Htr2a receptors in male Wistar rats` brain were also investigated. Rats were reared in isolation or in groups of five for nine weeks after weaning. Isolated and socially reared rats were tested for exploratory activity in the open field test for 5 min and brains were processed for reverse-transcription coupled to quantitative polymerase chain reaction (qRT-PCR). Isolated reared rats showed decreased exploratory activity in the open field. Compared to socially reared rats, isolated rats showed reduced Htr2a mRNA expression in the striatum and brainstem and reduced Eag2 mRNA expression in all examined regions except cerebellum. To our knowledge, this is the first work to show that isolation rearing can change Eag2 gene expression in the brain. The involvement of this channel in stress-related behaviors is discussed.

Nicorandil protects cardiac mitochondria against permeability transition induced by ischemia-reperfusion

Ischemia followed by reperfusion is known to negatively affect mitochondrial function by inducing a deleterious condition termed mitochondrial permeability transition. Mitochondrial permeability transition is triggered by oxidative stress, which occurs in mitochondria during ischemia-reperfusion as a result of lower antioxidant defenses and increased oxidant production. Permeability transition causes mitochondrial dysfunction and can ultimately lead to cell death. A drug able to minimize mitochondrial damage induced by ischemia-reperfusion may prove to be clinically effective. We aimed to analyze the effects of nicorandil, an ATP-sensitive potassium channel agonist and vasodilator, on mitochondrial function of rat hearts and cardiac HL-1 cells submitted to ischemia-reperfusion. Nicorandil decreased mitochondrial swelling and calcium uptake. It also decreased reactive oxygen species formation and thiobarbituric acid reactive substances levels, a lipid peroxidation biomarker. We thus confirm previous reports that nicorandil inhibits mitochondrial permeability transition and demonstrate that nicorandil inhibits this process by preventing oxidative damage and mitochondrial calcium overload induced by ischemia-reperfusion, resulting in improved cardiomyocyte viability. These results may explain the good clinical results obtained when using nicorandil in the treatment of ischemic heart disease.

Channels underlying neuronal calcium-activated potassium currents

In many cell types rises in cytosolic calcium, either due to influx from the extracellular space, or by release from an intracellular store activates calcium dependent potassium currents on the plasmalemma. In neurons, these currents are largely activated following calcium influx via voltage gated calcium channels active during the action potentials. Three types of these currents are known: I-c. I-AHP and I-sAHP. These currents can be distinguished by clear differences in their pharmacology and kinetics. Activation of these potassium currents modulates action potential time course and the repetitive firing properties of neurons. Single channel studies have identified two types of calcium-activated potassium channel which can also be separated on biophysical and pharmacological grounds and have been named BK and SK channels. It is now clear that BK channels underlie Ic whereas SK channels underlie I-AHP. The identity of the channels underlying I-sAHP are not known. In this review, we discuss the properties of the different types of calcium-activated potassium channels and the relationship between these channels and the macroscopic currents present in neurons. (C) 2002 Elsevier Science Ltd. All rights reserved.

Tertiapin-Q blocks recombinant and native large conductance K+ channels in a use-dependent manner

Tertiapin, a short peptide from honey bee venom, has been reported to specifically block the inwardly rectifying K+ (Kir) channels, including G protein-coupled inwardly rectifying potassium channel (GIRK) 1 + GIRK4 heteromultimers and ROMK1 homomultimers. In the present study, the effects of a stable and functionally similar derivative of tertiapin, tertiapin-Q, were examined on recombinant human voltage-dependent Ca2+-activated large conductance K+ channel (BK or MaxiK; alpha-subunit or hSlo1 homomultimers) and mouse inwardly rectifying GIRK1 + GIRK2 (i.e., Kir3.1 and Kir3.2) heteromultimeric K+ channels expressed in Xenopus oocytes and in cultured newborn mouse dorsal root ganglion (DRG) neurons. In two-electrode voltage-clamped oocytes, tertiapin-Q (1-100 nM) inhibited BK-type K+ channels in a use- and concentration-dependent manner. We also confirmed the inhibition of recombinant GIRK1 + GIRK2 heteromultimers by tertiapin-Q, which had no effect on endogenous depolarization- and hyperpolarization-activated currents sensitive to extracellular divalent cations (Ca2+, Mg2+, Zn2+, and Ba2+) in defolliculated oocytes. In voltage-clamped DRG neurons, tertiapin-Q voltage- and use-dependently inhibited outwardly rectifying K+ currents, but Cs+-blocked hyperpolarization-activated inward currents including I-H were insensitive to tertiapin-Q, baclofen, barium, and zinc, suggesting absence of functional GIRK channels in the newborn. Under current-clamp conditions, tertiapin-Q blocked the action potential after hyperpolarization (AHP) and increased action potential duration in DRG neurons. Taken together, these results demonstrate that the blocking actions of tertiapin-Q are not specific to Kir channels and that the blockade of recombinant BK channels and native neuronal AHP currents is use-dependent. Inhibition of specific types of Kir and voltage-dependent Ca2+-activated K+ channels by tertiapin-Q at nanomolar range via different mechanisms may have implications in pain physiology and therapy.

Chaotic model and memory in single calcium-activated potassium channel kinetics

Ion channels are pores formed by proteins and responsible for carrying ion fluxes through cellular membranes. The ion channels can assume conformational states thereby controlling ion flow. Physically, the conformational transitions from one state to another are associated with energy barriers between them and are dependent on stimulus, such as, electrical field, ligands, second messengers, etc. Several models have been proposed to describe the kinetics of ion channels. The classical Markovian model assumes that a future transition is independent of the time that the ion channel stayed in a previous state. Others models as the fractal and the chaotic assume that the rate of transitions between the states depend on the time that the ionic channel stayed in a previous state. For the calcium activated potassium channels of Leydig cells the R/S Hurst analysis has indicated that the channels are long-term correlated with a Hurst coefficient H around 0.7, showing a persistent memory in this kinetic. Here, we applied the R/S analysis to the opening and closing dwell time series obtained from simulated data from a chaotic model proposed by L. Liebovitch and T. Toth [J. Theor. Biol. 148, 243 (1991)] and we show that this chaotic model or any model that treats the set of channel openings and closings as independent events is inadequate to describe the long-term correlation (memory) already described for the experimental data. (C) 2008 American Institute of Physics.

Spider toxins: A new group of potassium channel modulators

Spider toxins that target potassium channels constitute a new class of pharmacological tools that can be used to probe the structure and function of these channels at the molecular level. The limited studies performed to date indicate that these peptide toxins may facilitate the analysis of K+ channels that have proved insensitive to peptide inhibitors isolated from other animal sources. Thus far, two classes of K+ channel-selective spider toxins have been isolated, sequenced, and pharmacologically characterised - the hanatoxins (HaTx) from Grammastola spatulata and heteropodatoxins (HpTx) from Heteropoda venatoria. The hanatoxins block Kv2.1 and Kv4.2 voltage-gated K+ channels. In Kv2.1 K+ channels this occurs as a consequence of a depolarising shift in the voltage dependence of activation and not by occlusion of the channel pore. These toxins show minimal sequence homology with other peptide inhibitors of K+ channels, but they do share some homology with other ion channel toxins from spiders, particularly with regard to the spacing between cysteine residues. We have recently isolated three K+ channel antagonists from the venom of the Australian funnel-web spider Hadronyche versuta; at least two of these toxins are likely to constitute a new class of spider toxins active on K+ channels as they are approximately twice as large as HaTx and HpTx.

Calcium-activated potassium currents in mammalian neurons

1. Influx of calcium via voltage-dependent calcium channels during the action potential lends to increases in cytosolic calcium that can initiate a number of physiological processes. One of these is the activation of potassium currents on the plasmalemma. These calcium-activated potassium currents contribute to action potential repolarization and are largely responsible for the phenomenon of spike frequency adaptation. This refers to the progressive slowing of the frequency of discharge of action potentials during sustained injection of depolarizing current. In some cell types, this adaptation is so marked that despite the presence of depolarizing current, only a single spike (or a few spikes) is initiated, Following cessation of current injection, slow deactivation of calcium-activated potassium currents is also responsible for the prolonged hyperpolarization that often follows, 2. A number of macroscopic calcium-activated potassium currents that can be separated on the basis of kinetic and pharmacological criteria have been described in mammalian neurons. At the single channel level, several types of calcium-activated potassium channels also have been characterized. While for some macroscopic currents the underlying:single channels have been unambiguously defined, for other currents the identity of the underlying channels is not clear. 3. In the present review we describe the properties of the known types of calcium-activated potassium currents in mammalian neurons and indicate the relationship between macroscopic currents and particular single channels.

T-type Ca2+ channels, SK2 channels and SERCAs gate sleep-related oscillations in thalamic dendrites.

T-type Ca2+ channels (T channels) underlie rhythmic burst discharges during neuronal oscillations that are typical during sleep. However, the Ca2+-dependent effectors that are selectively regulated by T currents remain unknown. We found that, in dendrites of nucleus reticularis thalami (nRt), intracellular Ca2+ concentration increases were dominated by Ca2+ influx through T channels and shaped rhythmic bursting via competition between Ca2+-dependent small-conductance (SK)-type K+ channels and Ca2+ uptake pumps. Oscillatory bursting was initiated via selective activation of dendritically located SK2 channels, whereas Ca2+ sequestration by sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs) and cumulative T channel inactivation dampened oscillations. Sk2-/- (also known as Kcnn2) mice lacked cellular oscillations, showed a greater than threefold reduction in low-frequency rhythms in the electroencephalogram of non-rapid-eye-movement sleep and had disrupted sleep. Thus, the interplay of T channels, SK2 channels and SERCAs in nRt dendrites comprises a specialized Ca2+ signaling triad to regulate oscillatory dynamics related to sleep.

Renal tubular NEDD4-2 deficiency causes NCC-mediated salt-dependent hypertension.

The E3 ubiquitin ligase NEDD4-2 (encoded by the Nedd4L gene) regulates the amiloride-sensitive epithelial Na+ channel (ENaC/SCNN1) to mediate Na+ homeostasis. Mutations in the human β/γENaC subunits that block NEDD4-2 binding or constitutive ablation of exons 6-8 of Nedd4L in mice both result in salt-sensitive hypertension and elevated ENaC activity (Liddle syndrome). To determine the role of renal tubular NEDD4-2 in adult mice, we generated tetracycline-inducible, nephron-specific Nedd4L KO mice. Under standard and high-Na+ diets, conditional KO mice displayed decreased plasma aldosterone but normal Na+/K+ balance. Under a high-Na+ diet, KO mice exhibited hypercalciuria and increased blood pressure, which were reversed by thiazide treatment. Protein expression of βENaC, γENaC, the renal outer medullary K+ channel (ROMK), and total and phosphorylated thiazide-sensitive Na+Cl- cotransporter (NCC) levels were increased in KO kidneys. Unexpectedly, Scnn1a mRNA, which encodes the αENaC subunit, was reduced and proteolytic cleavage of αENaC decreased. Taken together, these results demonstrate that loss of NEDD4-2 in adult renal tubules causes a new form of mild, salt-sensitive hypertension without hyperkalemia that is characterized by upregulation of NCC, elevation of β/γENaC, but not αENaC, and a normal Na+/K+ balance maintained by downregulation of ENaC activity and upregulation of ROMK.

mitoKATP channel activation in the postanoxic developing heart protects E-C coupling via NO-, ROS-, and PKC-dependent pathways.

Whereas previous studies have shown that opening of the mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channel protects the adult heart against ischemia-reperfusion injury, it remains to be established whether this mechanism also operates in the developing heart. Isolated spontaneously beating hearts from 4-day-old chick embryos were subjected to 30 min of anoxia followed by 60 min of reoxygenation. The chrono-, dromo-, and inotropic disturbances, as well as alterations of the electromechanical delay (EMD), reflecting excitation-contraction (E-C) coupling, were investigated. Production of reactive oxygen species (ROS) in the ventricle was determined using the intracellular fluorescent probe 2',7'-dichlorofluorescin (DCFH). Effects of the specific mitoK(ATP) channel opener diazoxide (Diazo, 50 microM) or the blocker 5-hydroxydecanoate (5-HD, 500 microM), the nitric oxide synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 50 microM), the antioxidant N-(2-mercaptopropionyl)glycine (MPG, 1 mM), and the PKC inhibitor chelerythrine (Chel, 5 microM) on oxidative stress and postanoxic functional recovery were determined. Under normoxia, the baseline parameters were not altered by any of these pharmacological agents, alone or in combination. During the first 20 min of postanoxic reoxygenation, Diazo doubled the peak of ROS production and, interestingly, accelerated recovery of ventricular EMD and the PR interval. Diazo-induced ROS production was suppressed by 5-HD, MPG, or L-NAME, but not by Chel. Protection of ventricular EMD by Diazo was abolished by 5-HD, MPG, L-NAME, or Chel, whereas protection of the PR interval was abolished by L-NAME exclusively. Thus pharmacological opening of the mitoK(ATP) channel selectively improves postanoxic recovery of cell-to-cell communication and ventricular E-C coupling. Although the NO-, ROS-, and PKC-dependent pathways also seem to be involved in this cardioprotection, their interrelation in the developing heart can differ markedly from that in the adult myocardium.

Increased voltage-dependent K+ channel Kv1.3 and Kv1.5 expression correlates with leiomyosarcoma aggressiveness

Voltage-dependent K+ channels (Kv) are involved in the proliferation and differentiation of mammalian cells, since Kv antagonists impair cell cycle progression. Although myofibers are terminally differentiated, some myoblasts may re-enter the cell cycle and proliferate. Since Kv1.3 and Kv1.5 expression is remodeled during tumorigenesis and is involved in smooth muscle proliferation, the purpose of this study was to analyze the expression of Kv1.3 and Kv1.5 in smooth muscle neoplasms. In the present study, we examined human samples of smooth muscle tumors together with healthy speci­mens. Thus, leiomyoma (LM) and leiomyosarcoma (LMS) tumors were analyzed. Results showed that Kv1.3 was poorly expressed in the healthy muscle and indolent LM specimens, whereas aggressive LMS showed high levels of Kv1.3 expression. Kv1.5 staining was correlated with malignancy. The findings show a remodeling of Kv1.3 and Kv1.5 in human smooth muscle sarcoma. A correlation of Kv1.3 and Kv1.5 expression with tumor aggressiveness was observed. Thus, our results indicate Kv1.5 and Kv1.3 as potential tumorigenic targets for aggressive human LMS.

Vasorelaxant effects of the potassium channel opener SR 47063 on the isolated human saphenous vein and rat aorta

The vasorelaxant effects of SR 47063 (4-(2-cyanimino-1,2-dihydropyrid-1-yl)-2,2-dimethyl-6-nitrochromene), a new K+-channel opener structurally related to levcromakalim, were examined in isolated human saphenous vein (HSV) and rat aorta (RA). HSV or RA rings were precontracted with either KCl or noradrenaline and cumulative relaxant concentration-response curves were obtained for SR 47063 (0.1 nM to 1 µM) in the presence or absence of 3 µM glibenclamide. SR 47063 potently relaxed HSV and RA precontracted with 20 mM (but not 60 mM) KCl or 10 µM noradrenaline in a concentration-dependent manner, showing slightly greater activity in the aorta. The potency of the effect of SR 47063 on HSV and RA was 12- and 58-fold greater, respectively, than that reported for the structurally related K+-channel opener levcromakalim. The vasorelaxant action of SR 47063 in both blood vessels was strongly inhibited by 3 µM glibenclamide, consistent with a mechanism of action involving ATP-dependent K+-channels.

Calmodulin/KCa3.1 channel interactions as determinant to the KCa3.1 Ca2+ dependent gating : theoretical and experimental analyses

Differentes études ont montré que la sensibilité au Ca2+ du canal KCa3.1, un canal potassique indépendant du voltage, était conférée par la protéine calmoduline (CaM) liée de façon constitutive au canal. Cette liaison impliquerait la région C-lobe de la CaM et un domaine de $\ikca$ directement relié au segment transmembranaire S6 du canal. La CaM pourrait égalment se lier au canal de façon Ca2+ dépendante via une interaction entre un domaine de KCa3.1 du C-terminal (CaMBD2) et la région N-lobe de la CaM. Une étude fut entreprise afin de déterminer la nature des résidus responsables de la liaison entre le domaine CaMBD2 de KCa3.1 et la région N-lobe de la CaM et leur rôle dans le processus d'ouverture du canal par le Ca2+. Une structure 3D du complexe KCa3.1/CaM a d'abord été générée par modélisation par homologie avec le logiciel MODELLER en utilisant comme référence la structure cristalline du complexe SK2.2/CaM (PDB: 1G4Y). Le modèle ainsi obtenu de KCa3.1 plus CaM prévoit que le segment L361-S372 dans KCa3.1 devrait être responsable de la liaison dépendante du Ca2+ du canal avec la région N-lobe de la CaM via les résidus L361 et Q364 de KCa3.1 et E45, E47 et D50 de la CaM. Pour tester ce modèle, les résidus dans le segment L361-S372 ont été mutés en Cys et l'action du MTSET+ (chargé positivement) et MTSACE (neutre) a été mesurée sur l'activité du canal. Des enregistrements en patch clamp en configuration ``inside-out`` ont montré que la liaison du réactif chargé MTSET+ au le mutant Q364C entraîne une forte augmentation du courant, un effet non observé avec le MTSACE. De plus les mutations E45A et E47A dans la CaM, ont empêché l'augmentation du courant initié par MTSET+ sur le mutant Q364C. Une analyse en canal unitaire a confirmé que la liaison MTSET+ à Q364C cause une augmentation de la probabilité d'ouverture de KCa3.1 par une déstabilisation de l'état fermé du canal. Nous concluons que nos résultats sont compatibles avec la formation de liaisons ioniques entre les complexes chargés positivement Cys-MTSET+ à la position 364 de KCa3.1 et les résidus chargés négativement E45 et E47 dans la CaM. Ces données confirment qu'une stabilisation électrostatique des interactions CaM/KCa3.1 peut conduire à une augmentation de la probabilité d'ouverture du canal en conditions de concentrations saturantes de Ca2+.