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1
Mason, Amy. "Single-Channel Characterisation of Potassium Channels with High Temperature Studies." Thesis, University of Oxford, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.491373.
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Potassium channels control the conduction of K+ across cell membranes, down their electrochemical gradient. This rapid and highly selective movement of K+ is essential to many biological processes. K+ channels are largely alpha helical, tetrameric proteins that span the lipid bilayer. Diversity in K+ channels arises primarily in the mechanism of gating by various ligands or voltage; however, the basic structural elements, notably the selectivity filter are conserved within the family. Studies ~n this thesis focus on the single-channel behaviour of the K+ channels KcsA and Kcv. KcsA is a proton-activated channel from the bacterium Streptomyces lividans and its crystal structure was the first of a K+ channel to be solved. Kcv expressed by the Paramecium bursaria Chlorella virus is the smallest known K+ channel and thus represents the minimal structural entity necessary to form a functional and selective pore. Studies on the bacterial inward rectifying channels, KirBacs, have also been initiated. The KirBacs are a superfamily of prokaryotic channels homologous to eukaryotic Kir channels. In this work, KcsA and Kcv were found to form stable tetramers, which can be expressed by coupled in vitro transcription and translation and purified by polyacrylamide gel electrophoresis. The purified tetramers were reconstituted into planar lipid bilayers and studied at the single-channel level. Through single-channel recordings the ionic selectivity, gating behaviour, functional effects of site-directed mutagenesis and the interaction between Kcv and blockers have been studied. In addition, single-channel studies at elevated temperatures have revealed the remarkable thermostability of Kcv, as well as insight into the transport of ions through a narrow and selective pore. Through temperature studies, it has been possible to obtain the thermodynamic and kinetic parameters describing Kcv activity.
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Alexander, Sian. "Modulation of voltage-gated potassium channels: a pathophysiological mechanism of potassium channel antibodies in limbic encephalitis?" Thesis, University of Oxford, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.487139.
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Limbic encephalitis (LE) is a central nervous system disorder that is characterised by memory impairments, confusion, agitation and seizures, and associated with hyperintense lesions of the medial temporal lobe, seen with MRI. Anti-voltage-gated potassium channel (VOKC) antibodies have been been detected in the plasma of a subset of LE patients using a 125I-a-dendrotoxin (a-DTX) radioimmunoprecipitation assay, suggesting that the likely antigens are VOKC Kv1.l, 1.2 and 1.6 subunits. Symptoms of the disease improve markedly with immunosuppression, correlating with similarly dramatic falls in the titre of anti-VOKC antibodies, thus implicating anti-VOKC antibodies in the pathogenesis of LE. Circumstantial evidence from studies of inherited channelopathies and animal models of reduced VOKC activity suggests that VOKC dysfunction may contribute to the pathogenesis of LE. This thesis addresses whether anti-VOKC antibodies (i) bind to a-DTX-sensitive subunits and (ii) affect VOKC function. Immunofluorescence data show that binding of LE patient IgO to the surface of primary neurons and Kvl-expressing HEK-293/HEKTSA cells could not be detected with indirect immunofluorescence. Comparison of intracellular labelling with patient and control IgO showed that no additional labelling could be detected with LE patient IgO. Electrophysiological data show first, that a-DTXsensitive currents could not be reliably isolated from primary cultured hippocampal neurons; second, that NMT or LE samples did not affect VOKCs expressed by neuroblastoma-l cells; third, that none of the LE samples affected potassium currents in Kvl-transfected HEK-293 cells. These data suggest that 'anti-VOKC' antibodies may not bind directly to Kv1.111.2/1.6 homomers, or to a range of Kv1.I/1.2/1.6 subunit-containing heteromers in transfected cells. The findings instead suggest that 'anti-VOKC' antibodies in LE patient plasma may bind to a Kvl-associated protein that contributes to a-DTX-sensitive complexes in the radioimmunoprecipitation assay, but is absent from Kv1.111.2/1.6-transfected cells. Future work to characterise whether another antigen is bound by LE patient anti-VOKC antibodies will be important in determining how these antibodies contribute to the pathogenesis of LE.
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Hodge, J. J. L. "Shaw potassium channel genes in Drosophila." Thesis, University of Cambridge, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.604121.
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Potassium (K+) channels shape the electrical activities of cells by changing the frequency and duration of action potentials and by setting the resting potential. The huge diversity of K+ channels is conserved across species and is thought to be required to uniquely customise both the active excitable and passive properties of different cell types. When this study began, Drosophila voltage-gated K+ currents were thought to be encoded by the four cloned members of this family Shaker, Shab, Shaw and Shal. Mutant analysis had only been performed with Shaker. In order to investigate the in vivo function of another member of the family, a number of methods of making Shaw mutants were used. A more accurate genomic structure and location of Shaw was determined, and I attempted to identify P-element inserts in Shaw. However all the candidate inserts identified were located too far from Shaw to be of practical use for generating mutations affecting Shaw. A dominant negative strategy to remove Shaw function was therefore performed. PCR mutagenesis was used to generate both epitope-tagged full-length and truncated mutant versions of Shaw. Additional control constructs were also made: a full-length wildtype Shaw and a dominant negative version of Shaker. Transgenic lines were generated containing the constructs whose expression was GAL4 inducible. Expression of mutant Shaw, using a number of GAL4 lines, caused an ether-sensitive leg-shaking phenotype.
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Choi, Eun Kyung. "Regulation of KCNQ1 potassium channel trafficking and gating by KCNE1 and KCNE3 /." Access full-text from WCMC, 2009. http://proquest.umi.com/pqdweb?did=1692648191&sid=1&Fmt=2&clientId=8424&RQT=309&VName=PQD.
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Zhang, Hailin. "ATP-sensitive potassium channels and their modulation by nucleotides and potassium channel openers in vascular smooth muscle cells." Thesis, St George's, University of London, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.309744.
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Ellis, Lee David. "Potassium channel control of neuronal frequency response." Thesis, McGill University, 2007. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=103381.
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The processing of sensory signals is an important, yet complex task in which a system must extract behaviorally relevant stimulus patterns from a vast array of sensory cues. When a neuron within a major sensory area is presented with a stimulus, one of the important characteristics used to distinguish between types of input is frequency. Often sensory neurons are tuned to narrow stimulus frequency ranges and are thus charged with the processing of subtypes of sensory signals. The weakly electric fish Apteronotus lepthorhynchus senses it's environment through modulations of a self-generated electric field. Two main types of sensory signals can be distinguished based on their frequency patterns. Prey stimuli cause low frequency perturbations of the electric field, while communication signals often result in high frequency signals. Pyramidal neurons in the electrosensory lateral line lobe (ELL) encode the low frequency signals with bursts, while the high frequency signals are relayed with single spikes. This thesis describes how a pyramidal neuron's response patterns can be tuned to specific frequencies by the expression of distinct classes of potassium channels.I have cloned 3 small conductance (SK) calcium activated potassium channels from cDNA libraries created from the brain of Apteronotus. I have subsequently localized the AptSK channels throughout the brain using both in situ hybridization (AptSK1, 2 & 3) and immunohistochemical (AptSK1 & 2) techniques. The 3 channels showed distinct expression patterns, with the AptSK1 & 2 channels showing a partially overlapping expression pattern, while AptSK3 appears to be expressed in unique areas of the brain. In the ELL AptSK1 & 2 show a partially overlapping expression pattern, appearing in similar pyramidal neurons. However, their distribution within individual cell is unique, with AptSK1 showing a dendritic localization, while AptSK2 is primarily somatic. We have demonstrated that the unique expression pattern of the somatic AptSK2 channel in the ELL coincides with the functional SK currents evaluated through in vitro electrophysiology. Further we have shown that neurons that encode low frequencies do not possess functional SK channels. It thus appears that the presence of the AptSK2 channel subtype can predispose a neuron to respond to specific types of sensory signals.
In an attempt to evaluate if second messengers could modify the AptSK control of frequency tuning I investigated the consequences of muscarinic acetylcholine receptor (mAChR) activation on a pyramidal neurons response patterns. While it had been shown in vivo that mAChR activation increased a pyramidal neuron's response to low frequencies, I have found that this was not due to a decrease in AptSK current, but rather appears to be the result of a down-regulation of an A-type potassium channel.
Taken together the studies that comprise this thesis show how the selective expression of a single potassium channel subtype can control a sensory neurons response to specific environmental cues. The secondary modulation of the A-type current highlights the potential for a second messenger to control a neuron's sensory response through the down-regulation of constitutively expressed potassium current.
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Appenrodt, Peter. "Single-channel recordings of potassium channels from guinea-pig inner hair cells." Thesis, University of Sussex, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.390054.
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Styrczewska, Katarzyna. "Turnover of voltage-gated potassium channel Kv 1.3." Doctoral thesis, Universitat de Barcelona, 2017. http://hdl.handle.net/10803/456990.
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Voltage-gated K channels (Kv) is large family of channels that are expressed in both excitable and non-excitable cells. In excitable cells they contribute to the control of resting membrane potential and action potentials frequency and duration. In non-excitable tissues they are involved in many processes such as secretion to cell proliferation. Kv1.3 channel plays a key role in a wide range of physiological phenomenon. Regulation of this transmembrane protein is therefore essential for a correct function of the living cell. The balance between synthesis and degradation is highly important and must be tightly regulated. The present dissertation is focused in investigating endocytosis mechanisms of Kv1.3, as a process controlling number of the channel on the cell surface and the possible implication in cell destiny.
We deciphered major endocytosis mechanisms triggered by EGF and Adenosine (ADO) in HeLa and HEK 293 heterologous cell systems as well as in native cell lines (macrophages, dendritic or neuronal precursor). These studies pointed out the impact of endocytosis in turnover and homeostasis of Kv1.3 and possible physiological relevance of these finding. Our experiments showed two different ways to control abundance of the Kv1.3 channel by EGF: via tyrosine phosphorylation and unconventional ERK1/2-dependent mechanisms. EGF triggered clathrin-dependent lysosomal degradation of Kv1.3. Moreover, this study show a high physiological relevance, pointing to EGF as a Kv1.3 inhibitor that might therefore reduce radiation-induced brain injury by targeting the key cells involved in the inflammatory process. As next, study was to investigating PMA-induced PKC-dependent endocytosis and ubiquitination. We revealed that PMA triggered PKC-dependent ubiquitin-mediated lysosomal degradation of Kv1.3. Next, we show that adenosine (ADO), which is a potent endogenous modulator, similar to PMA, stimulates PKC, thereby causing immunosuppression. PKC activation triggers down-regulation of Kv1.3 by inducing a clathrin-mediated endocytosis which targets the channel to lysosomal-degradative compartments. Therefore, the abundance of Kv1.3 at the cell surface decreases, which is clearly compatible with an effective anti-inflammatory response. This mechanism requires ubiquitination of Kv1.3, catalyzed by the E3 ubiquitin-ligase Nedd4-2. However, we discover that ADO activates both PKC and PKA signaling pathways. To further investigate the molecular mechanisms of the Kv1.3 internalization in response to ADO, we have examined the effects of PKA antagonists. Our results, for the first time, provided evidence on the effect of PKA activation on the Kv1.3 trafficking. Our findings indicated that PKA adenosine activation triggered Kv1.3 endocytosis redundantly to PKC. In addition, we put a hypothesis that PKA downregulated Kv1.3 in an ubiquitin-independent manner. In the last part of this dissertation we concentrated at molecular determinants involved in Kv1.3 ubiquitination. We found that complementary and redundant lysines participate in the ubiquitin-dependent PKC and PKA regulation of Kv1.3.
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Thomson, Steven James. "Deactivation gating and pharmacology of hERG potassium channel." Thesis, University of Leicester, 2012. http://hdl.handle.net/2381/11071.
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hERG (Kv11.1) encodes the α-subunit of the potassium (K+) channel that carries IKr, an important current for repolarisation of the cardiac action potential. Alterations of hERG current, either through inherited mutations that alter gating or through drugs that block the pore, are associated with Long QT syndrome, cardiac arrhythmias and sudden death. The N-terminus has an important role in regulating deactivation, a gating process that is important for timing of the hERG current during cardiac action potentials. Removing the entire N-terminus accelerates deactivation. A crystal structure of part of the N-terminus (residues 26-135) was solved in 1998 and showed it contained a PAS domain, but it did not resolve the structure of the functionally important first 26 residues (NT 1-26). Here we present an NMR structure of residues 1-135. The structure reveals that residues 1-10 are unordered and residues 11-24 form an amphipathic helix one face of which is positively charged. Neutralising the positive charge accelerates deactivation to similar rates as if the whole of the N-terminus has been removed. Neutralising negative charge in the C-terminus also accelerates deactivation. We propose a model where the N and C-termini interact to stabilise the open state of the channel and slow deactivation. Exactly how changes in membrane voltage are transduced into movement of the activation gate is not fully understood. In hERG, the mutation V659A dramatically slows deactivation. Val659 is located in a region where hERG’s activation gate is believed to lie. From the structure of Kv2.1 it can be seen the S4-S5 linker forms a cuff around S6 where the activation gate is thought to be. Using cysteine cross-linking experiments we show that V659C interacts with E544C and Y545C in the S4-S5 linker to lock the channel in the open state. Trapping of drugs in the inner cavity of hERG has been an important model used to help explain why hERG is blocked by so many drugs and with high potency. A series of derivatives of E-4031, a well characterised high-affinity hERG blocker, were made that progressively increased the length of the molecule. Results in this thesis showed these compounds had binding kinetics completely different from E-4031 and none were trapped in the inner cavity. An alternative model of strongly state-dependent drug binding rather than drug-trapping is proposed. Together, the results in this thesis present new insights on the structural basis for deactivation gating and drug binding in hERG channels.
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Alvis, Simon. "Interactions of phospholipids with the potassium channel KcsA." Thesis, University of Southampton, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.417416.
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Roncoroni, Laura. "Functional role of the background potassium channel K2P15.1." Thesis, University of Southampton, 2012. https://eprints.soton.ac.uk/360274/.
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hK2P15.l, a two-pore domain potassium channel (K2P) was first identified in 2001 by four independent groups (Ashmole et aI., 2001; Karschin et aI., 2001; Kim and Gnatenco, 2001; Vega-Saenz de Miera et aI., 2001). Although hK2P15.l fails to show functional current in recombinant expression systems it was included in the Acid Sensitive subgroup (TASK) of K2P channels primarily due to its sequence homology with the other two family members K2P3.l and K2P9.1. K2P channels are active over physiological voltage ranges resulting in constituent leak of K+ from the cell. These channels are fundamental in setting and regulating the resting membrane potential of cells, are regulated by physiological stimuli and play key roles in several physiological processes. At the messenger RNA level, KCNK15 has a wide tissue distribution and shows high levels of expression specifically in pancreas and adrenal glands (Ashmole et aI., 2001; Kim and Gnatenco, 2001). However, while hK2P 15.1 is anticipated to play an important role in both adrenal and pancreatic function to date the physiological function and pharmacological profile of this channel has been elusive (Ashmole et aI., 2001; Karschin et aI., 2001; Kim and Gnatenco, 2001). Data presented here provide the first evidence of hK2P15.1 retention within the ER and inability to achieve surface expression under control conditions. Significantly, hK2P15.l does localise to other intracellular organelles including the nuclear membrane, mitochondria, endocytic vesicles and lysosomes. Consequently, the physiological role of hK2P15.1 was examined. By analysing hK2P15.1 post-translational modification and binding proteins, the retention of hK2P 15.1 within the ER was found to be dependent on its interaction with BIP. BIP is a chaperon protein involved in cellular response to stress. Removal of BIP-binding or cellular stress enable hK2P15.l release from the ER and targeting at the cell membrane. This links the BIP-dependent ER retention of hK2P 15.1 with the role of BIP in response to cellular stress. Hence, hK2P 15.1 is here suggested to play a role in the control of mitochondrial potential and activity in control conditions and to achieve surface expression and control the cell membrane potential under conditions of cellular stress.
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Rogers, Nik. "The structure of a voltage gated potassium channel." Thesis, University of Southampton, 2010. https://eprints.soton.ac.uk/187983/.
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Voltage gated potassium channels (Kv channels) are tetrameric ion channels, responsible for regulating the potassium component of the membrane potential in a large range of cell types ranging from mammalian excitable cells to bacteria. Attempts have been made to elucidate the structure of voltage gated potassium channels using X-ray crystallography, however due to the inherent flexibility of the voltage sensors, removal of the channels from their native lipid environment causes distortion of the channels, and as a result much controversy remains over their exact structure. KvAP is a voltage gated potassium channel from the thermophilic archaea Aeropyrum pernix which contains a single cysteine residue, which can be removed by site directed mutagenesis to give a template for cysteine scanning mutagenesis. Fluorescence spectroscopy utilising cysteine reactive probes can be used to probe the membrane topology of proteins in the context of a lipid bilayer. Single cysteine mutants within the pore domain outer helix (S5 helix) of KvAP were generated and labelled with thiol reactive fluorescent probes. These probes were used to report on the polarity of the surrounding environment using a combination of the environmental sensitivity of the probes and fluorescence quenching from both aqueous and lipid phases. Fluorescence results fit well to a hypothetical model describing a trough like variation in dielectric constant of the membrane, allowing the determination of the position of the hydrophobic interface of the membrane at each end of the helix. A mutant of KvAP with no voltage sensing domains was also generated and subjected to cysteine scanning mutagenesis of the S5 helix. Again results fitted well to a hypothetical profile of the dielectric constant of the membrane, and the shift in fluorescence properties at some positions within the helix in the absence of the voltage sensor shows the residues of the pore domain which are in close contact with the voltage sensor.
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Rea, Ruth. "Ion channel dysfunction in neurological disease : mutations of potassium channels and glycine receptors." Thesis, University College London (University of London), 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.271822.
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Prole, David L. "Intrinsic functional properties of neuronal KCNQ2/KCNQ3 potassium channels : insights into channel structure." Thesis, University of Bristol, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.400272.
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Carraretto, Luca. "Functional characterization of AtTPK3 potassium channel of Arabidopsis thaliana." Doctoral thesis, Università degli studi di Padova, 2013. http://hdl.handle.net/11577/3426295.
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My Ph.D. project has focused on the characterization of TPK3, a putative channel selective for potassium (K+) with a predicted chloroplast localization in higher plants, from biochemical, physiological and electrophysiological point of view. This protein belongs to the TPK channel family (from Tandem-Pore K+ channels) and displays amino acid sequence homology with another K+ channel studied in our laboratory, called SynK (Zanetti et al., 2010). SynK shows thylakoid localization in Cyanobacteria. The SynK channel has been shown to be critical for photosynthetic performances in Cyanobacteria, given the photosensitive phenotype displayed by the mutants lacking the SynK protein. Given the homology, we hypothesized that similarly, TPK3 might be involved in the regulation of photosynthetic processes in higher plants. So far, no information is available about the properties of TPK3, nor about its physiological roles, neither about its possible involvement in photosynthesis; the work presented in this thesis had the aim of clarifying some important aspects of the functions of TPK3.
Following subcellular localization studies carried out using biochemistry and confocal microscopy techniques, the TPK3 channel was expressed in E. coli cells for subsequent electrophysiological characterization in a planar lipid bilayer setup in order to prove its function as K+ channel. The unavailability of commercial mutants for tpk3 gene required setting up of a silencing procedure via RNA interference of the messenger for the protein, in order to analyze the possible physiological roles of TPK3. The resulting silenced plants have been studied under different growth conditions to determine changes in physiology of the plants including their photosynthetic parameters.
In parallel with the TPK3 project, the most important part of my Ph.D., I also followed two other major areas of research: one concerning the study of the functions of two members of plant Glutamate Receptors (GluRs) and the other one concerning the characterization of the plant homologous of the recently identified MCU (Mitochondrial Calcium Uniporter) of mammals. This thesis also includes a manuscript (Checchetto et al., 2012) to which I contributed with the heterologous expression of a calcium-activated K+ channel, SynCaK, of Cyanobacteria.Il mio progetto di dottorato si è focalizzato sulla caratterizzazione, dal punto di vista biochimico ed elettrofisiologico, di una proteina denominata TPK3 che è predetta di funzionare come canale selettiva per il potassio (K+) ed essere localizzata nei cloroplasti nelle piante superiori,. Questa proteina appartiene alla famiglia dei canali TPK (da Tandem-Pore K+ channels) e mostra omologia di sequenza a un altro canale del K+ studiato nello stesso nostro laboratorio, denominato SynK (Zanetti et al., 2010), a localizzazione tilacoidale ed appartenente al phylum dei Cianobatteri. È stato dimostrato in più esperimenti che il canale SynK è fondamentale per la regolazione della fotosintesi nei Cianobatteri, in considerazione del fenotipo fotosensibile mostrato dai mutanti per il gene synk. Visto la localizzazione predetta del TPK3, è stato ipotizzato in partenza che TPK3 potesse svolgere un ruolo simile nelle piante superiori. Finora nulla si conosceva sulle proprietà di TPK3, ne sui suoi ruoli fisiologici, ne su di un suo eventuale coinvolgimento nella fotosintesi nelle piante superiori; il lavoro contenuto nel progetto presentato ha cercato di chiarire alcuni aspetti salienti delle funzioni di TPK3. Dopo studi di localizzazione subcellulare condotti con tecniche di biochimica e microscopia confocale, il canale TPK3 è stato espresso in E. coli per la successiva caratterizzazione elettrofisiologica in bilayer lipidico planare allo scopo di determinare la sua funzione come canale di K+. L’assenza di mutanti commerciali per il gene tpk3 ha necessitato la messa a punto del suo silenziamento tramite RNA interference del messaggero per la proteina suddetta, al fine di analizzarne i possibili ruoli fisiologici. Le piante silenziate risultanti, sottoposte a differenti condizioni di crescita, sono state studiate in vari esperimenti atti a determinarne vari parametri inclusi quelli fotosintetici. Contemporaneamente allo studio del TPK3, quello di maggior rilievo nel mio dottorato, ho seguito anche altri due filoni di ricerca principali, riguardanti l’uno l’approfondimento delle funzioni di due membri dei Recettori di Glutammato vegetali (GluRs) e l’altro la caratterizzazione degli omologhi del recentemente identificato MCU (Mitochondrial Calcium Uniporter) di Mammiferi. Nella presente tesi è inoltre incluso un manoscritto (Checchetto et al., 2012) per il quale ho collaborato nell’espressione eterologa del canale di K+ calcio-dipendente (SynCaK) di Cianobatteri.
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Stirling, Lee. "Dual Roles for Rhoa/Rho-Kinase in the Regulated Trafficking of a Voltage-Sensitive Potassium Channel." ScholarWorks @ UVM, 2009. http://scholarworks.uvm.edu/graddis/223.
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Kv1.2 is a member of the Shaker family of voltage-sensitive potassium channels and contributes to regulation of membrane excitability. The electrophysiological activity of Kv1.2 undergoes tyrosine kinase-dependent suppression in a process involving RhoA. We report that RhoA elicits suppression of Kv1.2 ionic current by modulating channel endocytosis. This occurs through two distinct pathways, one clathrin-dependent and the other cholesterol-dependent. Activation of RhoA downstream effectors Rho-kinase (ROCK) or protein kinase N (PKN) via the lysophosphatidic acid (LPA) receptor elicits clathrin-dependent Kv1.2 endocytosis and consequent attenuation of its ionic current. LPA-induced channel endocytosis is blocked by ROCK inhibition , dominant negative PKN, or by clathrin RNAi. In contrast, steady-state endocytosis of Kv1.2 in un-stimulated cells is cholesterol-dependent. Inhibition of basal ROCK with Y27632 or basal PKN with HA1077 increases steady-state surface Kv1.2. The Y27632-induced increase persists in the presence of clathrin RNAi and, in the presence of the sterol-binding agent filipin, does not elicit an additive effect. Temperature block experiments in conjunction with studies that perturb trafficking of newly synthesized proteins from the Golgi demonstrate that basal ROCK affects cholesterol-dependent trafficking by modulating the recycling of constitutively endocytosed Kv1.2 back to the plasma membrane. Both receptor-stimulated and steady-state Kv1.2 trafficking modulated by RhoA/ROCK require the activation of dynamin as well as the ROCK effector LIM kinase, indicating a key role for actin remodeling in RhoA-dependent Kv1.2 regulation.
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Corsaro, Veronica Carmen. "Cooperation between potassium channels and gap junctions: interaction between Kv1.1 channel and Pannexin 1." Doctoral thesis, Università di Catania, 2012. http://hdl.handle.net/10761/1037.
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The beta 3 subunit of voltage gated has been recently identified as a modulatory macromolecule of pannexin 1. Our interest has focused on the possible interaction between the alpha subunit of Kv1.1 channel and pannexin 1. Through voltage clamp studies we have analyzed the electrical activity of the single channels and their behavior when they were coexpressed. With our results we have demonstrated that pannexin 1 was less susceptible to its inhibitors, like probenecid and DTT, when it was coexpressed with Kv1.1 channel, on the contrary pannexin 1 did not seem to influence the activity of Kv1,1 channel. Through immunocytochemistry on HEK-hBK1 cells expressing in stable way Kv1.1 channel we have observed the colocalization of the two channels but through coimmunoprecipitation we proved the lack of a physical interaction between these proteins, therefore the interaction should be functional. Moreover previous studies has reported an involvement of pannexin 1 in apoptosis at elevated concentrations of extracellular potassium. So we wanted to estimate the cell death in presence and absence of pannexin 1 inhibitors, using like control SH-SY5Y cells, being a cell line that does not express Kv1.1 channel. With our results we have obtained a decreased in the cell death when HEK-hBK1 cells were treated with probenecid 1 mM in presence of KCl 140 mM suggesting that this
behavior was the consequence of pannexin 1 inhibition, therefore in these conditions Kv1.1 channel did not influence in some way its activity; on the contrary the treatment with 10 mM DTT did not produce any benefical effects in HEK-hBK1 cells. These findings confirmed that Kv1,1 channel influenced the sensibility of pannexin 1 to its inhibitors only when redox potential was altered and then in presence of reducing agents, or when a depolarization of the membrane was induced like in oocytes, but this phenomenon didn t occur in other conditions. Probably this indirect interaction is mediated by other proteins, such as calmodulin and kinase proteins or by lipids of the membrane such as PIP2; it could represent a regulatory mechanism that replaces or enhances that exercised by beta 3 subunit on pannexin 1, in order to control the potassium buffering' and then the cell excitability and survival, both in pathological and physiological conditions.
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Connors, Emilee. "Positive Trafficking Pathways of a Voltage Gated Potassium Channel." ScholarWorks @ UVM, 2009. http://scholarworks.uvm.edu/graddis/52.
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ABSTRACT The voltage-gated potassium channel Kv1.2 is a key determinant of cellular excitability in the nervous and cardiovascular systems. In the brain, Kv1.2 is strongly expressed in neurons of the hippocampus, a structure essential for learning and memory, and the cerebellum, a structure essential for motor control and cognition. In the vasculature, Kv1.2 is expressed in smooth muscle cells where it contributes to the regulation of blood flow. Dynamic regulation of Kv1.2 is fundamental to its role in these tissues. Disruption of this regulation can manifest in a range of pathological conditions, including seizure, hypertension and neuropathic pain. Thus, elucidating the mechanisms by which Kv1.2 is regulated addresses fundamental aspects of human physiology and disease. Kv1.2 was the first voltage gated ion channel found to be regulated by tyrosine phosphorylation. The ionic current of Kv1.2 is suppressed following tyrosine phosphorylation by a process involving channel endocytosis. Movement of channel away from the plasma membrane involves many proteins associated with the cytoskeleton, including dynamin, cortactin and RhoA. Because trafficking of Kv1.2 away from the cell surface has emerged as the primary mechanism for its negative regulation, we hypothesized that trafficking of the channel to the cell surface could be a mechanism for positive regulation of the Kv1.2 ionic current. Activation of the cAMP/PKA pathway enhances the ionic current of Kv1.2. We hypothesized that a mechanism for this positive regulation is an increase in the amount of channel protein present at the cell surface. Our data show that cAMP can regulate Kv1.2 surface levels by two opposing trafficking pathways, one PKA-dependent and one PKA-independent. Channel homeostasis is preserved by the dynamic balance between these two pathways. Accordingly, any change in the levels of cAMP causes a net increase in the amount of Kv1.2 present at the cell surface. Specific C-terminal phosphorylation sites of Kv1.2 were identified and shown to have a role in maintaining basal surface channel levels. These findings demonstrate channel trafficking as a mechanism for the positive regulation of the Kv1.2 ionic current. In addition to Kv1.2 trafficking at the plasma membrane, movement of the channel from the biosynthetic pathway to the cell surface is another checkpoint for its regulation. Here we show that the protein arginine methyltransferase 8 (PRMT8) is able to promote the ER exit of Kv1.2, resulting in an increase in Kv1.2 surface expression. PRMT8 not only promoted surface expression of the high mannose glycosylated form of Kv1.2, characteristic of immature, ER-localized channels, but also enhanced Kv1.2 total protein levels, most likely by decreasing the amount of channel protein available for ER-associated degradation (ERAD). These findings highlight biosynthetic trafficking of Kv1.2 as a crucial part of its regulation and identify a novel role for PRMT8, as a regulator of biosynthetic protein trafficking.
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Beatch, Gregory N. "Antifibrillatory actions of K+ channel blocking drugs." Thesis, University of British Columbia, 1991. http://hdl.handle.net/2429/30907.
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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
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Angué, Lauriane. "Single molecule studies of a voltage-gated potassium channel." Thesis, University of Oxford, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.669847.
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This thesis aims to study the activation and deactivation mechanism of voltage-gated K+ channels (Kv), through fluorescence and electrical measurements. Transmembrane voltage changes are detected by the voltage sensor (helices S3b-S4) among Kv, a phenomenon which triggers a conformational change to open or close the pore. By attaching a fluorescent tag on helices S3b-S4, voltage sensor movement can be followed by fluorescence and pore opening or closure by electrical recordings. KvAP, a channel from an archaea organism was selected for this task. Channels were expressed, purified and fluorescently labelled before being reconsituted into droplet interface bilayers (DIB). The degree of labelling was determined by photobleaching measurements. Electrical activity and diffusion were recorded at the single molecule level. Further developments of the technique need to be achieved before successfully correlating fluorescence and electrical recordings at the single molecule level. However, both signals could be simultaneously recorded at bulk and single molecule levels, paving the way for future experiments.
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Campbell, Jeffery. "The structural biology of the ATP-sensitive potassium channel." Thesis, University of Oxford, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.443602.
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Du, Chunyun. "The effects of acidosis on the hERG potassium channel." Thesis, University of Bristol, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.555619.
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The human ether-a-go-go-related gene (hERG) encodes channels mediating the rapid delayed rectifier K+ current (IKr). IKr participates in cardiac action potential (AP) repolarisation and may also protect the ventricles against premature stimulation. The heart is exposed to acidosis (low pH) in a number of pathological conditions including myocardial ischemia. Extracellular acidosis is known to modulate hERG current (IhERG) function, although a number of aspects of the modulation remain incompletely understood. The aims of this investigation were to establish the effects of acidosis on: (i) IhERG amplitude, kinetics and the response to premature stimulation at mammalian physiological temperature; (ii) the hERG blocking potency of selected anti arrhythmic drugs. Whole-cell patch-clamp recordings of IhERG were made from mammalian cells (CHO or HEK 293) at 37 QC. Lowering external pH from 7.4 to 6.3 reduced the magnitude of IhERG by reducing macroscopic hERG conductance and modulating IhERG kinetics, with positively shifted activation and accelerated deactivation. Results from experiments using an acidic pipette solution showed that the actions of protons occurred from the external surface and not from secondary intracellular acidosis. Experimental and computer simulation work demonstrated that acidosis impairs the protective role of IhERG against premature stimulation. The effects of extracellular acidosis on IhERG kinetics were preserved when the shortened hERG I b isoform was studied, indicating that a full-length N-terminus is not necessary for acidic modulation of hERG channel function. Interestingly, the inhibitory effect of acidosis on IhERG was greater for hERG 1 band hERG lall b than for hERG la. Extracellular acidosis decreased the hERG blocking potency of flecainide, dofetilide and ranolazine, whilst the potency of amiodarone was unaffected. IhERG inactivation was found to be important for ranolazine's inhibitory action and a series of S6 and inner helix residues (Y652, F656, T623, S624 and V625) were identified as contributing to ranolazine binding. I.
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Roosild, Tarmo P. "Studies of prokaryotic potassium channel structures and regulatory mechanisms /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2002. http://wwwlib.umi.com/cr/ucsd/fullcit?p3055798.
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Ferrer, Patricia Preston. "Functional analysis of the potassium channel beta subunit KCNE3." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2011. http://dx.doi.org/10.18452/16264.
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KCNE-Hilfsuntereinheiten assoziieren mit Spannungs-abhängigen K+-Kanälen und verändern dadurch deren subzelluläre Lokalisation, Regulation sowie deren biophysikalische Eigenschaften. Bei heterologer Expression interagiert KCNE3 mit mehreren Poren-bildenden K+-Kanal-Hauptuntereinheiten, deren Ströme dadurch stark modifiziert werden. Aufgrund dieser in vitro-Experimente wurden verschiedenste Funktionen von KCNE3 in den verschiedenen Geweben, wie Gehirn, Herz, Muskel, Kolon und Niere, vermutet. Außerdem wurden Variationen im kcne3-Gen mit menschlichen Skelettmuskelpathologien in Verbindung gesetzt (Abbott et al. 2001). In der gegenwärtigen Literatur wird die physiologische Funktion von KCNE3 eher als komplex und heterogen dargestellt. Auch die direkte Beteiligung von KCNE3 an Krankheiten ist immer noch spekulativ. Zur Untersuchung der physiologischen Funktion von KCNE3 in vivo sowie der potentiellen Rolle bei Krankheiten generierten wir ein kcne3-/- Mausmodell. Die vorliegende Arbeit unterstützt die kritische Rolle der KCNQ1/KCNE3-Kanäle beim Salz- und Flüssigkeitstransport über intestinale und respiratorische Epithelien. Insbesondere fanden wir für die KCNQ1/KCNE3-Heteromere eine basolaterale Lokalisation in Darm- und Trachea-Epithelzellen, wo sie die transepitheliale Cl--Sekretion über basolaterales Recycling von K+-Ionen sowie über Erhöhung der elektrochemischen Triebkraft für apikalen Cl--Austritt fördern. Da weder Veränderungen in der KCNQ1-Expressionsmenge noch in dessen subzellulärer Lokalisation festgestellt wurden, ist die durch KCNE3 verursachte Modifikation der KCNQ1-Kanaleigenschaften essenziell für die hier beschriebene physiologische Rolle im Intestinal- und Trachealtransport. Ferner wird von unserer Arbeit die postulierte Funktion von KCNE3-Heteromeren im Skelettmuskel, Herz und zentralen Nervensystem nicht unterstützt und erweckt somit erhebliche Zweifel über den Beitrag von KCNE3 zu menschlichen Krankheiten, die mit diesen Organen in Verbindung stehen.When overexpressed in heterologous systems, KCNE3 is able to interact with several pore-forming K+ channel alpha subunits greatly modifying their currents. Based on these in vitro evidences, KCNE3 has been proposed to serve different roles in multiple tissues, including brain, heart, muscle, colon and kidney. Additional reports have also linked sequence variations in the KCNE3 gene to cardiac and skeletal muscle pathologies in human. Based on the literature, the overall picture of KCNE3 physiological function is rather complex and heterogeneous, and its direct involvement in pathologies is still speculative and far from being conclusively proven. In order to study the physiological role of KCNE3 in vivo and to address its potential pathological implications, we generated kcne3-/- mice. The present analysis of kcne3-/- mice strongly supports a crucial role of KCNQ1/KCNE3 channels in salt- and fluid secretion across intestinal and airway epithelia. In particular, we found that KCNQ1/KCNE3 heteromers are present in basolateral membranes of intestinal and tracheal epithelial cells where they facilitate transepithelial Cl- secretion through basolateral recycling of K+ ions and by increasing the electrochemical driving force for apical Cl- exit. Because the abundance and subcellular localization of KCNQ1 was unchanged in kcne3-/- mice, the modification of biophysical properties of KCNQ1 by KCNE3 is essential for its role in intestinal and tracheal transport. In addition, our work does not support the postulated role of KCNE3 heteromers in skeletal muscle, heart and CNS physiology, and raises considerable doubts concerning its implication in human pathologies which affect these tissues.
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Paggio, Angela. "Molecular identification of ATP sensitive Mitochondrial Potassium Channel (mitoKATP)." Doctoral thesis, Università degli studi di Padova, 2016. http://hdl.handle.net/11577/3424388.
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Mitochondria ATP-sensitive potassium channels (mitoKATP) were first described in 1991 by direct patch clamp of isolated mitoplasts (i.e. mitochondria lacking the outer membrane). Since then, a growing amount of evidences showed that they are implicated in the control of a variety of mitochondrial functions. Most importantly, pharmacological modulation of the mitoKATP can efficiently protect the heart from ischemia/reperfusion injury. However, despite its huge therapeutic potential, the molecular identity of the mitoKATP is still unknown. Here we identify a protein complex that is sufficient to reconstitute a functional K+ permeant channel with the same pharmacological profile of the mitoKATP. In particular, we demonstrate that the mitoKATP is a heterooligomer composed by two different components. One is a previously uncharacterized protein of unknown function that we now name “mitoK”. The other is a protein belonging to the ABC superfamily here named “mitoSUR”. It possesses a nucleotide-binding site (a standard Walker motif) and provides ATP sensitivity to the mitoK pore-forming subunit through direct protein interaction.
In detail, we demonstrated that mitoK is a mitochondrial protein located in the inner mitochondrial membrane (IMM), with two transmembrane domains and both its N- and C-termini exposed to the organelle matrix. Its overexpression induces a decrease in mitochondrial Ca2+ transients evoked by agonist stimulation, a drastic reduction of mitochondria membrane potential, fragmentation of the mitochondrial network and a derangement of the IMM ultrastructure, with the total collapse of the cristae. However, all these effect can be rescued by the concomitant overexpression of mitoSUR, but not by the expression of the mitoSURK513A mutant that lacks ATP binding. Most importantly, in vitro reconstitution of purified mitoK and mitoSUR in a planar lipid bilayer form a channel i) selective for monovalent cations, ii) blocked by tetraethyl ammonium (TEA, a general inhibitor of K+ channels), iii) sensitive to ATP, iv) activated by diazoxide and v) inhibited by 5-HD.
Finally, we focused our efforts to understand the physiological role of mitoKATP. According to the available literature, mitoKATP channels are believed to be protective in ischemia/reperfusion injury. However, the broad conservation profile among all vertebrates suggests that the mitoKATP should also have a primary housekeeping function. In order to analyze the genuine physiological role of the mitoKATP, we generated Hela cells knockout for mitoK by using the Crispr/Cas9 technology. Two different guides targeting different gene regions were validated and several Hela mitoKKO clones were obtained. Overall, ablation of mitoK leads to no gross defects in mitochondria morphology, although an impairment of cristae structure becomes evident through electron microscopy. Mitochondrial membrane potential is overall intact and expression of respiratory chain complexes is unaffected. However, we noticed that Hela mitoKKO cells undergo to asynchronous, rapid and transient depolarizations of single mitochondria, a phenome known as “flickering” of mitochondria membrane potential. Therefore, oxygen consumption rate (OCR) is greatly impaired in Hela mitoKKO cells when compared to their wild type counterparts, in both basal, leaky (oligomycin induced) and maximal (i.e. FCCP induced) respiration. As already reported, mitochondrial K+ homeostasis is fundamental to regulate mitochondrial matrix volume: while excessive accumulation leads to organelle swelling, a decrease in K+ uptake is predicted to cause matrix contraction, with obvious consequences on the performance of the oxidative phosphorylation. Overall, our data indicates that mitoKATP is a central regulator of mitochondrial function that modulates the efficiency of energy production according to the ATP availability through the regulation of matrix volume.I canali mitocondriali del potassio sensibili all’ATP (mitoKATP) sono stati descritti per la prima volta nel 1991 in seguito ad esperimento di patch clamp su mitoplasti (mitocondri privi della membrana mitocondriale esterna) isolati. Da allora, un numero crescente di esperimenti ha dimostrato il coinvolgimento di questi canali nella regolazione di numerose funzioni mitocondriali. In particolare, la modulazione farmacologia di questi canali può efficacemente proteggere il cuore dal danno da ischemia/riperfusione. Tuttavia, nonostante il loro enorme potenziale terapeutico, la struttura molecolare dei canali mitoKATP è ancora oggi sconosciuta. In questo lavoro abbiamo identificato un complesso proteico permeabile al K+ con lo stesso profilo farmacologico dei canali mitoKATP. In particolare, abbiamo dimostrato come questi canali siano composti da due differenti subunità. Una di queste è una proteina non ancora caratterizzata dal punto di vista biologico e con funzione sconosciuta, che d’ora in poi chiameremo “mitoK” . L’altra componente del canale è una proteina appartenente alla superfamiglia delle proteine ABC, che chiameremo “mitoSUR”. Quest’ultima possiede un dominio in grado di legare ATP (chiamato “dominio Walker”) attraverso cui fornisce sensibilità ai canali mitoKATP attraverso una interazione diretta con mitoK. In particolare, abbiamo dimostrato che mitoK è una proteina mitocondriale sita sulla membrana mitocondriale interna, con due domini transmembrana e le rispettive porzioni N- e C- terminale esposte nella matrice. La sovraespressione di mitoK induce una diminuzione dell’accumulo mitocondriale dello ione calcio in risposta a stimoli, una drastica riduzione del potenziale di membrana mitocondriale, frammentazione della morfologia mitocondriale ed una perdita totale delle cristae. Tuttavia, le normali funzioni mitocondriali possono essere recuperate attraverso la contemporanea sovraespressione di mitoSUR, ma non dall’espressione del mutante mitoSURK513A mancante del dominio che lega ATP. Inoltre, l’espressione in vitro delle proteine purificate mitoK e mitoSUR ed il loro inserimento in una membrana artificiale ci ha permesso di studiarne le caratteristiche elettrofisiologiche, dimostrando che mitoK e mitoSUR formano un canale i) selettivo per cationi monovalenti, ii) inibito da ammonio tetraetile (TEA, un inibitore generico per i canali K+), iii) sensibile ad ATP, iv) attivato da diazossido e v) inibito da 5-HD. Infine abbiamo cercato di comprendere il ruolo fisiologico dei canali mitoKATP. Secondo la letteratura disponibile, questi canali hanno un ruolo protettivo nel danno da ischemia/riperfusione. Tuttavia, l’ampio profilo di conservazione tra tutti i vertebrati suggerisce che i canali mitoKATP abbiano prima di tutto un ruolo nel controllo delle normali funzioni mitocondriali. Al fine di analizzare il vero e proprio ruolo fisiologico di questi canali mitoKATP, abbiamo generato cellule Hela prive del gene che codifica per mitoK utilizzando la tecnologia Crispr/Cas9. Utilizzando due differenti guide in grado di riconoscere due diverse regioni del gene sono stati ottenuti alcuni cloni cellulari privi di mitoK a livello proteico. Nel complesso, la mancanza di mitoK non comporta alcun difetto in termini di morfologia mitocondriale, anche se è evidente una diversa strutture delle cristae attraverso la microscopia elettronica. Il potenziale di membrana mitocondriale è complessivamente intatto e l’espressione dei complessi della catena respiratoria è inalterata. Tuttavia, abbiamo notato che le cellule Hela mitoKKO vanno incontro a depolarizzazioni asincrone, rapide e transitorie, caratteristiche di un fenomeno conosciuto come “flickering” del potenziale di membrana mitocondriale. Abbiamo inoltre osservato che il consumo di ossigeno (OCR) è notevolmente ridotto in questi cloni rispetto alle cellule di controllo, sia in termini di respirazione basale che massimale. Dalla letteratura è infatti noto che l’omeostasi mitocondriale del potassio è fondamentale per regolare il volume della matrice mitocondriale; mentre un accumulo eccesivo di K+ comporta un aumento del volume, una diminuzione dell’accumulo di K+ può causare la contrazione della matrice, con ovvie conseguenze sulle prestazioni della fosforilazione ossidativa. Nel complesso, i nostri dati indicano che i canali mitoKATP regolano le funzioni mitocondriali, modulando l’efficienza della produzione di energia secondo la disponibilità di ATP attraverso la regolazione del volume della matrice.
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Konstas, Angelos Aristeidis. "The regulation and functional interaction of the epilethial sodium channel (ENaC) and renal potassium channels." Thesis, University of Oxford, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.249463.
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Doczi, Megan Anne. "Subcellular Distribution of a Voltage-Gated Potassium Channel: the Effect of Localization on Channel Function." ScholarWorks @ UVM, 2010. http://scholarworks.uvm.edu/graddis/69.
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Voltage-gated potassium channels are primary determinants of cellular excitability in the mammalian nervous system. The localization of these channels to distinct cellular compartments influences components of neuronal function, including resting membrane potential, action potential characteristics and neurotransmitter release. Thus, understanding the mechanistic basis of ion channel localization can provide fundamental insight into human physiology. The overall goal of this dissertation was to elucidate the regulatory mechanisms governing localization and function of the Kv1.3 voltage-gated potassium channel. The sympathetic branch of the autonomic nervous system innervates many organ systems including the kidneys, heart and blood vessels and was used as a model to study endogenous Kv1.3. We found that postganglionic sympathetic neurons express Kv1.3 and that the channel exhibits a striking pattern of localization to the Golgi apparatus in the soma of these cells. Kv1.3 ionic current was also isolated from the soma of these neurons, indicating the channel is a determinant of the electrophysiological properties of sympathetic neurons. In addition, the specific inhibition of Kv1.3 with margatoxin was found to depolarize neuronal resting membrane potential, decrease the latency to action potential firing and increase nicotinic agonist-induced neurotransmitter release. Collectively, these findings demonstrate that Kv1.3 influences the function of postganglionic sympathetic neurons and led to the hypothesis that regulating channel localization may be a mechanism for modulating the activity of these cells. In this dissertation, we propose that the observed Golgi retention of Kv1.3 may be a trafficking-dependent mechanism of channel regulation. To test this hypothesis, we used HEK293 cells as our model system. Our data show that the degree of Kv1.3 Golgi localization is inversely correlated with the amount of channel at the plasma membrane. In addition, the amplitude of Kv1.3 ionic current measured in cells with low Kv1.3 Golgi localization was significantly greater than the current measured in cells with high Kv1.3 Golgi localization. One mechanism for localizing ion channels to the Golgi apparatus involves the Class I PDZ-binding motif (X-S/T-X-Φ). Deletion of the C-terminal PDZbinding motif of Kv1.3 decreased the intracellular Golgi localization of the channel and increased channel localization at the cell surface. Disrupting this canonical binding motif also increased the amplitude of Kv1.3 ionic current. These findings indicate that regulated subcellular distribution of the channel may be a determinant of Kv1.3 surface expression and function.
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Werry, Daniel. "Single channel properties of the slow cardiac potassium current, IKs." Thesis, University of British Columbia, 2011. http://hdl.handle.net/2429/38968.
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The slow potassium current, IKs, abbreviates the cardiac action potential by repolarizing the membrane to a resting state. Mutations in the pore-forming IKs subunit, KCNQ1, cause long QT syndrome type 1 (LQT1), which increases risk of fatal arrhythmia. Despite the physiological and clinical importance of IKs, little is known about the elementary events that underlie the unique biophysical properties of the channel, and how these elementary events are altered in the face of disease. This thesis investigates single channel recordings of IKs with and without mutations that cause LQT1 using patch clamp electrophysiology. Single channel IKs is described by slow and fast gating processes. The channel is slow to open, but flickers rapidly between open and closed states in non-deactivating bursts. Long latency periods to opening underlie the slow activation of IKs at depolarized potentials. Channel activity is cyclic with periods of high activity followed by quiescence, leading to an overall low open probability. The mean single channel conductance was determined to be 3.2 pS and long-lived subconductance levels coupled to activation were observed. Single channel properties of IKs with LQT1 mutations in the S3 helix of the voltage sensing domain in KCNQ1 were investigated to uncover pathogenic mechanisms at the single molecule level. Open probability was reduced in loss-of-function mutations (D202H, I204F and V205M) and increased in a unique gain-of-function mutation (S209F) that may cause LQTS from a reduced number of functional channels at the cell surface. The mean duration of open events correlated well with deactivation rate in all mutants and first latency to opening determined activation rate. From these results, we attributed the pathogenic mechanisms of LQT1 mutations to alterations in the stability of specific channel states.
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Neill, Colin Gardner. "Synthesis of pyridothiadiazine dioxides as potential potassium ion channel openers." Thesis, Heriot-Watt University, 1995. http://hdl.handle.net/10399/1347.
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Miller, Paula. "Oxygen sensing by hTREK1, a twin-pore-domain potassium channel." Thesis, University of Leeds, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.403031.
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Spruce, Austen Edwin. "Potassium conductances of skeletal muscle investigated using single channel recording." Thesis, University of Leicester, 1986. http://hdl.handle.net/2381/33614.
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This thesis describes studies of unitary currents flowing through two different potassium (K) channels present in sarcolemmal vesicles of the frog, Rana temporaria. The ATP-regulated K-channel is described first and the results are divided into three parts. Firstly, ATP applied to the cytoplasmic face of a membrane patch closes the channels in a dose-dependent fashion. Different nucleotides and other metabolic substances are used in order to find chemicals which can substitute for ATP or which regulate its effect. Secondly, the permeability properties of the channel are described. Ion flux is non-independent. Rubidium (Rb) is permeant, and anomalous mole-fraction behaviour is demonstrated in mixtures of K+and Rb+. The final part investigates the kinetic properties of the channel. Both voltage and ATP affect the rate constants regulating transitions between closed and open states of the channel. In particular, ATP causes the channel to occupy a very long-lived closed state. Block of the channel by tetraethylammonium (TEA) ions applied to either membrane surface is described as well. Block by external TEA+ is very fast and it is suggested that the channel cannot close when blocked. The block by internal TEA+ is slower and some evidence of voltage dependency is seen. The delayed rectifier K-channel is investigated. The first of two parts describes Rb+ permeability of the channel and its effect on open and closed times. The Hodgkin-Huxley model of the channel is questioned by the very different times of occupancy of closed states and differing voltage dependencies of the steps leading to opening of the channel. The second part describes block of the channel by externally applied TEA+ and the blocking reaction is shown to be very fast and voltage dependent.
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Kirby, Robert William. "Investigation of the pharmacophore of BK[ca] potassium channel openers." Thesis, Sheffield Hallam University, 2008. http://shura.shu.ac.uk/19920/.
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Large conductance voltage-activated calcium-sensitive potassium channels (BK[ca]) are fundamental in the control of cellular excitability which is critical in the regulation of many physiological processes. Agents that activate the channel (openers) have been proposed to be potential therapeutics for a number of un-met clinical conditions. Several heterogeneous classes of compounds have been described as BK[ca] channel openers and preliminary pharmacophore data has arisen for synthetic molecules based on the structure of benzimidazolones. The project aimed to explore a series of novel compounds based on a benzanilide template for BK[ca] channel opener properties and to further probe the pharmacophore of BK[ca] channel openers. This was achieved by the validation and optimisation of a medium through-put, non-radioactive, rubidium (Rb[+]) efflux assay using recombinant HEK293 cells expressing BK[ca] channel subunits. From which novel benzanilides have been identified as BKca channel openers that display varying degrees of potency, efficacy and co-operativity. The Rb[+] efflux stimulated by each compound was blocked by use of the BKca channel blockers paxilline and iberiotoxin demonstrating specificity to BK[ca] channels in the cell lines studied. Furthermore initial data demonstrates that some of these compounds showed selectivity for the BK[ca] channel alpha-subunit. The benzanilide compounds were also examined using whole cell electrophysiology; all compounds tested, relative to control, produced shifts in V50 values in the hyperpolarising direction, indicative of BK[ca] channel activation. The benzanilides did not affect the kinetics of activation or deactivation. Furthermore, the order of effectiveness determined using whole cell electrophysiology showed good correlation with that obtained using the Rb[+] efflux assay. Using both the electrophysiology and Rb+ efflux techniques BKOEt1 was identified as a novel and potent BKca channel opener and selected as a lead compound. In single channel electrophysiology recordings of cloned cell lines expressing BK[ca] channel subunits application of BKOEt1 promoted significant, paxilline sensitive, BK[ca] channel activation. BKOEt1 increased open pore probability rapidly for small changes in voltage and increased the voltage-sensitivity of the channel. In addition, BKOEt1 did not affect single channel conductance. BKOEt1 activated BKca channels in the near absence of intracellular calcium and its effects were not additive. Furthermore, the compound did not affect the level of intracellular calcium. It was concluded that BKOEt1 acts directly on the channel at a site located on the alpha-subunit. A Hill slope of unity suggested one binding site per tetrameric channel complex and either an intracellular or transmembrane site of interaction was proposed. BKOEt1 also stimulated paxilline sensitive Rb+ efflux from rat bladder myocytes and initial Rb+ efflux studies demonstrated that BKOEt1 could activate K[v] and SK channels. Molecular modelling of the series of benzanildies provided clues as to the chemical or structural features required, in particular for BKOEt1, to retain potent channel opener properties. In addition, physicochemical properties were determined and revealed commonalities for compounds to retain potent BKca channel opener properties. A confirmatory pharmacophore was proposed with compounds requiring two substituted phenyl rings, the presence of an oxygen containing group, an amide group provided by the linker region and hydrophobic moieties.
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Carvalho, Maria João Marques de. "Characterization of a C-terminal domain from eag potassium channel." Master's thesis, Universidade de Aveiro, 2010. http://hdl.handle.net/10773/4343.
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Mestrado em Métodos BiomolecularesDomínios que ligam nucleotideos cíclicos (CNBD) regulam muitas vias de sinalização em células procarióticas e eucarióticas. Os ligandos AMP cíclico ou GMP cíclico ligam-se a estes domínios e induzem uma alteração conformacional que é propagada ao domínio efector, como uma cinase ou um canal iónico. Os canais de potássio da família ether-a-go-go (EAG) estão envolvidos em muitos processos fisiológicos que incluem repolarização cardíaca e neuronal, proliferação tumoral e secreção de hormonas. Estes canais são tetraméricos e cada subunidade inclui seis hélices transmembranares e dominios citoplasmáticos em N- e C-terminal. O domínio em C-terminal tem homologia com domínios que ligam nucleotídeos cíclicos mas foi demonstrado que os canais EAG não são afectados por nucleotídeos e o domínio não liga nucleotideos. O objectivo deste projecto foi resolver a estrutura de um domínio C-terminal de um canal EAG por cristalografia de raios-X e compreender o seu papel funcional. Determinei a estrutura de um destes domínios à resolução de 2,2 Å; a estrutura tem a topologia de um CNBD mas a cavidade de ligação apresenta várias diferenças relativamente à de domínios que ligam nucleotideos cíclicos. Mais ainda, os canais EAG são inibidos por calmodulina e há dois locais de ligação de calmodulina a seguir ao CNBD. A estrutura mostrou que um destes locais se encontra sobreposto com uma região do domínio levantando a possibilidade da calmodulina regular o canal através da alteração conformacional do domínio C-terminal dos canais EAG. Esta possibilidade começou a ser explorada com recurso a ensaios de cross-linking químico e espectroscopia de fluorescência.
Cyclic nucleotide binding domains (CNBD) are regulatory domains that participate in many signaling pathways in prokaryotic and eukaryotic cells. The ligand cAMP or cGMP binds these domains and induces a conformational change that is propagated to an effector domain, like a kinase or an ion channel. The ether-a-go-go (EAG) potassium channel family is involved in important physiological roles that include cardiac and neuronal repolarization, tumor proliferation and hormone secretion. These channels are tetramers, where each subunit includes six transmembrane helices and N- and C-terminal cytoplasmic domains. The C-terminal domain has strong homology to CNBDs but it has been demonstrated that EAG channels are not affected by cyclic nucleotides and that the domain does not bind nucleotides. The ultimate goal of this project was to solve the structure of an EAG family C-terminal domain by X-ray crystallography and to understand its functional role. I have determined the structure of one of these domains at 2.2 Å; the structure has the canonical CNBD fold but it shows a ligand pocket that has several differences relative to a cyclic nucleotide binding site. Furthermore, EAG currents are inhibited by calmodulin binding and there are two calmodulin binding sites C-terminal to the CNBD. The structure reveals that one of these sites overlaps with a region of the domain raising the possibility that calmodulin affects channel function by changing the EAG C-terminal domain conformation. I have conducted preliminary tests on this hypothesis by using biochemical cross-linking experiments and fluorescence spectroscopy.
FCT
FCOMP-010124-FEDER-007427/PTDC/QUI/66171/2006
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Yamauchi, Tomofusa. "Mitochondrial ATP-sensitive potassium channel : A novel site for neuroprotection." Kyoto University, 2003. http://hdl.handle.net/2433/148749.
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Allen, Margaret Louise. "Post-transcriptional regulation of expression of the potassium channel, Kv1.1 /." Thesis, Connect to this title online; UW restricted, 1998. http://hdl.handle.net/1773/6264.
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Bohm, Rudy Ashish. "Transcriptional control of slowpoke, a calcium activated potassium channel gene /." Full text (PDF) from UMI/Dissertation Abstracts International, 2000. http://wwwlib.umi.com/cr/utexas/fullcit?p3004218.
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Pan, Geng. "Potassium channel expression and function in the N9 murine microglial cell line." Thesis, University of Edinburgh, 2012. http://hdl.handle.net/1842/8191.
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Microglia are immunocompetent cells in the central nervous system that have many similarities with macrophages of peripheral tissues. Their activation protects local cells from foreign microbial infection in the CNS. However, “over-activated“ microglia become a “Double-edged sword” which show neuronal toxicity and are implicated in a variety of neurodegenerative diseases. Previous studies have suggested that potassium channels play a role in regulating microglial activation, migration and proliferation. However what kinds of potassium channel subunits are expressed in microglia, whether their expression changes after microglial activation and the functional role of most potassium channels expressed in microglia are still not fully characterized. To address these questions, we used the N9 mouse microglial cell line as a cell model for experiments in vitro. We first optimized the cell culture and lipopolysaccharide (LPS), the endotoxin of gram-negative bacteria, mediated stimulation of microglial activation that results in subsequent nitric oxide (NO) release. Using qRT-PCR, we analyzed mRNA expression of >80 potassium channel pore-forming subunits and their regulatory subunits in both LPS-treated (1μg/ml, 24hr) and untreated microglia. The subunits which displayed the highest mRNA expression in resting N9 cells included Kcnma1 (KCa1.1), Kcnk6 (K2p6.1), Kcnc3 (Kv3.3) and Abcc8 (SUR1). In addition, N9 cells also expressed the mRNAs for other channel subunits previously reported in microglia such as Kcnn4 (KCa3.1), Kcna3 (Kv1.3) and Kcna5 (Kv1.5) subunits. Of these channel subunits LPS had no significant effect on mRNA expression except for Kcnk6 which was significantly reduced. We then examined whether pharmacological manipulation of these channels controlled LPS-induced NO release. It was found out that the KCa3.1 selective blocker Tram34 and the Kv1.5 inhibitor propafenone (PPF) significantly decreased LPS-induced NO in agreement with data in primary microglia. Ba2+ that inhibits inwardly rectifying potassium channels as well as K2p6.1 also significantly attenuated LPS-induced microglial activation. Inhibition or activation of KCa1.1 channels by paxilline and NS1619 respectively had no significant effect. However, paxilline significantly attenuated the effect of Tram34, PPF and Ba2+ to control LPSinduced NO release while NS1619 significantly facilitated the effect of Tram34 and PPF. To investigate the major ionic currents expressed in N9 microglia with and without LPS application, we examined whole-cell ionic currents using the patch-clamp technique. Resting N9 cells display a small outward current at positive potentials but a large inwardly rectifying component at negative potentials in physiological potassium gradients. The outward current was dramatically increased by LPS application that was dependent upon the intracellular free calcium concentration. Paxilline or Tram34 was then applied to acutely block this apparent outward KCa current. The result indicated that the LPS triggered KCa current was mainly paxilline sensitive supporting a role for an LPS-induced increase in KCa1.1 channel current. In addition, by using current clamp the mean resting membrane potential of N9 cells was -50.6±6.6mV (N=7) determined in the presence of 1μM [Ca2+]i and -59.4±8.5mV (N=10) with 10nM [Ca2+]i. N9 cells did not display any spontaneous action potentials and the resting membrane potential was not significantly affected by LPS. To conclude, the work presented in this thesis extends the current knowledge regarding potassium channel mRNA expression in microglia and their function in microglial NO release. What is more, it was found that KCa1.1 current expression was increased in LPS-activated N9 cells and revealed KCa1.1 channels as a modulator of NO release by activated microglia.
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Vaid, Moninder. "Structural examination of voltage gated potassium channels by voltage clamp fluorometry." Thesis, University of British Columbia, 2007. http://hdl.handle.net/2429/443.
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Voltage clamp fluorometry (VCF) was first developed in the mid 1990s by Isacoff and his colleagues. In this approach fluorophores are attached to substituted cysteine residues that are engineered by site-directed mutagenesis. Changes in the dielectric environment of the fluorophore report local transitions that are associated with electrically-related and electrically-silent transitions. VCF provides a powerful technique to observe real time reports of ion channel gating conformations. It has proven to be a useful technique because it adds insight that is not available using other techniques. X-ray crystallography studies give a predominantly static picture of the channel, while patch clamping of channels gives information only about residues that effect ionic current flow. Similarly, gating current provides insight only about residues that are charged and move across the membrane electric field.
In this thesis we examined the structural rearrangements of the Shaker channel and the effect of 4-AP on channel gating. We also examined for the first time the structural rearrangements of the Kv1.5 gating and the how the channel responds to depolarization pulses. This work is instrumental in the examination of the potassium channel gating.
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Männikkö, Roope. "Voltage sensor movements in shaker and HCN channels /." Stockholm, 2003. http://diss.kib.ki.se/2003/91-7349-739-8.
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Wilson, Stacey. "Modulation of the hERG potassium channel function by extracellular acidosis : single channel effects and underlying basis." Thesis, University of Bristol, 2018. http://hdl.handle.net/1983/7ef42e09-9a08-4da4-8762-e6797cbad57e.
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Human ether-à-go-go-related gene (hERG) potassium channels underlie the rapid delayed rectifier K+ current (IKr) and play an important role in repolarisation of cardiac action potentials (APs). Pathological events such as cardiac ischaemia can lead to a decrease in extracellular pH (acidosis). Extracellular acidosis is known to modulate the hERG current (IhERG) but the underlying mechanism(s) are not completely known. The aims of this study were: (1) to establish the effects of acidosis on macroscopic and single-channel IhERG, investigating both hERG1a and hERG1b isoforms; (2) to use an amino acid modifying reagent and site-directed mutagenesis to probe the molecular basis of proton modulation of IhERG, focusing on the hERG1a isoform. Whole-cell and cell-attached patch-clamp recordings were made at ambient temperature of wild-type IhERG from mammalian cell lines (HEK-293 or CHO). When external pH (pHe) was reduced from 7.4 to 6.3, macroscopic IhERG amplitude and conductance decreased, activation was positively shifted, and deactivation kinetics were accelerated. Results obtained in the cell-attached configuration showed a reduction at pHe 6.3 in single-channel IhERG amplitude and conductance, decreased open- and burst-durations and increased closed-time durations. These effects at the single-channel level account for the modulation of macroscopic IhERG by acidic pHe. The first known single-channel recordings from hERG1b showed that this isoform retained sensitivity to acidic pHe, indicating that N-terminal differences between the two isoforms are not critical for proton sensitivity. Experiments completed with a range of extracellular pH values (4.5 – 8.0) revealed that different features of IhERG have distinct pKa values, suggesting multiple sites of proton modulation. Titratable residues located in the pore region of the hERG1a channel were mutated to determine if they were responsible for pH sensitivity. The double mutation E575Q/H578N appeared to remove the proton reduction of channel conductance, thus identifying a novel proton sensor on the hERG channel.
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McGuinness, James. "Implications of potassium channel heterogeneity for model vestibulo-ocular reflex response fidelity." Thesis, University of Stirling, 2014. http://hdl.handle.net/1893/21844.
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The Vestibulo-Ocular Reflex (VOR) produces compensatory eye movements in response to head and body rotations movements, over a wide range of frequencies and in a variety of dimensions. The individual components of the VOR are separated into parallel pathways, each dealing with rotations or movements in individual planes or axes. The Horizontal VOR (hVOR) compensates for eye movements in the Horizontal plane, and comprises a linear and non-linear pathway. The linear pathway of the hVOR provides fast and accurate compensation for rotations, the response being produced through 3-neuron arc, producing a direct translation of detected head velocity to compensatory eye velocity. However, single neurons involved in the middle stage of this 3-neuron arc cannot account for the wide frequency over which the reflex compensates, and the response is produced through the population response of the Medial Vestibular Nucleus (MVN) neurons involved. Population Heterogeneity likely plays a role in the production of high fidelity population response, especially for high frequency rotations. Here we present evidence that, in populations of bio-physical compartmental models of the MVN neurons involved, Heterogeneity across the population, in the form of diverse spontaneous firing rates, improves the response fidelity of the population over Homogeneous populations. Further, we show that the specific intrinsic membrane properties that give rise to this Heterogeneity may be the diversity of certain slow voltage activated Potassium conductances of the neurons. We show that Heterogeneous populations perform significantly better than Homogeneous populations, for a wide range of input amplitudes and frequencies, producing a much higher fidelity response. We propose that variance of Potassium conductances provides a plausible biological means by which Heterogeneity arises, and that the Heterogeneity plays an important functional role in MVN neuron population responses. We discuss our findings in relation to the specific mechanism of Desynchronisation through which the benfits of Heterogeneity may arise, and place those findings in the context of previous work on Heterogeneity both in general neural processing, and the VOR in particular. Interesting findings regarding the emergence of phase leads are also discussed, as well as suggestions for future work, looking further at Heterogeneity of MVN neuron populations.
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Garg, Vivek. "Regulation of ATP-Sensitive Potassium Channels in the Heart." The Ohio State University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=osu1238179085.
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Rezazadeh-Roudsari, Saman. "Structural and genetic modulators of voltage-gated potassium channel activation kinetics." Thesis, University of British Columbia, 2007. http://hdl.handle.net/2429/31467.
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Voltage-gated potassium (Kv) channels regulate membrane excitability and are therefore critical determinants of cellular function. However, the detailed mechanisms by which Kv channel activity is modulated are not well understood. This thesis investigates the modulation of activation of Kv1.2 and KCNQ1 channels. These studies reveal that Kv1.2 can activate via two different pathways that produce two distinct gating phenotypes/modes. In the 'slow' gating mode, the activation V 1/2 was shifted by +30 mV and activation kinetics were at least 20-fold slower than those of channels gating through the 'fast' mode. This offers an explanation for the wide variations in the reported activation kinetics of Kv1.2 in the literature. Introduction of a positive charge at or around threonine 252 (T252) in the S2-S3 cytoplasmic linker of Kv1.2 trapped channels in the 'fast' activation mode, suggesting that this region may act as the molecular switch in Kv1.2. Consistent with this, the S2-S3 linker was shown to mediate the gating-modifying effect of a mutation (T46V) in the cytoplasmic T1 domain of Kv1.2. Excision of patches containing Kv1.2 also trapped channels in the 'fast' gating mode, indicating cytoplasmic regulators may also modify the gating mode via the S2-S3 linker. We have ruled out cytoplasmic regulation by PIP₂, polyamines and phoshporylation. Interestingly, one kinase inhibitor, KN-93, a commonly used calcium/calmodulin-dependent protein kinase II inhibitor, was found to be a direct extracellular blocker of many different Kv channels including Kv1.2. Finally, a novel missense mutation at the intracellular end of the S3 helix in a mutant KCNQ1 channel (V205M), detected in an aboriginal community with a high prevalence of long QT syndrome and sudden death, was shown to cause a depolarizing shift in the voltage dependence of activation and a slowing of activation kinetics. This resulted in reduced repolarization reserve during the cardiac action potential and a likely increased susceptibility to the initiation of arrhythmias. The close positioning of this mutation to the S2-S3 linker provides a putative structural working model for the gating switch in Kv1.2 that involves changes in the hydrophobic packing of the S3 helix and its influence on S4 voltage-sensor movement.Medicine, Faculty of
Cellular and Physiological Sciences, Department of
Graduate
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Brasko, Csilla. "Expression of inward rectifier potassium channel subunits in optic nerve glia." Thesis, University of Portsmouth, 2013. https://researchportal.port.ac.uk/portal/en/theses/expression-of-inward-rectifier-potassium-channel-subunits-in-optic-nerve-glia(941b2bcc-471e-4ad4-affd-22547ba7533f).html.
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In glia inward rectifying potassium channels (Kir) are predominantly responsible for the high selective membrane permeability to K+, for the maintenance of the RMP close to the EK and for the clearance of excess K+ released during action potentials by the process of K+ buffering. In this study I have investigated the expression, subcellular localisation and heteromer forming ability of Kir4.1, Kir5.1 and Kir2.1 subunits in optic nerve glia. Immunocytochemistry results demonstrated the expression of Kir4.1, Kir5.1 and Kir2.1 subunits in the optic nerve astrocytes and oligodendrocytes. I used immunocytochemical approach and Western blot analysis of the optic nerve plasma membrane fraction to prove the in vitro and in vivo functional expression of Kir4.1, Kir5.1 and Kir2.1 subunits in optic nerve glia. As Kir4.1 is known to form heteromeric channels with the Kir5.1 and Kir2.1 subunit I investigated the in vivo and in vitro heteromer forming ability of these Kir subunits using co-immunoprecipitation and immunocytochemistry techniques and found evidence for heteromeric Kir4.1/Kir5.1, Kir4.1/Kir2.1 and Kir5.1/Kir2.1 and homomeric Kir4.1 channels in optic nerve astrocytes and oligodendrocyte. By using shRNA and genetic ablation of the Kir4.1 subunit I found that Kir4.1 regulates the expression and subcellular localisation of the Kir5.1 and Kir2.1 subunits. With Western blot analysis I demonstrated that in the absence of the Kir4.1 subunit Kir2.1, Kir6.1 and Na/K ATPase α1, the subunits involved in the K+ buffering, compensate for the lack of functional Kir4.1. However, as a result of the metabolic stress due to the increased ATP consumption by the Na/K ATPase the mitochondrial fraction and the activity of KATP channels were found to be increased in the brain of the Kir4.1 knock out mice. The increased expression of Na/K ATPase α1 subunit, known to be involved in K+ buffering, in the the absence of the Kir4.1 subunit affirms the role of Kir4.1 in K+ buffering. In astrocytes homomeric Kir4.1, Kir2.1 and heteromeric Kir4.1/Kir2.1, Kir4.1/Kir5.1 channels are believed to be involved in K+ buffering and their disfunction has been associated with epilepsy. Heteromeric Kir4.1/Kir5.1 channels are suggested to be involved in CO2/H+ chemosensation in the optic nerve. Due to the unique pH sensitivity of the Kir4.1/Kir5.1 channel, it has been identified as an astrocytic CO2/H+ chemoreceptor in the retrotrapezoid nucleus, associated with the control of respiration. Silent Kir5.1/Kir2.1 channels may regulate these processes. As optic nerve oligodendrocytes also express Kir4.1, Kir5.1 and Kir2.1 subunits, they may also be involved in K+ buffering and/or CO2/H+ chemosensation via these Kir channels, and therefore important for maintaining ionic homeostasis and normal cellular physiology.
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Knight, Jennifer Lynn. "Molecular dynamics simulation of ion permeation in a potassium ion channel." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape3/PQDD_0021/MQ54463.pdf.
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Deng, Qingwei 1968. "Identification of dendritic targeting signals of voltage-gated potassium channel 3." Thesis, McGill University, 2004. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=82219.
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Members of voltage-gated potassium channel subfamily 3 (Kv3) have been extensively demonstrated to play a significant role in facilitating function of "fast-firing" neurons in the central nervous system. Kv3.1 and Kv3.3 channels, members of Kv3 channel subfamily, have different distribution profiles on the regional level of brain and on the subcellular level of neurons in mammals and in weakly electric fish, according to mRNA hybridizations in situ and immunohistochemical analysis. In mammals, Kv3.1 channels are expressed in soma, axon and proximal dendrites as well as presynaptic membrane of "fast-firing" neurons. In weakly electric fish (Apteronotus), Kv3.1 channels are distributed in the soma, in the basilar dendrites and in the proximal apical dendrites of pyramidal neurons; on the other hand, Kv3.3 channels are expressed in a larger region: soma, basilar dendrites and entire apical dendrites of these cells. Mechanisms underlying differential subcellular distribution of Kv3.1 and Kv3.3 channels in the apical dendritic compartment of pyramidal neurons are unknown. In order to identify peptide sequences responsible for the differential subcellular localization, I have used Semliki Forest virus as a modified viral expression system (PDE) in vivo to study dendritic targeting mechanisms in the pyramidal neurons of electrosensory lateral line lobe (ELL), where the primary processing for afferent input occurs in the apteronotid electrosensory system.
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Ketchum, Karen Ann. "A calcium-dependent potassium channel in corn (Zea mays) suspension cells /." Thesis, McGill University, 1990. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=74658.
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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.
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Thomas, J. "The effects of homocysteine on potassium channel function in human platelets." Thesis, University of Oxford, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.270751.
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Davies, Lowri Meryl. "Caveolins in the vasculature : a role in regulating potassium channel function." Thesis, University of Liverpool, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.548771.
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McPate, Mark John William. "hERG potassium channel electrophysiology and pharmacology in the short QT syndrome." Thesis, University of Bristol, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.486078.
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K+ channels mediating the rapid delayed rectifier current (lKr) are encoded by human ether-a-go-go-related gene (hERG) and play an important role in determining cardiac action potential (AP) repolarisation and the QT interval of the electrocardiogram. A gain-of-function hERG mutation (N588K) is associated with variant 1 of the Short QT syndrome (SQT1), which is characterised by short QT intervals «320 ms) and increased risk of cardiac arrhythmias and sudden death. Using whole-cell patch-clamp recordings ofhERG current (lhERG) from Chinese Hamster Ovary cells expressing WildType (WT) or N588K-hERG at 37°C, I investigated the effects of the N588K mutation on hERG channel electrophysiology and pharmacology. The N588K mutation produced a -+60 mV shift in IhERG availability, without concomitant alterations ofthe voltage-dependence of activation or deactivation kinetics OfIhERG. N588K IhERG peaked much earlier than did WT IhERG during guinea-pig and human ventricular AP command waveforms. The N588K mutation also resulted in IhERG peaking earlier during atrial and Purkinje fibre AP commands. MiRP1 co-expression with hERG had little effect on the timing ofpeak repolarising current during AP commands, but did affect maximal IhERG density. Results of experiments using paired AP waveforms raise the possibility that the potentially protective role ofhERG against premature depolarisations may to an extent be compromised for N588K-hERG. The N588K mutation differentially attenuated IhERG block of selected antiarrhythmic drugs. My data indicate that in addition to quinidine, disopyramide and amiodarone may be useful pharmacological treatments for SQT1. Co-expression ofhERG-1a with hERG-1b accelerated WT and N588K IhERG deactivation kinetics and for N588K increased attenuation of inactivation compared to hERG-1a alone. The differences in IhERG-Iallb kinetics as a result of the N588K mutation did not greatly influence currents during AP command waveforms, except that peak repolarising current occurred earlier during the AP than for hERG 1a alone.