@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} }