Rozprawy doktorskie na temat „Potassium Ion Cells”

Three distinct K$ sp+$ currents were identified in corn (Zea mays) protoplasts using the whole-cell patch-clamp technique. Inward-rectifying K$ sp+$ currents were evoked at membrane potentials more negative than $-$100 mV. The activation range was sensitive to external K$ sp+$ and shifted in the positive direction as the K$ sp+$ concentration was elevated. The second K$ sp+$ current was voltage-independent and contributed to the resting membrane conductance of the protoplast. Finally, a voltage- and Ca$ sp{2+}$-dependent K$ sp+$ current was observed at potentials positive to $-$60 mV. This current was inhibited by reagents which antagonize plasmalemma Ca$ sp{2+}$ influx (e.g. nitrendipine, verapamil). In contrast, currents were enhanced by increasing the cytosolic free Ca$ sp{2+}$ concentration from 40 to 400 nM. The Ca$ sp{2+}$-dependent K$ sp+$ current was inhibited by tetraethylammonium ions, Cs$ sp+$, Ba$ sp{2+}$, and charybdotoxin which suggested that the channel protein has structural similarities to the high conductance Ca$ sp{2+}$-dependent K$ sp+$ channel observed in animal systems.

Previous studies have provided evidence that endothelial cell-based potassium ion selective electrodes possess the ability to quantify substances that have permeability-altering effects on those endothelial cells. The capability of these so-called biosensors to detect elevated concentrations of certain chemical agents found following tumor formation make them useful in the application as an alternative tumor angiogenesis assay. In this study an epithelial cell line, human colon adenocarcinoma epithelial cells (Caco-2), was used to fabricate membranes that were used to test concentrations of these chemical agents, known as cytokines, mimicking the concentrations that have been observed in the serum of healthy individuals as well as the higher concentration found in individuals with cancer. Additionally background information is provided related to the development of whole cell-based biosensors, metabolic pathways related to tumor angiogenesis and the subsequent increase in cytokine concentration, properties of the Caco-2 cell line that make them useful for the application in cell-based biosensors, and the ultimate effect the cytokines have on the permeability of the cells.

Dans cette thèse nous avons utilisé des méthodes de systèmes dynamiques et des simulations numériques pour étudier les mécanismes d'oscillations d'épilepsie associés à des concentrations d’ions dynamiques et au comportement bimodal des cellules Purkinje du cervelet. Le propos général de ce travail est l'interaction entre les propriétés intrinsèques des neurones simple et la structure d'entrée synaptique contrôlant l'excitabilité neuronale. Dans la première partie de la thèse nous avons développé un modèle de transition de crise épileptique dans le lobe temporal du cerveau. Plus précisément nous nous sommes concentrés sur le rôle du cotransporteur KCC2, qui est responsable de la maintenance du potassium extracellulaire et du chlorure intracellulaire dans les neurones. Des données expérimentales récentes ont montré que cette molécule est absente dans un groupe significatif de cellules pyramidales dans le tissue neuronal de patients épileptiques suggérant son rôle épileptogène. Nous avons trouvé que l'addition d’une quantité critique de cellules pyramidale KCC2 déficient au réseau de subiculum, avec une connectivité réaliste, peut provoquer la génération d’oscillations pathologiques, similaire aux oscillations enregistrées dans des tranches de cerveau épileptogène humaines. Dans la seconde partie de la thèse, nous avons étudié le rôle du bruit synaptique dans les cellules de Purkinje. Nous avons étudié l'effet de l'inhibition de la génération du potentiel d’action provoquée par injection de courant de bruit, un phénomène connu comme résonance stochastique inverse (RSI). Cet effet a déjà été trouvé dans des modèles neuronaux, et nous avons fournis sa première validation expérimentale. Nous avons trouvé que les cellules de Purkinje dans des tranches de cerveau peuvent être efficacement inhibées par des injectionsde bruit de courant. Cet effet est bien reproduit par le modèle phénoménologique adapté pour différentes cellules. En utilisant des méthodes de la théorie de l'information, nous avons montré que RSI prend en charge une transmission efficace de l'information des cellules de Purkinje simples suggérant son rôle pour les calculs du cervelet
In this thesis we used dynamical systems methods and numericalsimulations to study the mechanisms of epileptic oscillations associated with ionconcentration changes and cerebellar Purkinje cell bimodal behavior. The general issue in this work is the interplay between single neuron intrinsicproperties and synaptic input structure controlling the neuronal excitability. In the first part of this thesis we focused on the role of the cellular intrinsicproperties, their control over the cellular excitability and their response to thesynaptic inputs. Specifically we asked the question how the cellular changes ininhibitory synaptic function might lead to the pathological neural activity. We developed a model of seizure initiation in temporal lobe epilepsy. Specifically we focused on the role of KCC2 cotransporter that is responsible for maintaining the baseline extracellular potassium and intracellular chloride levels in neurons. Recent experimental data has shown that this cotransporter is absent in the significant group of pyramidal cells in epileptic patients suggesting its epileptogenic role. We found that addition of the critical amount of KCC2-deficient pyramidal cells to the realistic subiculum network can switch the neural activity from normal to epileptic oscillations qualitatively reproducing the activity recorded in human epileptogenic brain slices. In the second part of this thesis we studied how synaptic noise might control the Purkinje cell excitability. We investigated the effect of spike inhibition caused by noise current injection, so-called inverse stochastic resonance (ISR). This effect has been previously found in single neuron models while we provided its first experimental evidence. We found that Purkinje cells in brain slices could be efficiently inhibited by current noise injections. This effect is well reproduced by the phenomenological model fitted for different cells. Using methods of information theory we showed that ISR supports an efficient information transmission of single Purkinje cells suggesting its role for cerebellar computations

Cell survival is modulated by a cocktail of ion channels engaging cell life and death decisions through controlling key cellular messages such as apoptosis and proliferation. Unnatural regulation of these processes results in various disorders, for example neurodegenerative diseases, as well as the cancers. Nowadays, these pathologies are affecting millions of people per year in the world. Potassium (K+) ion channels appear to play a potent role in such illnesses since they can control many cellular gates in cell physiology such as ionic homeostasis and signalling cascades. Amongst the K+ channels, small (SK1-3) and intermediate (SK4) conductance Ca2+-activated potassium ion channels have recently been shown to save cells, thereby protecting mitochondrial function which serves as a cell survival platform. In the case of other ion channels, for instance transient receptor potential melastatin 7 (TRPM7), it is also repeatedly stated that such membrane channels shows an impressive and differential role in excitable and non-excitable cell survival. This channel also modulates ionic homeostasis of crucial ions in cellular physiology such as Ca2+. This study reveals that central nervous system (CNS) and breast cancer cells differentially express SK1-4 ion channel subtypes, and their functional presence is pharmacologically confirmed, however, in most cases these results were further clarified through small interference RNA (siRNA) method. Similarly, functional TRPM7 channel expression in CNS cells is also confirmed. In the CNS, SK1-4 channel activation rescues neurons from oxidative stress, whereas, TRPM7 channel inhibition protects CNS cells from this hydrogen peroxide (H2O2) harmful effect, as well as hypoxia and apoptosis, so improving cell survival. Excitingly, SK1-4 channels differentially exist between wild-type and Huntington’s affected mouse striatal cells, where diseased cells lack SK1-3 channels, key players in action potential activity. Interestingly, SK2 or SK3 channel subtypes are also functionally expressed in breast cancer cells with various phenotypes. This study established that these ion channels are powerful agents in a survival role, in fact controlling growth through cross-talk with an apoptotic avenue “intrinsic pathway”. SK2 or SK3 channel activation enhances cell viability, while its inhibition dampens cell growth. It is very noteworthy that SK2 and SK3 channels are not expressed in non-tumorigenic breast cells. In brief, SK1-4 and TRPM7 molecules are clearly implicated in the survival of diverse cell types through an apoptotic route, indicating that these ionic regulators are promising targets in channelopathies related to cellular degeneration and growth.

A recent X-ray analytical technique for the measurement of inorganic solutes in dried vacuolar sap has been improved and tested and was used to measure the content of barley leaf epidermal vacuoles. This method was compared with a technique which measured inorganic solutes in vacuoles of frozen leaf sections. The methods gave comparable results. They were used in conjunction with a range of other microanalytical methods to investigate the role of potassium as an osmoticum in individual leaf epidermal cells of barley grown in either low (0.2 mM) or control (4.0 mM) levels of potassium. In the low potassium plants, both turgor and osmotic pressure were at first relatively low, but eventually rose to levels similar to the control plants. In both treatments, vacuolar potassium and its counterion accounted for all the osmotic pressure in young leaves. However, during leaf development, the potassium was replaced to . varying extents by calcium. During this time gradients of ion concentrations developed between adjacent cells, depending on their proximity to vascular tissue. In older leaves, the cellular concentrations of calcium and potassium were negatively correlated. The nature of this relationship was affected by potassium nutrition. In control plants, the ratio of calcium to potassium concentration was 2: 3 (0.67). This value is consistent with the maintenance of osmotic pressure which was observed. In the low potassium plants, the Ca: K ratio was 0.76, which explained the increase in osmotic pressure over time in these plants. It is proposed that the epidermis behaves as a storage tissue for potassium which is retranslocated to younger tissue as the leaf ages. The rate of export depends on the potassium nutrition of the plant. These events are also consistent with the hypothesis that turgor and osmotic pressure are regulated to compensate for the arrival of soluble calcium in the leaf.

The aim of this thesis was to study the effects of extremely low frequency (ELF) electromagnetic magnetic fields on potassium currents in neural cell lines ( Neuroblastoma SK-N-BE ), using the whole-cell Patch Clamp technique. Such technique is a sophisticated tool capable to investigate the electrophysiological activity at a single cell, and even at single channel level. The total potassium ion currents through the cell membrane was measured while exposing the cells to a combination of static (DC) and alternate (AC) magnetic fields according to the prediction of the so-called ‘ Ion Resonance Hypothesis ’. For this purpose we have designed and fabricated a magnetic field exposure system reaching a good compromise between magnetic field homogeneity and accessibility to the biological sample under the microscope. The magnetic field exposure system consists of three large orthogonal pairs of square coils surrounding the patch clamp set up and connected to the signal generation unit, able to generate different combinations of static and/or alternate magnetic fields. Such system was characterized in term of field distribution and uniformity through computation and direct field measurements. No statistically significant changes in the potassium ion currents through cell membrane were reveled when the cells were exposed to AC/DC magnetic field combination according to the afore mentioned ‘Ion Resonance Hypothesis’.

The aim of this thesis was to study the effects of extremely low frequency (ELF) electromagnetic magnetic fields on potassium currents in neural cell lines ( Neuroblastoma SK-N-BE ), using the whole-cell Patch Clamp technique. Such technique is a sophisticated tool capable to investigate the electrophysiological activity at a single cell, and even at single channel level. The total potassium ion currents through the cell membrane was measured while exposing the cells to a combination of static (DC) and alternate (AC) magnetic fields according to the prediction of the so-called ‘ Ion Resonance Hypothesis ’. For this purpose we have designed and fabricated a magnetic field exposure system reaching a good compromise between magnetic field homogeneity and accessibility to the biological sample under the microscope. The magnetic field exposure system consists of three large orthogonal pairs of square coils surrounding the patch clamp set up and connected to the signal generation unit, able to generate different combinations of static and/or alternate magnetic fields. Such system was characterized in term of field distribution and uniformity through computation and direct field measurements. No statistically significant changes in the potassium ion currents through cell membrane were reveled when the cells were exposed to AC/DC magnetic field combination according to the afore mentioned ‘Ion Resonance Hypothesis’.

This study aims to determine the effect of shear stress on membrane potassium (K$\sp+$) permeability in vascular endothelial cells. Cultured monolayers of calf pulmonary artery endothelial cells, preloaded with $\rm \sp{86}Rb\sp+$, were subjected to different levels of fluid shear stress in the range 1-10 dynes/cm$\sp2$, in a parallel-plate geometry flow chamber, and the radioactivity of the effluent was monitored with time. Increase in laminar shear stress, from 1 dyne/cm$\sp2$ to a higher level, resulted in a rapid transient increase in the rate constant for $\rm \sp{86}Rb\sp+$ release. The difference of efflux rate coefficients, between the peak and the baseline at 1 dyne/cm$\sp2$, varied with shear stress in a "dose-dependent" manner. Changes in K$\sp+$ permeability may occur via activation of shear-stress-activated K$\sp+$ channels, Ca$\sp{2+}$-activated K$\sp+$ channels, or both. The Ca$\sp{2+}$-activated K$\sp+$ channels were shown to respond to bradykinin stimulation under flow conditions. The significance of such flow studies is that they provide more knowledge about the flow-associated changes in ionic channels and intracellular second messengers in endothelial cells.

The commercialization of Li-ion battery (LIB) in 1990s by Sony Corporation has led to its applications in portable electronic devices such as mobile phones, cameras, laptop computers, etc. Initially, the energy density of commercial LIB was only about 120 Wh Kg-1. However, with sustained improvements in properties of various cell components, the present-day LIB provides energy density of about 250 Wh Kg-1. With future use envisaged for mobility applications such as electric vehicles, research activities have gained momentum for development of high energy density Li-S and Li-O2 batteries. However, due to limited sources of lithium (0.007 % in earth’s crust and 0.2 ppm in sea water) and uneven distribution, concerns arise about its cost and availability which would inhibit bulk production and utilization of lithium-based batteries. Hence, there is an urgent need to switch over to battery systems employing earth abundant and environmentally benign materials. Sodium and potassium-based batteries have received attention in research laboratories as alternatives to lithium-based batteries due to their natural abundance and low cost. Na and K are the metals below Li in the periodic table and their physical and chemical properties are similar to those of Li. Na and K are the sixth and seventh most abundant elements, constituting 2.6 % and 2.4 %, respectively of the earth’s crust. Sea water contains about 10800 ppm Na and 400 ppm K. Although, the standard potentials of Na/Na+ (-2.71 V vs. standard hydrogen electrode (SHE)) and K/K+ (-2.93 V vs. SHE) are less than Li/Li+ (-3.04 V vs. SHE) by about 300 and 100 mV, respectively, the cost and availability factors overweigh the marginal reduction in energy density. The quest for new electrode materials for Na- and K-based batteries, their physicochemical characterizations and electrochemical investigations are described in the thesis. It consists of a comprehensive review of the literature on the evolution of battery systems with a focus on the next generation Na- and K-based batteries. The cathode and anode materials for Na- and K-ion batteries are reviewed along with the current research activities in Na- and K-sulphur, and Na- and K-O2 batteries. It furnishes a brief description of various experimental techniques and procedures adopted at different stages of the present thesis. The amorphous MnO2 has been prepared by two different methods: (i) reduction of KMnO4 using ethylene glycol (EG) and (ii) the redox reaction between KMnO4 and MnSO4.H2O at ambient conditions. The as prepared MnO2 samples in both cases are amorphous in nature and on heating in the temperature range of 300 – 800 °C, they convert to α-MnO2. The MnO2 prepared by reduction by EG has been studied for Na/MnO2 and Li/MnO2 laboratory scale primary cells in non-aqueous electrolytes. The specific capacity of amorphous MnO2 is 300 mAh g-1 in both Na/MnO2 and Li/MnO2 cells. Na/MnO2 cell shows a nominal voltage less than Li/MnO2 cell by 0.35 V, as expected. MnO2 prepared by the redox reaction between KMnO4 and MnSO4.H2O has a specific surface area of 184 m2 g-1 with narrowly distributed mesopores of 3.5 nm pore diameter. The crystallinity increases and specific surface area decreases upon heating. The as prepared sample provides the first discharge capacity of about 300, 200 and 80 mAh g-1 for Li-, Na- and K-MnO2 cells, respectively, at a specific current of 50 mA g-1. The attractively high discharge capacity of the as prepared amorphous MnO2 is attributed to the large specific surface area and mesoporosity. However, the crystalline samples exhibit low specific discharge capacity in comparison with amorphous samples. It deals with electrochemical impedance spectroscopy (EIS) study of Na/MnO2 primary cell fabricated in a non-aqueous electrolyte of Na salt. The EIS data provides a high resistance of Na metal due to the surface passive film. On subjecting the cell for discharge, the surface film causes a delay response of the cell voltage and the closed-circuit voltage reaches the normal discharge level following dielectric break-down of the film. The EIS data measured at different stages of cell discharge are subjected to non-linear least squares fitting with the aid of an appropriate equivalent circuit. The impedance parameters are examined to throw light on state-of-charge of Na/MnO2 primary cells. The study has been further extended to analyze the delay-time behaviour of the non-aqueous Na/MnO2 cells and quantifying the film resistance and break-down field for the film formed on the Na surface. P2-type Na0.67Mn0.65Fe0.20Ni0.15O2 is studied as a cathode material for Na-ion battery and presented. It is synthesized in microspherical and disc-like morphologies using two different synthetic procedures. Microspheres of FeCO3 are first prepared and used as a template to synthesize Mn0.65Fe0.20Ni0.15CO3, followed by its thermal decomposition to the corresponding oxide and finally, thermal fusion of the oxide with Na2CO3 to produce P2-type Na0.67Mn0.65Fe0.20Ni0.15O2. However, disc-like Na0.67Mn0.65Fe0.20Ni0.15O2 is synthesized by sintering the product obtained using a low temperature solution combustion method using aqueous solution of stoichiometric quantities of corresponding metal nitrates and sucrose as the fuel at 800 °C. Cyclic voltammograms in both the samples are characterized by well-defined two pairs of current peaks corresponding to the oxidation and reduction processes in two different stages. The sodiated microspherical oxide provides an initial discharge capacity of about 216 mAh g-1 at C/15 rate cycling with an excellent cycling stability (Fig. 3a). The rate capability is also high, and the discharge capacity is about 100 mAh g-1 at 2C rate. The high discharge capacity and high rate capability are attributed to porous microspherical morphology. When the cells with disc-like morphology cathode sample are cycled at a current density of 35 mA g-1, a specific discharge capacity of 178 mAh g-1 is obtained with close to 100 % coulombic efficiency. Capacity retention of more than 70 % is observed after 50 charge-discharge cycles Potassium tetratitanate (K2Ti4O9) is synthesized by solid-state method using K2CO3 and TiO2 and studied as an anode material for potassium ion batteries (KIB) for the first time. A discharge capacity of 97 mAh g-1 has been obtained at a current density of 30 mA g-1 (0.2 C rate) and 80 mAh g-1 at 100 mA g-1 (0.8 C rate), initially (Fig. 4a). The proposed mechanism of charging involves reduction of two Ti ions from 4+ oxidation state to 3+ oxidation state, which facilitates insertion of two K+ ions per formula unit in the zig-zag layer of TiO6 octahedra separated with K+ ions with interlayer spacing of 0.85 nm. For KIB cathode, K0.27Mn0.65Fe0.35-xNixO2 (0.00 ≤ x ≤ 0.35) is synthesized in microspherical morphology. The potassiated mixed metal oxide formed in microspherical morphology is in pure crystalline phase. The oxide with the composition x = 0.35 i.e., K0.27Mn0.65Ni0.35O2 provides the highest first specific discharge capacity of 97 mAh g-1 at C/10 rate (Fig. 4b). A good cycling stability is observed. It deals with carbonization of milk-free coconut kernel pulp carried out at low temperatures. The carbon samples are activated using KOH and electrical doublelayer capacitor (EDLC) properties are studied (Fig. 5a). Among the several samples prepared, activated carbon prepared at 600 °C has a large specific surface area (1200 m2 g-1). Cyclic voltammetry and galvanostatic charge-discharge studies suggest that activated carbons derived from coconut kernel pulp are appropriate materials for EDLC studies in acidic, alkaline and non-aqueous electrolytes. Specific capacitance (SC) of 173 F g-1 is obtained in 1 M H2SO4 electrolyte for the activated carbon prepared at 600 °C. The supercapacitor properties of activated carbon sample prepared at 600 °C are superior to the samples prepared at higher temperatures. Electrochemical studies are also undertaken for the prepared and activated samples for sodium ion intercalation/deintercalation. It is found that various factors such as surface area, mesoporosity, inter-layer spacing, electrolyte diffusion, solid electrolyte interface formation for high surface area carbon, etc. contribute to the capacity and cycle life of the material. Carbon sample synthesized at 600 °C and having a specific surface area of about 280 m2 g-1 provides the highest discharge capacity of about 200 mAh g-1 with good cycling stability. The thesis ends with a short summary and prospects of the investigations described here in. The work presented in it is carried out by the candidate as a part of Int. Ph.D. program. Some of the results are published in the literature and some more manuscripts are in preparation. A list of publications is enclosed. It is hoped that the studies reported in the thesis are worthy contributions.

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Introduction:The human ether-a-go-go related gene (hERG) encodes the pore forming protein which mediates the rapid delayed rectifier K+ current in the heart (IKr). Together with other ion channels hERG determines the cardiac action potential and regulates the heart beating. Dysfuction of the hERG ion channel will lead to acquired long QT syndrome (LQTS). Therefore, new drug candidates must pass the test for a potential inhibitory effect on the hERG current as a first step in a nonclinical testing strategy. Arrhythmias in patients with LQTS are typically triggered during physical or emotional stress, suggesting a link between sympathetic stimulation and arrhythmias. It is well known that potassium level can affect the QT interval through affecting IhERG both in vivo and in vitro.In this study, we try to find out whether the trigger effect still exist when K+ changes violently in a short time period. In other words, whether the risk of TdP aggravate when patients suffer from acute water electrolyte balance disorder, which is a common symptom in hot weather. Methods: HEK293 Cell line stably expressing hERG channel were cultured in DMEM supplemented with 10% of fetal bovine serum.Whole-cell patch-clamp method was applied for ionic current recordings. The compositions of pipette was (in mM) 125 KCl, 5 MgCl2, 5 EGTA-K, 10 HEPES-K and 5 Na-ATP adjusted to pH 7.2 with KOH. The bath solutions for recording the IhERG currents was 136 NaCl, 4 KCl, 1 MgCl2, 10 HEPES-Na, 1.8 CaCl2 and 10 glucose, pH 7.4 with NaOH. The low extracellular K+ solution was 115 KCl, 5 MgCl2, 5 EGTA-K, 10 HEPES-K and 10 Na-ATP adjusted to pH 7.2 with NaOH. Patch-clamp experiments were performed at room temperature (22 ± 1°C). The recording of low K+ current was carried out immediately after the original normal K+ solution has been totally replaced. Isoproterenol (ISO) 100nM was added into both kinds of K+ solution to apply the effect of β1-AR stimulation. Results: We found that low K+ solution increased IhERG from 907.39±18.68to 1620.08±249.44pA(n=30,P<0.05); Low K+also shifted the I-V curve to the left. IC50 in control is 10.31±5.52 mV, low K+ is -6.15±1.58 mV. When adding ISO 100nM to extracellular solution, same effects were shown for both groups.ISO decreased Imax for both group. In control group, Imax reduced from 907.39±18.68to493.16±54.41pA (n=30, P<0.01), while in low K+ group, I max decreased Imax from 1620.08±29.44to 488.48±81.87pA(n=30,P<0.05). At the same time, ISO shifts the I-V curve to the right for the control group and shift the curve to the left for low K+ group. IC50 in control when added ISO is 22.25±3.80 mV, while IC50 in low K+ group after adding 100nM ISO is -31.00±5.73 mV. Conclusion: The results from this study is contradict to those in our previous study where low K+ combined with ISO can lead to temporarily increase of QT interval in vivo.It is reported that an increase in net outward repolarizing current, due to a relatively large increase of IKs, is responsible for the changes of QT interval in response to beta-adrenergic stimulation in vivo(2). Therefore future studies need to co-transfect IKs channel to confirm this. References: 1. Guo J, Massaeli H, Xu J, Jia Z, Wigle JT, Mesaeli N, et al. Extracellular K+ concentration controls cell surface density of IKr in rabbit hearts and of the HERG channel in human cell lines. The Journal of clinical investigation. 2009;119(9):2745- 57. 2. Shimizu W, Antzelevitch C. Differential effects of beta-adrenergic agonists and antagonists in LQT1, LQT2 and LQT3 models of the long QT syndrome. Journal of the American College of Cardiology. 2000;35(3):778-86.

碩士
長庚大學
電子工程學系
100
Inflammasomes are wellknown as the key regulators of the innate immune response triggered by tissue damage or other microbial stimulator, and the activity of these multi-protein complexes has been linked to common autoinflammatory . Inflammasomes activate the proinflammatory cytokines interleukin IL-1β. In addition, potassium efflux has been linked to the activation of inflammasomes. In this research, a novel method was proposed to detect the inflammasome activation from extracellular potassium ion concentration. The sensor platform was designed as the electrolyte insulator semiconductor (EIS) structure with fluorinated HfO2 sensing membrane. The potassium (K+) sensitivity of HfO2-EIS structure is 2.23 mV/pK in the concentration between 10-5 M and 1 M. For the samples with fluorine (F19+) ion implantation, the pK sensitivity can be effectively improved to 75.58mV/pK. The variation of K+ concentration under treatment with the nigericin was 65.92 mV. It takes the advantage of directly monitoring the alteration of K+ as the measurement indicator of inflammasome activation.

Breast cancer is the most prevalent cancer type among Canadian women. Breast cancers originate from the malignant transformation of mammary epithelial cells, which causes them to adopt an uncontrolled cell proliferation phenotype. My research suggests that the activity of specific ion channels (KV10.1, KCa3.1 and TRPM8) contribute to the proliferation of MCF-7 cells, a cell line commonly used to study breast cancer in vitro. Pharmacologically inhibiting the activities of KV10.1 or KCa3.1 channels decreased basal, but not estrogen-stimulated [3H]-thymidine incorporation, demonstrating that these channels contribute to MCF-7 cell proliferation. One way K+ channel activity is hypothesized to control cell proliferation is via regulation of membrane potential-dependent Ca2+ influx. Inhibition of KCa3.1 but not KV10.1 channel activity resulted in a membrane potential-dependent decrease in basal Ca2+ influx, suggesting that the way in which KCa3.1 channels contribute to cell proliferation is via regulating Ca2+ influx. In addition, my research also demonstrated that TRAM-34 increased or decreased cell proliferation depending on the concentration used and mitogenesis by TRAM-34 was blocked by estrogen receptor antagonists. TRAM-34 increased progesterone receptor mRNA expression, decreased estrogen receptor-alpha mRNA expression and reduced the binding of radiolabelled estrogen to estrogen receptor protein, in each case mimicking the effects of estrogen. Our finding that TRAM-34 is able to activate the estrogen receptor suggests a novel action of this supposedly specific K+ channel inhibitor and raises concerns of interpretation in its use. TRPM8 channels were also identified in MCF-7 cells, where they appeared to be important Ca2+ entry pathways. Inhibiting the activity of TRPM8 pharmacologically, as well as knocking down TRPM8 mRNA expression decreased cell proliferation, indicating that TRPM8 also contributed to MCF-7 cell proliferation. In conclusion, my research demonstrates that the activities of KV10.1, KCa3.1 and TRPM8 channels contribute to basal breast cancer cell proliferation. These findings suggest that the activity of specific ion channels may be potential targets for future therapeutic agents to treat breast cancer.

Background potassium currents play an important role in the regulation of the resting membrane potential and excitability of mammalian neurons. Recently cloned two- pore domain potassium channels (K2p) are believed to underlie these currents. The roles of K2P channels in general anesthesia and neuroprotection have been proposed recently. In view of this, we investigated the ability of trichloroethanol (an active metabolite of the non-volatile general anesthetic cldoral hydrate, widely used as a pediatric sedative) to modulate the activity of human TREK-1 and TRAAK channels. We found that trichloroethanol potently activates both hTREK-1 and hTRAAK channels at pharmacologically relevant concentrations. The parent compound chloral hydrate was also found to augtnent the activity of both the channels reversibly. Studies with carboxy- terminal deletion mutants (hTREK-1A89, hTREK-1 A100 and hTREK-1 A1 19), suggested that C-terminal tail is not essential for the activation of TREK-1 by trichloroethanol. Our findings identify TREK-1 and TRCL4K channels as molecular targets for trichloroethanol and we propose that activation of both these channels might contribute to the CNS depressant effects of chloral hydrate. Another channel TASK-2, which is essentially absent in the human brain was also found to be potently activated by both trichloroethanol and chloral hydrate. In another series of experiments, we studied the effects of methyl xanthines caffeine and theophylline on hTREK-1 channels. Caffeine and theophylline are used for therapeutic purposes and frequently cause life-threatening convulsive seizures due to systemic toxicity. The mechanisms for the epileptogenicity of caffeine and theophylline are not clear. Recent experiments using knockout mice provided direct evidence for a role for TREK-1 in the control of epileptogenesis. We hypothesized that the epileptogenicity of caffeine and theophylline may be related to the inhibition of TREK-1 channels. We investigated this possibility and observed massive inhibition of TREK-1 channels at toxicologically relevant concentrations. Experiments with the mutant TREK-1 channel (S348A mutant) suggested the involvement of cANP/PKA pathway in the inhibition of TREK-1 channels by caffeine and theophylline. We suggest that inhibition of TREK-1 channels may contribute to the convulsive seizures induced by toxic levels of caffeine and theophylline. Local anesthetics exhibit their clinical effects not only by binding to voltage-gated sodium channels, but also by interacting with other ion channels such as potassium channels. Because of the physiological significance of TREK-1 channels and their abundant expression in peripheral sensory neurons, we investigated the effects of lidocaine to see whether its interaction with 'REK-1 channels contribute to the conduction blockade. Lidocaine caused dose-dependent inhibition of TREK-1channels and the inhibition was voltage-independent. Cytoplasmic C-terminal tail is critically required for lidocaine action. Inhibition of TREK-1 channels is achieved at concentrations for iiz vivo action and this effect may have implications for the clinically observed drug action of lidocaine.

Background potassium currents play an important role in the regulation of the resting membrane potential and excitability of mammalian neurons. Recently cloned two- pore domain potassium channels (K2p) are believed to underlie these currents. The roles of K2P channels in general anesthesia and neuroprotection have been proposed recently. In view of this, we investigated the ability of trichloroethanol (an active metabolite of the non-volatile general anesthetic cldoral hydrate, widely used as a pediatric sedative) to modulate the activity of human TREK-1 and TRAAK channels. We found that trichloroethanol potently activates both hTREK-1 and hTRAAK channels at pharmacologically relevant concentrations. The parent compound chloral hydrate was also found to augtnent the activity of both the channels reversibly. Studies with carboxy- terminal deletion mutants (hTREK-1A89, hTREK-1 A100 and hTREK-1 A1 19), suggested that C-terminal tail is not essential for the activation of TREK-1 by trichloroethanol. Our findings identify TREK-1 and TRCL4K channels as molecular targets for trichloroethanol and we propose that activation of both these channels might contribute to the CNS depressant effects of chloral hydrate. Another channel TASK-2, which is essentially absent in the human brain was also found to be potently activated by both trichloroethanol and chloral hydrate. In another series of experiments, we studied the effects of methyl xanthines caffeine and theophylline on hTREK-1 channels. Caffeine and theophylline are used for therapeutic purposes and frequently cause life-threatening convulsive seizures due to systemic toxicity. The mechanisms for the epileptogenicity of caffeine and theophylline are not clear. Recent experiments using knockout mice provided direct evidence for a role for TREK-1 in the control of epileptogenesis. We hypothesized that the epileptogenicity of caffeine and theophylline may be related to the inhibition of TREK-1 channels. We investigated this possibility and observed massive inhibition of TREK-1 channels at toxicologically relevant concentrations. Experiments with the mutant TREK-1 channel (S348A mutant) suggested the involvement of cANP/PKA pathway in the inhibition of TREK-1 channels by caffeine and theophylline. We suggest that inhibition of TREK-1 channels may contribute to the convulsive seizures induced by toxic levels of caffeine and theophylline. Local anesthetics exhibit their clinical effects not only by binding to voltage-gated sodium channels, but also by interacting with other ion channels such as potassium channels. Because of the physiological significance of TREK-1 channels and their abundant expression in peripheral sensory neurons, we investigated the effects of lidocaine to see whether its interaction with 'REK-1 channels contribute to the conduction blockade. Lidocaine caused dose-dependent inhibition of TREK-1channels and the inhibition was voltage-independent. Cytoplasmic C-terminal tail is critically required for lidocaine action. Inhibition of TREK-1 channels is achieved at concentrations for iiz vivo action and this effect may have implications for the clinically observed drug action of lidocaine.