Dissertations / Theses on the topic 'Kca1.1'

KCa3.1, a potassium channel protein, mediates cellular signaling processes associated with dysfunction of vasculature. However, the role of KCa3.1 in diabetic nephropathy has not been studied. In this thesis, human proximal tubular cells and fibroblasts as well as two diabetic mouse models: KCa3.1-/- and eNOS-/- treated with KCa3.1 inhibitor TRAM34, were used to determine the therapeutic effect of KCa3.1 in diabetic tubulointerstitial fibrosis. Our results show that blockade of KCa3.1 by gene knockout or by concurrent exposure to the inhibitor TRAM34 reversed the diabetic-induced upregulation of inflammation and fibrotic responses through a TGF-β1/Smad dependent signaling pathway. We also demonstrated a key role of KCa3.1 in mediating TGF-β1 induced MCP-1 expression in renal proximal tubular cells via Smad3, p38 and ERK1/2 MAPK signaling pathways. In addition, our studies also demonstrate that blocking the KCa3.1 channel inhibits the NF-κB pathway, resulting in downregulation of the inflammatory marker CCL20. Our preliminary studies have further shown that blockade of KCa3.1 is likely to exert its anti-fibrotic effects through normalization of the autophagocytic pathways, and by inhibition of fibroblast activation. Collectively, the data shown in the thesis uniquely demonstrates that blockade of KCa3.1 is able to prevent the development of diabetic nephropathy.

Idiopathic pulmonary fibrosis (IPF) is a common disease with a median survival of only 3 years. There is no effective treatment. IPF is characterized by myofibroblast accumulation and progressive lung scarring. The Ca²⁺-activated K⁺ channel KCa3.1 modulates the activity of several structural and inflammatory cells which play important roles in model diseases characterized by tissue remodelling and fibrosis. We hypothesise that KCa3.1-dependent cell processes are a common denominator in IPF. KCa3.1 expression and function were examined in human myofibroblasts derived from IPF and non-fibrotic (NFC) donors. Myofibroblasts grown in vitro were characterised by western blot, immunofluorescence, RT-PCR and patch clamp electrophysiology to determine KCa3.1 channel expression. Wound healing, collagen secretion and contraction assays were performed using the pro-fibrotic mediators TGFβ1 and bFGF and two specific KCa3.1 blockers (TRAM-34, ICA-17043 [Senicapoc]). Both NFC and IPF myofibroblasts expressed KCa3.1 channel mRNA and protein. Using the KCa3.1 channel opener 1-EBIO, KCa3.1 ion currents were elicited in 59% of NFC and 77% of IPF myofibroblasts tested (P=0.0411). These currents were blocked by TRAM-34 (200 nM). The 1-EBIO-induced currents were significantly larger in IPF cells compared to NFC cells (P=0.0078). TGFβ1 and bFGF increased KCa3.1 channel expression. TRAM-34 and ICA-17043 dose-dependently attenuated wound healing, TGFβ1-dependent collagen secretion and bFGF- and TGFβ1-dependent contraction. We show for the first time that human lung myofibroblasts express the KCa3.1 K⁺ channel. KCa3.1 channel block attenuates pro-fibrotic myofibroblast function. These findings raise the possibility that blocking the KCa3.1 channel will inhibit pathological myofibroblast function in IPF, and thus offer a novel approach to IPF therapy.

En France, le cancer du sein constitue l’affection tumorale maligne la plus fréquemment rencontrée chez la femme. Malgré les progrès thérapeutiques, il représente le premier cancer en termes de mortalité féminine. De nombreux travaux ont montré que le calcium et le potassium jouent un rôle prépondérant dans la régulation du cycle cellulaire et donc dans la prolifération. Ils permettent la progression en phase G1 et la transition G1/S. Dans ce travail, nous avons étudié le rôle du canal calcique Orai3 dans la régulation de la prolifération et la survie cellulaire des cellules cancéreuses du sein. Nous montrons que la prolifération et la survie des cellules cancéreuses mammaires non invasives et invasives est dépendante du canal Orai3 qui constitue un acteur majeur de l’entrée calcique dans ces cellules. Par contre, l’inhibition du canal Orai3 n’a aucun effet sur les cellules mammaires normales. Par ailleurs, nous montrons que le rôle du canal Orai3 dans la prolifération et la survie des cellules cancéreuses implique la régulation de l’oncogène c-myc. Le canal Orai3 serait, aussi, à l’origine d’une régulation, transcriptionnelle, du canal KCa3. 1 via une voie de signalisation Myc-dépendante. Finalement, nous montrons que les canaux Orai3 et KCa3. 1 ainsi que la protéine c-Myc sont surexprimés dans les tissus mammaires cancéreux par rapport aux tissus adjacents non tumoraux. L’ensemble de nos résultats nous permet de supposer que les canaux calciques Orai3, potassiques KCa3. 1 et le facteur de transcription c-Myc interagissent ensemble et constituent des éléments clés dans la cancérogenèse mammaire
In France, breast cancer (BC) is the most frequent malignant disease among women. Despite the undeniable advances achieved in the therapy, BC still the leading cause of death. Several studies have reported that calcium and potassium ions are involved in the regulation of cell cycle progression and cell proliferation. We have previously reported that KCa3. 1 potassium channels are responsible for BC cell cycle progression by regulating calcium influx. In this study, we investigated the role of Orai3 channels in BC cell proliferation and survival. We found that Orai3 channels regulate calcium influx, cell proliferation and survival of both non-invasive and invasive BC cell lines. In contrast, Orai3 silencing did affect neither cell proliferation and mortality nor calcium entry in normal cells. Furthermore, we showed that Orai3 effects on BC cell proliferation and survival involve the oncogenic transcription factor c-Myc. Moreover, we also demonstrated that Orai3 channels regulate KCa3. 1 expression via Myc pathway. Finally, we showed that Orai3, KCa3. 1 and c-Myc are all together overexpressed in breast cancer tissues as compared to their adjacent normal tissues. In conclusion, our results suggest that Orai3 channels interact with KCa3. 1 channels and c-Myc transcription factor forming together key players in BC development

Neuronal calcium-activated potassium currents are intimately involved in the regulation of cellular membrane potential. Different currents have been identified with diverse pharmacological and physiological properties and in many cases the molecular correlates of the currents have been identified. This thesis will seek to provide information about the molecular biology of two distinct Ca2+-activated K+ currents. In this study the distribution of the small conductance Ca2+-activated K+ channel SK3\alpha-subunit in rat brain during development will be described. Firstly, a polyclonal anti-CSK3 antibody was developed and affinity purified. Secondly, this antibody along with a commercial polyclonal antibody against the N-terminus of SK3 have been used to assess the developmental expression pattern of SK3 at five distinct post-natal time points (P1, P3, P6, P12 and P25). The distribution and level of SK3 expression remains constant throughout development and is in accordance with the pattern previously described for adult rat brain. SK3 is highly expressed in many different neuronal populations but of most interest are the high levels of expression seen in monoaminergic regions such as the substantia nigra pars compacta, locus coeruleus and the dorsal raphe nucleus, regions that have been examined in detail here. The slow afterhyperpolarisation is a Ca2+-activated potassium current present in a restricted number of cell types, chiefly the pyramidal cells of the hippocampus and neocortex. A study recently published identified the neuronal calcium sensor hippocalcin, whose expression is also limited to these cell types, as a key molecule responsible for gating the calcium activation of this current (Tzingounis et al. 2007). I have undertaken a yeast two-hybrid screen to identify potential interacting partners of hippocalcin in an attempt to elucidate the molecular pathway underlying the sAHP. This thesis will present the novel interaction between hippocalcin and the Mitogen- Activated Protein Kinase Kinase Kinase (MAP3K), Mixed Lineage Kinase 2 (MLK2 or MAP3K10).

一氧化氮（NO）和內皮源性超極化因子（EDHFs）是內皮衍生的血管舒張因子兩大類。 EETs是構成EDHFs的主要類型，這是由花生四烯酸通過細胞色素P450 （CYP）表氧化酶的催化活性得到。雖然這兩個EET和NO誘導血管舒張，從而降低血壓，許多報告表明，NO對EET引起的血管舒張起抑製作用。然而，不管它的重要性，有關一氧化氮對EETs的抑制作用的機理尚未完全了解。
在本研究中，我調查了一氧化氮對EET的負調控。通過膜電位和動脈張力測量，我們發現， 11,12-EET可引起內皮剝脫豬冠狀動脈平滑肌細胞膜超極化和血管舒張。該反應被S-亞硝基-N-乙酰青黴胺（SNAP）和8-Br-cGMP，一個NO的供體和cGMP的膜穿透物類似物，分別抑制。 SNAP和8-Br-cGMP對11,12-EET引起的細胞膜超極化和血管舒張的抑製作用被羥鈷胺，一氧化氮清除劑; ODQ ，鳥苷酸環化酶抑製劑;和KT5823 ，蛋白激酶G（PKG）抑製劑逆轉。 SNAP和8-Br-cGMP對EET反應的抑製作用也被過度供應外源性激酶底物， TAT-TRPC1S¹⁷²和TAT -TRPC1T³¹³廢除。羥鈷胺，ODQ， KT5823， TAT -TRPC1，和TAT -scrambled獨自使用不影響11,12-EET引起的細胞膜超極化和血管舒張作用。然而，獨自使用14，15-EEZE（EET的拮抗劑）抑制了11,12-EET的作用。 此外，磷酸化試驗表明， PKG可以直接在Ser172和Thr313位點磷酸化TRPC1 。此外，TRPV4 ， TRPC1 ，或KCa1.1被選擇性地抑制時，11,12-EET未能引起細胞膜超極化和血管舒張。免疫共沉澱研究表明， TRPV4 ， TRPC1和KCa1.1物理上彼此相關聯。
以上結果表明，NO-cGMP-PKG通路可通過TRPC1的磷酸化來抑制11,12- EETs在冠狀動脈血管平滑肌細胞上的作用。此外，TRPV4，TRPC1和KCa1.1參與11,12-EET誘導平滑肌超極化和血管舒張，他們可能互相關聯。從本研究的結果表明，NO和cGMP可通過PKG-介導的TRPC1的磷酸化，抑製EET誘導的平滑肌超極化和血管舒張。
Nitric oxide (NO) and endothelium-derived hyperpolarizing factors (EDHFs) are two main classes of endothelium-derived vascular relaxant factors. EETs constitute a major type of EDHFs, which are derived from arachidonic acids via the catalytic activity of cytochrome P450 (CYP) epoxygenases. Although both EET and NO induce vascular relaxation, thus reduce blood pressure, numerous reports demonstrated that NO exerts an inhibitory action on EET-induced vascular relaxation. However, despite of its importance, the mechanisms related to the inhibitory effects of NO on EETs are incompletely understood.
In the present study, I investigated the scheme for negative regulation of NO on EET action. Through measurements of membrane potential and arterial tension, we showed that 11,12-EET could induce membrane hyperpolarization and vascular relaxation in endothelium-denuded porcine coronary arteries. The responses were suppressed by S-nitroso-N-acetylpenicillamine (SNAP) and 8-Br-cGMP, a NO donor and a membrane-permeant analogue of cGMP, respectively. The inhibitory actions of SNAP and 8-Br-cGMP on 11,12-EET-induced membrane hyperpolarization and vascular relaxation were reversed by hydroxocobalamin, a NO scavenger; ODQ, a guanylyl cyclase inhibitor; and KT5823, a protein kinase G (PKG) inhibitor. The inhibitory actions of SNAP and 8-Br-cGMP on EET responses were also abrogated by shielding TRPC1-PKG phosphorylation sites with excessive supply of exogenous PKG substrates, TAT-TRPC1S¹⁷² and TAT-TRPC1T³¹³. Hydroxocobalamin, ODQ, KT5823, TAT-TRPC1 and TAT-scrambled alone has no effect on 11,12-EET-induced membrane hyperpolarization and vascular relaxation. However, 14,15-EEZE (a selective EET antagonist) alone inhibits the action of 11,12-EET. Furthermore, phosphorylation assay was performed and it demonstrated that PKG could directly phosphorylate TRPC1 at Ser¹⁷² and Thr³¹³. In addition, 11,12-EET failed to induce membrane hyperpolarization and vascular relaxation when TRPV4, TRPC1, or KCa1.1 was selectively inhibited. Co-immunoprecipitation studies demonstrated that TRPV4, TRPC1 and KCa1.1 physically associated with each other in smooth muscle cells.
Taking together, our findings demonstrated that the NO-cGMP-PKG pathway may act through the phosphorylation of TRPC1 to inhibit the action of 11,12-EETs in coronary arterial smooth muscle cells. Furthermore, TRPV4, TRPC1 and KCa1.1 are critically involved in the 11,12-EET-induced smooth muscle hyperpolarization and relaxation and that they may physically associate with each other. The results from this study demonstrated that NO and cGMP could lead to PKG-mediated phosphorylation of TRPC1, resulting in an inhibition of EET-induced smooth muscle hyperpolarization and vascular relaxation.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Zhang, Peng.
"Ca" on title page is subscript.
Thesis (Ph.D.) Chinese University of Hong Kong, 2014.
Includes bibliographical references (leaves 115-133).
Abstracts also in Chinese.

Platelets are nowadays more and more investigated to better characterise their role in many different pathophysiological processes. The present thesis summarises explore NAFLD (Non-Alcoholic Fatty Liver Disease) is a chronic epatopathy degenerating in NASH syndrome and in the worst scenario to liver cancer. Using a Cellix Microfluidic platform with collagen coated microchip and a Flow Cytometry (FACS) on Platelet derived Microvesicles (PMVs) essay, a stronger platelet activity and higher release of PMVs in such patients, is highlighted, probably due to a preactivation platelet status. A platelet activation, leading to wider platelets aggregates, is observed after incubation with C6O4 (100-200 ng/mL), a new compound belonging to Perfluoroalkyl substances (PFAS), used in detergent, coatings for cookware etc. Aspirin 100µM (ASA) shows a neutralisation of C6O4 procoagulant properties. Similar results are observed in FACS analysis on PMVs, regardless the co-stimulation with ADP 7.5µM, TRAP 10µM or in resting condi

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+.
The Ca2+ sensitivity of the voltage-insensitive calcium activated potassium channel of intermediate conductance KCa3.1 is conferred by calmodulin (CaM) constitutively bound to the membrane-proximal region of the channel intracellular C-terminus. A study was performed to investigate the nature of the residues involved in the CaM/KCa3.1 interactions and determine how these interactions could modulate the channel gating properties. A 3D-structure of the KCa3.1/CaM complex was first generated by homology modeling with MODELLER using as template the crystal structure of SK2.2/CaM complex (PDB: 1G4Y). The resulting structural model of KCa3.1 plus CaM predicts that the segment L361-S372 in KCa3.1 should be responsible for the Ca2+-dependent binding of the channel to the CaM-N lobe, with residues L361 and Q364 facing residues E45, E47 and D50 of CaM. To test this model residues in L361-S372 segment were substituted by Cys and the action of MTSET+ (positive charged) and MTSACE (neutral charged) measured on channel activity. Inside-out patch clamp recordings showed that the binding of the charged MTSET+ reagent to the Q364C mutant resulted in a strong current increase, an effect not seen with the neutral MTSACE. The mutations E45A and E47A in CaM prevented the current increase initiated by MTSET+ on the Q364C mutant. A single channel analysis confirmed that the binding of MTSET+ to Q364C caused an increase in the channel open probability by a destabilization of the channel closed state. Altogether, our results are compatible with the formation of ionic bonds between the positively charged Cys-MTSET+ complex at position 364 in KCa3.1 and the negatively charged E45 and E47 residues in CaM, and confirm that an electrostatic stabilization of the CaM/KCa3.1 interactions can lead to an increase in the channel open probability at saturating Ca2+.

Amyotrophic lateral sclerosis (ALS) is a multifactorial disease characterized by the progressive degeneration of motor neurons (MN) and muscle paralysis. Despite current treatments, patients survive less than 3–5 years after the initial diagnosis. Most ALS cases are sporadic (sALS), and only 5-10% have a familial origin (fALS). Among the latter, about 20% express a dominant mutant form of the Cu, Zn superoxide dismutase (SOD1) (Rothstein, 2009). Transgenic mice expressing a mutant SOD1 develop MN pathology, with muscle denervation and weakness similar to ALS patients (Fischer et al., 2004). Many evidence demonstrate that ALS is non-cell autonomous, with multiple co-players involved in disease progression (Robberecht et al, 2013). In particular, signals from both glial cells and muscles initiate and sustain MN degeneration (Boillée et al., 2006; Dobrowolny et al, 2008). Recent studies described a critical role for microglia in amyotrophic lateral sclerosis (ALS), where these CNS-resident immune cells participate in the establishment of an inflammatory microenvironment that contributes to motor neuron degeneration. Understanding the mechanisms leading to microglia activation in ALS could help to identify specific molecular pathways which could be targeted to reduce or delay motor neuron degeneration and muscle paralysis in patients. The intermediate-conductance calciumactivated potassium channel KCa3.1 has been reported to modulate the “pro-inflammatory” phenotype of microglia in different pathological conditions. We here investigated the effects of blocking KCa3.1 activity in the hSOD1G93AALS mouse model, which recapitulates many features of the human disease. We report that treatment of hSOD1G93A mice with a selective KCa3.1 inhibitor, 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34), attenuates the “pro-inflammatory” phenotype of microglia in the spinal cord, reduces motor neuron death, delays onset of muscle weakness, and increases survival. Specifically, inhibition of KCa3.1 channels slowed muscle denervation, decreased the expression of the fetal acetylcholine receptor γ subunit and reduced neuromuscular junction damage. Taken together, these results demonstrate a key role for KCa3.1 in driving a pro-inflammatory microglia phenotype in ALS.

The aim of the present work was that of study two strategies to overcome Cisplatin chemoresistance in colorectal cancer: (1) to exploit K+ ion channels modulating agents as new therapeutic tools in order to increase Cisplatin therapeutic potential in colorectal cancer cells (2) to test two new Cisplatin-analogues, cis-Pt-I2 and cis-Pt-Br2, with increased selectivity in order to overcome resistance in a panel of tumor cell lines.

Les cellules épithéliales des voies aériennes respiratoires sécrètent du Cl- via le canal CFTR. La fibrose kystique est une maladie génétique fatale causée par des mutations de ce canal. La mutation la plus fréquente en Amérique du Nord, ∆F508, met en péril la maturation de la protéine et affecte les mécanismes d’activation du canal. Au cours des dernières années, plusieurs molécules ont été identifiées par criblage à haut débit qui peuvent rétablir l’activation de protéines CFTR mutées. Ces molécules sont nommées potentiateurs. Les canaux K+ basolatéraux, dont KCa3.1, jouent un rôle bien documenté dans l’établissement d’une force électromotrice favorable à la sécrétion de Cl- par CFTR dans les cellules épithéliales des voies aériennes respiratoires. Il a par exemple été démontré que l’application de 1-EBIO, un activateur de KCa3.1, sur des monocouches T84 résulte en une augmentation soutenue de la sécrétion de Cl- et que cette augmentation était réversible suite à l’application de CTX, un inhibiteur de KCa3.1(Devor et al., 1996). Dans le cadre d’une recherche de potentiateurs efficaces en conditions physiologiques et dans un contexte global de transport trans-cellulaire, il devient essentiel de considérer les effets des potentiateurs de CFTR sur KCa3.1. Une caractérisation électrophysiologique par la méthode du patch clamp et structurelle via l’utilisation de canaux modifiés par mutagenèse dirigée de différents potentiateurs de CFTR sur KCa3.1 fut donc entreprise afin de déterminer l’action de ces molécules sur l’activité de KCa3.1 et d’en établir les mécanismes. Nous présentons ici des résultats portant sur les effets sur KCa3.1 de quelques potentiateurs de CFTR possédant différentes structures. Un criblage des effets de ces molécules sur KCa3.1 a révélé que la genisteine, le SF-03, la curcumine et le VRT-532 ont des effets inhibiteurs sur KCa3.1. Nos résultats suggèrent que le SF-03 pourrait agir sur une protéine accessoire et avoir un effet indirect sur KCa3.1. La curcumine aurait aussi une action inhibitrice indirecte, probablement via la membrane cellulaire. Nos recherches sur les effets du VRT-532 ont montré que l’accessibilité au site d’action de cette v molécule est indépendante de l’état d’ouverture de KCa3.1. L’absence d’effets inhibiteurs de VRT-532 sur le mutant constitutivement actif V282G indique que cette molécule pourrait agir via l’interaction CaM-KCa3.1 et nécessiter la présence de Ca2+ pour agir. Par ailleurs, un autre potentiateur de CFTR, le CBIQ, a des effets potentiateurs sur KCa3.1. Nos résultats en canal unitaire indiquent qu’il déstabilise un état fermé du canal. Nos travaux montrent aussi que CBIQ augmente la probabilité d’ouverture de KCa3.1 en conditions sursaturantes de Ca2+, ainsi que son affinité apparente pour le Ca2+. Des expériences où CBIQ est appliqué en présence ou en absence de Ca2+ ont indiqué que l’accessibilité à son site d’action est indépendante de l’état d’ouverture de KCa3.1, mais que la présence de Ca2+ est nécessaire à son action. Ces résultats sont compatibles avec une action de CBIQ déstabilisant un état fermé du canal. Finalement, des expériences en Ba2+ nous ont permis d’investiguer la région du filtre de sélectivité de KCa3.1 lors de l’action de CBIQ et nos résultats pointent vers une action de CBIQ dans cette région. Sur la base de nos résultats nous concluons que CBIQ, un potentiateur de CFTR, aurait un effet activateur sur KCa3.1 via la déstabilisation d’un état fermé du canal à travers une action sur sa ‘gate’ au niveau du filtre de sélectivité. De plus, les potentiateurs de CFTR ayant montré des effets inhibiteurs sur KCa3.1 pourraient agir via la membrane ou via une protéine accessoire du canal ou sur l’interaction CaM-KCa3.1. Dans l’optique de traitements potentiels de la fibrose kystique, nos résultats indiquent que le CBIQ pourrait être un potentiateur efficace pusiqu’il est capable de trimuler à la fois KCa3.1 et CFTR. Par contre, dans les cas du VRT-532 et du SF-03, une inhibition de KCa3.1 pourraient en faire des potentiateurs moins efficaces.
Airway epithelial cells are the site of Cl- secretion through CFTR. Cystic fibrosis is a fatal genetic disease caused by mutations in CFTR. The most frequent mutation in North America (∆F508) results in impaired maturation and altered channel gating of the protein. In the last years, several small molecules were identified by high throughput screening that could restore mutated CFTR function. Compounds addressing CFTR gating defects are referred to as potentiators. The basolateral K+ channel KCa3.1 has been documented to play a prominent role in establishing a suitable driving force for CFTR-mediated Clsecretion in airway epithelial cells. It has been shown, for example, that the application of 1-EBIO on T84 monolayers results in a sustained increase of Clsecretion and that this current can be reversed by application of CTX, a KCa3.1 inhibitor (Devor et al., 1996). Thus, in a global approach of transepithelial transport, the research for physiologically relevant CFTR potentiators should also consider their effects on the KCa3.1 channel. Electrophysiological patch clamp measurements and channel structural modification by site directed mutagenesis were used to characterize the action of CFTR potentiators on KCa3.1 and study their molecular mode of action. In this work we present results on the effects on KCa3.1 of several CFTR potentiators of different structures. We observed that the CFTR potentiators genistein, curcumin, SF-03 and VRT-532 could inhibit KCa3.1 activity at concentrations known to activate CFTR. Our results suggest that SF- 03 could act indirectly on KCa3.1 through a mechanism involving an accessory protein. Curcumin would also have an indirect inhibitory effect, probably mediated by the plasma membrane, as documented for other ion channels. A detailed study of VRT-532 revealed that this molecule has access to its binding site in a state independent manner, and is poorly effective on the V282G mutant of KCa3.1, which is constitutively active. These results suggest that VRT-532 could act through the CaM/KCa3.1 complex and require the presence of Ca2+ to inhibit channel activity. In contrast, CBIQ, another CFTR potentiator, succeeded to activate KCa3.1. Our results in single channel show that CBIQ vii destabilizes a non conducting state of the channel. We also showed that this molecule increases the apparent Ca2+ affinity as well as the channel open probability, even in saturating Ca2+ conditions. Experiences in which Ba2+ was used as a probe were also performed to determine if the action mechanism of CBIQ involves an effect on the selectivity filter. Our results showed that Ba2+ could displace CBIQ from its interacting site, suggesting that the increases in channel activity induced by CBIQ could result from a change in the energetics of the channel at the level of the selectivity filter. On the basis of our results, we conclude that CBIQ, a CFTR potentiator, could activate KCa3.1 by destabilizing a non conducting state of the channel, probably through an action near the selectivity filter region. Also, CFTR potentiators having an inhibitory effect on KCa3.1 are likely to act through the plasmic membrane, the CaM/KCa3.1 interaction or an accessory protein of the channel. In a perspective of future treatments for CF, our results indicate that CBIQ could be an efficient potentiator since this product stimulates KCa3.1 as well as CFTR. Conversly, the VRT-532 and SF-03 could be less efficient than on CFTR alone, due to their inhibition of KCa3.1.

博士
國防醫學院
生命科學研究所
104
Cisplatin is the most widely used as a chemotherapeutic agent for several solid tumor types. Despite its effectiveness for cancer therapy, the clinical use of cisplatin is frequently limited by its toxicity against normal tissues, particular kidney toxicity. Accumulating evidence supports that tubular cell apoptosis significantly contributes to the pathogenesis of cisplatin-induced acute kidney injury (AKI). KCa3.1, a calcium-activated potassium channel, is widely expressed in both excitable and non-excitable cells. It is activated by the increase of intracellular calcium to regulate intracellular potassium efflux, membrane potential, and calcium signaling in various cellular processes. It has also been reported to contribute to various pathological events. Recently, KCa3.1 has been reported to participate in the regulation of apoptosis. However, its involvement in cisplatin-induced AKI is unknown. The aim of this thesis is that to determine the involvement of KCa3.1 in the pathogenesis of cisplatin-induced AKI. First, we determined the causal relationship between KCa3.1 expression and cisplatin-induced AKI in vitro and in vivo. We found that cisplatin treatment triggered an early induction of KCa3.1 expression associated with apoptosis in HK-2 cells. We also found that the induction of KCa3.1 expression was associated with the development of renal tubular damage and apoptosis in cisplatin-treated mice. We then used the highly selective KCa3.1 blocker TRAM-34 treatment to evaluate whether KCa3.1 induction was involved in cisplatin-induced apoptosis in vitro. We found that treatment with TRAM-34 suppressed cisplatin-induced apoptosis in HK-2 cells. We further assessed whether KCa3.1 mediated cisplatin-induced AKI in genetic knockout and pharmacological blockade mouse models. We found that KCa3.1 deficiency reduced renal function loss, renal tubular damage, and the induction of the apoptotic marker caspase-3 in the kidneys of cisplatin-treated KCa3.1-/- mice. Pharmacological blockade of KCa3.1 by TRAM-34 similarly attenuated cisplatin-induced AKI in mice. Furthermore, we dissected the mechanisms underlying cisplatin-induced apoptosis reduction via KCa3.1 blockade. We found that KCa3.1 blockade attenuated cytochrome c release and the increase in the intrinsic apoptotic mediators Bax, Bak, and caspase-9 after cisplatin treatment. KCa3.1 blocking inhibited the cisplatin-induced activation of the endoplasmic reticulum (ER) stress mediator caspase-12, which is independent of calcium-dependent protease m-calpain activation. In conclusion, KCa3.1 blockade protects against cisplatin-induced AKI through the attenuation of apoptosis by interference with intrinsic apoptotic and ER stress-related mediators, providing a potential target for the prevention of cisplatin-induced AKI.

碩士
長庚科技大學
健康產業科技研究所
106
Diabetic nephropathy (DN) is the leading cause of end-stage kidney disease (ESKD) in chronic kidney disease (CKD). Reducing mesangial expansion in early pathological stage of DN can ameliorate glomerular filtration and attenuate the progressing of DN. Hyperglycemia in diabetic patients stimulates the expression of growth factor TGF-β1, oxidative stress and KCa3.1 potassium channel, all of these are the important factors in renal fibrosis. The role of KCa3.1 in the activation of mesangial expansion is unclear. Using KCa3.1 inhibitor TRAM-34, caffeic acid and clinical drug pentoxifylline to invest relative proteins expression and oxidation stress by TGF-β1 stimulation. Our results demonstrated that blockade KCa3.1 via TRAM-34 and caffeic acid inhibit the upregulation of phosphorylation of Smad2/3, and expression of collagen-I/III which associated with extracellular matrix (ECM). TRAM-34 and pentoxifylline also reduced oxidative stress. In summary, we demonstrated the blockade of KCa3.1 to reduce ECM associate collagens which stimulated by TGF-β1. This therapeutic intervention will ameliorate the mesangial expansion to delay the process of diabetic nephropathy in further study.

Le syndrome de détresse respiratoire aiguë (SDRA) est caractérisé par des dommages au niveau de la barrière alvéolo-capillaire, résultant en la formation d’un œdème pulmonaire et une réponse inflammatoire exacerbée. Sans résolution rapide de ces paramètres, le syndrome progresse vers le développement de fibrose menant à l’insuffisance respiratoire. Or, il a été établi que la réparation de l’épithélium alvéolaire est une étape cruciale pour la résolution du SDRA. Une meilleure compréhension des mécanismes de réparation de l’épithélium alvéolaire est donc nécessaire afin de proposer de nouvelles thérapies pour le SDRA, pour lequel aucun traitement efficace n’existe. Il a été montré que les mécanismes de réparation sont régulés par des protéines membranaires, non seulement par les récepteurs aux facteurs de croissance et les intégrines, mais également par les canaux ioniques, en particulier les canaux potassiques. L’objectif principal de cette étude était donc de caractériser l’impact de la modulation des canaux potassiques KCa3.1 et KvLQT1 dans la résolution du SDRA. Dans un premier temps, nos résultats ont montré le rôle coopératif du canal potassique KCa3.1, de la matrice extracellulaire et de l’intégrine-β1 dans les processus de réparation de l’épithélium alvéolaire in vitro. Nous avons montré que la matrice de fibronectine et le KCa3.1 étaient impliqués dans la migration et dans la réparation de monocouches de cellules alvéolaires de cultures primaires de rat. Dans un deuxième temps, nous avons étudié l’impact de la modulation du canal potassique KvLQT1 dans certains aspects physiopathologiques du SDRA à l’aide de modèles in vivo. Nous avons montré que KvLQT1 n’était pas seulement impliqué dans les mécanismes de réparation de l’épithélium alvéolaire, mais également dans la résorption de l’œdème pulmonaire et la résolution de la réponse inflammatoire. Nos résultats démontrent que les canaux potassiques, tels que KCa3.1 et KvLQT1, pourraient être identifiés en tant que cibles thérapeutiques potentielles pour le SDRA.
Acute respiratory distress syndrome (ARDS) is characterized by alveolar-capillary barrier damage, resulting in the formation of pulmonary oedema and an exacerbated inflammatory response. Without rapid recovery of these parameters, there is a gradual development of fibrosis, leading to respiratory failure. It has been established that alveolar regeneration is a critical step for the resolution of ARDS. A better understanding of alveolar epithelial repair mechanisms is hence necessary to identify new therapies for ARDS, for which no effective treatment exist. It has been shown that repair mechanisms are regulated by membrane proteins, not only by growth factor receptors and integrins, but also by ion channels, in particular potassium channels. Therefore, the main objective of this study was to characterize the impact of KCa3.1 and KvLQT1 potassium channels modulation in the resolution of ARDS. First, our results have shown the cooperative role of the potassium channel KCa3.1, the extracellular matrix and the β1-integrin in alveolar epithelial repair processes in vitro. We have shown that the fibronectin matrix and KCa3.1 are involved in the migration and repair of primary cultures of rat alveolar cell monolayers. Our data also revealed a putative relationship between Kca3.1 and the β1-integrin. Second, we studied the impact of KvLQT1 potassium channel modulation on ARDS pathophysiological aspects with in vivo models. We showed that KvLQT1 was not only involved in alveolar epithelial repair, but also in the resolution of pulmonary oedema and inflammatory response. Taken together, our data demonstrate that potassium channels, such as KCa3.1 and KvLQT1, may be identified as potential therapeutic targets for the resolution of ARDS.