Oxidative stress causes neural damage and inhibits essential cellular processes, such as synaptic transmission. Despite this knowledge, currently available pharmaceutical agents cannot effectively protect neural cells from acute oxidative stress elicited by strokes, heart attacks, and traumatic brain injuries in a real life clinical setting. Our lab has developed an electrophysiology protocol to identify novel drugs that protect an essential cellular process (neurotransmission) from acute oxidative stress-induced damage. Through this doctoral dissertation, we have identified three new drugs, including a Big K+ (BK) K+ channel blocker (iberiotoxin), resveratrol, and a custom made resveratrol-like compound (fly2) that protect synaptic function from oxidative stress-induced insults. Further developing these drugs as neuroprotective agents may prove transformative in protecting the human brain from acute oxidative stress elicited by strokes, heart attacks, and traumatic brain injuries. Inhibiting the protein kinase G (PKG) pathway protects neurotransmission from acute oxidative stress. This dissertation has expanded upon these findings by determining that the PKG pathway and BK K+ channels function through independent biochemical pathways to protect neurotransmission from acute oxidative stress. Taken together, this dissertation has identified two classes of compounds that protect neurotransmission from acute oxidative stress, including resveratrol-like compounds (resveratrol, fly2) and a BK K+ channel inhibitor (iberiotoxin). Further developing these drugs in clinical trials may finally lead to the development of an effective neuroprotective agent. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection

Small conductance Ca2+-activated K+ (SK) channels have been shown to alter the encoding of spatial and non-spatial memory in the hippocampus by shaping glutamatergic postsynaptic potentials and modulating NMDA receptor-dependent synaptic plasticity. When activated, dendritic SK channels reduce hippocampal neuronal excitability and LTP. Similar SK channel properties have been demonstrated in lateral amygdala (LA) pyramidal neurons. Additionally, induction of synaptic plasticity and beta-adrenoreceptor activation in LA pyramidal neurons causes PKA-mediated internalization of SK channels from the postsynaptic density. Chronic activation of the amygdala through repetitive stressful stimuli can lead to excitatory synaptic strengthening that may create permanent hyper-excitability in its circuitry. This mechanism may contribute to a number of mood and anxiety disorders. The selective influence of SK channels in the LA on anxiety and fear conditioning are not known. The thesis project outlined herein examined whether SK channel blockade by bee venom peptide, apamin, during a repetitive acute fear conditioning paradigm was sufficient to alter fear memory encoding and the resulting behavioral outcome. Following the final fear memory test session, mice were tested in the open field immediately after the second fear conditioning test session. The findings indicate that intracranial LA microinfusions of apamin did not affect memory encoding or subsequent anxiety. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2015. / FAU Electronic Theses and Dissertations Collection

The pulmonary vein (PV) constricts to hypoxia however little is known about the underlying mechanisms. Hypoxic PV constriction is proposed to recruit upstream capillary beds and optimise gas exchange in healthy humans and may play a role in high altitude pulmonary oedema. The PV is also intrinsic to disease states including pulmonary hypertension and pulmonary veno-occlusive disease. Blood vessel culture can be a powerful tool to enable assessment of the impact of environmental factors on vessel function and as a disease model. However culture conditions alone affect vessel contractility; the effect of culture conditions on PV function remained to be established. The aim of this project was to investigate hypoxic responses of porcine PVs including the impact of maintenance in culture. Maintenance of PVs in culture conditions for 24 hours increased contraction to hypoxia and inhibited hypoxic relaxation post-contraction. These changes to PV hypoxic responses were thought to result from endothelial dysfunction. However, the endothelial nitric oxide synthase inhibitor L-NAME inhibited PV hypoxic contraction and enhanced relaxation. The impact of K+ channel inhibitors on hypoxic contraction was also investigated. Penitrem A, 4AP, DPO-1, ZnCl2 and glyburide had no significant effect however TEA and BDM inhibited the hypoxic contraction. This suggested that TASK, KV1.5, BKCa and KATP do not play a role in the mechanism of hypoxic pulmonary venoconstriction however KV channels containing KV2.1 α subunits may modulate the response. Results with L-NAME suggested endothelial dysfunction may not fully account for the change in PV function after exposure to culture. Therefore the impact of PV maintenance in culture was further explored using an isolated PV smooth muscle cell (PVSMC) model. Maintenance of PVs in culture conditions had minimal impact on morphology and electrical properties of PVSMCs. Notably, resting membrane potential and hypoxia-induced depolarisation were not significantly different. Based on the findings of this study, the endothelium in PVs appears to a) play a major role in modulation of the hypoxic response b) be sensitive to short-term exposure to culture conditions. K+ channels appear to play a minor role in PV hypoxic contraction and SMCs isolated from PVs maintained in culture conditions have similar morphological and electrophysiological characteristics to freshly isolated PVSMCs. Taking all this into account, endothelial regulation of contractility should be a key focus for future PV research.

Lau Chui Pik. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 104-122). / Abstracts in English and Chinese. / Chapter Chapter 1 --- --- Introduction --- p.1 / Chapter 1.1 --- Importance of sinoatrial node in heart functions --- p.3 / Chapter 1.2 --- The importance of Adenosine 5'-triphosphate (ATP) --- p.5 / "ATP as a neurotransmitter, cotransmitter and neuromodulator" --- p.5 / Role of ATP in the heart --- p.7 / Chapter 1.3 --- Importance of delayed rectifier potassium channels (Ik) in the heart --- p.9 / Delayed rectifier potassium channel --- p.10 / Properties of Ik channels in the sinoatrial nodal (SAN)cells --- p.11 / Importance of Ik on heart function --- p.14 / Chapter 1.4 --- Drug/hormone/neurotransmitter modulation of Ik --- p.15 / Drugs modulations of Ik --- p.15 / Hormones/neurotransmitters modulations of Ik --- p.18 / Chapter 1.5 --- Problems encountered in using extracellular ATP on experiments --- p.23 / Chapter 1.6 --- Classification of P2-purinergic receptors --- p.24 / Major nucleotide receptors --- p.24 / p2X receptors --- p.26 / p2Y receptors --- p.28 / 1.7Objectives of the experiment --- p.30 / Chapter Chapter 2 --- --- Materials & Methods --- p.31 / Chapter 2.1 --- Materials --- p.32 / Chapter 2.1.1 --- Solutions --- p.32 / Chapter 2.1.2 --- Enzymes --- p.34 / Chapter 2.1.3 --- Drugs --- p.34 / Chapter 2.2 --- Methods --- p.35 / Chapter 2.2.1 --- Isolation of guinea pig SAN cells --- p.35 / Chapter 2.2.2 --- Identification of SAN region --- p.36 / Chapter 2.2.3 --- Obtaining of single SAN cells --- p.38 / Chapter 2.2.4 --- Preparation of micro-pipettes --- p.40 / Chapter 2.2.5 --- The Patch Clamp Technique --- p.40 / Recording configurations --- p.41 / Electrical recordings --- p.44 / Formation of gigaseal on cell membrane and the development of whole-cell configuration --- p.45 / The changing of bathing solution and addition of drugs --- p.46 / The voltage clamp protocol --- p.47 / Data acquisition and analysis --- p.48 / Statistics --- p.48 / Chapter Chapter 3 --- --- Results --- p.49 / Chapter 3.1 --- The modulatory effect of different concentrations of [ATP]0 on IKs in guinea pig SAN cells --- p.50 / Chapter 3.1.1 --- Characterization of IKs currents --- p.50 / Chapter 3.1.2 --- Stimulatory effect of extracellular A TP on IKs current --- p.51 / Chapter 3.1.3 --- Current-Voltage relationship of ATP on IKs current --- p.57 / Chapter 3.1.4 --- Percentage increase of IKs current in the presence of different [ATP] o --- p.63 / Chapter 3.2 --- Investigation on whether the enhancement effect on IKs is due to ATP or its metabolite adenosine --- p.71 / Chapter 3.2.1 --- Effect of 100 μMATP-γS and adenosine on IKs --- p.71 / Chapter 3.2.2 --- Percentage increase of IKs in the presence of adenosine and ATP-γS --- p.76 / Chapter 3.3 --- Investigation on whether or not G-protein signalling pathway involved in ATP-mediated response on SAN IKs --- p.80 / Chapter 3.3.1 --- Effects of GTP-γS alone on IKs --- p.80 / Chapter 3.3.2 --- Effect of 100 μM ATP in the presence of GTP-yS on IKs --- p.83 / Chapter Chapter 4 --- --- Discussion --- p.86 / Chapter 4.1 --- The modulatory effect of different concentrations of [ATP]0 on IKs in guinea pig SAN cells --- p.87 / Chapter 4.2 --- Investigation on whether the enhancement effect on IKs is due to ATP or its metabolite adenosine --- p.92 / Chapter 4.3 --- Investigation on whether or not G-protein signalling pathway involved in ATP-mediated response on SAN IKs --- p.97 / Chapter 4.4 --- Limitations of this study --- p.102 / Chapter 4.5 --- Future studies --- p.102 / Chapter Chapter 5 --- --- References --- p.104

Ion channels are membrane proteins that facilitate electrical signaling in important physiological processes, such as the rhythmic contraction of the heart. KCNQ1 is the pore-forming subunit of a voltage-gated potassium channel that assembles with the β-subunit KCNE1 in the heart to generate the IKs current, which is critical to cardiac action potential repolarization and electrical conduction in the heart. Mutations in IKs subunits can cause potentially lethal arrhythmia, including long QT syndrome, short QT syndrome, and atrial fibrillation. Each channel consists of four voltage-sensing domains and a central pore through which ions permeate. Voltage-dependent gating occurs when movement of voltage sensors cause pore opening/closing through coupling mechanisms. Although KCNQ1 by itself is able to form a voltage-dependent potassium channel, its assembly with KCNE1 is essential to generating the physiologically critical cardiac IKs current, characterized by a delay in the onset of activation, an increase in current amplitude, and a depolarizing shift in the current-voltage relationship. KCNE1 is thought to have multiple points of contact with KCNQ1 that reside within both the voltage-sensing domain and the pore domain, allowing for extensive modulation of channel function. Atrial fibrillation is the most common cardiac arrhythmia and affects more than 3 million adults in the United States. Much rarer, genetic forms of atrial fibrillation have been associated with gain-of-function mutations in KCNQ1, such as two adjacent mutations, S140G and V141M. Both mutations drastically slow channel deactivation, which underlies their pathophysiology. Deactivation slowing causes accumulation of open channels in the context of repeated stimulation, which abnormally increases the repolarizing K+ current, excessively shortens the action potential duration, and predisposes to re-entry arrhythmia such as atrial fibrillation. Although both mutations are located in the voltage-sensing domain, their mechanisms of action remain unknown. Understanding the gating mechanisms underlying deactivation slowing may provide key insights for the development of mechanism-based pharmacologic therapies for arrhythmias associated with KCNQ1 mutations. In addition to gain-of-function mutations, molecular activators of KCNQ1 can slow deactivation and increase channel activity. An existing problem in the pharmacologic treatment of arrhythmia is that many antiarrhythmic drugs do not have specific targets and cause undesired side effects such as additional arrhythmia. Thus, developing mechanism-based therapies may optimize clinical treatment for patients with specific forms of channel dysfunction. Two KCNQ1 activators, ML277 and R-L3, have been previously shown to slow current deactivation, but the underlying gating mechanisms remain known. Although these modulators are unlikely to serve directly as antiarrhythmic therapy, investigating their mechanisms will likely provide fundamental insights on channel modulation and guide future efforts to develop personalized therapies for arrhythmia, such as congenital long QT syndrome. Given the central importance of deactivation slowing in both pathophysiology and pharmacology, we focused on investigating gating mechanisms that underlie deactivation slowing. To this end, we utilized voltage clamp fluorometry, a technique that simultaneously assays for voltage sensor movement and ionic current through the channel pore. In Chapter 1, we begin our study by examining the gating mechanisms of KCNQ1 atrial fibrillation mutations in the absence of KCNE1. We show that S140G slows voltage sensor deactivation, which indirectly slows current deactivation. On the other hand, V141M neither slows voltage sensor nor current deactivation. This is followed by Chapter 2, where we examine the gating mechanisms underlying deactivation slowing by atrial fibrillation mutations in the presence of KCNE1. We show that both S140G and V141M slow IKs deactivation by slowing pore closing and altering voltage sensor-pore coupling. Based on these findings, we proposed a molecular mechanism in which both mutations disrupt the orientation of KCNE1 relative to KCNQ1 and thus impede pore closing, implying that future efforts to modulate KCNQ1 function can benefit from targeting the β-subunit. Finally, in Chapter 3, we explore the gating mechanisms underlying deactivation slowing for two small-molecule activators of KCNQ1. We show that ML277 predominantly slows pore transitions, whereas R-L3 slows voltage sensor deactivation, which indirectly slows current deactivation. Taken together, these studies guide future efforts to develop mechanism-based therapies for arrhythmia.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / INTRODUÃÃO: O papel do LPS na defesa da mucosa gÃstrica ainda nÃo està estabelecido. OBJETIVOS: 1-Verificar o efeito protetor do LPS na lesÃo gÃstrica (LG), na infiltraÃÃo de neutrÃfilos (IN), no aumento da adesÃo leucocitÃria, na diminuiÃÃo dos nÃveis de GSH induzidos por indometacina (INDO) em ratos; 2-Investigar o papel da COX-2, NOSi e dos canais de K sensÃveis ao ATP (KATP) na gastroproteÃÃo do LPS na gastropatia por INDO. MÃTODOS: Os ratos foram tratados com LPS da E. coli (30, 100 ou 300 g/Kg, e.v.). ApÃs 6 hs, foi administrado INDO (20mg/Kg, p.o.). Decorridas 3 hs, o sangue foi colhido para determinaÃÃo do leucograma. Posteriormente, os ratos foram sacrificados e a LG foi aferida. Fragmentos do estÃmago foram retirados para avaliaÃÃo da atividade de mieloperoxidase (MPO) e determinaÃÃo dos nÃveis de glutationa (GSH). A adesÃo e o rolling dos leucÃcitos foram avaliados por microscopia intravital. Diferentes grupos foram tratados com rofecoxib, L-NAME, aminoguanidina, dexametasona, glibenclamida, diazÃxido ou glibenclamida + diazÃxido. ApÃs 3 horas da administraÃÃo de INDO (20mg/Kg, p.o.), foram avaliadas a LG, a MPO e GSH. RESULTADOS: LPS reduziu a LG e o aumentou a MPO induzidas por INDO de forma dose-dependente, com o efeito mÃximo na dose de 300 g/Kg e no tempo de 6 hs. O prÃ-tratamento com LPS induziu uma neutrofilia na gastropatia induzida pela INDO. LPS reverteu à queda dos nÃveis de GSH no estÃmago com INDO. O tratamento com LPS diminui a adesÃo e aumentou o rolling dos leucÃcitos quando comparado com o tratado com INDO. Rofecoxib, L-NAME, aminoguanidina ou dexametasona nÃo reverteram o efeito protetor do LPS. Glibenclamida, mas nÃo diazÃxido, reverteu o efeito protetor do LPS na gastropatia induzida por INDO, aumentando de forma significativa a LG, MPO e diminuindo a GSH. A associaÃÃo de glibenclamida com diazÃxido nÃo reverteu o efeito protetor do LPS. CONCLUSÃES: LPS protege contra a LG por INDO, atravÃs da inibiÃÃo da IN por uma diminuiÃÃo da adesÃo de leucÃcitos ao endotÃlio e por um aumento dos nÃveis de GSH no estÃmago. Este evento dependente da abertura de KATP. Nossos dados tambÃm sugerem que a atividade de COX-2 e NOSi nÃo estÃo envolvidos no efeito protetor do LPS. / INTRODUCTION: The role of the LPS in the defense of the gastric mucosa is still not established. AIMS: To verify the protective effect of the LPS in the gastric damage (GD), in the neutrophil infiltration (NI), in the increase of the leukocyte of adhesion, in the reduction of the induced glutathione levels for indomethacin (INDO) in rats and to investigate the role of the COX-2, NOSi and of ATP-sensitive k channels (KATP) in the protective effect of LPS administration on INDO- induced gastropathy. METHODS: The rats were treated with LPS of E. coli (30, 100 or 300 mg/Kg, e.v.). After 6 hs, INDO was administrated (20mg/Kg, p.o.). 3 hs later, the blood was harvested for determination the total and differential number of white blood cell counts. Later, the rats had been sacrificed and the GD was surveyed. Piece of the stomach had been removed for evaluation of the MPOactivity and determination of the GSH levels. The adhesion and rolling of the leukocytes had been evaluated by intravital microscopy. Different groups were treated with rofecoxib, L-NAME, aminoguanidine, dexamethasone, glibenclamide, diazoxide or glibenclamide + diazoxide. After 3 hs of the administration of INDO (20mg/Kg, p.o.), had been evaluated the GD, MPO and GSH. RESULTS: LPS reduced dose- dependently INDO- induced GD and increase in MPO, with the maximal effect at the dose of 300 g/kg and in the time of 6 hs. The LPS treatment neutrophilia induced in INDO induced gastropathy. LPS reverted to the fall of the GSH levels in the stomach with INDO. The LPS treatment decreased the adhesion and increased rolling of the leukocytes when compared with the INDO treated. Rofecoxib, L-NAME, aminoguanidine or dexamethasona had not reverted the protective effect of the LPS. Glibenclamide, but not diazoxide, reverted the protective effect of the LPS in the induced gastropathy for INDO, increasing of significant form the GD, MPO and decreasing the GSH. The diazoxide + glibenclamide association of with did not revert the protective effect of the LPS. CONCLUSIONS: LPS protects against INDO induced GD, through the inhibition of the NI for a reduction of the adhesion of leukocytes to the endothelin and for an increase of the GSH levels in the stomach. This dependent event of the KATP opening. Our data also suggest that the activity of COX-2 and NOSi are not involved in the protective effect of the LPS.

Schizophrenia is a devastating psychiatric disorder that affects approximately 1% of the population. The main treatments for schizophrenia are antipsychotic drugs that target dopamine receptors, yet the underlying biological mechanisms through which they alleviate the symptoms of schizophrenia remain ill defined. In this study, we used microarray analysis to profile the expression changes of thousands of genes simultaneously, following antipsychotic drug treatment of mice. Mice were treated chronically (28 days), or for a novel intermediate time-point (7 days), with one of three antipsychotic drugs: clozapine, haloperidol or olanzapine. The use of three drugs enabled us to discern antipsychotic-specific effects co-regulated by multiple drugs, rather than the side effects of individual compounds. Transcript profiling and validation by quantitative PCR of whole brain tissue revealed antipsychotic drug regulation of genes in diverse biological pathways, including: dopamine metabolism, neuropeptide and second-messenger signalling, neurogenesis, synaptic plasticity, cell adhesion, myelination, and voltage-gated ion channels. The regulation of voltage-gated channels by antipsychotic drugs has been suggested previously by electrophysiological studies, although thorough analysis has not been undertaken in vivo. Therefore, the second aim of this study was to characterise the regional mRNA and protein expression of two genes altered by multiple APDs, the voltage-gated potassium channel ??-subunit (Kcna1) and voltage-gated potassium channel interacting protein (Kchip3). Regional characterisation and expression analyses were carried out by immunohistochemistry, in situ hybridisation, and Western blot analysis of mouse brain regions of interest to schizophrenia and its treatment. Following 7-day haloperidol treatment we observed up-regulation of Kcna1 in the striatum and dentate gyrus, with increased protein in the striatum, hippocampus and midbrain; and down-regulation of Kchip3 in the striatum, with decreased protein in the cortex, hippocampus and midbrain. These studies implicate voltage-gated potassium channels in the antipsychotic drug regulation of midbrain dopaminergic neuronal activity, adult neurogenesis and/or striatothalamic GABAergic neuronal inhibition. These findings indicate that regulation of potassium channels may underlie some of the mechanisms of action of antipsychotic drugs, and that voltage-gated ion channels may provide alternative drug targets for the treatment of schizophrenia.

Voltage-gated ion channels are fundamental for electrical signaling in living cells. They are composed of four subunits, each holding six transmembrane helices, S1-S6. Each subunit contains a voltage-sensor domain, S1-S4, and a pore domain, S5-S6. S4 contains several positively charged amino-acid residues and moves in response to changes in membrane voltage. This movement controls the opening and closing of the channel. The structure of the pore domain is solved and demonstrates principles of channel selectivity. The molecular mechanism of how the voltage sensor regulates the opening of the channel is still under discussion. Several models have been discussed. One of the models is the paddle model where S3b and S4 move together. The second one is the helical-twist where S4 makes a small rotation in order for the channel to open. The third one is the helical-screw model where S4 twists around its axis and moves diagonally towards the extracellular side of the channel. The aim of this PhD project was to study the molecular movement of the voltage sensor in the depolarization-activated Shaker K channel. Cloned channels were expressed in Xenopus laevis oocytes, and investigated with several electrophysiological techniques. 1. We show that S4 moves in relation to both S3b and S5. The formation of some disulfide bonds between S4 and neighboring positions, in only the open state, shows that the paddle model cannot be correct. Furthermore, electrostatic and steric effects of residues in S3b suggest that S3b is tilted, with the intracellular part close to S4. 2. We show that the relatively Mg-sensitive Shaker K channel is changed into the less Mg-sensitive Kv2.1 K channel with respect to its sensitivity to extracellularly applied Mg2+ by changing the charge of three extracellularly positioned amino acid residues. One of the residues, F425C, mediates its effect through the neighboring residue K427. 3. We show that oxaliplatin, an anti-cancer drug, has no effect on the Shaker K channel. It has been suggested that a negatively charged monochloro complex of oxaliplatin is the active substance, and also causes the neurotoxic side effects. Neither this complex shows any effect on the channel. Our experiments point towards the helical-screw model. The other models for voltage-sensor movements are incompatible with the results in this study.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 108-119).

Thesis (Ph. D.)--University of Nevada, Reno, 1999. / Includes bibliographical references. Online version available on the World Wide Web.