15 research outputs found
COMBINING FLUORESCENCE AND ELECTROPHYSIOLOGY MEASUREMENTS TO STUDY ASIC FUNCTION
Acid-Sensing Ion Channels (ASICs) are trimeric proton-gated and sodium-conducting channels widely expressed in neurons of the central and peripheral nervous systems. They contribute as pH sensors to a number of physiological and pathological conditions, such as learning, neurodegeneration after ischemia, and pain sensation. ASICs open transiently upon a lowering of the extracellular pH, before they enter a non-conductive desensitized state. Chicken ASIC1a structures have been solved in the closed, open and desensitized states. Despite the large amount of available information on the structure and function of ASICs, the precise proton binding sites and the mechanisms by which protonation promotes channel opening are still poorly defined. The acidic pocket, a cavity in the extracellular domain containing several negatively charged amino acids, has been proposed as the primary site for proton sensing. Concerning the molecular mechanisms, it is generally thought that protonation events on extracellular residues induce conformational changes that transmit the “activation signal” to the pore to control the opening of the channel gate. In the first part of this project, we asked whether protonation in the acidic pocket and palm domain is required for channel activation. We found that combination of neutralizing mutations of a large number of titratable acidic pocket residues produced channels that retained their proton sensitivity, suggesting that the residues that are essential for proton sensing are located in other domains. Concomitant with these experiments, we employed the voltage-clamp fluorometry (VCF) technique to investigate the structural rearrangements in the acidic pocket. Our VCF analysis indicates that this region undergoes conformational changes during both activation and desensitization. In the palm, neutralizing mutations of several acidic residues impaired channel desensitization, leading, in some cases, to the disappearance of the transient ASIC component and to the appearance of a sustained current component. In the second part of the project, we elucidated the structural rearrangements occurring in key channel regions during ASIC1a activity. Our VCF experiments reveal the presence of conformational changes in the wrist and palm domain consistent with a role of these regions in channel activation and desensitization.
In summary, our studies suggest that the acidic pocket is not the primary site for proton sensing in ASIC1a, but has, rather, a modulatory role. We show, in addition, that proton binding to the extracellular domain of ASIC1a induces conformational changes in the palm and in the wrist regions most likely important for transmitting the transduction signal to the channel gate. Our findings provide new insights on the basic mechanisms controlling ASIC activity and may be relevant for other members of ENaC/DEG family, to which ASICs belong.
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Les canaux sensibles aux protons (ASICs) sont des canaux sodiques activés par les protons. Ils sont largement exprimés dans les neurones du système nerveux central et périphérique. Ils contribuent en tant que senseurs de pH à un certain nombre de conditions physiologiques et pathologiques, telles que l'apprentissage, la neurodégénérescence après l'ischémie et la sensation de douleur. Les ASICs s'ouvrent de manière transitoire lors d'un abaissement du pH extracellulaire, avant d'entrer dans un état désensibilisé non conducteur. Les structures d’ASIC1a de poulet ont été résolues dans des états fermés, ouverts et désensibilisés. Malgré la grande quantité d'informations disponible sur la structure et la fonction des ASIC, les sites précis de liaison des protons et les mécanismes par lesquels la protonation induit l'ouverture du canal ASIC sont encore très peu définis. La poche acide, une cavité dans le domaine extracellulaire contenant plusieurs acides aminés chargés négativement, a été proposée être le site primaire pour la détection des protons. En ce qui concerne les mécanismes moléculaires de l’activation des ASICs, on pense généralement que les évènements de protonation sur les résidus extracellulaires induisent des changements conformationnels qui transmettent le «signal d'activation» au pore afin de contrôler l'ouverture du canal. Dans la première partie de ce projet, nous avons investigué si la protonation dans la poche acide et dans le domaine de la paume était nécessaire pour l'activation du canal. Nous avons constaté que la combinaison de mutations neutralisantes d'un grand nombre de résidus titrables dans la poche acide produisait des canaux qui conservaient leur sensibilité aux protons, suggérant que les résidus essentiels à la détection de protons se situaient dans d'autres domaines. Parallèlement à ces expériences, nous avons utilisé la technique de “voltage-clamp fluorometry” (VCF) pour étudier les réarrangements structurels dans la poche acide. Notre analyse par VCF indique que cette région subit des changements conformationnels lors de l’activation et de la désensibilisation. Dans la paume, des mutations neutralisantes de plusieurs résidus acides ont altéré la désensibilisation des canaux, conduisant, dans certains cas, à la disparition de la composante transitoire du courant d’ASIC et à l’apparition d’une composante soutenue. Dans la deuxième partie du projet, nous avons élucidé les réarrangements structurels intervenant dans des régions clés pendant l’activité de ASIC1a. Nos expériences de VCF révèlent la présence de changements conformationnels dans les domaines du poignet et de la paume au cours de l'activation et la désensibilisation du canal.
En résumé, nos études suggèrent que la poche acide n'est pas le principal site de liaison de protons dans ASIC1a, mais joue plutôt un rôle de modulateur. Nous montrons, en outre, que la
liaison de protons au domaine extracellulaire de ASIC1a induit des changements de conformation dans la paume et dans les régions du poignet, probablement importants pour la transmission du signal d’activation au pore du canal. Nos résultats fournissent de nouvelles informations sur les mécanismes de base contrôlant l'activité des ASICs et peuvent être pertinents pour d'autres membres de la famille des canaux épithéliaux sodiques / dégénerines, à laquelle les ASICs appartiennent
Structural and Functional Analysis of Gly212 Mutants Reveals the Importance of Intersubunit Interactions in ASIC1a Channel Function
Acid-sensing ion channels (ASICs) act as pH sensors in neurons. ASICs contribute to pain sensation, learning, fear behavior and to neuronal death after ischemic stroke. Extracellular acidification induces a transient activation and subsequent desensitization of these Na+-selective channels. ASICs are trimeric channels made of identical or homologous subunits. We have previously shown that mutation of the highly conserved Gly212 residue of human ASIC1a to Asp affects the channel function. Gly212 is located in the proximity of a predicted Cl- binding site at a subunit interface. Here, we have measured the function of a series of Gly212 mutants. We show that substitution of Gly212 affects the ASIC1a pH dependence and current decay kinetics. Intriguingly, the mutations to the acidic residues Asp and Glu have opposing effects on the pH dependence and the current decay kinetics. Analysis of molecular dynamics simulation trajectories started with the coordinates of the closed conformation indicates that the immediate environment of residue 212 in G212E, which shifts the pH dependence to more alkaline values, adopts a conformation closer to the open state. The G212D and G212E mutants have a different pattern of intersubunit salt bridges, that, in the case of G212E, leads to an approaching of neighboring subunits. Based on the comparison of crystal structures, the conformational changes in this zone appear to be smaller during the open-desensitized transition. Nevertheless, MD simulations highlight differences between mutants, suggesting that the changed function upon substitution of residue 212 is due to differences in intra- and intersubunit interactions in its proximity
Kinetic analysis of ASIC1a delineates conformational signaling from proton-sensing domains to the channel gate
Accelerated Current Decay Kinetics of a Rare Human Acid-Sensing ion Channel 1a Variant That Is Used in Many Studies as Wild Type
Acid-sensing ion channels (ASICs) are neuronal Na+-permeable ion channels that are activated by extracellular acidification and are involved in fear sensing, learning, neurodegeneration after ischemia, and in pain sensation. We have recently found that the human ASIC1a (hASIC1a) wild type (WT) clone which has been used by many laboratories in recombinant expression studies contains a point mutation that occurs with a very low frequency in humans. Here, we compared the function and expression of ASIC1a WT and of this rare variant, in which the highly conserved residue Gly212 is substituted by Asp. Residue 212 is located at a subunit interface that undergoes changes during channel activity. We show that the modulation of channel function by commonly used ASIC inhibitors and modulators, and the pH dependence, are the same or only slightly different between hASIC1a-G212 and -D212. hASIC1a-G212 has however a higher current amplitude per surface-expressed channel and considerably slower current decay kinetics than hASIC1a-D212, and its current decay kinetics display a higher dependency on the type of anion present in the extracellular solution. We demonstrate for a number of channel mutants previously characterized in the hASIC1a-D212 background that they have very similar effects in the hASIC1a-G212 background. Taken together, we show that the variant hASIC1a-D212 that has been used as WT in many studies is, in fact, a mutant and that the properties of hASIC1a-D212 and hASIC1a-G212 are sufficiently close that the conclusions made in previous pharmacology and structure-function studies remain valid
Sulfonamides incorporating heteropolycyclic scaffolds show potent inhibitory action against carbonic anhydrase isoforms I, II, IX and XII
Three series of polycyclic compounds possessing either primary sulfonamide or carboxylic acid moieties as zinc-binding groups were investigated as inhibitors of four physiologically relevant CA isoforms, the cytosolic hCA I and II, as well as the transmembrane hCA IX and XII. Most of the new sulfonamides reported here showed excellent inhibitory effects against isoforms hCA II, IX and XII, but no highly isoform-selective inhibition profiles. On the other hand, the carboxylates selectively inhibited hCA IX (KIs ranging between 40.8 and 92.7 nM) without inhibiting significantly the other isoforms. Sulfonamides/carboxylates incorporating polycyclic ring systems such as benzothiopyranopyrimidine, pyridothiopyranopyrimidine or dihydrobenzothiopyrano[4,3-c]pyrazole may be considered as interesting candidates for exploring the design of isoform-selective CAIs with various pharmacologic applications
Efficacy of residual site radiation therapy (ISRT) in patients with primary mediastinal lymphoma with Deauville Score 4 following R-CHT: results of a retrospective mono institutional study
Background: In order to evaluate the efficacy of residual site radiation therapy (RSRT) in
terms of progression-free survival (PFS) and overall survival (OS) in patients with primary mediastinal
lymphoma (PMBCL) with Deauville Score 4 (DS 4) following rituximab and chemotherapy treatment
(R-ICHT). Methods: Thirty-one patients with PMBCL were recruited. After completion of R-ICHT,
patients were staged with 18F-fluorodeoxyglucose positron-emission tomography, showing DS 4, and
were treated with adjuvant RSRT. The chosen techniques for RT delivery were intensity-modulated
radiation therapy (IMRT) or three-dimensional conformal RT (3D-CRT). Most patients underwent
the first one using cone-beam computed tomography (CBCT). All patients were evaluated every
3 months for the first 2 years and every 6 months afterwards for a period of at least 5 years, with
clinical and radiological procedures as required. Results: All patients received RSRT with a dose of
30 Gy in 15 fractions. The median follow-up time of 52.7 months (IQR: 26–64.1 months). The 5-year
OS rate was 100%. The 2-year and 5-year PFS rates were 96.7% and 92.5%, respectively. Patients with
relapsed disease had been treated with high-dose chemotherapy (HDC) and autologous stem cell
transplantation (auto-SCT). Conclusion: RSRT in patients with PMBCL treated with ICHT and DS 4
did not impact unfavorably on patient survival
1,2-Benzisothiazole Derivatives Bearing 4-, 5-, or 6-Alkyl/arylcarboxamide Moieties Inhibit Carbonic Anhydrase Isoform IX (CAIX) and Cell Proliferation under Hypoxic Conditions
Three novel series of 1,2-benzisothiazole derivatives have been developed as inhibitors of carbonic anhydrase isoform IX. Compounds 5c and 5j, tested in vitro on the human colon cell line HT-29, blocked the growth of cells cultured under chemically induced hypoxic conditions, displaying a specific activity against cancer cells characterized by CAIX up-regulation. Moreover, a synergistic activity of 5c with SN-38 (the active metabolite of irinotecan) and 5-fluorouracil on cell proliferation under hypoxic conditions was demonstrated
Residual Site Radiotherapy After Immunochemotherapy in Primary Mediastinal B-Cell Lymphoma: A Monoinstitutional Retrospective Study
Aim: To evaluate the efficacy of residual site radiation therapy (RSRT) on local control (LC), progressionfree (PFS) and overall (OS) survival in patients with primary mediastinal lymphoma (PMBCL), following rituximab and chemotherapy treatment (ICHT). Patients and Methods: The study included 34 patients with PMBCL treated between 2006 and 2014 with ICHT with/without autologous stem cell transplantation and RSRT. Between the end of ICHT/stem cell transplantation and RSRT, patients were evaluated with F-18-fluorodeoxyglucose positron-emission tomography. The gross tumor volume included morphological mediastinal residual disease after ICHT/SCT. The percentage of LC, PFS and OS were assessed. Results: All patients received RSRT with a median dose of 30 Gy. Median follow-up was 82 months. One patient out of 34 (3%) showed progressive disease 9 months from diagnosis. The 10-year PFS and OS were 97% and 97% respectively. Conclusion: RSRT in patients with PMBCL treated with ICHT did not impact unfavorably on LC and patient survival
Conformational dynamics and role of the acidic pocket in ASIC pH-dependent gating
Acid-sensing ion channels (ASICs) are proton-activated Na(+) channels expressed in the nervous system, where they are involved in learning, fear behaviors, neurodegeneration, and pain sensation. In this work, we study the role in pH sensing of two regions of the ectodomain enriched in acidic residues: the acidic pocket, which faces the outside of the protein and is the binding site of several animal toxins, and the palm, a central channel domain. Using voltage clamp fluorometry, we find that the acidic pocket undergoes conformational changes during both activation and desensitization. Concurrently, we find that, although proton sensing in the acidic pocket is not required for channel function, it does contribute to both activation and desensitization. Furthermore, protonation-mimicking mutations of acidic residues in the palm induce a dramatic acceleration of desensitization followed by the appearance of a sustained current. In summary, this work describes the roles of potential pH sensors in two extracellular domains, and it proposes a model of acidification-induced conformational changes occurring in the acidic pocket of ASIC1a