VSOP/Hv1 proton channels sustain calcium entry, neutrophil migration, and superoxide production by limiting cell depolarization and acidification

Neutrophils kill microbes with reactive oxygen species generated by the NADPH oxidase, an enzyme which moves electrons across membranes. Voltage-gated proton channels (voltage-sensing domain only protein [VSOP]/Hv1) are required for high-level superoxide production by phagocytes, but the mechanism of this effect is not established. We show that neutrophils from VSOP/Hv1−/− mice lack proton currents but have normal electron currents, indicating that these cells have a fully functional oxidase that cannot conduct protons. VSOP/Hv1−/− neutrophils had a more acidic cytosol, were more depolarized, and produced less superoxide and hydrogen peroxide than neutrophils from wild-type mice. Hydrogen peroxide production was rescued by providing an artificial conductance with gramicidin. Loss of VSOP/Hv1 also aborted calcium responses to chemoattractants, increased neutrophil spreading, and decreased neutrophil migration. The migration defect was restored by the addition of a calcium ionophore. Our findings indicate that proton channels extrude the acid and compensate the charge generated by the oxidase, thereby sustaining calcium entry signals that control the adhesion and motility of neutrophils. Loss of proton channels thus aborts superoxide production and causes a severe signaling defect in neutrophils

Etude fonctionnelle des conductances chlorures et protonique sur les fibroblastes cardiaques humains

Le but de notre étude était de caractériser les propriétés électrophysiologiques des fibroblastes cardiaques humains (FCs) et déterminer leur rôle fonctionnel possible.En utilisant plusieurs approches, combinant les techniques de patch-clamp, des efflux de radiotraceurs, de RT-PCR, de dosage ELISA et de fluorescence calcique, nous avons pu mettre en évidence sur les FCs un canal chlorure activé par le calcium.En effet, nous avons montré que la perfusion d Ag II ou de l A23187 (ionophore calcique) favorisait l activation d une conductance chlorure. Cette composante ionique est bloquée en présence d acide flufénamique connu pour inhiber les courants chlorures activés par le calcium. L activation de ce canal, par l augmentation transitoire de calcium, serait impliquée dans le couplage excitation-sécrétion des fibroblastes cardiaques humains. Parallèlement à la sécrétion de facteurs fibroblastiques, Yan et Kleber (1992) ont observé durant l ischémie cardiaque une diminution du pH intracellulaire dans les myocytes. Les mécanismes précis impliqués dans cette variation de pH ne sont pas complètement démontrés. Dans ce contexte, il nous a semblé intéressant d évaluer, dans les conditions ischémiques, sur les fibroblastes cardiaques, l existence de canaux protons qui, avec d autres co-transporteurs (Na+/H+ et Na+/HCO3-) exprimés sur ces cellules, pourraient contribuer au rétablissement du pH intracellulaire.En effet, nous avons mis en évidence pour la première fois sur l atrium humain, l existence d un canal à protons dépendant du voltage sur les FCs. Cette conductance sélective aux protons fait partie des mécanismes membranaires alcalinisants qui sont particulièrement sollicités dans une situation pathologique telle que l ischémie du myocarde. En conclusion, cette étude a mis en évidence d importantes propriétés électrophysiologiques des FCs méconnues à ce jour, et le rôle probable qu elles peuvent jouer durant le remodelage et l ischémie du myocarde.Taking into account the experimental data obtained on human cardiac fibroblasts, the goal of our study was to characterize the electrophysiological properties of these cells and to determine their possible functional role. By using several approaches, combining the techniques of patch-clamp, iodides effluxes, RT-PCR, ELISA and calcium fluorescence, we were able to report a calcium activated chloride channel on FCs. Indeed, we showed that the perfusion of Ag II or A23187 (a calcium ionophore) activated a chloride conductance. This ionic component is blocked by flufenamic acid which is known to inhibit calcium activated chloride currents. The activation of this channel, following an increase in the transitory calcium, could be implied in the excitation-secretion coupling of the human cardiac fibroblasts. In parallel to the secretion of fibroblastic factors, Yan and Kleber (1992) observed a reduction in the intracellular pH in the myocytes, during cardiac ischemia. The precise mechanisms implied in this variation of pH are not completely demonstrated. In this context, it seemed interesting to evaluate, under ischemic conditions, on the cardiac fibroblasts, the existence of proton channels which, with other co-transporters (Na+/H+ and Na+/HCO3-) expressed on these cells, could contribute to the re-establishment of the intracellular pH. Indeed, we report for the first time on the human atrium, the existence of a proton channel on FCs. This selective conductance to protons is part of the alkalizing membrane mechanisms which are particularly requested in a pathological situation such as cardiac ischemia. In conclusion, this study highlighted important electrophysiological properties of FCs ignored to date, and the probable role that they could play during ischemia and heart remodeling process.POITIERS-BU Sciences (861942102) / SudocSudocFranceF

ANO1 contributes to angiotensin-II-activated Ca2+-dependent Cl- current in human atrial fibroblasts.

International audienceCardiac fibroblasts are an integral part of the myocardial tissue and contribute to its remodelling. This study characterises for the first time the calcium-dependent chloride channels (CaCC) in the plasma membrane of primary human atrial cardiac fibroblasts by means of the iodide efflux and the patch clamp methods. The calcium ionophore A23187 and Angiotensin II (Ang II) activate a chloride conductance in cardiac fibroblasts that shares pharmacological similarities with calcium-dependent chloride channels. This chloride conductance is depressed by RNAi-mediated selective Anoctamine 1 (ANO1) but not by Anoctamine 2 (ANO2) which has been revealed as CaCC and is inhibited by the selective ANO1 inhibitor, T16inh-A01. The effect of Ang II on anion efflux is mediated through AT1 receptors (with an EC50 = 13.8 ± 1.3 nM). The decrease of anion efflux by calphostin C and bisindolylmaleimide I (BIM I) suggests that chloride conductance activation is dependent on PKC. We conclude that ANO1 contributes to CaCC current in human cardiac fibroblasts and that this is regulated by Ang II acting via the AT1 receptor pathway

Physiological roles of voltage-gated proton channels in leukocytes

Voltage-gated proton channels are designed to extrude large quantities of cytosolic acid in response to depolarising voltages. The discovery of the Hvcn1 gene and the generation of mice lacking the channel molecule have confirmed several postulated functions of proton channels in leukocytes. In neutrophils and macrophages, proton channels are required for high-level production of superoxide anions by the phagocytic NADPH oxidase, a bactericidal enzyme essential for host defence against infections. In B lymphocytes, proton channels are required for low-level production of superoxide that boosts the production of antibodies. Proton channels sustain the activity of immune cells in several ways. By extruding excess cytosolic acid, proton channels prevent deleterious acidification of the cytosol and at the same time deliver protons required for chemical conversion of the superoxide secreted by membrane oxidases. By moving positive charges across membranes, proton channels limit the depolarisation of the plasma membrane, promoting the electrogenic activity of NADPH oxidases and the entry of calcium ions into cells. Acid extrusion by proton channels is not restricted to leukocytes but also mediates the intracellular alkalinisation required for the activation of spermatozoids. Proton channels are therefore multitalented channels that control male fertility as well as our innate and adaptive immunity