Intracellular protons control the affinity of skeletal muscle ATP-sensitive K+ channels for potassium-channel-openers

AbstractLevcromakalim, a potential antihypertensive agent, is known to activate potassium channels dependent on intracellular ATP (K-ATP channels). In inside-out patches excised from frog skeletal muscle, levcromakalim or a related compound, SR 47063, caused a strong and persistent activation of K-ATP channels at a cytoplasmic pH of 7.1. However, at pH 6, these activators could no longer affect the K-ATP currents. Conversely, in the continuous presence of activator, lowering pH from 7.1 to 6 returned channel activity to its level in pH 6 alone. After wash-out of the activator, recovery from activation took minutes at pH 7.1 but only seconds at pH 6, thus ruling out an effect of protons on the activators in solution. These experiments suggest that K-channel-activators are unable to bind to their receptor when it is protonated, and more generally, they provide evidence at the microscopic level for proton-induced allosteric modulation of drug-receptor interaction

Hourglass SiO2 coating increases the performance of planar patch-clamp.

International audienceObtaining high-throughput electrophysiological recordings is an ongoing challenge in ion channel biophysics and drug discovery. One particular area of development is the replacement of glass pipettes with planar devices in order to increase throughput. However, successful patch-clamp recordings depend on a surface coating which ideally should promote and stabilize giga-seal formation. Here, we present data supporting the use of a structured SiO(2) coating to improve the ability of cells to form a "seal" with a planar patch-clamp substrate. The method is based on a correlation study taking into account structure and size of the pores, surface roughness and chip capacitance. The influence of these parameters on the quality of the seal was assessed. Plasma-enhanced chemical vapour deposition (PECVD) of SiO(2) led to an hourglass structure of the pore and a tighter seal than that offered by a flat, thermal SiO(2) surface. The performance of PECVD chips was validated by recording recombinant potassium channels, BK(Ca), expressed in stable HEK-293 cell lines and in inducible CHO cell lines and low conductance IRK1, and endogenous cationic currents from CHO cells. This multiparametric investigation led to the production of improved chips for planar patch-clamp applications which allow electrophysiological recordings from a wide range of cell lines

eeFit: a Microsoft Excel-embedded program for interactive analysis and fitting of experimental dose–response data

International audienceWe present here a software program dedicated to the fitting of experimental dose-response data, which integrates seamlessly with Excel and allows curve fitting plots and results to reside alongside data within Excel spreadsheets. The program, named eeFit, for Excel-Embedded Fitting software, requires no advanced knowledge of Excel or non-linear least-squares fitting. Any experimental data present in an Excel file, such as dose-effect data obtained with membrane receptor or ion channel ligands, can be graphed and fitted interactively with standard Hill models for activation or inhibition, or with more complex models for biphasic effects resulting from combinations of activation and inhibition. When benchmarked against the commercial program Origin, eeFit yielded equivalent or better results, in terms of accuracy and convergence, and proved much easier to learn and use

Etude structure/fonction d'une protéine ABC (SUR, le récepteur des sulfonylurées)

Le canal KATP résulte de l'assemblage d'un canal potassique inhibé par l'ATP intracellula ire (Kir6.2) et d'un transporteur ABC, le récepteur des sulfonylurées (SUR) de la famille MRP/ABCC. SUR a un rôle régulateur essentiel : il confère au canal une sensibilité accrue à l'inhibition par l'ATP, provoque son activation lorsque l'ADP augmente, et est la cible des activateurs et bloqueurs pharmacologiques du canal. Nous nous sommes intéressés à divers aspects structure/fonction de SUR en tant que modèle de transporteur ABC eucaryote. Son couplage naturel à un canal ionique en facilité gradement l'étude gra ce à la technique électrophysiologique du patch-clamp. La poursuite des travaux pour déterminer la nature moléculaire de la sélectivité des isoformes de SUR aux ouvreurs pharmacologiques nous a permis de conclure que seul le faible encombrement de la Thr1253 de SUR2A, contre la Met 1290 de SURI, serait le critère important pour l'activation pharmacologique des canaux KATP. Nos travaux ont ensuite porté sur un domaine de la sous-unité SUR riche en acides aminés chargés négativement (succession de 15 résidus glutamates ou aspartates) qui s'est avéré ne pas être impliquée dans la fonction du canal dans notre système d'expression. Nous avons étudié l'effet des ions Zn2+ et Cd2+ intracellulaires sur les canaux KATP et montré que ces ions peuvent activer les canaux via leur liaison à SUR. Ce site de liaison reste encore à déterminer. Nous avons enfin essayé de comprendre le rôle de chacun des domaines de liaison des nucléotides et nous avons pour cela conçu des protéines SUR2A possédant des NBD identiques (NBDl-NBDI et NBD2-NBD2) ou inversés (NBD2-NBDl). Nos résultats suggèrent que (1) les NBD sont interchangeables (2) l'activation pas le Mg-ADP requiert les deux NBD (3) l'action des ouvreurs est indépendante du NBD2.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Antiobesity strategy targets energy economy safeguards.

Remerciements EcofectInternational audienceNipah virus and Hendra virus are emerging, highly pathogenic, zoonotic paramyxoviruses that belong to the genus Henipavirus. They infect humans as well as numerous mammalian species. Both viruses use ephrin-B2 and -B3 as cell entry receptors, and following initial entry into an organism, they are capable of rapid spread throughout the host. We have previously reported that Nipah virus can use another attachment receptor, different from its entry receptors, to bind to nonpermissive circulating leukocytes, thereby promoting viral dissemination within the host. Here, this attachment molecule was identified as heparan sulfate for both Nipah virus and Hendra virus. Cells devoid of heparan sulfate were not able to mediate henipavirus trans-infection and showed reduced permissivity to infection. Virus pseudotyped with Nipah virus glycoproteins bound heparan sulfate and heparin but no other glycosaminoglycans in a surface plasmon resonance assay. Furthermore, heparin was able to inhibit the interaction of the viruses with the heparan sulfate and to block cell-mediated trans-infection of henipaviruses. Moreover, heparin was shown to bind to ephrin-B3 and to restrain infection of permissive cells in vitro. Consequently, treatment with heparin devoid of anticoagulant activity improved the survival of Nipah virus-infected hamsters. Altogether, these results reveal heparan sulfate as a new attachment receptor for henipaviruses and as a potential therapeutic target for the development of novel approaches against these highly lethal infections. The Henipavirus genus includes two closely related, highly pathogenic paramyxoviruses, Nipah virus and Hendra virus, which cause elevated morbidity and mortality in animals and humans. Pathogenesis of both Nipah virus and Hendra virus infection is poorly understood, and efficient antiviral treatment is still missing. Here, we identified heparan sulfate as a novel attachment receptor used by both viruses to bind host cells. We demonstrate that heparin was able to inhibit the interaction of the viruses with heparan sulfate and to block cell-mediated trans-infection of henipaviruses. Moreover, heparin also bound to the viral entry receptor and thereby restricted infection of permissive cells in vitro. Consequently, heparin treatment improved survival of Nipah virus-infected hamsters. These results uncover an important role of heparan sulfate in henipavirus infection and open novel perspectives for the development of heparan sulfate-targeting therapeutic approaches for these emerging infections

Conception et caractérisation de biocapteurs basés sur l'association de récepteurs et canaux ioniques

Les canaux sensibles à l'ATP (KATP) résultent de l'association unique d'une protéine ABC (le récepteur des sulfonylurées, SUR) et d'un canal potassique rectifiant entrant (Kir6.x). Suite à la liaison de divers effecteurs (nucléotides, molécules pharmacologiques), SUR module l'activité de Kir6.x. Dans l'organisme, les canaux KA TP lient le niveau énergétique de la cellule au potentiel membranaire. De ce fait, leur implication dans diverses fonctions physiologiques telles que la sécrétion pancréatique d'insuline ou le contrôle du tonus vasculaire en fait des cibles thérapeutiques. Ainsi, dans le cadre d'une collaboration, nous avons testé des composés (dérivés benzothiazine) sur le canal KATP. Quatre d'entre eux se sont avérés être des ouvreurs des canaux KATP. Nous avons également exploré l'effet de deux insecticides, l'amitraz et le diflubenzuron sur le canal KATP. En outre, dans une optique de généralisation du concept d'Ion Channel-Coupled Receptor (ICCR) mis au point par l'équipe, nous avons élaboré des biocapteurs reposant sur l'assemblage de récepteurs couplés aux protéines G (GPCR) et Kir6.2. Cette association, inspirée par le canal KATP, est réalisée de telle manière que la fixation d'un ligand sur le GPCR entraîne la modulation de l'activité de Kir6.2. L'ingénierie moléculaire nous a permis de fusionner Kir6.2 aux récepteurs t3J. adrénergique, dopaminergique D3, cannabinoïde 1 (CBl), des CC-chimiokines 2 et à l'opsine. La méthode de double-microélectrodes nous a permis d'identifier trois ICCR fonctionnels basés sur les récepteurs t3J., D3 et CBl. Ce~ biocapteurs présentent des applications dans le cadre du criblage haut débit, la caractérisation fonctionnelle des GPCR Ol la compréhension de la régulation de l'activité de Kir6.2.A TP sensitive potassium channels (KA TP) comprise two proteins: The sulfonylurea receptor (SUR. an ABC protein) and an inward rectifier potassium channel (Kir6.2). Upon binding of different types of effectors (nucleotides, pharmacological compounds), SUR. the regulatory subunit, modulates the gating ofKir6.2. The KATP channel links the metabolic state of the cell to its electrical excitability. Thereby, it acts as a metabolic sensor and is involved in physiological functions such as insulin secretion, cardioprotection, vascular tone control... Thus, the KA TP channel constitutes a therapeutic target. ln this work, we studied the effects ofbenzothiazine derivatives on SURI + Kir6.2 and SUR2A + Kir6.2. We also investigated the effects oftwo pesticides, amitraz and diflubenzuron on the KATP channel. To extend the concept ofIon Channel-Coupled Receptor (ICCR) initiated in the laboratory, we designed new constructs by engineering fusion between Kir6.2 and five GPCRs: the t3J. adrenergic, cannabinoid 1 (CBl), dopaminergic D3, CC chemokine (CCR2) receptors and opsin. The receptor C-ter and Kir6.2 N-ter extremities were pared to promote efficient coupling and joined covalently. The fusions were heterologously expressed in Xenopus oocytes and characterized by the two-electrode voltage clamp technique. Three ICCR t3J. adrenergic-basecl, CBl-based and D3-based) out of five were shown to be functional. Thus, the ICCR concept is readily applicable to class A GPCRs. Besides their obvious interest in drug screening, the new ICCRs should be valuable tools to investigate the intermolecular events involved in the modulation of Kir6.2 gating and the nature of the GPCR conformational changes evoked by their ligands.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Les bases moléculaires du mécanisme d'action des ouvreurs potassiques sur les canaux Katp

GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

The unusual stoichiometry of ADP activation of the KATP channel.

International audienceKATP channels, oligomers of 4 pore-forming Kir6.2 proteins and 4 sulfonylurea receptors (SUR), sense metabolism by monitoring both cytosolic ATP, which closes the channel by interacting with Kir6.2, and ADP, which opens it via SUR. SUR mutations that alter activation by ADP are a major cause of KATP channelopathies. We examined the mechanism of ADP activation by analysis of single-channel and macropatch recordings from Xenopus oocytes expressing various mixtures of wild-type SUR2A and an ADP-activation-defective mutant. Evaluation of the data by a binomial distribution model suggests that wild-type and mutant SURs freely co-assemble and that channel activation results from interaction of ADP with only 2 of 4 SURs. This finding explains the heterozygous nature of most KATP channelopathies linked to mutations altering ADP activation. It also suggests that the channel deviates from circular symmetry and could function as a dimer-of-dimers