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1
Roberts, Dewi. "Calcium-dependent inactivation of Cav1.3 calcium channels". Thesis, University of Bristol, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.446186.
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Peterson, Blaise. "Molecular determinants of dihydropyridine binding on L-type calcium channels /". Thesis, Connect to this title online; UW restricted, 1996. http://hdl.handle.net/1773/6269.
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Farrington, Jasmine. "Calcium release activated calcium channels in human lung mast cells". Thesis, University of Sheffield, 2014. http://etheses.whiterose.ac.uk/6609/.
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Nakayama, Shinsuke. "Calcium channels in detrusor smooth muscle". Thesis, University of Oxford, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.334328.
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Xie, Mian. "Calcium Channel Beta Subunits and SCA6-Type Calcium Channel Alpha Subunits C-Termini Regulate Targeting and Function of Presynaptic Calcium Channels in Hippocampal Neurons". Case Western Reserve University School of Graduate Studies / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=case1188326628.
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Yasuda, Takahiro. "Modulation of calcium channel function and toxin sensitivity by auxiliary subunits /". [St. Lucia, Qld.], 2004. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe18052.pdf.
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Doughty, Stephen William. "Molecular modelling of voltage-gated calcium channels". Thesis, University of Oxford, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.362014.
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Warburton, Steven Peter Marc. "Calcium ion channels in insect skeletal muscle". Thesis, University of Nottingham, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.363592.
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Richardson, C. Mark. "Presynaptic calcium channels in skate electric organ". Thesis, University of Nottingham, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.319614.
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Pearson, Hugh Anthony. "Physiology and pharmacology of insect calcium channels". Thesis, University College London (University of London), 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.308295.
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Brosnan, James N. "Biochemical characterization of red beet calcium channels". Thesis, University of York, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.304314.
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Burgess, Alison J. "Immunological characterization of voltage-sensitive calcium channels". Thesis, University of Leicester, 1988. http://hdl.handle.net/2381/34291.
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A panel of monoclonal antibodies were raised against the 1,4-dihydropyridine sensitive Ca2+ channel of rabbit skeletal muscle. When tested on immunoblot assay of denatured and reduced transverse tubule membranes, four of the antibodies specifically recognized a polypeptide of Mr 140,000. This component co-migrated with the large glycoprotein ?2 subunit of purified Ca2+ channel preparations. On immunoblots of nonreducing gels the antibodies detected a component that migrated more slowly in the gel, with a Mr of 170,000, consistent with the disulphide-linkage of the ?2 subunit to a small component of Mr 30,000. Additionally, three of the antibodies also recognized high molecular weight components of Mr 310,000-330,000 under these conditions. Crossreactive polypeptides of similar apparent molecular weight were detected in immunoblot assays of rabbit heart and brain membranes and of skeletal muscle membranes from different species. Further similarities between the ?2 components of Ca2+ channels from different species were investigated by immunoblot assay, following the limited tryptic digestion of the skeletal muscle membranes. A similar pattern of immunoreactive peptides were detected in each case, suggesting that the ?2 subunits of Ca2+ channels from different species are similar, not only in terms of antibody binding sites but also with respect to similarly positioned trypsin cleavage sites. The extent of glycosylation of the ?2 component was investigated using enzymatic and chemical deglycosylation techniques. Chemical deglycosylation resulted in a core polypeptide of Mr 105,000, consistent with a carbohydrate content of approximately 25%. Enzymatic treatments, although insufficient to completely deglycosylate the ?2 component, reduced the maximal 1,4-dihydropyridine binding capacity of transverse tubule membranes by 73-77%. The co-development of the ?2 subunit with 1,4- dihydropyridine binding activity was shown in rat skeletal muscle. These results indicate that the '2 subunit is an integral structural component of the 1,4-dihydropyridine sensitive Ca2+ channel.
13
Georgiou, Panayiotis Paulou. "Calcium-activated potassium-channels in mammalian eggs". Thesis, University of Edinburgh, 1985. http://hdl.handle.net/1842/29774.
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Ruchala, Iwona. "EXPANDING MONOAMINE TRANSPORTERS PHARMACOLOGY USING CALCIUM CHANNELS". VCU Scholars Compass, 2017. http://scholarscompass.vcu.edu/etd/5032.
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Research in drug development meets many challenges including lengthy, complex and costly procedures to identify novel pharmacotherapies. In our lab, we developed a method for fast screening of small molecules that interact with monoamine transports – dopamine and serotonin (DAT, SERT). These membrane proteins play important roles in brain neurotransmission responsible for cognition, motion and pleasure. Dysfunction in dopaminergic and serotonergic systems result in neurological disorders such as depression, Attention Deficit Hyperactivity Disorder (ADHD), schizophrenia and addiction.
DAT and SERT are responsible for uptake of dopamine (DA) or serotonin (5HT) into the synapse and they limit neurotransmitter signaling. Drugs that mimic or antagonize actions of endogenous neurotransmitters (DA and 5HT) increase the concentrations of DA and/or 5HT either by blocking the transporter (blockers) or by competing uptake with neurotransmitter (substrate). The uptake of substrates is associated to an inward current that depolarizes the cell membrane. Voltage-gated calcium channels (CaV) can respond to small changes in membrane potential. In our method, we combined permanent cell line expressing the human dopamine transporter (hDAT) or the human serotonin transporter (hSERT) (FlpIn TREx expression system) with transient transfection of CaV. This system works as a tightly electrically coupled system. Cells challenged with substrate of the transports produce detectable Ca2+ signal while monoamine transporter blockers can inhibit these Ca2+ signals. The novelty of this method relies on the ability to discriminate between substrate and blockers of monoamine transporters.
Preliminary experiments measuring our optimized cell system in a Flex Station 3 plate reader suggest that the co-expression of a voltage-gated Ca2+ channel, a monoamine transporter and a genetically encoded Ca2+ sensor constitute a rapid screening biosensor to identify active drugs at monoamine transporters.
Our novel methodology can rapidly assess drug-effect profile on monoamine transporters and benefit development of new psychotherapeutics for treatment of mental illnesses. It can also be used to characterize mechanism of action of emerging drug of abuse, as well as to discover small molecules with novel drug-effect profile useful in basic neuroscience research.
15
Wang, Shiyi. "Regulation of voltage-gated calcium channels Cav1.2". Diss., University of Iowa, 2017. https://ir.uiowa.edu/etd/6009.
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Voltage-gated Ca2+ (Cav) channels are activated upon depolarization. They specifically allow Ca2+ ions to come into the cell. These Ca2+ ions are bi-functional because they not only control cell excitability but also couple electrical activity to complex downstream signaling events, such as excitation-contraction coupling in muscles and neurotransmitter release in neurons. In the brain, Cav channels are expressed in the pre- or post-synaptic membrane of most excitable cells, neurons. In the past few years, their expression and function have also been characterized in many nonexcitable cells such as astrocytes. This dissertation focuses on the regulation of one subtype of postsynaptic Cav channels, Cav1.2, in neurons. In the first part of chapter I, I provide a literature overview of Cav channels in terms of their subtypes, localizations, physiological functions, and biophysical properties.
For years, Cav channels were studied as single entities. But now, based on multiple proteomic studies, we know that these channels actually do not live alone. They interact with numerous proteins depending on the physiological conditions. Such interactions can anchor the channels to optimal sites of action, and tether Cav channels to their modulatory molecules. Therefore, it is crucial to understand how Cav channels are regulated by their macromolecular assembly. Among these protein partners, our lab studied the regulation of Cav channels by a subset of PDZ-domain containing proteins. Because these proteins play an important role in scaffolding and they colocalize with both pre- and post-synaptic Cav channels. Indeed, previous studies from our lab and other groups have revealed that PDZ proteins participate in a multitude of Cav regulation. The second part of chapter I introduces the diverse modulation of neuronal Cav channels by numerous PDZ proteins.
In neurons, Cav1.2 channels regulate neuronal excitability and synaptic plasticity. Their functions have been implicated in learning, memory, and mood regulation. A study published in the journal Lancet showed that the gene encoding Cav1.2 is a common risk factor for five major psychiatric disorders. A PDZ protein, densin-180 (densin) is an excitatory synapse protein that promotes Ca2+-dependent facilitation of voltage-gated Cav1.3 Ca2+ channels in transfected cells. Mice lacking densin exhibit similar behavioral phenotypes that closely match those in mice lacking Cav1.2. In chapter II and III, we investigated the functional impact of densin on Cav1.2 channels and their auxiliary subunit β2a.
Besides the regulation of Cav channels by their interactome, we have also known for a long time that Ca2+ currents undergo a negative feedback regulation. This regulation is called Ca2+-dependent inactivation (CDI) and it is mediated by Ca2+ that directly traverses the pore. CDI has been described for Cav channels in multiple cell types. In the heart, CDI prevents excessively long cardiac action potentials, which in turn can prevent activity-dependent arrhythmia. In neurons, CDI may be neuroprotective by preventing excitotoxic Ca2+ overloads. In the last 18 years, two essential components have been revealed in the mechanism of CDI. One is the protein calmodulin (CaM). CaM interacts directly with sites on the C-terminus of Cav channels. It binds to the incoming Ca2+ ions and produces a mysterious conformational change that determines the conductance of the channel. The other molecular player is Cavβ protein family. Cavβ comprises four subfamilies β1 through β4, which generally enhance the channel inactivation, except β2a. In chapter IV, Xiaohan Wang from Roger Colbran’s lab in Vanderbilt University, and I identified a new molecular determinant for Cav1.2 CDI.
The α2δ subunit is an extracellular component of the Cav channel complex. Similar to Cavβ subunits, α2δ subunits are essential for the biophysical properties, surface level, and trafficking of Cavα1 subunits. There are four isoforms of α2δ subunits (α2δ1 to α2δ4). They display distinct tissue distributions. Although the roles of α2δ subunits in Cav channel regulation were studied extensively, studies have proposed that the function of α2δ subunits may be in part or entirely independent of Cav channel complex, such as synaptogenesis. Considering the important role of α2δ in physiology and pathology, it is imperative to identify the factors that regulate the properties of α2δ. In chapter V, I explored the trafficking dynamics of α2δ1 and revealed a potential regulator of α2δ1 for its protein stability and localization.
One beauty of doing research is that it always motivates us to think and ask more questions on our journey of demystifying nature. While looking at the evidence that I find, I realize how much more we could do in the future. In chapter VI, I conclude the findings of each chapter and share my perspectives on the future direction for these research projects.
16
Pifferi, Simone. "Calcium Activated Chloride Channels In Olfactory Transduction". Doctoral thesis, SISSA, 2008. http://hdl.handle.net/20.500.11767/4668.
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Ca2+-activated Cl ̄ channels are an important component of olfactory transduction. Odorant binding to odorant receptors in the cilia of olfactory sensory neurons (OSNs) leads to an increase of intraciliary Ca2+ concentration by Ca2+ entry through cyclic nucleotide-gated channels. Ca2+ activates a Cl ̄ channel that leads to an efflux of Cl ̄ from the cilia, contributing to the amplification of the OSN depolarization. The molecular identity of this Cl ̄ channel remains elusive. Recent evidences have indicated that bestrophins are able to form Ca2+-activated Cl ̄ channels channels in heterologous systems. Immunohistochemistry revealed that mBest2 was expressed on the cilia of OSNs, the site of olfactory transduction, and co-localized with the main subunit of cyclic nucleotide-gated channels, CNGA2. We performed a functional comparison of the properties of Ca2+-activated Cl ̄ channels from native channels expressed in dendritic knob/cilia of mouse OSNs with those induced by heterologous expression of mBest2 in HEK-293 cells. Even if the two channels did not display identical characteristics, they have many similar features such as the same anion permeability, the Ca2+ sensitivity in micromolar range and the same side-specific blockage of the two Cl ̄ channel blockers commonly used to inhibit the odorant-induced Ca2+-activated Cl ̄ channels in OSNs, niflumic acid and 4-acetamido-4’-isothiocyanato-stilben-2,2’-disulfonate (SITS). However electroolfactogram recording from mBest2 null mice showed a normal sensitivity to odorant stimulation. Therefore mBest2 is a good candidate for being a molecular component of the olfactory Ca2+-activated Cl ̄ channels but its precise role in olfactory transduction remains to be clarified.
17
Muller, Yunhua Li 1963. "Developmentally regulated expression of the calcium-dependent potassium channel and calcium channels during maturation of the rat cerebellum". Diss., The University of Arizona, 1996. http://hdl.handle.net/10150/282231.
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Potassium channels govern the duration and frequency of excitable membrane events, and thus may regulate voltage-dependent signals that are important in neuronal development. This study assesses the developmental expression of two classes of K⁺ channels in vivo and in vitro in the rat cerebellum. In vivo, the level of mslo-related transcript for the Ca²⁺-dependent K⁺ channel (KCa) was shown by Northern analysis to be upregulated during development, whereas transcripts for delayed rectifier (KD) channels remained fairly constant. The same pattern of in vivo development was demonstrated with functional assays by expression in Xenopus oocytes of poly A-enriched RNA isolated from postnatal rat cerebella. In vitro, single channel studies of Purkinje neurons showed that KCa channel activity was increased during development and KD channel activity remained stable. Although the semi-quantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR) showed that the level of transcripts of the KCa channel sequence remained constant in control culture, the developmental pattern that was seen in vivo was mimicked in vitro when cultures were treated chronically with tetraethylammonium (TEA, 1mM). Chronic treatment with 10 mM extracellular KCl resulted in an upregulation of KCa transcripts similar to that seen with chronic TEA. The stimulatory effects of TEA or KCl were negated in low external calcium (0.1 mM), suggesting that KCa transcript levels were influenced by depolarization and calcium entry. The KCa channels may in part contribute to the mature electrical properties of Purkinje neurons. This was supported by evidence that developmental trends in cellular firing activity were antagonized by decreased KCa channel abundance caused by chronic treatment with TEA. Voltage-gated Ca²⁺ channels (N, R and P type) were developmentally down-regulated at the transcriptional level in control cultures. Chronic treatment with TEA increased the transcript levels for N and R type Ca²⁺ channels, but not for P type, suggesting that the various types of Ca²⁺ channels were differentially regulated. Ca²⁺ signaling plays a key role in neuronal development in many cells. The KCa and Ca²⁺ channels regulate Ca²⁺-entry, and may thus influence the neuronal differentiation.
18
Hagen, Brian M. "Regulation of calcium-activated potassium channels by localized calcium transients in murine colon". abstract and full text PDF (free order & download UNR users only), 2005. http://0-gateway.proquest.com.innopac.library.unr.edu/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3209955.
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Victory, Jason G. G. "Ion channels and the myocardium : interactions between general anaesthetics and calcium channel blockers". Thesis, University of Oxford, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.253420.
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Balasubramanian, Bharathi. "Regulation of cloned cardiac channels". Texas A&M University, 2005. http://hdl.handle.net/1969.1/2525.
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Activation of a5??1 integrin potentiates L-type calcium current in vascular smooth
muscle, which is partly mediated by tyrosine phoshorylation of the a1c channel subunit.
Expressed rabbit VSM and neuronal isoforms are also potentiated by a5??1 integrin
activation and require dual phosphorylation of a1c by PKA and c-Src. To explore
common mechanisms of regulation by a5??1 integrin, whole cell patch clamp experiments
were used to investigate the effects of a5??1 integrin antibody on expressed cardiac
calcium channels. In HEK cells transfected with a1c, ??2a and a2-d1 subunits alone,
currents increased 1.8 ?? 2.0 fold on application of a5??1 antibody. The potentiation was
almost completely abolished on the application of PKI, a highly specific Protein Kinase
A (PKA) inhibitor. The expressed currents increased 2.0 ?? 2.2 fold on application of
PKA activator 8-Br-cAMP, and abolished by PKI. Our results suggest that regulation of
L-type calcium channels by a5??1 integrin is a general mechanism shared by VSM,
neuronal and cardiac channels. However, in the cardiac isoform, only PKA
phosphorylation is involved.
21
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.
22
Yassine, Maya. "Calcium, Calcium-permeable channels and autophagy modulators in control of autophagy and cancer". Thesis, Lille 1, 2013. http://www.theses.fr/2013LIL10159/document.
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L'autophagie est une voie cellulaire strictement régulée dont le but principal est la dégradation lysosomale et le recyclage ultérieur du matériel cytoplasmique afin de maintenir l'homéostasie cellulaire normale. Des défauts dans l'autophagie sont liés à une variété d'états pathologiques, dont le cancer. Le cancer est une maladie associée aux modifications des processus cellulaires fondamentaux tels que l'apoptose et l'autophagie. Le calcium régule une série de processus physiologiques et pathologiques tels que le vieillissement, la neurodégénérescence et le cancer. Si le rôle du calcium et des canaux calciques dans le cancer est bien établi, l'information sur la nature moléculaire des canaux régulant l’autophagie ainsi que les mécanismes de cette régulation reste encore limitée. Le rôle de l'autophagie dans le cancer est complexe. En effet, elle peut favoriser à la fois la prévention tumorale et la résistance aux traitements. Elle est souvent détectée dans les cellules cancéreuses en réponse aux expositions aux rayons et la chimiothérapie. Elle semble contribuer à la résistance thérapeutique de certains cancers. Il est maintenant bien établi que sa modulation peut potentiellement contribuer à la mise en œuvre des méthodes de traitement du cancer. Dans cette étude, nos travaux ont permis d’identifier le calcium intracellulaire, comme un régulateur important de l'autophagie. Ainsi, nous proposons un lien possible entre le calcium, les canaux calciques, l’autophagie et la progression du cancer. De plus, nous avons mis en évidence un nouveau modulateur de l’autophagie, le ML-9. Cet outil pourrait cibler l'autophagie et être utilise dans le traitement des cancersAutophagy is a tightly regulated cellular pathway the main purpose of which islysosomal degradation and subsequent recycling of cytoplasmic material to maintain normal cellular homeostasis. Defects in autophagy are linked to a variety of pathological states,including cancer. Cancer is the disease associated with abnormal tissue growth following an alteration in such fundamental cellular processes as apoptosis, proliferation, differentiation,migration and autophagy. Calcium is a ubiquitous secondary messenger which regulates plethora of physiological and pathological processes such as aging, neurodegeneration and cancer. The role of calcium and calcium-permeable channels in cancer is well-established, whereas theinformation about molecular nature of channels regulating autophagy and the mechanisms of this regulation is still limited. The role of autophagy in cancer is complex, as it can promoteboth tumor prevention and survival/treatment resistance. Elevated autophagy is often detected in cancer cells in response to radiation and chemotherapy. Furthermore, autophagy seems to contribute to the therapeutic resistance of some cancers. It's now clear that modulation of autophagy has a great potential in cancer diagnosis and treatment. Our findings identified intracellular calcium as an important regulator of autophagy. We propose a possible link between calcium, calcium permeable ion channels, autopohagy and cancer progression. Further, our results revealed a new autophagy modulator ML-9 as an attractive tool for targeting autophagy in cancer therapy
23
Gelli, Angie Cristina. "The electrophysiological characterization of plant calcium channels and their role in calcium signaling". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0007/NQ27928.pdf.
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Calcraft, Peter James. "Two-pore channels and NAADP-dependent calcium signalling". Thesis, St Andrews, 2010. http://hdl.handle.net/10023/888.
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Conrad, Rachel [Verfasser]. "Trafficking of voltage-activated calcium channels / Rachel Conrad". Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2017. http://d-nb.info/1136717919/34.
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Millership, Joanne Ella. "Regulation and function of calcium-activated potassium channels". Thesis, University of Manchester, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.503016.
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Muir, Shelagh R. "Ligand-gated calcium channels in higher plant membranes". Thesis, University of York, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.319717.
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Wajdner, Hannah. "Characterisation of calcium channels in human immune cells". Thesis, University of Sheffield, 2015. http://etheses.whiterose.ac.uk/12414/.
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Stevens, Louisa Rebecca. "Structure and function of potassium and calcium channels". Thesis, University of Leeds, 2005. http://etheses.whiterose.ac.uk/382/.
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In this thesis, potassium channels human Kv2.1 and rat Kv2.1, along with calcium channels Ca, 1.2, Ca, 3.1, and chimeras have been studied. These channels were expressed in Xenopuso ocytesf or electrophysiological experiments using two electrode voltage clamp. For protein expression studies,DNA was expressed in BL21 or COS-7 cells and purified using glutathione or an ID4 affinity column. Firstly, the roles of the N- and C- terminal domains in the activation kinetics of rat and human forms of Kv2.1 were investigated. A mutant in the N- terminal domain and chimeras between the rat and human forms were constructed. All clones were expressed in Xenopuso ocytesa nd activation times obtained. The results suggested that key residues in the N- and C- terminal domains are involved in determining the activation kinetics of rat and human Kv2.1. Further experiments were carried out using GST fusion proteins, Biacore surface plasma resonance and FRET investigations. These confirmed that the N and C- terminal domains are important in determining activation kinetics, and that these regions interact. To determine the positions of both the N- and C- terminal domains in the folded channel, rat Kv2.1 and a C-terminal deleted protein were expressed purified, and shown to have correct protein folding. These samples were sent for electron microscopy experiments and a preliminary picture obtained. No studies of S4 movement in calcium channels have been reported previously. Cysteine residues were substituted into the domain I S4 of a calcium channel chimera (with domain I of Ca, 3.1 replaced by Caj. 2). Cysteine residues at positions 263,265,266,268,269 and 271 were characterized by electrophysiology. Cysteine mutants at residues 263,265,266 and 268 reacted when extracellular PCMBS was applied, but mutants at residues 269 and 271 did not. This suggests that under depolarising conditions the S4 segment is exposed to the extracellular environment up to and including residue 268, with residues 269 and 271 remaining buried. Further investigation of residue 263 indicated that this movement occurs at potentials more negative than the resting membrane potential of -80mV. The data suggests that under depolarisation, the S4 becomes exposed to the extracellular solution and this movement of the S4 occurs before the ionic flow.
30
Pardo, Pastor Carlos 1989. "Piezo ion channels in cancer cell mechanotransduction". Doctoral thesis, Universitat Pompeu Fabra, 2018. http://hdl.handle.net/10803/664209.
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The mechanical dependence of transformation and metastasis is an emerging field, but the role of mechanosensitive channels has been largely omitted. This thesis focuses on the roles played by the mechanosensitive ion channels Piezo1 and Piezo2 in the transduction of mechanical stimuli (confinement, adhesion, substrate rigidity, adhesive ligand concentration) by cancer cells.
In a first chapter, we show that confinement triggers Piezo1-mediated calcium entry. This activates phosphodiesterase 1, reducing cAMP levels and, consequently, PKARac1 activity, relieving Myosin II from its inhibition. We also find a parallel, direct activation of Myosin II by confinement. As a combined result, cells stiffen and optimize their adhesion-free migration mode, usually responsible for in vivo migration during metastatic invasion. Piezo1 knockdown supresses confinement-induced calcium entry and impairs the underlying circuitry in ovarian epithelial (CHO) or melanoma (A375) cells. As a result, siPiezo1 cells show reduced migratory capacity under confinement.
In the second chapter, we discover an essential role for Piezo2 as a transducer of environmental mechanical cues into RhoA activation to modulate the mechanobiological responses of MDA-MB-231-BrM2 brain metastatic breast cancer cells. Piezo2 knockdown disturbs stress fibre formation, adhesion orientation, force transmission and nuclear accumulation of the malignant co-transcriptional activator YAP, and this is phenocopied by extracellular calcium suppression. Promoting Actin polymerization with jasplakinolide or by over-expressing constitutively active forms of Rho or mDia1 restores stress fibres and nuclear YAP accumulation. In addition, Piezo2 knockdown disrupts several pro-metastatic functions: cell proliferation, migration, invadopodia formation, extracellular matrix degradation, and secretion of SERPINB2, a protein needed for protecting invasive cells from brain parenchymal defence mechanisms.
The works presented in this thesis unveil important roles for Piezo channels as a first line of mechanical input detectors in distinct cells. These discoveries are relevant for several fields, e.g. cancer research, and highlight the importance of ion channels as transducers of environmental stimuli.La dependència mecànica de la transformació i la metàstasi és un camp d’estudi / de recerca emergent, però el paper que hi juguen els canals iònics mecanosensibles s’ha omès fins ara. Aquesta tesi se centra en els rols dels canals Piezo1 i Piezo2 en la transducció d’estímuls mecànics per cèl·lules canceroses, com ara confinament, adhesió, rigidesa del substrat, concentració de lligands adhesius. En un primer capítol, mostrem que el confinament dispara l’entrada de calci per mitjà de Piezo1. Això activa la fosfodiesterasa 1, que redueix els nivells d’AMPc i, en conseqüència, l’activitat PKARac1, que deixen d’inhibir Miosina II. També trobem una activació paral·lela de Miosina II directament per confinament. Com a resultat final, les cèl·lules guanyen rigidesa i optimitzen el seu mode migratori independent d’adhesions, que és el preponderant in vivo durant la invasió metastàtica. Reduir els nivells de Piezo1 suprimeix l’entrada de calci induïda per confinament i desactiva el circuit subjacent en cèl·lules ovàriques epitelials (CHO) i de melanoma (A375). Això minva la capacitat migratòria de les cèl·lules siPiezo1. En un segon capítol, descobrim un rol essencial per a Piezo2 com a activador de RhoA en resposta a estímuls mecànics. Això modula les respostes mecanobiològiques de les cèl·lules MDA-MB-231-BrM2, de càncer de mama metastàtic a cervell. La reducció dels nivells de Piezo2 destorba la formació de fibres d’estrès, l’orientació de les adhesions, la transmissió de forces i l’acumulació nuclear del regulador transcripcional prometastàtic YAP. Suprimir el calci extracel·lular fenocòpia aquests resultats. Promoure la polimerització d’Actina amb jasplaquinolida o mer mitjà de la sobreexpressió de formes constitutivament actives de RhoA o mDia1 restableix les fibres d’estrès i l’acumulació nuclear de YAP. A més, la reducció de Piezo2 suspèn diverses funcions prometastàtiques: proliferació cel·lular, migració, formació d’invadopodis, degradació de la matriu extracel·lular i secreció de SERPINB2, una proteïna necessària per protegir les cèl·lules invasores dels mecanismes de defensa del parènquima cerebral. Els treballs presentats en aquesta tesi desvelen rols importants pels canals Piezo com a una primera línia de detectors d’estímuls mecànics en diferents tipus cel·lulars. Aquests descobriments són rellevants per a diversos àmbits, com ara la recerca en càncer, i remarquen la importància dels canals iònics com a transductors d’estímuls ambientals.
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Lao, Yuanzhi. "Calcium signaling in apoptotic mammalian cells /". View abstract or full-text, 2008. http://library.ust.hk/cgi/db/thesis.pl?BIOL%202008%20LAO.
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Zhang, Hongling. "Sigma Receptors Modulation of Voltage-gated Ion Channels in Rat Autonomic Neurons". [Tampa, Fla.] : University of South Florida, 2005. http://purl.fcla.edu/fcla/etd/SFE0001183.
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Thrower, Edwin C. "A study of the inositol (1,4,5) triphosphate-sensitive Ca'2'+ channel". Thesis, University of East Anglia, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.361749.
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Forristal, Ailish. "MRS studies on the role and function of divalent cations in the cerebral cortex". Thesis, University of Nottingham, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.299709.
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Cifelli, Carlo. "Impairment of force development in K(ATP) channel deficient skeletal muscle involves calcium ion influx through L-type calcium ion channels". Thesis, University of Ottawa (Canada), 2006. http://hdl.handle.net/10393/27342.
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ATP-sensitive potassium (KATP) channels link membrane excitability to metabolism. They are regulated by intracellular nucleotides and other factors, and have been shown to play a role in development of skeletal muscle force, but controversy surrounds their role during fatigue. The aim of this research project was to determine the role of KATP channel under conditions that allow for better assessment of changes in force during fatigue, by virtue of using a smaller whole muscle model less subject to anoxia. Thus, the first objective was to determine the effect of the loss of KATP channel activity on force during fatigue in small FDB muscle bundles. KATP channel deficient fibers had faster and greater decreases in peak tetanic force during fatigue, developed greater resting tension, and had lower force recovery following fatigue compared to control wild type muscles. The second objective was to determine whether the functional impairment in skeletal muscle without KATP channel activity was due to an increase in Ca 2+ influx. When [Ca2+]e was reduced or L-type Ca2+ channels partially blocked, Kir6.2-/- FDB muscle had slower fatigue development, less resting tension, and had an improved force recovery.
A novel phenomenon was observed while studying the effect of KATP channel activity in vitro. During a second bout of fatigue the decrease in peak tension was significantly lower than the decrease during the first bout of fatigue. Furthermore, the deleterious effects of the loss of KATP channel activity during an initial fatigue were absent during the second fatigue in FDB exposed to glibenclamide.
It is concluded (i) that the KATP channel is important to prevent impairment of function during fatigue, (ii) that this impairment of function is due to an increase in Ca2+ influx through L-type Ca2+ channels, causing Ca2+ overload, and (iii) that fatigue resistance increases while the dependency on the KATP channel to prevent function impairment and fiber damage decreases following one fatigue bout at 37°C; a phenomenon here termed fatigue pre-conditioning.
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Kouzai, Daisuke. "Chemical biological studies on oxidation status-sensitive calcium channels". 京都大学 (Kyoto University), 2014. http://hdl.handle.net/2433/188546.
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Skeer, Jacqueline Mary. "Calcium channels of insect nervous system and skeletal muscle". Thesis, University of Cambridge, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.239592.
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Harding, Louise M. "The effects of locusta peptides on mammalian calcium channels". Thesis, Oxford Brookes University, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.389106.
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Hendrich, Janek K. "Alpha-2-delta subunits of voltage-gated calcium channels". Thesis, University College London (University of London), 2008. http://discovery.ucl.ac.uk/1444197/.
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The calcium channel alpha-2-delta (0,28) subunit is an auxiliary subunit associated with voltage-dependent calcium channels. It is implicated in the trafficking and functional expression of the calcium channel complex. This study expands the functional role of the VWA domain and the RRR motif of the 0:26 subunit, and the interaction between this subunit and the anti-epileptic drug, gabapentin The VWA domain is normally found in integrins, where it mediates binding to extracellular proteins. A mutation in the 0:28-2 subunit VWA domain (uMIDAS) did not produce the increase in current amplitude elicited by the wildtype (WT) 0:28-2 control. Co-immunoprecipitation studies using tsA-201 cells (stably expressing OC28-2 containing a mid-HA tag) co-cultured with cerebellar granule cells identified potential proteins from the cerebellar cultures that co-immunoprecipitate with the 0:28-2 protein. This suggests that protein from cerebellar cultures may bind the 0:28-2 subunit. Secondly, an RRR motif found in 0128-I subunit has been implicated as important for binding of the anticonvulsant drug gabapentin (Wang et al., 1999). The electrophysiological properties of the RRA mutant OC28 proteins were examined, and found not to enhance current amplitude to the full extent seen by WT 0:28-1 and 0:28-2 coexpression. Binding studies using both membrane and lipid raft of tsA-201 cells expressing 0:28-1 confirmed a lack of 3H-gabapentin in the R217A mutant condition. Chronic exposure of tsA-201 cells expressing Cav2.1, p4, 0:28-2 or Cav2.2, pib, 0:28-1 to gabapentin resulted in a reduction in size of the resultant currents. The inhibitory action of gabapentin was prevented by pre-incubation of cells with the system-L amino-acid transport inhibitor, suggesting gabapentin acts intracellularly after uptake via this transport mechanism. The inhibitory effects of gabapentin exposure were not replicated using co-expression of 0:28-3, or the non gabapentin-binding mutant 0128-I R217A or a28-2 R282A proteins. This indicated the inhibitory effect was mediated through gabapentin-binding calcium channel a28 subunits.
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Wang, Ming Chuan. "Structural studies of L-type voltage-gated calcium channels". Thesis, University of Manchester, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.525174.
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Dedman, Alexandra Margaret. "Regulation of expression and function of TRPC calcium channels". Thesis, University of Leeds, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.436392.
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Brice, Nicola. "Voltage dependent calcium channels : subcellular subunit localisation and targeting". Thesis, University College London (University of London), 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.299977.
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Gillard, Samantha Ellen. "Voltage-dependent calcium channels of cerebellar neurones in culture". Thesis, Royal Veterinary College (University of London), 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.268052.
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Zeng, Bo. "Pharmacological regulation and function of store-operated calcium channels". Thesis, University of Hull, 2012. http://hydra.hull.ac.uk/resources/hull:6432.
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Store-operated Ca²⁺ entry (SOCE) is an important Ca²⁺ influx pathway existing in almost all types of mammalian cell. STIM1, ORAI and TRPC have been regarded as the molecular basis of SOCE. Once the endoplasmic reticulum (ER) Ca²⁺ store is depleted, STIM1 proteins move to the plasma membrane and activate ORAI and TRPC channels to allow Ca²⁺ influx. In this thesis, the pharmacological aspects and regulatory mechanisms of SOCE were investigated using HEK293 cells overexpressing STIM1, ORAI or TRPC genes. The expression and function of TRPC channels and their spliced variants in native cells were also examined. Using live-cell imaging, the cytosolic clustering of STIM1-EYFP was observed after challenging with the compounds including 2-APB, flufenamic acid, 4-chloro- 3-ethylphenol, U73122 and FCCP. The aggregation of STIM1 in the cytosol coud be a novel mechanism for the inhibition of SOCE, and the process did not rely on the depletion of ER Ca²⁺ store. The ryanodine receptor agonist 4-chloro-3-ethylphenol not only caused Ca²⁺ release and cytosolic STIM1 clustering, but also nonselectively inhibited ORAI1/2/3 and TRPC3/4/5/6 channels. The sensitivity of TRP channels to metal ions was also investigated using patch clamp. Micromolar Cu²⁺ significantly increased the currents of TRPC3/4/5/6 channels. The glutamic acid (E542/E543) and cysteine (C554) residues in the extracellular pore region of TRPC4 were involved in the channel opening by Cu²⁺. Moreover, Cu²⁺ showed inhibitory effect on TRPM2 channel. TRPC1/3/4/6 and multiple alternatively spliced variants were detected in the human ovarian adenocarcinoma-derived SKOV3 cells. Blockade of TRPC channel activity by 2-APB, SKF-96365, TRPC pore-blocking antibodies or transfection with TRPC siRNA significantly inhibited SKOV3 cell proliferation. Overexpression of TRPC genes promoted colony growth of SKOV3 cells. It is concluded that cytosolic STIM1 movement could be a new pharmacological target for SOCE. 4-Chloro-3-ethylphenol and Cu²⁺ are new modulators of ORAI and TRP channels. TRPC channels and their spliced variants are important for cancer cell growth. These findings provide novel insights into the pharmacology and pathophysiology of store-operated channels.
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Mulgrew, Christopher James. "T-type calcium channels and human mesangial cell proliferation". Thesis, King's College London (University of London), 2012. https://kclpure.kcl.ac.uk/portal/en/theses/ttype-calcium-channels-and-human-mesangial-cell-proliferation(8d963ed0-7b21-4f73-bc94-ec5a76205bdd).html.
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Aberrant proliferation of human mesangial cells (MC) is a critical step in the pathogenesis of mesangioproliferative renal diseases. The T-type calcium channel (T-CaCN) has been proposed to play an important role in the proliferation of a number of non-excitable cell types, but T-CaCN expression and functional significance in MC is not known. The aim of this thesis was to investigate the hypothesis that T-CaCN may play an important role in the proliferation of MC in primary culture. Expression of mRNA encoding the α1H isoform (Cav3.2 clone) (but not the α1G nor α1I T-CaCN isoforms) was demonstrated in human MC by RT-PCR. Expression of α1H T-CaCN protein was difficult to assess directly due to the lack of a highly sensitive and specific antibody, despite attempts at upregulation of the protein. Electrophysiological studies of MC demonstrated an inward calcium current in a proportion of cells with characteristics consistent with T-CaCN. Culture of MC with the T-CaCN antagonists mibefradil, Ni2+ and TTL-1177 resulted in a significant reduction in the growth velocity of MC. This effect was not seen upon incubation with verapamil, an L-type calcium channel antagonist. DNA synthesis in MC treated with each of the T-CaCN antagonists was significantly reduced by up to 50% as shown by BrdU incorporation. This anti-proliferative effect was not associated with direct drug-induced cytotoxicity or apoptosis. FACS analysis of MC incubated with T-CaCN antagonists illustrated an increased proportion of cells remaining in G1 and not progressing into S-phase. Treatment of cultured MC with TTL-1177 resulted in a significant reduction in the signalling protein p-ERK within 30 minutes, an effect not seen with verapamil, suggesting a possible mechanism needing further investigation. MC transfection with siRNA targeting the α1H isoform resulted in significant knockdown of T-CaCN α1H mRNA and a reduction in the growth velocity of cultured MC of approximately 50% compared to control siRNA transfection, with an associated 37% reduction in DNA synthesis. These results demonstrate evidence for an important role for T-type calcium channels in the proliferation of human mesangial cells, justifying further study in in greater detail. This could potentially lead to a novel therapy in the treatment of proliferative renal diseases.
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DeRemigio, Hilary. "Markov chain models of instantaneously coupled intracellular calcium channels". W&M ScholarWorks, 2008. https://scholarworks.wm.edu/etd/1539623334.
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Localized calcium elevations known as calcium puffs or sparks are cellular signals arising from cooperative activity of clusters of inositol 1,4,5-trisphosphate receptors (IP3Rs) or ryanodine receptors (RyRs) located at calcium release sites on the endoplasmic or sarcoplasmic reticulum membrane. When Markov chain models of these intracellular calcium-regulated calcium channels are coupled via a mathematical representation of the calcium microdomain, simulated calcium release sites may exhibit the phenomenon of "stochastic calcium excitability" where the IP3Rs or RyRs open and close in a concerted fashion. Although the biophysical theory relating the kinetics of single channels to the collective phenomena of puffs and sparks is only beginning to be developed, Markov chain models of coupled intracellular channels give insight into the dynamics of calcium puffs and sparks.;Interestingly, under some conditions simulated puffs and sparks can be observed even when the single channel model used does not include slow calcium inactivation or any long-lived closed state. In this case termination of the localized calcium elevation occurs when all of the intracellular channels at a release site simultaneously close through a process called stochastic attrition. This dissertation investigates the statistical properties of stochastic attrition viewed as an absorption time on a terminating Markov chain that represents a calcium release site composed of two-state channels that are activated by calcium. Assuming that the local calcium concentration experienced by a channel depends only on the number of open channels at the calcium release site, the probability distribution function for the time until stochastic attrition occurs is derived and an analytical formula for the expectation of this random variable is presented. Also explored is how the contribution of stochastic attrition to the termination of calcium puffs and sparks depends on the number of channels at a release site, the source amplitude of the channels, the background calcium concentration, channel kinetics, and the cooperativity of calcium binding.;This dissertation also studies whether single channel models with calcium inactivation are less sensitive to the details of release site ultrastructure than models that lack a slow calcium-inactivation process. Release site dynamics obtained from simulated calcium release sites composed of instantaneously coupled calcium-regulated calcium channels whose random spatial locations were chosen from a uniform distribution on a disc of specified radius are compared to simulations with channels arranged on hexagonal lattices. Analysis of puff/spark statistics confirms that puffs and sparks are less sensitive to the spatial organization of release sites when the single channel model includes a slow inactivation process. The validity of several different mean-field reductions that do not explicitly account for the details of release site ultrastructure is also investigated.;Calcium release site models are stochastic automata networks that involve many functional transitions, that is, the transition probabilities of each channel depend on the local calcium concentration and thus the state of the other channels. A Kronecker structured representation for calcium release site models is presented and benchmark stationary distribution calculations using both exact and approximate iterative numerical solution techniques that leverage this structure are performed. When it is possible to obtain an exact solution, response measures such as the number of channels in a particular state converge more quickly using the iterative numerical methods than occupation measures calculated via Monte Carlo simulation. When an exact solution is not feasible, iterative approximate methods based on the Power method may be used, with performance similar to Monte Carlo estimates.
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Amjad, Asma. "Calcium-activated chloride channels in mouse vomeronasal sensory neurons". Doctoral thesis, SISSA, 2013. http://hdl.handle.net/20.500.11767/3899.
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In vomeronasal sensory neurons, signal transduction occurs in microvilli that are present at the neuron’s apical surface. The binding of pheromone to vomeronasal receptors causes an increase of the intracellular calcium concentration by calcium entry through TRPC2. An important issue is the impact of Ca2+ entry in pheromonal transduction.
In the first part of this thesis we have investigated the functional role played by the increase in intracellular Ca2+ concentration in the apical region of vomeronasal sensory neurons. By taking advantage of flash photolysis of caged calcium restricted to the apical region of neurons, we have measured a calcium-activated current with the whole-cell voltage-clamp technique. Our results demonstrated that a large current is indeed activated by calcium in the apical region of mouse vomeronasal sensory neurons and our immunohistochemistry data has revealed the presence of the proteins TMEM16A and TMEM16B, responsible for calcium-activated chloride channels, in the microvilli of vomeronasal sensory neurons. Therefore we have concluded that calcium-activated chloride channels are present at high density in the region where signal transduction occurs and therefore may play an important role in vomeronasal transduction.
In the second part of this thesis we have characterized in more detail the calcium activated currents in mouse vomeronasal sensory neurons using the whole-cell voltage-clamp technique in the presence of various intracellular Ca2+ concentrations. From the dose-response relation we determined that the Ca2+ concentration necessary to activate 50% of the maximal current was 1.4 µM at -100 mV and 0.6 µM at +100 mV. From ion selectivity experiments, we found that the current is carried by anions. Moreover, we demonstrated that some of the commonly used Cl- channel blockers, NFA and CaCCinh-A01, do inhibit the Ca2+-activated current in vomeronasal sensory neurons. Further studies with knockout mice for TMEM16A or TMEM16B will be necessary to establish the physiological role of these channels in vomeronasal transduction.
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Thomas, Balbir. "A model of mitochonrial [sic] calcium induced calcium release". The Ohio State University, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=osu1190053132.
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Sharma, Aarushi. "HUMAN CLCA2 MODULATES THE CONDUCTANCE OF CALCIUM-ACTIVATED CHLORIDE CHANNELS BY REGULATION OF INTRACELLULAR CALCIUM". OpenSIUC, 2016. https://opensiuc.lib.siu.edu/dissertations/1252.
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Chloride channels play an essential role in the physiology of the respiratory system, the gastrointestinal tract, and secretory glands. Their dysregulation underlies debilitating pathologies such as cystic fibrosis, asthma, and certain cancers. The CLCA (Chloride Channel Accessory) gene family is thought to determine severity of these diseases by modulating an unidentified Calcium-activated Chloride Channel (CaCC). Recent evidence indicates Ano1 to be the mediator of strong quintessential calcium-activated chloride current in several cell types. Ano1 is highly expressed in airway epithelium and downregulated in cystic fibrosis patients. Human CLCA2 is also expressed in epithelium of airways and mammary glands, and there it promotes calcium-activated chloride current. Hence, we hypothesized that CLCA2 modulates the conductance of Ano1. We tested this by introducing Ano1 and CLCA2 together or separately into HEK293 cells, which express endogenous Ano1 at a low level. Using whole-cell voltage clamp, we found that CLCA2 enhanced the conductance of the endogenous CaCC. This current was inhibited by a specific inhibitor of Ano1, tannic acid. CLCA2 also increased both the amplitude and the onset rate of the Ano1-mediated current. To determine the mechanism by which CLCA2 amplifies Ano1 mediated current, we used co-immunoprecipitation with or without a protein cross-linking agent and to test whether the interaction if any, was stable or transient, respectively. Neither any interaction, nor any change in Ano1 multimerization was found. We next tested whether CLCA2 enhanced Ano1 conductance by increasing its stability or surface localization. Surface-labelling the cells expressing Ano1 alone or both proteins with biotin, no difference in Ano1 level or surface expression was detected. Ano1 has recently been shown to be activated by intracellular calcium released from endoplasmic reticulum (ER) stores and by subsequent store-operated calcium entry (SOCE). Therefore, we investigated whether CLCA2 could increase intracellular calcium levels. With Fluo-4 dye calcium imaging, we found that CLCA2 expression enhanced both ER calcium stores and SOCE upon exhaustion of intracellular stores, and the SOCE response could be abolished by a specific inhibitor of SOCE, BTP-2. This inhibitor also abolished CLCA2-induced chloride current, establishing that CLCA2 enhances CaCC via SOCE. Moreover, knockdown of CLCA2 in MCF10A cells, that naturally express both proteins, reduced both ER calcium stores and SOCE. Mutations that abolished the metalloprotease activity of CLCA2 or deleted the cytoplasmic tail had little effect on its enhancement of chloride current or intracellular calcium, suggesting that the uncleaved ectodomain was responsible for both effects of CLCA2. Since, the ectodomain is the most conserved region of the protein, we found that another member of the CLCA family, CLCA1, was also effective in enhancing intracellular calcium storage and SOCE. Co-immunoprecipitation studies further revealed that CLCA2 interacts in a ternary complex with mediators of SOCE, STIM1 and ORAI1. These results explain the CaCC-enhancing effects of CLCA family members and suggest a broader role in other calcium-dependent processes. Understanding the modulatory relationship between these molecules may lead to better therapies for airway diseases and Ano1-dependent cancers. Furthermore, the discovery that CLCA2 regulates intracellular calcium levels may explain its effects on cellular differentiation, stress response, and cell death.
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Douglas, Sophie Georgina. "Regulation of CRAC channels and agonist-induced Ca2+ signals". Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:ae94ca14-ac95-4ea6-b14a-14f9f7bafd63.
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Calcium ions (Ca2+) are extremely important intracellular messengers, activating a plethora of cellular processes. Growing evidence now points to a major role for the local Ca2+ signal in driving specific cellular responses. The simplest and most fundamental local Ca2+ signal is the Ca2+ microdomain, which rapidly forms when Ca2+ permeable ion channels open. In non-excitable cells the dominant Ca2+ entry channels are store-operated Ca2+ channels (SOCCs). The best characterised is the Ca2+ release activated Ca2+ (CRAC) channel. How local Ca2+ entry through CRAC channels impacts on channel function however is unclear. I have investigated the interaction between the Ca2+ binding protein calmodulin and CRAC channel activity and subsequent agonist-induced Ca2+ signals. Furthermore, I have investigated a role for mitofusin 2 (a protein that is known to tether the ER and mitochondria) on these Ca2+ signals. Using three different calmodulin mutant constructs with alterations to their Ca2+ binding sensitivities, I have shown that calmodulin facilitates CRAC channel dependent Ca2+ entry and maintains agonist-induced cytosolic Ca2+ oscillations in a lobe-specific manner. Calmodulin has four Ca2+ binding sites, two on the N-lobe and two on the C-lobe. I found a dominant negative calmodulin mutant (CAM4M, where all four binding sites had been mutated), or one where the C-lobe could not bind Ca2+ (CAM2C), impaired both Ca2+ influx through CRAC channels and maintenance of cytosolic Ca2+ oscillations. In contrast, a Ca2+-insensitive N-lobe mutant had little effect, (CAM2N). Knockdown of the mitochondrial Ca2+ uniporter regulator (MICU1) or mitochondrial membrane depolarization had similar effects to those seen with CAM4M or CAM2C, suggesting that at least in part, the action of calmodulin was through regulation of mitochondrial Ca2+ dynamics. This was confirmed by directly measuring the mitochondrial matrix Ca2+ concentration in intact RBL-1 cells using the mitochondrial targeted, fluorescent protein, pericam. Both CAM4M and disruption of mitochondrial Ca2+ buffering impaired agonist-induced mitochondrial Ca2+ uptake, suggesting that the modulation of CRAC channels occurred through Ca2+-calmodulin facilitation of mitochondrial Ca2+ uptake. Using a mutant Orai1 (A73E) that cannot bind calmodulin, I have shown that calmodulin tethered to the CRAC channel provides a major source of calmodulin for effective mitochondrial Ca2+ uptake. Physiological relevance of my proposed pathway was provided from experiments where I showed knockdown of MICU1 impaired agonist-induced CRAC channel dependent NFAT-1-driven gene expression. In addition, I establish a crucial role for mitochondrial MFN2 and presumably its ability to properly link the mitochondria and ER in the control of CRAC channels and agonist-induced Ca2+ signals.