@phdthesis{Fei2023,
  author    = {Fei, Lin},
  title     = {Optogenetic regulation of osmolarity and water flux},
  doi       = {10.25972/OPUS-32309},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-323092},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Optogenetics is a powerful technique that utilizes light to precisely regulate physiological activities of neurons and other cell types. Specifically, light-sensitive ion channels, pumps or enzymes are expressed in cells to enable their regulation by illumination, thus allowing for precise control of biochemical signaling pathways. The first part of my study involved the construction, optimization, and characterization of two optogenetic tools, KCR1 and NCR1. Elena Govorunova et al. discovered a lightgated potassium channel, KCR1, in the protozoan Hyphochytrium catenoides. Traditional potassium ion channels are classified as either ligand-gated or voltage-gated and possess conserved pore-forming domains and K+ -selective filters. However, KCR1 is unique in that it does not contain the signature sequence of previously known K+ channels and is a channelrhodopsin. We synthesized the KCR1 plasmid according to the published sequence and expressed it in Xenopus oocytes. Due to the original KCR1 current being too small, I optimized it into KCR1 2.0 to improve its performance by fusing LR (signal peptide LucyRho, enhances expression) at the N-terminal and T (trafficking signal peptide) and E (ER export signal peptide) at the C-terminal. Additionally, I investigated the light sensitivity, action spectrum, and kinetics of KCR1 2.0 in Xenopus oocytes. The potassium permeability of KCR1 2.0, PK/Pna  24, makes KCR1 2.0 a powerful hyperpolarizing tool that can be used to inhibit neuronal firing in animals. Inspired by KCR1, we used the KCR1 sequence as a template for gene sequence alignment with the sequences in H. catenoides. We found that NCR1 and KCR1 have similar gene sequences. NCR1 was characterized by us as a light-gated sodium channel. This NCR1 was also characterized and published by Govorunova et al. very recently, with the name HcCCR. Due to the original NCR1 current being too small, I optimized it into NCR1 2.0 to improve its performance by fusing LR at the N-terminal and T and E at the C-terminal, which significantly improved the expression level and greatly increased the current amplitude of NCR1. Full-length NCR1 2.0 contains 432 amino acids. To test whether the number of amino acids changes the characteristics of NCR1 2.0, we designed NCR1 2.0 (330), NCR1 2.0 (283), and NCR1 2.0 (273) by retaining the number of amino acids at 330, 280, and 273 in NCR1 2.0, respectively. As the number of amino acids decreased, the current in NCR1 2.0 increased. I also investigated the light sensitivity, action spectrum, and kinetics of NCR1 2.0 (273) in the Xenopus Abstract 2 oocytes. We performed four point mutations at amino acid positions 133 and 116 of NCR1 2.0 and analyzed the reversal potentials of the mutants. The mutations were as follows: NCR1 2.0 (273 D116H), NCR1 2.0 (273 D116E), NCR1 2.0 (283 V133H), and NCR1 2.0 (283 D116Q). The second part of this study focuses on light-induced water transport using optogenetic tools. We explored the use of optogenetic tools to regulate water flow by changing the osmolarity in oocytes. Water flux through AQP1 is driven by the osmotic gradient that results from concentration differences of small molecules or ions. Therefore, we seek to regulate ion concentrations, using optogenetic tools to regulate the flux of water noninvasively. To achieve this, I applied the light-gated cation channels XXM 2.0 and NCR1 2.0 to regulate the concentration of Na+ , while K + channel KCR1 2.0 was used to regulate K + concentration. As Na+ flows into the Xenopus oocytes, the membrane potential of the oocytes becomes positive, and Clcan influx through the light-gated anion channel GtACR1. By combining these optogenetic tools to regulate NaCl or KCl concentrations, I can change the osmolarity inside the oocytes, thus regulating the flux of water. I co-expressed AQP1 with optogenetic tools in the oocytes to accelerate water flux. Overall, I designed three combinations (1: AQP1, XXM 2.0 and GtACR1. 2: AQP1, NCR1 2.0 and GtACR1. 3: AQP1, KCR1 2.0 and GtACR1) to regulate the flow of water in oocytes. The shrinking or swelling of the oocytes can only be achieved when AQP1, light-gated cation channels (XXM 2.0/NCR1 2.0/KCR1 2.0), and light-gated anion channels (GtACR1) are expressed together. The illumination after expression of either or both alone does not result in changes in oocyte morphology. In sum, I demonstrated a novel strategy to manipulate water movement into and out of Xenopus oocytes, non-invasively through illumination. These findings provide a new avenue to interfere with water homeostasis as a means to study related biological phenomena across cell types and organisms.},
  subject      = {Osmolarit{\"a}t},
  language  = {en}
}
@article{ZhuShabalaCuinetal.2016,
  author    = {Zhu, Min and Shabala, Lana and Cuin, Tracey A. and Huang, Xin and Zhou, Meixue and Munns, Rana and Shabala, Sergey},
  title     = {Nax loci affect SOS1-like Na\(^+\)/H\(^+\) exchanger expression and activity in wheat},
  series = {Journal of Experimental Botany},
  volume    = {67},
  journal   = {Journal of Experimental Botany},
  number    = {3},
  doi       = {10.1093/jxb/erv493},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-190908},
  pages     = {835-844},
  year      = {2016},
  abstract  = {Salinity stress tolerance in durum wheat is strongly associated with a plant's ability to control Na\(^+\) delivery to the shoot. Two loci, termed Nax1 and Nax2, were recently identified as being critical for this process and the sodium transporters HKT1;4 and HKT1; 5 were identified as the respective candidate genes. These transporters retrieve Na\(^+\) from the xylem, thus limiting the rates of Na\(^+\) transport from the root to the shoot. In this work, we show that the Nax loci also affect activity and expression levels of the SOS1-like Na\(^+\)/H\(^+\) exchanger in both root cortical and stelar tissues. Net Na\(^+\) efflux measured in isolated steles from salt-treated plants, using the non-invasive ion flux measuring MIFE technique, decreased in the sequence: Tamaroi (parental line)>Nax1=Nax2>Nax1:Nax2 lines. This efflux was sensitive to amiloride (a known inhibitor of the Na\(^+\)/H\(^+\) exchanger) and was mirrored by net H\(^+\) flux changes. TdSOS1 relative transcript levels were 6-10-fold lower in Nax lines compared with Tamaroi. Thus, it appears that Nax loci confer two highly complementary mechanisms, both of which contribute towards reducing the xylem Na\(^+\) content. One enhances the retrieval of Na\(^+\) back into the root stele via HKT1;4 or HKT1;5, whilst the other reduces the rate of Na\(^+\) loading into the xylem via SOS1. It is suggested that such duality plays an important adaptive role with greater versatility for responding to a changing environment and controlling Na\(^+\) delivery to the shoot.},
  language  = {en}
}
@article{BeckYuStrzelczykPaulsetal.2018,
  author    = {Beck, Sebastian and Yu-Strzelczyk, Jing and Pauls, Dennis and Constantin, Oana M. and Gee, Christine E. and Ehmann, Nadine and Kittel, Robert J. and Nagel, Georg and Gao, Shiqiang},
  title     = {Synthetic light-activated ion channels for optogenetic activation and inhibition},
  series = {Frontiers in Neuroscience},
  volume    = {12},
  journal   = {Frontiers in Neuroscience},
  number    = {643},
  doi       = {10.3389/fnins.2018.00643},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-177520},
  year      = {2018},
  abstract  = {Optogenetic manipulation of cells or living organisms became widely used in neuroscience following the introduction of the light-gated ion channel channelrhodopsin-2 (ChR2). ChR2 is a non-selective cation channel, ideally suited to depolarize and evoke action potentials in neurons. However, its calcium (Ca2\(^{2+}\)) permeability and single channel conductance are low and for some applications longer-lasting increases in intracellular Ca\(^{2+}\) might be desirable. Moreover, there is need for an efficient light-gated potassium (K\(^{+}\)) channel that can rapidly inhibit spiking in targeted neurons. Considering the importance of Ca\(^{2+}\) and K\(^{+}\) in cell physiology, light-activated Ca\(^{2+}\)-permeant and K\(^{+}\)-specific channels would be welcome additions to the optogenetic toolbox. Here we describe the engineering of novel light-gated Ca\(^{2+}\)-permeant and K\(^{+}\)-specific channels by fusing a bacterial photoactivated adenylyl cyclase to cyclic nucleotide-gated channels with high permeability for Ca\(^{2+}\) or for K\(^{+}\), respectively. Optimized fusion constructs showed strong light-gated conductance in Xenopus laevis oocytes and in rat hippocampal neurons. These constructs could also be used to control the motility of Drosophila melanogaster larvae, when expressed in motoneurons. Illumination led to body contraction when motoneurons expressed the light-sensitive Ca\(^{2+}\)-permeant channel, and to body extension when expressing the light-sensitive K\(^{+}\) channel, both effectively and reversibly paralyzing the larvae. Further optimization of these constructs will be required for application in adult flies since both constructs led to eclosion failure when expressed in motoneurons.},
  language  = {en}
}
@article{ZhuShabalaCuinetal.2016,
  author    = {Zhu, Min and Shabala, Lana and Cuin, Tracey A and Huang, Xin and Zhou, Meixue and Munns, Rana and Shabala, Sergey},
  title     = {Nax loci affect SOS1-like Na\(^{+}\)/H\(^{+}\) exchanger expression and activity in wheat},
  series = {Journal of Experimental Botany},
  volume    = {67},
  journal   = {Journal of Experimental Botany},
  number    = {3},
  doi       = {10.1093/jxb/erv493},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-150236},
  pages     = {835-844},
  year      = {2016},
  abstract  = {Salinity stress tolerance in durum wheat is strongly associated with a plant's ability to control Na\(^{+}\) delivery to the shoot. Two loci, termed Nax1 and Nax2, were recently identified as being critical for this process and the sodium transporters HKT1;4 and HKT1;5 were identified as the respective candidate genes. These transporters retrieve Na\(^{+}\) from the xylem, thus limiting the rates of Na\(^{+}\) transport from the root to the shoot. In this work, we show that the Nax loci also affect activity and expression levels of the SOS1-like Na\(^{+}\)/H\(^{+}\) exchanger in both root cortical and stelar tissues. Net Na\(^{+}\) efflux measured in isolated steles from salt-treated plants, using the non-invasive ion flux measuring MIFE technique, decreased in the sequence: Tamaroi (parental line)>Nax1=Nax2>Nax1:Nax2 lines. This efflux was sensitive to amiloride (a known inhibitor of the Na\(^{+}\)/H\(^{+}\) exchanger) and was mirrored by net H\(^{+}\) flux changes. TdSOS1 relative transcript levels were 6-10-fold lower in Nax lines compared with Tamaroi. Thus, it appears that Nax loci confer two highly complementary mechanisms, both of which contribute towards reducing the xylem Na\(^{+}\) content. One enhances the retrieval of Na\(^{+}\) back into the root stele via HKT1;4 or HKT1;5, whilst the other reduces the rate of Na\(^{+}\) loading into the xylem via SOS1. It is suggested that such duality plays an important adaptive role with greater versatility for responding to a changing environment and controlling Na\(^{+}\) delivery to the shoot.},
  language  = {en}
}
@phdthesis{Eckert2011,
  author    = {Eckert, Michaela Brigitte},
  title     = {Die Wirkung von Antidepressiva auf neuronale und kardiale Tandemporen-Kaliumkan{\"a}le},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-65804},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2011},
  abstract  = {Die vorliegende Arbeit besch{\"a}ftigte sich mit der Wirkung von Antidepressiva auf K2P-Kan{\"a}le. Sie stellen wie spannungsabh{\"a}ngige Ca2+, Na+ und K+-Kan{\"a}le als neuronale Ionenkan{\"a}le aufgrund ihrer Expressionsmuster und physiologischen Eigenschaften potentielle Zielproteine f{\"u}r Antidepressiva dar. Darum werden K2P-Kan{\"a}le in heterologen Expressionssystemen von klinisch verabreichten Antidepressiva inhibiert. Die K2P-Kan{\"a}le TREK-1, TASK-1 und THIK-1 zeigten sich in dieser Arbeit alle sensitiv auf das Antidepressivum Fluoxetin, welches die Kaliumstr{\"o}me der Kan{\"a}le unterschiedlich stark inhibierte. Hierbei lieferten die vorliegenden Untersuchungen den Nachweis, dass TREK-1 auf Fluoxetin am meisten, THIK-1 am wenigsten sensitiv reagiert. Der humane TREK-1 wird durch Fluoxetin in den Expressionssystemen Oozyten und HEK-Zellen zu fast 80\% inhibiert, wobei bei der humanen Zelllinie nur ein Zehntel der vorher eingesetzten Antidepressivakonzentration f{\"u}r die gleiche Inhibition des Ausw{\"a}rtsstroms notwendig war. Die vorliegende Arbeit weist Inhibitionen des Kanals bei einer Fluoxetinkonzentration von 1 µM nach, was der Serumkonzentration von depressiven Patienten entspricht. Zudem wird TREK-1 durch die Antidepressiva Maprotilin, Mirtazapin, Citalopram, Doxepin und Venlafaxin inhibiert, wobei letzteres kaum eine Wirkung zeigt. Alle verwendeten Antidepressiva nutzen die gleichen Angriffspunkte am Kanalprotein, da es bei einer Koapplikation mit einem weiteren Antidepressivum oder Benzodiazepin zu keiner Inhibitionsverst{\"a}rkung kommt. Die Interaktion zwischen Antidepressivum und Kanalprotein verl{\"a}uft mit großer Wahrscheinlichkeit direkt und ohne „second-messenger-Wege". Hierbei konnten die porenformende Region und der C-Terminus des Kanals als Interaktionspartner ausgeschlossen werden. Der Mechanismus der alternativen Translations-Initiaton generiert zwei unterschiedliche Proteinprodukte aus einem TREK-1 Transkript, eine lange Version des Proteins mit 426 Aminos{\"a}uren und zus{\"a}tzlich eine kurze Version mit 374 Aminos{\"a}uren, welcher die ersten 52 N-terminalen Aminos{\"a}uren fehlen. Die Fluoxetin-Sensitivit{\"a}t von TREK-1 [N52] verringert sich um 70\%. Dies verdeutlicht, dass die ersten 52 Aminos{\"a}uren essentiell zur TREK-1 Interaktion mit Antidepressiva beitragen.},
  subject      = {Kaliumkanal},
  language  = {de}
}
@phdthesis{Buechsenschuetz2005,
  author    = {B{\"u}chsensch{\"u}tz, Kai},
  title     = {Struktur und r{\"a}umlich-zeitliches Expressionsverhalten von Kaliumkan{\"a}len bei Zea mays},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-17522},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2005},
  abstract  = {Bei Zea mays wurden neue Kaliumkan{\"a}le der Shaker-Familie isoliert, charakterisiert und zusammen mit bereits bekannten Vertretern dieser Familie hinsichtlich m{\"o}glicher Aufgaben und Interaktionen untersucht.},
  subject      = {Mais},
  language  = {de}
}
@phdthesis{Roemer2005,
  author    = {R{\"o}mer, Michael},
  title     = {Endozytose des Ca2+-sensitiven hIK1-Kanals in migrierenden MDCK-F-Zellen},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-15750},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2005},
  abstract  = {Zellmigration ist ein wichtiges Ph{\"a}nomen im gesamten Leben eines menschlichen Organismus. Erw{\"u}nschte physiologische Bedeutung hat die Zellmigration z. B. im Rahmen der Embryogenese, der Wundheilung und der Immunabwehr, w{\"a}hrend sie pathophysiologisch unter anderem bei der Metastasierung maligner Neoplasien in Erscheinung tritt. F{\"u}r optimale Migration ist eine Beteiligung von Ionenkan{\"a}len, Ionentransportern und zytoskeletalen Mechanismen in streng koordinierter Interaktion notwendig. Bei selektiver Blockade Ca2+-sensitiver K+-Kan{\"a}le (IK1) durch das Skorpiongift Charybdotoxin wird die Migrationsf{\"a}higkeit von Zellen erheblich gest{\"o}rt. Da dies ein m{\"o}glicher thera-peutischer Ansatzpunkt zur Malignit{\"a}tsreduzierung von Tumoren durch Hemmung der Metastasierung sein k{\"o}nnte, galt es in dieser Arbeit, den „Lebenslauf" dieses Kanalproteins genauer zu erforschen. Bisherige Erkenntnisse {\"u}ber Migrationsmechanismen, Membran-Umbau und Integrintransport wurden in einem neuen Modell zusammengefasst, das unter anderem die Theorie des K+-Kanal-Rezirkulierens beinhaltet. Zur {\"U}berpr{\"u}fung dieser Theorie wurde in der vorliegenden Doktorarbeit auf die Endo-zytose der Ca2+-abh{\"a}ngigen K+-Kan{\"a}le als ersten Schritt bei deren Rezirkulation fokussiert. Durch die Insertion eines viralen HA-Epitops in die Gensequenz des hIK1 mit PCR-Technik konnte der Kanal durch monoklonale anti-HA-Antik{\"o}rper [Maus] markierbar und durch Zweitantik{\"o}rper [anti-Maus] detektierbar gemacht werden. Nach der Transfektion des hIK1-HA-34-Plasmids in migrierende MDCK-F-Zellen war aufgrund der extrazellul{\"a}ren Lage des HA-34-Epitops im Kanalprotein eine Markierung der Kan{\"a}le mit Antik{\"o}rpern in lebenden Zellen m{\"o}glich. Die Immunfluoreszenzmikroskopie zeigte nach 37°C-in vivo-Inkubation der Zellen mit extrazellul{\"a}rer hIK1-HA-34-Markierung die Bildung von vesikel{\"a}hnlichen Strukturen, die auf Endozytose hindeuten konnten. Untransfizierte Kontrollzellen blieben ungef{\"a}rbt - die Aufnahme der Antik{\"o}rper in die Zellen mit hIK1-HA-34 musste also spezi-fisch {\"u}ber Endozytose der Antik{\"o}rper-Kanal-Komplexe geschehen sein. In der in vivo-Inkubation bei 4°C waren die Versuchszellen nur schemenhaft zu erkennen und auch das bei den 37°C-Experimenten deutlich sichtbare „ruffling" an der Lamellipodiumspitze stellte sich nicht dar. Dies erh{\"a}rtete den Verdacht auf eine Ansammlung von Vesikeln an der Lamellipodiumvorderkante. Mit dem Enzyme-Linked Immuno Sorbent Assay (ELISA) konnte die K+-Kanalpr{\"a}senz in der Plasmamembran quantifiziert werden. Gegen{\"u}ber den untransfizierten Kontrollzellen waren die Peroxidase-Werte um das achtfache erh{\"o}ht. Dies weist ebenfalls auf eine spezifische Endozytose der hIK1-markierenden Antik{\"o}rper hin. Zudem zeigte sich bei der 37°C-Inkubation im Zeitverlauf {\"u}ber 60 Minuten eine S{\"a}ttigungskinetik, die ebenfalls f{\"u}r ein Rezirkulieren des hIK1 sprechen k{\"o}nnte. Zusammengefasst scheinen die Ergebnisse der verschiedenen Untersuchungstechniken die Endozytose Ca2+-empfindlicher K+-Kan{\"a}le als ersten Schritt eines vermuteten Rezirkulierens der Kanalproteine zu best{\"a}tigen.},
  language  = {de}
}