Flufenamic acid as an ion channel modulator

Flufenamic acid has been known since the 1960s to have anti-inflammatory properties attributable to the reduction of prostaglandin synthesis. Thirty years later, flufenamic acid appeared to be an ion channel modulator. Thus, while its use in medicine diminished, its use in ionic channel research expanded. Flufenamic acid commonly not only affects non-selective cation channels and chloride channels, but also modulates potassium, calcium and sodium channels with effective concentrations ranging from 10(-6)M in TRPM4 channel inhibition to 10(-3)M in two-pore outwardly rectifying potassium channel activation. Because flufenamic acid effects develop and reverse rapidly, it is a convenient and widely used tool. However, given the broad spectrum of its targets, experimental results have to be interpreted cautiously. Here we provide an overview of ion channels targeted by flufenamic acid to aid in interpreting its effects at the molecular, cellular, and system levels. If it is used with good practices, flufenamic acid remains a useful tool for ion channel research. Understanding the targets of FFA may help reevaluate its physiological impacts and revive interest in its therapeutic potential. (C) 2013 Elsevier Inc. All rights reserved

Implication du canal cationique non-sélectif TRPM4 dans l'activité électrique cardiaque

La protéine TRPM4 porte un courant cationique non-sélectif activé par le calcium intracellulaire (NSCCa). Au niveau cardiaque, il est présent sur l oreillette (humain, rat et souris) et dans le noeud sinusal (souris), mais peu exprimé au niveau ventriculaire. Toutefois son expression ventriculaire est augmentée en cas d hypertrophie cardiaque. Malgré une cartographie bien établie l implication de TRPM4 dans l activité électrique cardiaque était jusqu à aujourd hui inconnue. Ce travail de thèse a eu pour objectif d évaluer l implication du canal TRPM4 dans l activité cardiaque, en utilisant des techniques d électrophysiologie. Nous avons combiné une approche pharmacologique, utilisant les inhibiteurs du TRPM4 et une approche de transgénèse, en utilisant des souris invalidées pour le gène TRPM4. Nous avons observé que le canal TRPM4 est impliqué dans la durée du potentiel d action de l oreillette de souris, puisque son inhibition diminue la durée du PA, et que les souris invalidées pour le gène TRPM4 présentent un PA plus court que les souris sauvages. Le canal participe également à certaines formes d arythmies ventriculaires. Nous avons développé un modèle d arythmies d hypoxie-réoxygénation sur du ventricule de souris, où l application des inhibiteurs du TRPM4 conduit à la disparition de ces arythmies. Enfin nous avons établi un lien entre des mutations du gène TRPM4 et le syndrome de Brugada. En particulier, une mutation K914X aboutissant à un canal non fonctionnel, a été identifiée chez un malade atteint du syndrome de Brugada. L ensemble de nos travaux identifiant, le TRPM4 comme une nouvelle cible pharmacologique dans la prévention des troubles du rythme cardiaque.The TRPM4 protein, is a member of the larger family "Transient Receptor Potentiel" channels, and supports a nonselective cationique current activated by intracellular calcium (NSCCa). TRPM4 presents a large tissular distribution. In the heart, it is present in the atrium (human, rat and mouse) and sinus node (mouse), but it is only slightly expressed in the ventricle. However its ventricular expression is increased in case of cardiac hypertrophy (SHR rat). While TRPM4 cardiac mapping is well established, its implication in the cardiac activity was still unknown. Our work had for objective to estimate the implication of the TRPM4 channel in the cardiac activity, by using electrophysiological technics (intracellular microelectrode and patch-clamp). We combined a pharmacological approach, using two inhibitors of the TRPM4 (the acid flufénamique and 9-phénanthrol) and an approach of transgenesis, by using mice invalidated for the TRPM4 gene. We observed that TRPM is implied in the mice atrial action potential duration (APD), because its inhibition decreases the APD, and TRPM4-/-transgenics mice present a shorter AP than TRPM4+/+ mice. TRPM4 channel also participates in ventricular arrhythmias. We developed a model of hypoxia-reoxygenation to produce arrhythmias. The application of TRPM4 inhibitors eliminates these arrhythmias. Finally we established a link between TRPM4 mutations and Brugada syndrome. In particular, the mutation K914X giving an unfunctional channel, was identified in a patient affected by Brugada Syndrome. Our study identifies, TRPM4 as a new promising pharmacological target in the prevention of cardiac electrical disturbance.CAEN-BU Médecine pharmacie (141182102) / SudocSudocFranceF

Caractérisation fonctionnelle et moléculaire du courant cationique non selectif activé par le calcium sur les cellules sinusales et auriculaires de mammifères

Recent studies, on cardiac tissue at the ventricular level, showed in pathological conditions the expression of a calcium-activated nonselective cationic channel (NSCCa) supposed to be implicated in the genesis of arrhythmias. The aim of the present study was to search for this current on other cardiac levels: atria and sino-atrial nodes. Using the inside-out configuration of the patch-clamp technique, we showed the presence, on human atrial cardiomyocytes and mouse SAN cells, of a nonselective cationic current, permeable to monovalents cations but impermeable to divalents cations and anions. The current/voltage relationship is linear with a unitary conductance close to 20 pS. This current is activated by rise of intracellular calcium and membrane depolarization but inhibited by internal ATP. It is blocked by flufenamic acid and glibenclamide. This current probably corresponds to the new cloned protein TRPM4, which shares the same electrophysiological properties. We detected its mRNA transcript on human atrium and mouse SAN tissues and its protein on mouse SAN tissue. If the physiological impact of this channel is unclear, it could be implicated on arrhythmias genesis during ischemia. As the calcium/ATP ratio is increased and then is in favour of its activation. Also, we described at the unitary level on human atrial cardiomyocytes, a new chloride current activated by cell swelling so called ICl.swell. It could be implicated in the regulatory volume decrease observed during atrial dilatation.POITIERS-BU Sciences (861942102) / SudocSudocFranceF

A calcium-permeable non-selective cation channel in the thick ascending limb apical membrane of the mouse kidney

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Pathophysiological Roles of the TRPV4 Channel in the Heart

The transient receptor potential vanilloid 4 (TRPV4) channel is a non-selective cation channel that is mostly permeable to calcium (Ca2+), which participates in intracellular Ca2+ handling in cardiac cells. It is widely expressed through the body and is activated by a large spectrum of physicochemical stimuli, conferring it a role in a variety of sensorial and physiological functions. Within the cardiovascular system, TRPV4 expression is reported in cardiomyocytes, endothelial cells (ECs) and smooth muscle cells (SMCs), where it modulates mitochondrial activity, Ca2+ homeostasis, cardiomyocytes electrical activity and contractility, cardiac embryonic development and fibroblast proliferation, as well as vascular permeability, dilatation and constriction. On the other hand, TRPV4 channels participate in several cardiac pathological processes such as the development of cardiac fibrosis, hypertrophy, ischemia–reperfusion injuries, heart failure, myocardial infarction and arrhythmia. In this manuscript, we provide an overview of TRPV4 channel implications in cardiac physiology and discuss the potential of the TRPV4 channel as a therapeutic target against cardiovascular diseases

Exploration of the Basolateral Chloride Channels in the Renal Tubule using

International audienceChloride channels located on the basolateral membrane are known to be involved in chloride absorption in several parts of the renal tubule, and particularly in the thick ascending limb and distal convoluted tubule. The data available suggest that the ClC-K channels play the major role in this process. We provide here a description of the electrophysiological properties of these channels, still very incomplete at this stage, and we attempt to compare ClC-Ks to three chloride channels that we have identified in the basolateral membrane of microdissected fragments of the mouse renal tubule using the patch-clamp technique. Based on anion selectivity and dependence on external pH and calcium shown by the ClC-Ks, we propose candidate ClC-K1 and ClC-K2 in native tissue. We also discuss the possibility that chloride channels that do not belong to the ClC family may also be involved in the absorption of chloride across the cortical thick ascending limb

9-Phenanthrol inhibits human TRPM4 but not TRPM5 cationic channels

(IF : 3,76)International audienceTRPM4 and TRPM5 are calcium-activated non-selective cation channels with almost identical characteristics. TRPM4 is detected in several tissues including heart, kidney, brainstem, cerebral artery and immune system whereas TRPM5 expression is more restricted. Determination of their roles in physiological processes requires specific pharmacological tools. TRPM4 is inhibited by glibenclamide, a modulator of ATP binding cassette proteins (ABC transporters), such as the cystic fibrosis transmembrane conductance regulator (CFTR). We took advantage of this similarity to investigate the effect of hydroxytricyclic compounds shown to modulate ABC transporters, on TRPM4 and TRPM5. EXPERIMENTAL APPROACH: Experiments were conducted using HEK-293 cells permanently transfected to express human TRPM4 or TRPM5. Currents were recorded using the whole-cell and inside-out variants of the patch-clamp technique. KEY RESULTS: The CFTR channel activator benzo[c]quinolizinium MPB-104 inhibited TRPM4 current with an IC(50) in the range of 2 x 10(-5) M, with no effect on single-channel conductance. In addition, 9-phenanthrol, lacking the chemical groups necessary for CFTR activation, also reversibly inhibited TRPM4 with a similar IC(50). Channel inhibition was voltage independent. The IC(50) determined in the whole-cell and inside-out experiments were similar, suggesting a direct effect of the molecule. However, 9-phenanthrol was ineffective on TRPM5, the most closely related channel within the TRP protein family.CONCLUSIONS AND IMPLICATIONS: We identify 9-phenanthrol as a TRPM4 inhibitor, without effects on TRPM5. It could be valuable in investigating the physiological functions of TRPM4, as distinct from those of TRPM5