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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
41

Expression of Trp gene family in vascular system.

January 2001 (has links)
Yip Ham. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 132-141). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abbreviations --- p.ii / Abstract --- p.iii / 摘要 --- p.v / Chapter Chapter 1: --- Introduction --- p.5 / Chapter 1.1 --- Calcium Signaling --- p.5 / Chapter 1.1.1 --- Importance of Calcium to Life Forms --- p.5 / Chapter 1.1.2 --- Calcium Channels in Excitable and Non-excitable Cells --- p.6 / Chapter 1.2 --- Vascular Endothelial Cells --- p.8 / Chapter 1.2.1 --- General Functions --- p.8 / Chapter 1.2.2 --- Calcium signaling in Endothelial Cells --- p.9 / Chapter 1.3 --- Capacitative Calcium Entry (CCE) or Store-operated Calcium Entry (SOC) --- p.10 / Chapter 1.3.1 --- Definition --- p.10 / Chapter 1.3.2 --- Endoplasmic Reticulum (ER) as the Main Intracellular Calcium Stores --- p.10 / Chapter 1.3.3 --- Types of Experiments leading to the Identification of SOCs --- p.11 / Chapter 1.3.4 --- Emptying the Internal Calcium Store --- p.11 / Chapter 1.3.4.1 --- Inhibition of Calcium ATPase --- p.11 / Chapter 1.3.4.2 --- IP3 Triggered Release of Calcium --- p.12 / Chapter 1.3.5 --- "Store-operated Calcium Current, Icrac" --- p.15 / Chapter 1.3.6 --- Different Types of SOCs in Animal Cells --- p.16 / Chapter 1.4 --- Transient Receptor Potential (Trp) Gene & Transient Receptor Potential Like (Trpl) Gene in Drosophila --- p.17 / Chapter 1.4.1 --- Discoverery of Trp and Trpl --- p.17 / Chapter 1.4.2 --- Expression Studies of Drosophila Trp and Trpl --- p.19 / Chapter 1.4.2.1 --- Trp and Trpl form Channels but only Trp is Store Operated --- p.19 / Chapter 1.4.2.2 --- Co-expression Studies of Trp and Trpl --- p.20 / Chapter 1.5 --- Molecular Cloning and Expression of Mammalian Trp Homologues --- p.21 / Chapter 1.5.1 --- Seven Human Homologus of Trp were found --- p.21 / Chapter 1.5.2 --- Expression Pattern of mammalian Trp Homologues in Different Tissues --- p.23 / Chapter 1.5.3 --- Expression Studies of Mammalian Trp Homologues Yields Contradictory Results --- p.27 / Chapter 1.5.3.1 --- Trpl --- p.27 / Chapter 1.5.3.2 --- Trp2 --- p.28 / Chapter 1.5.3.3 --- Trp3 --- p.29 / Chapter 1.5.3.4 --- Trp4 --- p.30 / Chapter 1.5.3.5 --- Trp5 --- p.31 / Chapter 1.5.3.6 --- Trp6 --- p.31 / Chapter 1.5.3.7 --- Trp7 --- p.31 / Chapter 1.5.3.8 --- "Activation of Trp3, Trp6 and Trp7 by Diacylglycerol (DAG)" --- p.32 / Chapter 1.5.3.9 --- Functional Consequence after Co-expression of Trp Homologues --- p.34 / Chapter 1.5.3.10 --- Antisense Strategy to Determine the Functional Subunits of Store-operated Channels --- p.35 / Chapter 1.5.3.11 --- Possible Reasons for the Contradictory Results of Trp Homologues When Expressed in a Heterologous System --- p.36 / Chapter 1.6 --- Aims Of Study --- p.37 / Chapter Chapter 2. --- Materials and Methods --- p.38 / Chapter 2.1 --- Cell Culture --- p.38 / Chapter 2.2 --- Total RNA extraction from HCAEC 5286 --- p.39 / Chapter 2.3 --- Reverse Transcription from Cultured Human Coronary Artery Endothelial Cell Line HCAEC 5286 --- p.40 / Chapter 2.4 --- Polymerase Chain Reaction (PCR) of Partial Trp Gene Fragments --- p.41 / Chapter 2.5 --- Separation and Purification of PCR Products --- p.43 / Chapter 2.5.1 --- Separation --- p.43 / Chapter 2.5.2 --- Purification --- p.43 / Chapter 2.6 --- Confirmation of PCR Products --- p.44 / Chapter 2.7 --- Molecular Cloning of Trp Gene Family --- p.45 / Chapter 2.7.1 --- "Cloning of HTrpl, HTrp3, HTrp4,HTrp5,HTrp6, HTrp7" --- p.45 / Chapter 2.7.1.1 --- Polishing the Purified PCR Products --- p.47 / Chapter 2.7.1.2 --- Determination of the Amount of Polished PCR Products --- p.47 / Chapter 2.7.1.3 --- Inserting the PCR Products into the pPCR-Script Amp SK(+)Cloning Vector (Ligation) --- p.48 / Chapter 2.7.1.4 --- Transformation --- p.48 / Chapter 2.7.1.5 --- Preparing Glycerol Stocks Containing the Bacterial Clones --- p.49 / Chapter 2.7.1.6 --- Plasmid DNA Preparation --- p.49 / Chapter 2.8.1.7 --- Clones Confirmation --- p.50 / Chapter 2.8 --- In situ Hybridization --- p.54 / Chapter 2.8.1 --- Probe Preparation --- p.54 / Chapter 2.8.1.1 --- Trp1 Probe --- p.54 / Chapter 2.8.1.2 --- Trp3 Probe --- p.58 / Chapter 2.8.1.3 --- Trp4 Probe --- p.61 / Chapter 2.8.1.4 --- Trp5 Probe --- p.62 / Chapter 2.8.1.5 --- Trp6 Probe --- p.63 / Chapter 2.8.1.6 --- Trp7 Probe --- p.65 / Chapter 2.8.1.7 --- Control Probe --- p.66 / Chapter 2.8.2 --- Testing of DIG-Labeled RNA Probes --- p.66 / Chapter 2.8.3 --- Paraffin Sections Preparation --- p.67 / Chapter 2.8.4 --- In Situ Hybridization: Pretreatment --- p.67 / Chapter 2.8.5 --- "Pre-hybridization, Hybridization and Post-hybridization" --- p.68 / Chapter 2.8.5.1 --- Pre-Hybridization --- p.68 / Chapter 2.8.5.2 --- Hybridization --- p.68 / Chapter 2.8.5.3 --- Post-Hybridization --- p.69 / Chapter 2.8.6 --- Colorimetric Detection of Human Trps mRNA --- p.69 / Chapter 2.9 --- Northern Hybridization --- p.70 / Chapter 2.9.2 --- Labelling of Riboprobe with 32P --- p.70 / Chapter 2.9.3 --- Prehybridization and Hybridization with Radiolabeled RNA Probes --- p.73 / Chapter Chapter 3. --- Results --- p.74 / Chapter 3.1 --- Polymerase Chain Reaction (PCR) of Partial Trp Gene Fragments --- p.74 / Chapter 3.2.1 --- Expression of TRPs RNA in Human Coronary Artery --- p.78 / Chapter 3.2.1.1 --- Expression of Trp Transcripts in Tunica Intima and Media --- p.79 / Chapter 3.2.1.2 --- Expression of Trp Transcripts in the Tunica Adventitia --- p.88 / Chapter 3.2.2 --- Expression of TRPs RNA in Human Cerebral Artery --- p.97 / Chapter 3.2.2.1 --- Expression of Trp Transcripts in Tunica Intima and Media --- p.97 / Chapter 3.3 --- Northern Blot Analysis of Human Trp5 RNA in Human Multiple Tissue Blot --- p.115 / Chapter Chapter 4: --- Discussion --- p.117 / Chapter 4.1 --- Co-expression of Trps in Vascular Tissues --- p.117 / Chapter 4.1.1 --- Expression of Trps in Endothelia --- p.117 / Chapter 4.1.2 --- In Smooth Muscle Cells --- p.118 / Chapter 4.2 --- Trp Channel and Store-operated Channel in Endothelial Cells --- p.119 / Chapter 4.3 --- Heteromultimerization of Trps Subtypes --- p.120 / Chapter 4.4 --- Northern Blot Analysis --- p.124 / Chapter 4.5 --- Potential Physiological Functions of Trps --- p.125 / Chapter 4.6 --- Trp Channels as a Therapeutic Target? --- p.128 / Chapter 4.7 --- Technical Aspects in the Present Studies --- p.129 / Chapter 4.8 --- Conclusion --- p.131 / Reference --- p.133
42

Conserved and non-conserved roles of the I-II loop of T-type Ca²+ channels /

Baumgart, Joel Philip. January 2008 (has links)
Thesis (Ph. D.)--University of Virginia, 2008. / Includes bibliographical references. Also available online through Digital Dissertations.
43

Calcium channel activity and force regulation in smooth muscle effects of polyamines and growth stimulation /

Gomez, Maria. January 1998 (has links)
Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Includes bibliographical references.
44

Calcium channel activity and force regulation in smooth muscle effects of polyamines and growth stimulation /

Gomez, Maria. January 1998 (has links)
Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Includes bibliographical references.
45

Calcium signaling pathways and cell proliferation in human cardiac fibroblast

Chen, Jingbo, 陳靜波 January 2008 (has links)
published_or_final_version / Medicine / Master / Master of Philosophy
46

Regulation of CRAC channels and agonist-induced Ca2+ signals

Douglas, Sophie Georgina January 2012 (has links)
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.
47

Mécanismes psysiopathologiques des ataxies épisodiques et progressives associées aux canaux calciques de type P/Q / Pathogenic Mechanisms of Cav2.1 calcium channels associated Ataxia and potential therapy approaches

Salvi, Julie 27 November 2012 (has links)
Dans de nombreuses maladies héréditaires monogéniques, les bases moléculaires du mode de transmission (dominant vs récessif) et l'origine de nombreux phénotypes associés restent obscurs. C'est le cas pour les ataxies épisodiques (EA) et progressives liées au canal calcique Cav2.1. La plupart des mutations présentes dans ces ataxies génèrent une protéine mal repliée retenue et dégradée dans le réticulum endoplasmique. J'ai exploré plusieurs approches afin d'associer les phénotypes dues à la présence des mutants et ceux dues à une « pure » perte de fonction. A l'aide de vecteurs viraux codants pour des ARNs interférents de Cav2.1, j'ai montré que la réduction chez la souris adulte du canal récapitulait de nombreux symptômes de l'EA2.J'étudie également un nouveau modèle murin knock-In pour l'EA2 produisant une forme tronquée mal repliée. L'étude de ces modèles permettra une meilleure compréhension des mécanismes physiopathologiques et l'exploration de nouvelles voies thérapeutiques. / Voltage-gated calcium channels (VGCC) regulate an array of physiological process. The P/Q-type VGCC is principally expressed in the cerebellum and at the neuromuscular junction, where it plays an essential role at the presynaptic neuronal nerve. Interestingly, mutations in a1 subunit (Cav2.1) of P/Q-type VGCC gene have been found to be linked for three autosomal dominant human disorders, familial hemiplegic migraine type 1 (FHM1), spinocerebellar ataxia 6 (SCA6) and episodic ataxia type 2 (EA2). Mutations causing EA2 lead to loss-of-function of Cav2.1 channels and are principally non-sense. The origin of dominant transmission and the heterogeneity of the symptoms are not known. Recent data from different groups have shown a dominant-negative effect of Cav2.1 mutants in cultured cell lines. Indeed, the molecular mechanism of this dominant-negative effect appears to be due to the interference of EA2 mutants with the folding of the wildtype subunit, thereby abolishing channel activity. This destructive interaction mechanism promoted by the EA2 mutants is likely to occur in the disease.The first part of my thesis was to monitor the effect of a “pure” silincing of P/Q-type channel in adult mice. Suppression of Cav2.1 channel by RNAi lentiviral based-vector approaches has produced episodic as well as permanent ataxia without signs of progressive ataxia. As a direct approach to understanding the physiologicalcontributions of misfolded truncated mutants in EA2 phenotypes, a Cav2.1 knock-inmutant, CACNA1AR1479x has been generated. This is a fundamental issue to understand the pathogenic mechanisms of EA2 and more generally to the other neuronal and neuromuscular diseases.
48

Les venins animaux comme outils de recherche et d'identification de nouveaux composés thérapeutiques / venoms animals as research tools and identification of new therapeutic compounds.

Al Khoury, Sawsan 07 July 2017 (has links)
Les venins issus d’animaux sont des mélanges complexes de substances toxiques ou non qui sont utilisées pour se défendre contre des prédateurs et/ou pour chasser. Le venin est une solution contenant de nombreux composés avec une part prédominante pour les protéines et les peptides. Le venin d’un seul animal peut contenir plusieurs centaines de substances différentes. Les peptides dérivés du venin peuvent cibler des récepteurs membranaires et des canaux ioniques avec une grande affinité et une haute spécificité. Les animaux venimeux les plus connus sont les serpents, les scorpions et les araignées, et leurs toxines sont largement utilisés dans la recherche comme outils moléculaires et pour le développement des nouveaux traitements. Par conséquent, les venins d’animaux sont des sources naturelles extrêmement riches de découverte de molécules biologiquement actives. Dans ces études, nous avons criblé le venin du serpent égyptien Walterinnesia aegyptia pour l’identification de novo de peptides capables d’activer la motilité des spermatozoïdes in vitro des souris OF1. La Spermatin et X sont deux peptides de 57 et 63 acides aminés respectivement, isolés du venin du Walterinnesia aegyptia par la technique de séparation RP-HPLC suivie par la chromatographie échangeuse de cations. La spermatin et l’actiflagelin sont deux activateurs de la motilité spermatique des souris OF1. La spermatin a été séquencé par séquençage de novo en utilisant i) la digestion des peptides par des enzymes protéases comme la trypsine, la chymotrypsine et la protéase V8, et ii) les techniques de spectrométrie de masse TOF/TOF MS/MS et LC-ESI-QTOF MS/MS. Par contre, c’est la complémentarité entre le séquençage de novo, la dégradation d’Edman et les analyses ESI MS/MS qui a permis l’identification de la séquence de l’actiflagelin. Il semble dès lors que la spermatin et l’actiflagelin peuvent être utilisés comme outils pharmacologiques pour diminuer le taux d’infertilité. De l’autre côté, la maurocalcine (MCa) est un peptide de 33 résidus issu du venin du scorpion tunisien Scorpio maurus palmatus. La MCa est un activateur des récepteurs de ryanodine (RyR1) des muscles squelettiques. Elle stimule fortement la liaison du [³H]-ryanodine aux RyR1 et assure le relâchement du calcium intracellulaire des vésicules du réticulum sarcoplasmique (RS). Différents mutants de la MCa ont été utilisés et les résultats montrent que l’Arg24 joue un rôle critique dans la liaison de la MCa aux RyR1 ou tout au moins dans son impact fonctionnel. D’autres mutations de la MCa sont capables de modifier la liaison du [³H]-ryanodine aux RyR1 mais avec une plus faible efficacité que la séquence sauvage. De plus, la MCa est capable de franchir la membrane plasmique et elle est considérée comme un peptide de pénétration cellulaire. Ici, nous avons montré que la MCa est un substrat de la protéine kinase A in vitro suite à la phosphorylation du résidu Thr26. Nous avons aussi démontré que la MCa P-Thr26 inverse l’effet de la MCa. La MCa P-Thr26 inhibe la liaison du [³H]-ryanodine aux RyR1 des muscles squelettiques du lapin et elle est incapable de favoriser la libération du calcium des vésicules du RS. Alors, la MCa peut être utilisée pour le développement des analogues résistants à la phosphatase. Par ailleurs, nous avons étudié l’effet de l’agent réducteur dithiothreitol ou les agents oxydants le diamide et le peroxyde d’hydrogène sur la stimulation du RyR1 par la MCa ou MC E12A. La maurocalcine améliore la liaison du [³H]-ryanodine aux RyR1 des vésicules seules ou pré-incubées avec le dithiothreitol ou les agents oxydants. L’interprétation des résultats indiquent que la MCa et son mutant sont plus efficaces à activer RyR1 sous des conditions de réduction. Par ailleurs, des résultats ont montré que MCa E12A réduit significativement l’inhibition de la liaison du [³H]-ryanodine aux RyR1 induite par Mg²⁺. / Animal venoms are complex mixtures of toxic substances that are used to defend themselves against predators and / or to hunt. Venom is a solution containing many compounds, especially proteins and peptides. The venom of a single animal may contain several hundred different substances. Peptides derived from venom can target membrane receptors and ion channels with strong affinity and high specificity. The best known venomous animals are snakes, scorpions and spiders, and their toxins are widely used in research as molecular tools and for the development of new treatments. Therefore, animal venoms are an extremely rich and complex source of biologically active molecules. In these studies, we screened the venom of the Egyptian snake Walterinnesia aegyptia for the discovery of peptides able to activate sperm motility in vitro of mice OF1. Spermatin and actiflagelin are two peptides of 57 and 63 amino acids, respectively, isolated from the venom of Walterinnesia aegyptia by the RP-HPLC separation technique followed by cation exchange chromatography. Spermatin and actiflagelin are two activators of the spermatic motility of OF1 mice. Spermatin was sequenced by de novo sequencing using digestion of enzymes proteases such as trypsin, chymotrypsin and V8 protease, and further TOF / TOF MS / MS and LC-ESI-QTOF MS / MS. While the complementarity between de novo sequencing, Edman degradation and ESI MS / MS analyzes allowed the identification of the sequence of actiflagelin. Then, Spermatin and actiflagelin can be used as pharmacological tools to decrease the rate of infertility. On the other hand, maurocalcin (MCa) is a 33-residue peptide derived from the venom of the scorpion Scorpio maurus palmatus. MCa is an activator of ryanodine receptors (RyR1) of skeletal muscles. It strongly stimulates the binding of [³H]-ryanodine to RyR1 and ensures the release of intracellular calcium from the vesicles of the sarcoplasmic reticulum (SR). Different MCa mutants were used and the results showed that Arg24 plays a critical role in the binding of MCa to RyR1. Other mutations were able to alter the binding of [³H]-rhyanodine to RyR1 but with low efficiency. In addition, MCa is able to cross the plasma membrane and is considered a cell-penetrating peptide. Here, we have shown that MCa is a substrate of PKA in vitro following the phosphorylation of Thr26. We have also demonstrated that MCa P-Thr26 reverses the effect of MCa. MCa P-Thr26 inhibits the binding of [³H]-ryanodine to RyR1 from the rabbit skeletal muscle, and is unable to promote the release of calcium from the SR vesicles. Then, MCa can be used for the development of phosphatase resistant analogs.Keywords: Animal venom, Walterinnesia aegyptia, RP-HPLC, ion exchange chromatography, de novo sequencing, Spermatin, X, maurocalcine (MCa), [³H]-ryanodine, ryanodine receptors (RyR1), calcium, phosphorylation, sarcoplasmic reticulum (SR).
49

The Regulation and Function of RGK Proteins on Voltage-Gated Calcium Channel Physiology

Chang, Donald Dao-Yuan January 2015 (has links)
Rad/Rem/Rem2/Gem/Kir (RGK) proteins are Ras-like GTPases with diverse (and expanding) functions including: regulating cytoskeleton dynamics, cell proliferation, synaptogenesis, and inhibition of high voltage-dependent calcium (CaV) channels. Furthermore, they have tissue-specific distribution with Rem and Rad most highly expressed in the heart. Indeed, the importance of Rem and Rad in the cardiovascular system is underscored by a number of studies linking them to disease states including cardiac hypertrophy, cardiac fibrosis, and inflammation. A hallmark feature of RGK proteins is their ability to inhibit current through CaV channels (ICa) and in fact, they are recognized as the most potent endogenous inhibitors of ICa. However, how RGK proteins are regulated and what their physiological role is are unknown. Understanding these points is critical for defining the patho-physiological roles of RGK proteins. My thesis work contributes towards the RGK field on two fronts: First, we demonstrate that RGK proteins are non-canonical G-proteins in the context of their ability to undergo nucleotide regulation and second, we reveal a novel paradigm of RGK-mediated inhibition on CaV channels. In Chapters 2 and 3, we show that Rem and Rad are are non-canonical G-proteins with respect to the regulatory role of their guanine nucleotide binding pocket (GNBP). Canonical Raslike G-proteins contain a conserved G-domain that encompass a GNBP and is important for guanine nucleotide binding and hydrolysis. Since RGK proteins also possess a G-domain and GNBP as well as demonstrate bona fide nucleotide binding, it was initially thought that they were regulated in a manner similar to other Ras proteins. However, subsequent studies suggested that RGK proteins may not obey such a classical model and as a result, the regulatory role of their GNBP in the G-domain was unclear. By using a wide range of functional measurements (CaV1.2 currents, Ca2+ transients, β-subunit binding), we demonstrate that RGK proteins Rem and Rad are non-canonical G-proteins. Utilizing point mutants that abolish GTPbinding and prevent GTPase activity (RemT94N and RadS105N), we show that only some cellular functions are dependent on an operational nucleotide binding pocket while others are unperturbed. Specifically, Rem- and Rad-mediated inhibition of ICa is independent of guanine nucleotide regulation whereas protein interactions with the b-subunit of CaV channels (CaVβ) and protein stability are sensitive to nucleotide regulation. We also discover skeletal and cardiac actin to be novel binding partners of Rem. And lastly, we observe differences between the effects of Rem and Rad on their degree of ICa inhibition in cardiac myocytes. Thus, Rem and Rad are non-canonical G-proteins with respect to the regulatory role of their GNBP. In collaboration with a close colleague, Akil Puckerin, Chapter 4 reveals a novel mechanism behind RGK-mediated inhibition of ICa. Together, we show RGK proteins display different modes of inhibition against specific CaV channels and that we can utilize this property to design calcium channel blockers which inhibit CaV channels in an isoform specific manner. We demonstrate this by designing Rem and Rad mutants which have diminished CaVβ capacity, termed Rem-βNULL and Rad-βNULL, respectively. Characterization of these mutants using wholecell patch clamp experiments revealed that Rem-βNULL inhibits only CaV1.2 whereas Rad-βNULL inhibits only CaV1.2 and CaV2.2. Thus, our results describe the first genetically encoded calcium channel blocker that can selectively distinguish amongst L-type channels. Altogether, this thesis work contributes towards our understanding of RGK protein regulation function and the underlying mechanisms by which they inhibit ICa. These findings advance the field both from a mechanistic and physiological standpoint, and will be of great importance towards investigating the patho-physiological role of RGK proteins.
50

Structural Studies of a Mammalian Epithelial Calcium Channel

Saotome, Kei January 2016 (has links)
Calcium plays an essential role in the physiology and biochemistry of many biological functions, including excitation-contraction coupling, neuronal signaling, and fertilization. In mammals, the calcium content in various tissues, organs, and cell types is tightly regulated to maintain homeostasis. A chief process controlling calcium levels is absorption of the ion from the lumen by epithelial cells that line organs including the intestines and kidney. Calcium entry at the apical membrane constitutes the first step of epithelial calcium absorption. Two highly calcium-selective transient receptor potential vanilloid (TRPV) channels, TRPV5 and TRPV6, are the pore-forming subunits responsible for epithelial calcium entry in kidney and intestine, respectively. Genetic knockout of TRPV5 or TRPV6 in animals leads to phenotypes related to defective calcium homeostasis, including lowered serum calcium levels, decreased calcium absorption, reduced bone density, impaired sperm motility, and decreased maternal-fetal calcium transfer. In humans, aberrant TRPV5/6 expression is associated with preeclampsia and calcium nephrolithiasis (kidney stones). Additionally, TRPV6 expression level is upregulated in carcinomas of prostate, colon, breast, thyroid, and ovary, suggesting a role for TRPV6 in cancer survival. A detailed understanding of epithelial calcium entry is hindered by a lack of high-resolution structural information on intact channels. This dissertation presents structural analyses of the epithelial calcium channel TRPV6. We applied modern membrane protein screening and expression techniques, including fluorescence-detection size exclusion chromatography (FSEC) and baculovirus mediated mammalian cell transduction (BacMam), to identify optimal TRPV6 constructs and purification schemes for crystallization. Using a surface mutagenesis approach guided by lower-resolution structural solutions, we engineered a rat TRPV6 mutant (TRPV6cryst) that permitted solving a 3.25 Å resolution crystal structure. We used fluorescent calcium indicator assays to show that TRPV6cryst retains the permeation and ionic block properties of the wild type channel. The tetrameric structure of TRPV6cryst reveals a transmembrane domain architecture similar to voltage gated ion channels, with the ion conducting pore coincident with the overall four-fold symmetry axis. A ring of aspartate (D541) residues, shown in previous studies as a critical determinant of calcium selectivity, forms a narrow constriction at the extracellular pore entrance, or selectivity filter. Methionine (M577) side chains in the lower portion of the channel pore plug the conduction pathway and define the closed state of the channel. The ankyrin repeat domain, linker domain, N-terminal helix, and C-terminal hook form an intracellular skirt surrounding a cavity that lies beneath the pore axis. Close interactions between these domains, in large part mediated by the N-terminal helix, suggest that they are involved in allosteric modulation or concerted movements associated with channel activation. To shed light on the structural bases of permeation and ionic block, we cocrystallized TRPV6cryst with the permeant cations Ca²⁺ and Ba²⁺, and the channel blocker Gd³⁺. We identified binding sites for these cations by exploiting their anomalous scattering properties. On the basis of the cation-binding sites, we propose a permeation mechanism in which cations are recruited toward the pore by electronegative side chains in the extracellular vestibule, followed by sequential binding at least three binding sites along the central pore axis. Ca²⁺ selectivity is apparently achieved by high-affinity binding to the ring of D541 side chains in the selectivity filter. Gd³⁺ blocks permeation by similarly binding to the D541 ring and outcompeting ions of lesser charge. The results described in this dissertation provide a structural framework to further study mechanisms of epithelial calcium entry in health and disease.

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