TY - THES A1 - Fei, Lin T1 - Optogenetic regulation of osmolarity and water flux T1 - Optogenetische Regulation der Osmolarität und des Wasserflusses N2 - 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. N2 - Die Optogenetik ist eine leistungsstarke Technik, die Licht zur präzisen Regulierung der physiologischen Aktivitäten von Neuronen und anderen Zelltypen einsetzt. Konkret werden Licht-empfindliche Ionenkanäle, Pumpen oder Enzyme in Zellen exprimiert, um ihre Regulierung durch Belichtung zu ermöglichen und so eine präzise Kontrolle biochemischer Signalwege zu ermöglichen. Der erste Teil meiner Studie umfasste die Konstruktion, Optimierung und Charakterisierung von zwei optogenetischen Werkzeugen, KCR1 und NCR1. Elena Govorunova und Mitarbeiter entdeckten einen lichtgesteuerten Kaliumkanal, KCR1, in dem Protozoen Hyphochytrium catenoides. Herkömmliche Kalium-Ionenkanäle werden entweder als ligandengesteuert oder spannungsgesteuert klassifiziert und verfügen über konservierte porenbildende Domänen und K+-selektive Filter. KCR1 ist jedoch insofern einzigartig, als er nicht die Signatursequenz der bisher bekannten K+-Kanäle enthält und ein Kanalrhodopsin ist. Wir synthetisierten das KCR1-Plasmid entsprechend der veröffentlichten Sequenz und exprimierten es in Xenopus-Oozyten. Da der ursprüngliche KCR1-Strom zu klein war, optimierte ich ihn zu KCR1 2.0, um seine Leistung zu verbessern, indem LR (Signalpeptid LucyRho, verbessert die Expression) am N-Terminus und T (Trafficking-Signalpeptid) und E (ER-Export-Signalpeptid) am C-Terminus fusioniert wurden. Außerdem untersuchte ich die Lichtempfindlichkeit, das Wirkungs-Spektrum und die Kinetik von KCR1 2.0 in Xenopus-Oozyten. Die Kaliumpermeabilität von KCR1 2.0, PK/PNa  24, macht KCR1 2.0 zu einem leistungsfähigen hyperpolarisierenden Werkzeug, das zur Hemmung von Nervenzellen in Tieren eingesetzt werden kann. Inspiriert von KCR1 verwendeten wir die KCR1-Sequenz als Vorlage für den Gen-Sequenzabgleich mit Sequenzen in H. catenoides. Wir fanden heraus, dass NCR1 und KCR1 ähnliche Gensequenzen haben. NCR1 wurde von uns als lichtgesteuerter Natriumkanal charakterisiert. NCR1 wurde ebenfalls von Govorunova et al. charakterisiert und vor kurzem unter dem Namen HcCCR veröffentlicht. Da der ursprüngliche NCR1-Strom zu gering war, optimierte ich ihn zu NCR1 2.0, um seine Leistung zu verbessern, indem ich LR am N-Terminus und T und E am C-Terminus fusionierte, was das Expressionsniveau erheblich verbesserte und die Stromamplitude von NCR1 stark erhöhte. NCR1 2.0 in voller Länge enthält 432 Aminosäuren. Um zu testen, ob die Anzahl der Aminosäuren die Eigenschaften von NCR1 2.0 verändert, haben wir NCR1 2.0 (330), NCR1 2.0 (283) und NCR1 2.0 (273) entwickelt, indem wir die Anzahl der Aminosäuren auf 330, 280 bzw. 273 in NCR1 2.0 verkürzt haben. Mit abnehmender Anzahl der Aminosäuren nahm der Strom in NCR1 2.0 zu. Ich untersuchte auch die Licht-Empfindlichkeit, das Wirkungsspektrum und die Kinetik von NCR1 2.0 (273) in Xenopus-Oozyten. Wir führten vier Punktmutationen an den Aminosäurepositionen 133 und 116 von NCR1 2.0 durch und analysierten die Umkehrpotentiale der Mutanten. Die Mutationen waren wie folgt: NCR1 2.0 (273 D116H), NCR1 2.0 (273 D116E), NCR1 2.0 (283 V133H), und NCR1 2.0 (283 D116Q). Der zweite Teil dieser Studie konzentriert sich auf den lichtinduzierten Wassertransport mit Hilfe optogenetischer Methoden. Wir untersuchten den Einsatz optogenetischer Werkzeuge zur Regulierung des Wasserflusses durch Veränderung der Osmolarität in Oozyten. Der Wasserfluss durch AQP1 wird durch den osmotischen Gradienten angetrieben, der durch Konzentrationsunterschiede kleiner Moleküle oder Ionen entsteht. Daher versuchen wir, die Ionenkonzentration mit optogenetischen Mitteln zu regulieren, um den Wasserfluss nicht-invasiv zu steuern. Zu diesem Zweck verwendete ich die lichtgesteuerten Kationenkanäle XXM 2.0 und NCR1 2.0 zur Regulierung der Na+-Konzentration, während der K+-Kanal KCR1 2.0 zur Regulierung der K+-Konzentration eingesetzt wurde. Wenn Na+ in die Xenopus-Oozyten fließt, wird das Membranpotential der Oozyten positiv, und Cl- kann durch den lichtgesteuerten Anionenkanal GtACR1 einströmen. Durch die Kombination dieser optogenetischen Werkzeuge zur Regulierung der NaCl- oder KCl-Konzentration kann ich die Osmolarität innerhalb der Oozyten verändern und so den Wasserfluss regulieren. Ich habe AQP1 zusammen mit optogenetischen Werkzeugen in den Oozyten exprimiert, um den Wasserfluss zu beschleunigen. Insgesamt habe ich drei Kombinationen entwickelt (1: AQP1, XXM 2.0 und GtACR1. 2: AQP1, NCR1 2.0 und GtACR1. 3: AQP1, KCR1 2.0 und GtACR1) zur Regulierung des Wasserflusses in den Eizellen. Das Schrumpfen oder Anschwellen der Oozyten kann nur erreicht werden, wenn AQP1, lichtgesteuerte Kationenkanäle (XXM 2.0/NCR1 2.0/KCR1 2.0) und lichtgesteuerte Anionenkanäle (GtACR1) gemeinsam exprimiert werden. Die Belichtung nach Expression von einem oder beiden allein führt nicht zu Veränderungen der Morphologie der Oozyten. Zusammenfassend lässt sich sagen, dass ich eine neuartige Strategie zur nicht-invasiven Beeinflussung der Wasserbewegung in und aus Xenopus-Oozyten durch Licht demonstriert habe. Diese Ergebnisse eröffnen einen neuen Weg zur Beeinflussung der Wasserhomöostase als Mittel zur Untersuchung verwandter biologischer Phänomene in verschiedenen Zelltypen und Organismen. KW - aquaporins KW - Osmolarität KW - optogenetic KW - sodium KW - potassium Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-323092 ER - TY - JOUR A1 - Beck, Sebastian A1 - Yu-Strzelczyk, Jing A1 - Pauls, Dennis A1 - Constantin, Oana M. A1 - Gee, Christine E. A1 - Ehmann, Nadine A1 - Kittel, Robert J. A1 - Nagel, Georg A1 - Gao, Shiqiang T1 - Synthetic light-activated ion channels for optogenetic activation and inhibition JF - Frontiers in Neuroscience N2 - 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. KW - optogenetics KW - calcium KW - potassium KW - bPAC KW - CNG channel KW - cAMP KW - Drosophila melanogaster motoneuron KW - rat hippocampal neurons Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-177520 VL - 12 IS - 643 ER - TY - JOUR A1 - Zhu, Min A1 - Shabala, Lana A1 - Cuin, Tracey A A1 - Huang, Xin A1 - Zhou, Meixue A1 - Munns, Rana A1 - Shabala, Sergey T1 - Nax loci affect SOS1-like Na\(^{+}\)/H\(^{+}\) exchanger expression and activity in wheat JF - Journal of Experimental Botany N2 - 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. KW - HKT transporter KW - potassium KW - salinity stress KW - sequestration KW - sodium KW - xylem loading Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-150236 VL - 67 IS - 3 ER - TY - JOUR A1 - Zhu, Min A1 - Shabala, Lana A1 - Cuin, Tracey A. A1 - Huang, Xin A1 - Zhou, Meixue A1 - Munns, Rana A1 - Shabala, Sergey T1 - Nax loci affect SOS1-like Na\(^+\)/H\(^+\) exchanger expression and activity in wheat JF - Journal of Experimental Botany N2 - 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. KW - HKT transporter KW - potassium KW - salinity stress KW - sequestration KW - sodium KW - xylem loading Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-190908 VL - 67 IS - 3 ER -