Class III antiarrhythmic drugs share the common mechanism of widening the cardiac action potential without affecting conduction velocity. This thesis reports on the actions of newly developed putative Class III antiarrhythmic drugs, tedisamil, KC 8851, RP 62719, UK 68798, and risotilide, as well as an ATP-sensitive K⁺ channel blocker, glibenclamide. Studies were performed to examine the actions of these drugs in acute myocardial ischaemia and possible mechanisms responsible for these actions. The hypothesis tested was that drug treatment prevented arrhythmias induced by acute myocardial ischaemia. Species dependent actions of these drugs on ECG and blood pressure were examined in rats, guinea pigs, pigs and primates. The five putative class III drugs listed above were assessed for antiarrhythmic activity in a conscious rat model of myocardial ischaemia. It was found that only tedisamil and KC 8851, which widened the Q-T[formula omitted] interval of the ECG (by up to 65%) , were effective at suppressing fibrillation in this species. None of the drug treatments decreased the incidence of ventricular premature beats. Tedisamil, but not glibenclamide, prevented tachycardias in a rat model of myocardial ischaemia- and reperfusion-induced arrhythmias. In an anaesthetized pig model of acute myocardial ischaemia, tedisamil and UK 68,798 were shown to mildly prolong the Q-T[formula omitted] interval by less than 20%, but protection against arrhythmias was equivocal. In further studies, tedisamil and UK 68,798 were compared to each other for effects on ventricular epicardial action potential morphology using intracellular recording in vivo, and effects on ventricular effective refractory period using electrical stimulation in vivo in both rats and guinea pigs. Tedisamil (4 mg/kg, i.v.) prolonged rat ventricular epicardial action potential duration fourfold in vivo, while UK68,798 (up to 1 mg/kg, i.v.) was ineffective in this species. Tedisamil (4 mg/kg, i.v.) widened guinea pig ventricular epicardial potentials by 80%, while UK 68,798 (25 μg/kg, i.v.) increased these by 30%. Action potential widening paralleled increases in ventricular refractoriness to electrical induction of premature beats. It was found that the species selective actions of these drugs was most likely related to differences in selectivity for K⁺ channels which contribute to repolarization in myocardium. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

Small conductance Ca2+-activated K+ (SK) channels are expressed throughout brain regions important for long-term memory. They constrain the intrinsic excitability of neurons by enhancing afterhyperpolarization, shape glutamatergic synaptic potentials and limit induction of NMDA receptor-dependent synaptic plasticity. Behaviorally, SK channels modulate learning and memory encoding. It is hypothesized that SK channels influence cognitive symptoms of psychosis including executive functioning, working memory, and selective attention. Theories of psychosis currently posit that symptoms of psychosis are a result of dopaminergic hyperfunction, and glutamatergic dysregulation which can be induced following administration of the NMDA receptor antagonist, ketamine. Initial experiments confirmed that sub-chronic treatment with KET produced significant impairment of object recognition memory, trace fear memory, and latent inhibition compared to SAL mice. A comparison of ketamine dosing regimens revealed the necessity for sub-chronic/chronic dosing on a consistent schedule with a wash out period, to obtain long-lasting attention and memory impairment. These experiments revealed for the first time that sub-chronic KET treatment elicited a new phenotype in male C57BL/6J mice: audible vocalizations. KET mice emitted audible vocalizations within 10 min of receiving KET injections, and vocalizations were detected up to 30 min after injection. Experiments conducted to determine the efficacy of SK channel agonists and antagonists on SK channels to modulate attention and memory in the ketamineinduced model of psychosis in C57BL/6J mice demonstrated for the first time that the SK2 channel activator, CyPPA, significantly reduced memory impairment and decreased the attention deficit of KET mice. A new method of analysis for trace fear conditioning freezing responses permitted a more accurate measurement of the ability of mice to discriminate the predicted delivery of shock during trace versus CS intervals. The application of the novel analytical method further demonstrated that KET mice failed to accurately discriminate these intervals, due to their impaired attention and acquisition of the trace conditioned response. This study examined the efficacy of SK channel drugs to rescue cognitive impairments in a pharmacological mouse model of schizophrenia. The results indicate that SK2 subunit activators and blockers, may provide a new therapeutic treatment for memory impairment and attention deficits seen in schizophrenic disorders. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2020. / FAU Electronic Theses and Dissertations Collection

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.

Reproductive science plays an important role in conservation biology. Quantitative studies of basic reproductive biology in wildlife are critical for the development of successful assisted reproductive technologies. Investigation of the volume regulatory mechanism of spermatozoa could produce options to improve the cryopreservation of spermatozoa and provide a non-hormonal contraceptive option for men, both of which could have significant impacts on global biodiversity preservation. Volume regulation of somatic cells involves the movement of osmolytes through various channels, including potassium channels. The potassium channels involved in volume regulation of human, monkey, and murine spermatozoa were investigated. Flow cytometry was used to gauge the sensitivity of the volume regulatory process of spermatozoa to various potassium channel inhibitors and a simultaneous hypotonic challenge. Channels potentially involved in regulatory volume decrease of spermatozoa varied with species but included voltage-gated (Kv) channels 1.4, 1.5, 1.7, 4.1, 4.2 and 4.3 as well as TWIK1, TWIK2, TASK1, TASK2, TASK3, TREK2 , and minK. The presence of some of these channels was confirmed by western blotting and immunocytochemistry. Changes in the motility patterns of human and monkey spermatozoa in the presence of potassium channel inhibitors during hypotonic stress were also observed, suggesting a relationship between volume regulation and motility. To evaluate potential organic osmolytes involved in, and compare effects of CPAs on, volume regulation, the isotonicity of murine epididymal spermatozoa was measured using a null point method. Spermatozoa were then exposed to high concentrations of various osmolytes and cryoprotective agents in isotonic medium to evaluate which compounds were able to penetrate the sperm plasma membrane. The osmotic responses of spermatozoa from strains of mice known to have spermatozoa of high (B6D2F1) and low (C57BL6) post-thaw fertility were compared during various osmotic challenges in various media. These experiments indicated that spermatozoa from B6D2F1 mice may have better volume regulation capabilities than spermatozoa from C57BL6 mice, suggesting that better post-thaw fertility of murine spermatozoa could be influenced by the volume regulatory process. The knowledge gained from these experiments could contribute to improved sperm handling and preservation techniques and be used to develop non-hormonal male contraceptives based on inhibiting volume regulation.

A debilitating illness, pulmonary arterial hypertension (PAH) arises from deleterious remodeling of pulmonary arterioles, leading to increased pulmonary artery pressure, a rise in pulmonary vascular resistance, right sided heart failure and death. The pathogenesis of the disease is incompletely understood; however, certain established pathological features have guided medical treatments to improve mortality rates. For instance, an imbalance of vasoconstrictor molecules, such as endothelin-1, to vasodilator compounds, such as nitric oxide, contributes to excessive pulmonary arterial constriction, and a propensity for pulmonary arterial smooth muscle and endothelial cell proliferation. Therapeutic strategies may aim to restore this imbalance with the use of endothelin receptor antagonists, prostacyclin analogs, and other vasodilating agents. Mutations in the BMPR2 gene, the most common genetic cause of PAH, leads to aberrant TGF-ß signaling, which promotes uncontrollable cell proliferation and pathological changes in pulmonary arterioles. Genetic studies have revealed PAH-associated mutations in several other genes within the TGF-ß signaling pathway. More recently, our research group discovered loss-of-function mutations in the KCNK3 gene encoding the KCNK3 two-pore domain potassium channel in patients with idiopathic and familial PAH. KCNK3 (also referred to as TASK-1, or K2P3.1) represents the first ion channelopathy as a cause of PAH. KCNK3 is expressed in human pulmonary artery smooth muscle and endothelial cells. Loss of KCNK3 channel currents leads to membrane depolarization and predisposes to deleterious pulmonary arterial remodeling. Chapter 1 of my thesis explores the impact of KCNK3 mutations on potassium channel function in cellular models of heterozygous conditions, as all patients with PAH-associated KCNK3 mutations in our study were heterozygous at the KCNK3 gene locus. Furthermore, we explored function of mutant and non-mutant KCNK3 channels in cultured human pulmonary artery smooth muscle cells to better define the electrophysiological consequence of KCNK3 dysfunction, and used a KCNK3-activating pharmacological agent, ONO-RS-082, to gauge the therapeutic potential of KCNK3 as a pharmacological target in PAH. Moreover, the study of KCNK3 channel activity when assembled with the closely related KCNK9 channel provided a platform for exploring the lung-specific phenotype in patients with heterozygous KCNK3 mutations, despite widespread tissue expression KCNK3 in the body. In Chapter 2 of my thesis work, the discovery of a second potassium channelopathy in PAH is characterized. Heterozygous mutations in the ABCC8 gene, encoding the sulfonylurea receptor 1 (SUR1) protein, were found in pediatric and adult patients with idiopathic and familial PAH. SUR1, a beta subunit of the ATP-sensitive potassium channel (KATP), assembles with the pore-forming Kir6.2 alpha subunit to form KATP, a channel sensitive to inhibition by intracellular ATP. At the plasma membrane, KATP inwardly rectifying potassium currents contribute to the resting potential, and may play a pathophysiological role in PAH via dysfunction in pulmonary artery smooth muscle and/or endothelial cells. In this chapter, eight ABCC8 mutations associated with PAH were functionally characterized, and pharmacological agents were employed to examine the therapeutic potential in targeting SUR1-containing KATP channels in PAH. Altogether, the research presented in this dissertation identifies and explores potassium channel dysfunction as a pathogenic mechanism in PAH, due to heterozygous genetic mutations in KCNK3 and ABCC8. Evidence of restoration of mutant KCNK3 and KATP channel function by pharmacological agents suggests that targeting potassium channels as a therapeutic strategy may alleviate the severe morbidity and mortality burden in patients with PAH.

Abstract not available

Sponges are filter feeders that lack nerves and muscle but are nonetheless able to respond to changes in the ambient environment to control their feeding current. Cellular sponges undergo coordinated contractions that effectively expel debris. Syncytial sponges propagate action potentials through their tissue, causing immediate flagellar arrest. Understanding the basis of this coordination in sponges is of interest for the insight it provides on mechanisms of coordination in early branching animals. However, when I began this thesis no ion channels had been described from the Porifera. I adopted a multifaceted approach to studying the conduction system of sponges. This included cloning and characterizing potassium channels as a means to understanding the underlying ionic currents, and monitoring regulation of the sponge feeding current in response to environmental stimuli. The latter experiments provided a functional context. The glass sponges Rhabdocalyptus dawsoni and Aphrocallistes vastus arrest feeding in response to mechanical disturbance and to sediment in the incurrent water suggesting a protective role. Monitoring patterns of feeding current arrests also revealed several features of the glass sponge conduction system: pacemaker activity, mechanosensitivity, distinct excitability thresholds, and tolerance to repeated stimuli. With access to the genome of the demosponge Amphimedon queenslandica I have also cloned and characterized the first sponge ion channels. Inward rectifier potassium (Kir) channels were prioritized for their role in regulating excitability. Kir channels cloned from A. queenslandica shared critical residues and a strong rectifying phenotype with Kir channels typically expressed in excitable cells. A variety of potassium channels from A. queenslandica indicate great diversity and a foundation for coordination at the dawn of the Metazoa / Physiology, Cell and Developmental Biology

Thesis (Ph. D.)--University of Nevada, Reno, 2002. / Includes bibliographical references. Online version available on the World Wide Web.

My PhD project investigated the regulation of human cardiac transient outward potassium current (Ito) by protein tyrosine kinases (PTKs) and the functional expression of transient receptor potential (TRP) channels in human atrial myocytes to make an advanced understanding of human cardiac electrophysiology and pathophysiology. The modulation of human cardiac Itoby PTKs was studied in human atrial myocytes and HEK 293 cells expressing hKv4.3 (coding human cardiac Ito). We found that the broad-spectrum PTK inhibitor genistein, the selective EGFR kinase inhibitor AG556, and the Src-family kinases inhibitor PP2 inhibited human atrial Itoand the inhibitory effect was countered by the protein tyrosine phosphatase (PTP) inhibitor orthovanadate. Similar results were observed in hKv4.3-HEK cells. Interestingly, tyrosine phosphorylation of hKv4.3channels was reduced by genistein, AG556, and PP2,and the reduction was antagonized by orthovanadate. The mutant Y136F of hKv4.3 lost the inhibitory response to AG556, whileY108F lost the response to PP2.The double mutant Y108F-Y136F hKv4.3 failed to respond to both AG556 and PP2, and exhibited a dramatic reduction of tyrosine phosphorylation. These results indicate that native cardiac Itois regulated by both EGFR and Src family kinases. In the second part, we studied whether TRPC channels would mediate the nonselective cation current described previously in human atrial myocytes. It was found that TRPC1 channel activator thapsigargin activated the current, and the effect was suppressed by La3+or prevented by intracellular anti-TRPC1 antibody. Endothelin-1 and angiotensin II stimulated the current, andthe effect was inhibited by La3+and/or 2-APB. RT-PCR and Western blot analysis revealed that in addition to the TRPC1 channels mediating the nonselective cation current, the components of store-operated Ca2+channels (SOCs), STIM1 and Orai1 were abundantly expressed in human atria. The interaction of TRPC1, STIM1, and Orai1 was confirmed by co-immunoprecipitation. Interestingly, we found that protein expression of TRPC1 and STIM1, but not Orai1, was up-regulated in human atria with atrial fibrillation. The third part of the project determined whether TRPM7 channels were expressed in human atrial myocytes, since this channel was reported in human atrial fibroblasts, conferring atrial fibrosis in human atria with atrial fibrillation. We found a TRPM7 -like current which was potentiated by acidic pH, and inhibited by La3+and 2-APB, and a Ca2+-activated TRPM4 current. RT-PCR and Western blot analysis confirmed the expression of TRPM7 and TRPM4 channels in human atria. Moreover, we found TRPM7 protein, but not TRPM4 protein was significantly up-regulated in human atria with atrial fibrillation, suggesting the potential participation of TRPM7 channels in atrial remodeling of human atria with atrial fibrillation. Collectively, this PhD thesis project has demonstrated for the first time that human cardiac Itois modulated by EGFR kinase and Src kinases via phosphorylating Y136and Y108, respectively. TRPC1 channels mediate the nonselective cation current and SOCs.TRPM7 channels are expressed in human atrial myocytes. The up-regulation of TRPC1, STIM1, and TRPM7 channels in human atria with atrial fibrillation suggest that they are likely involved in atrial electrical and/or structure remodeling in patients with atrial fibrillation. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

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