Kanalrhodpsine (ChRs) sind lichtaktivierbare Kationenkanäle, welche als primäre Fotorezeptoren in Grünalgen dienen. In der Optogenetik werden ChRs verwendet um neuronale Membranen zu depolarisieren und mit Licht Aktionspotentiale auszulösen. Das mit blauem Licht aktivierte Chlamydomonas Kanalrhodopsin 2 (C2) und effiziente Mutanten wie C2 H134R stellen die am häufigsten genutzten depolarisierenden, optogenetischen Werkzeuge dar. Komplementär zu ChRs werden Protonen- und Chloridpumpen aus Archaebakterien zur neuronalen Inhibierung durch lichtinduzierte Hyperpolarisation verwendet. In der vorliegenden Arbeit untersuchten wir die ChR-Chimäre C1V1, ein grünlichtaktiviertes ChR, das sich durch hervorragende Membranlokalisierung und hohe Fotoströme in HEK-Zellen auszeichnet. C1V1 und C1V1-Mutanten mit feinabgestimmten spektralen und kinetischen Eigenschaften ermöglichen die neuronale Aktivierung mit Wellenlängen bis 620 nm sowie die unabhängige Aktivierung zweier Zellpopulationen in Kombination mit C2. Um die strukturelle Basis von Kanalöffnung und Ionentransport in ChRs zu verstehen, wurden gezielt Mutationen in C2 und C1V1 eingeführt. Die Fotoströme der entsprechenden Mutanten wurden auf Kationenselektivität und kinetische Veränderungen untersucht. Während Aminosäuren, die den Kanal an der zytosolischen Seite begrenzen, die Kationenfreisetzung und Einwärtsgleichrichtung der ChRs bestimmen, spielen zentral im Kanal gelegende Aminosäuren ein entscheidende Rolle für Kationenselektivität und -kompetition. Ein enzymkinetisches Modell ermöglichte außerdem die Zerlegung der Fotoströme in Beiträge der verschiedenen, konkurrierenden Kationen. Im letzten Teil der Arbeit wurde pHoenix, ein optogenetisches Werkzeug zur Ansäuerung synaptischer Vesikel, entwickelt. In Neuronen des Hippocampus wurde pHoenix verwendet, um die treibenden Kräfte für die vesikuläre Neurotransmitteraufnahme sowie den Zusammenhang zwischen Vesikelfüllstand und Freisetzungswahrscheinlichkeit zu analysieren. / Channelrhodopsins (ChRs) are light-activated cation channels functioning as primary photoreceptors in green algae. In the emerging field of optogenetics, ChRs are used to depolarize neuronal membranes, thus allowing for light-induced action-potential firing. The blue light-activated Chlamydomonas channelrhodopsin 2 (C2) and high-efficiency mutants such as C2 H134R represent the most commonly used depolarizing optogenetic tools. Complementary to ChRs, green to yellow light-activated proton and chloride pumps originating from archea enable neuronal inhibition by membrane hyperpolarization. In the present work, we developed the chimeric ChR C1V1, a green-light activated ChR with excellent membrane targeting and high photocurrents in HEK cells. Action spectrum and kinetic properties of C1V1 were further fine-tuned by site-directed mutagenesis. The ensemble of C1V1 variants allows for neuronal activation with wavelengths up to 620 nm and can be used in two-color optogenetic experiments in combination with C2 derivatives. In order to understand the structural motifs involved in channel gating and ion transport, conserved residues in C2 and C1V1 were mutated and photocurrents of the respective mutants were analyzed for kinetic characteristics and cation selectivity. In these experiments, residues of the inner gate region were shown to alter cytosolic cation release and inward rectification, whereas central gate residues determine cation competition and selectivity, as well as the equilibrium between the two open channel conformations. Moreover, an enzyme-kinetic model was used to quantitatively dissect ChR photocurrents into the contribution of different competing cations. Finally, we designed pHoenix, an optogenetic tool enabling green-light induced acidification of synaptic vesicles. In hippocampal neurons, pHoenix was used to study both the energetics of vesicular neurotransmitter uptake and the impact of the vesicular contents on synaptic vesicle release.

Endotelinas (ETs) e sarafotoxinas (SRTXs) pertencem a uma família de peptídeos vasconstritores que podem regular a migração e/ou produção de pigmentos em células pigmentares de vertebrados (cromatóforos). Em peixes teleósteos, ETs/SRTXs induzem a migração de pigmentos. Em melanócitos humanos, as ETs promovem a melanogênese e mitogênese. ETs também regulam a transcrição de diversos genes. Esses efeitos são mediados por diferentes vias de sinalização intracelular, dentre elas a via da fosfolipase C (PLC), da proteína quinase C (PKC) e da cascata de sinalização por proteína quinases ativadas por mitógeno (MAPKs). A rodopsina é um fotopigmento responsável pela detecção de fótons presente nos bastonetes dos olhos dos vertebrados. A modulação da transcrição do gene para rodopsina em peixes teleósteos e mamíferos parece ocorrer através de elementos conservados. Cromatóforos podem responder diretamente à luz, resultando no deslocamento dos grânulos de pigmentos através dos processos dendríticos das células. Essas respostas evocadas por luz são provavelmente mediadas por moléculas fotorreceptoras expressas por essas células. A linhagem celular GEM-81, proveniente de eritroforoma do peixe teleósteo Carassius auratus, assim como os melanócitos B16 de Mus musculus expressam rodopsina e receptores para ETs dos subtipos ETB e ETA, respectivamente. O objetivo deste trabalho foi determinar se: 1) os níveis do RNAm para rodopsina poderiam ser modulados por SRTX S6c em GEM-81 e por ET-1 em B16 e quais os mecanismos de sinalização intracelular envolvidos nessa modulação; 2) os níveis protéicos de rodopsina também poderiam ser modulados por SRTX S6c em GEM-81 e por ET-1 em B16. Através de PCR em tempo real (quantitativo), demonstrou-se que SRTX S6c e ET-1 modulam os níveis do RNAm para rodopsina em GEM-81 e B16, respectivamente, de forma temporal e dose-dependente. Em GEM-81, essa modulação envolve a ativação de uma PKC e da cascata das MAPKs. Já em B16, há o envolvimento de PLC, cálcio como mensageiro intracelular, calmodulina, quinase dependente de cálcio/calmodulina e PKC. Através de ensaios de Western blotting, foi demostrado que na linhagem GEM-81 os níveis protéicos de rodopsina não são significativamente alterados por 24 horas de tratamento com SRTX 10-9M S6c, sugerindo o envolvimento de mecanismos de controle pós-transcricional na modulação da expressão protéica de rodopsina. Nas células B16 cuja extração de proteína total ocorreu 0 ou 6h após o fim do tratamento de 24h com ET-1 10-10M, os níveis protéicos de rodopsina não são significativamente alterados. Já nas células cuja proteína total foi extraída 3h após o fim do tratamento com ET-1, observou-se uma diminuição significativa dos níveis da proteína rodopsina. Esses resultados sugerem o envolvimento de mecanismos de controle pós-transcricional na modulação da expressão protéica de rodopsina, mecanismos estes exacerbados nas células B16 cuja extração de proteína ocorreu 3h após o fim do tratamento. / Endothelins (ETs) and sarafotoxins (SRTXs) belong to a family of vasoconstrictor peptides, which can regulate pigment migration and/or production in vertebrate pigment cells (chromatophores). In teleostean fish, ETs/SRTXs induce pigment migration. In human melanocytes, ETs promote melanogenesis and mitogenesis. ETs also regulate the transcription of several genes. These effects are mediated by different intracellular signaling pathways, such as the phospholipase C (PLC), protein kinase C (PKC) and the mitogen-activated protein kinase (MAPK) cascade. Rhodopsin is a photopigment responsible for photon detection, found in vertebrate rod cells. Rhodopsin gene transcription regulation in teleostean fish and mammals seems to occur through conserved elements. Chromatophores can respond directly to light, promoting the migration of pigment granules along the cells dedritic processes. These light-evoked responses are probably mediated by photoreceptive molecules expressed by these cells. The teleost Carassius auratus erythrophoroma cell line, GEM-81 and Mus musculus B16 melanocytes express rhodopsin, as well as the ET receptors, ETB and ETA, respectively. The aim of this study was to determine whether 1) rhodopsin mRNA levels could be modulated by SRTX S6c in GEM-81 cells and ET-1 in B16 cells and the intracellular signaling mechanisms involved; 2) rhodopsin protein levels could also be modulated by SRTX S6c in GEM-81 and ET-1 in B16 cells. Using real time (quantitative) PCR, we demonstrated that SRTX S6c and ET-1 modulate rhodopsin mRNA levels in GEM-81 and B16, respectively, in a time and dose-dependent way. In GEM-81, this modulation involves the activation of a PKC and the MAPK cascade. In B16, it involves PLC, calcium as a second messenger, calmodulin, a calcium/calmodulin dependent kinase and PKC. The Western blotting assays demonstrated that in GEM-81 cells rhodopsin protein levels are not significantly altered by a 24-hour treatment with 10-9M SRTX S6c, suggesting the involvement of post-transcriptional mechanisms in the modulation of rhodopsin expression. In B16 cells, whose total protein was extracted 0 or 6 hours after the 24-hour treatment with 10-10M ET-1, rhodopsin protein levels were not significantly altered. When the cells total protein was extracted 3 hours after the 24-hour treatment with ET-1, a significant reduction in rhodopsin protein levels was observed. These results also suggest the involvement of post-transcriptional mechanisms in the modulation of rhodopsin expression in this cell line. These mechanisms could be somehow exacerbated in B16 cells whose protein was extracted 3 hours after the treatment.

L’horloge circadienne permet de régler avec précision les anticipations physiologiques et comportementales face à un environnement perpétuellement oscillant entre jour et nuit. Cette capacité endogène n’est utile que si les processus biologiques restent synchronisés sur le temps solaire. La lumière représente le stimulus le plus efficace pour informer l’horloge des cycles environnementaux.
Chez la drosophile (Drosophila melanogaster) la synchronisation des rythmes veille/sommeil par la lumière est assurée par la molécule photosensible CRYPTOCHROME et par le système visuel. Alors que le cryptochrome agit dans les neurones d'horloges, le système visuel renseigne ces derniers par des voies qui restent à découvrir. La drosophile possède trois organes photorécepteurs, l'oeil composé, les ocelles et l'eyelet de Hofbauer-Buchner, qui expriment chacun une ou plusieurs rhodopsines. La cascade de phototransduction activée par la lumière dépend de la phospholipase C-ß NORPA et conduit à une libération d’histamine.
Dans notre étude, nous avons tenté de caractériser la contribution de chaque rhodopsine dans l’entraînement circadien, mais également de déterminer leur contribution norpA-dépendante en condition de faible lumière.
L’analyse de mutants a montré que les 6 rhodopsines du système visuel constituaient les seules molécules photosensibles capables d’informer l’horloge et que la RH2 et la RH5 seules étaient capables d’entraîner l’horloge en fonction des conditions expérimentales. Nous avons également pu mettre en évidence le fait que les RH1, RH3, RH4 et RH6 utilisaient une voie NORPA-dépendante pour informer l’horloge, alors que la RH2 ne semblait pas le faire. Des doutes subsistent quant à l’existence d’une voie NORPA- dépendante de la RH5 pour informer l’horloge. Nous avons également caractérisé la contribution des récepteurs à l’histamine ORT et HISCL1 dans les processus circadiens: en l'absence de cryptochrome, chacun des deux récepteurs suffit à synchroniser l'horloge et la perte des deux rend les mouches circadiennement aveugles De plus, nous avons constaté que la connexion des photorécepteurs à l’horloge ne se faisait pas directement mais par l’intermédiaire de voies glutamatergiques ou cholinergiques. L’ensemble de ce travail a permis de faire une 1er ébauche des circuits nécessaires à la transmission de l’information lumineuse à l’horloge cérébrale et d’identifier les opsines ainsi que les interneurones impliqués. / The circadian clock allowed physiologic and behavioural anticipation against the day/night oscillation. Light is the most powerful clue for living organism. In the fly Drosophila melanogaster, the rest-activity is synchronized by light and pass through the cryptochrome and the visual system. CRYPTOCHROME act directly in the clock neurons to inform the clock but little is known about the visual system. Drosophila posses tree structures: the ocelli, the compound eye and the eyelet of Hofbauer-Buchner, each structure expressed one or multiple rhodopsins. The phototransduction cascade is activated by light and depend one a phospholipase C-ß NORPA, this lead to histamine realised. Study of mutants show that the 6 rhodopsines represent the only photo-sensible molecule for the clock and the RH2 and the RH5 alone could entrain the clock. We have also find that the RH1, RH3, RH4 and RH6 use a NORPA-dependant way to inform the clock whereas the RH2 does not. Some doubt is still present regarding the RH5 NORPA-dependant way. We have determined that the two-histamines receptor ORT and HISCL1 are involved in the circadian process. Besides, we have shown that there is no direct connexion between the clock and the photoreceptors but the information is relay on a glutamatergique and a cholinegique pathway. This thesis draws the circuit by which the light informed the clock and identified the opsines and the interneurons involved.

Rhodopsin, a visual pigment protein found in retinal photoreceptors, mediates vision at low-light levels. Recent studies focusing primarily in human and mouse have challenged the assumption of neutral evolution of synonymous substitutions in mammals. Using recently developed likelihood-based codon models accounting for mutational bias and selection, we find significant evidence for selective constraint on synonymous substitutions in mammalian rhodopsins, and a preference for cytosine at 3rd codon positions. A second project investigated adaptive evolution in rhodopsin, in view of theories of nocturnality in early mammals. We detected a significant acceleration of non-synonymous substitution rates at the origins of therian mammals, and a tendency of synonymous substitutions towards C-ending codons prior to that. These findings suggest an evolutionary scenario in which synonymous substitutions that increase mRNA stability and/or translation efficiency may have preceded adaptive non-synonymous evolution in early mammalian rhodopsins. These findings have important implications for theories of early mammalian nocturnality.

Thesis (M.S.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 68 pages. Includes Vita. Includes bibliographical references.

La leucine zipper de la rétine neurale (Nrl) joue un rôle central dans le développement et l'homéostasie des bâtonnets en activant I'expression de gènes tels que le photopigment Rhodopsine. Nrl est aussi associé à la Rétinite Pigmentaire, faisant ainsi de ce gène un modèle intéressant pour la compréhension des programmes contrôlant le développement et I'homéostasie des photorécepteurs.Ce travail de thèse vise à caractériser les mécanismes régulateurs de I'expression de Nr/ au cours du développement rétinien. L'électroporation in vivo de vecteurs rapporteurs dans des rétines de souris en développement, a révélé des séquences minimales de promoteur Nr/ nécessaires à une expression spécifique dans les photorécepteurs. Nous avons identifié RORI3 comme facteur requis pour cette expression, et montré que les facteurs OTX2, CRX et CREB s'accrochent aussi directement à des régions régulatrices particulières du promoteur. Nous avons construit un virus adéno-associé (AAV) contenant un promoteur minimal Nrl de 0.3 kb, et montré qu'il est adapté à la délivrance de gène spécifiquement dans les photorécepteurs.Nous avons montré que NRL, CRX et NR2E3, les régulateurs principaux de la Rhodopsine, ont une expression rythmique au cours de 24 h, et que l'expression cyclique de Nr/ peut être due à l'activation par RORp, un composant l'horloge circadienne. Enfin, nous avons identifié un nouveau facteur de transcription, NonO, au niveau de la région du promoteur proximal de la Rhodopsine, qui en combinaison avec NRL et CRX, active le promoteur de la Rhodopsine. L'invalidation de NonO au cours du développement rétinien a prouvé son implication pour le développement et I'homéostasie des bâtonnets. / The Neural Retina Leucine zipper transcription factor (Nrl) plays a central role in rod photoreceptor development and homeostasis, by activating the expression of rod-specific genes such as the visual photopigment, Rhodopsin. Nrlhave been also associated with Retinitis Pigmentosa, making this gene an interesting model for understanding genetic programs controlling photoreceptors development and homeostasis.This thesis work aimed at characterizing regulatory mechanisms of Nr/ expression during retinal development. Using in vivo electroporation of reporter vectors carrying distinct portions of Nrlpromoter into neonatal mouse retina, we identified minimal sequences required for expression photoreceptors-specific expression. We identified RORI3 as being required for this expression and showed that OTX2, CRX and CREB transcription factors also directly bind to the defined regulatory regions.We designed a novel adeno-associated virus (AAV) vector containing a minimal Nrl promoter fragment of 0.3 kb, and showed that it is well-suited for gene delivery specifically into photoreceptors.We also showed that NRL, CRX, and NR2E3, the main transcriptional regulators of Rhodopsin, display rhythmic expression over 24 h. and that Nrl might undergo cyclic activation by RORB which is part of the photoreceptor circadian clock. Finally, we investigated the role of a novel Rhodopsin transcriptional regulator, NonO, identified in theRhodopsin proximal promoter region. We demonstrated that NonO co-activates Rhodopsin promoter along with NRL and CRX. By knocking down this gene during retinal development we provided evidence for its role in rod development and homeostasis.

Endotelinas (ETs) e sarafotoxinas (SRTXs) pertencem a uma família de peptídeos vasconstritores que podem regular a migração e/ou produção de pigmentos em células pigmentares de vertebrados (cromatóforos). Em peixes teleósteos, ETs/SRTXs induzem a migração de pigmentos. Em melanócitos humanos, as ETs promovem a melanogênese e mitogênese. ETs também regulam a transcrição de diversos genes. Esses efeitos são mediados por diferentes vias de sinalização intracelular, dentre elas a via da fosfolipase C (PLC), da proteína quinase C (PKC) e da cascata de sinalização por proteína quinases ativadas por mitógeno (MAPKs). A rodopsina é um fotopigmento responsável pela detecção de fótons presente nos bastonetes dos olhos dos vertebrados. A modulação da transcrição do gene para rodopsina em peixes teleósteos e mamíferos parece ocorrer através de elementos conservados. Cromatóforos podem responder diretamente à luz, resultando no deslocamento dos grânulos de pigmentos através dos processos dendríticos das células. Essas respostas evocadas por luz são provavelmente mediadas por moléculas fotorreceptoras expressas por essas células. A linhagem celular GEM-81, proveniente de eritroforoma do peixe teleósteo Carassius auratus, assim como os melanócitos B16 de Mus musculus expressam rodopsina e receptores para ETs dos subtipos ETB e ETA, respectivamente. O objetivo deste trabalho foi determinar se: 1) os níveis do RNAm para rodopsina poderiam ser modulados por SRTX S6c em GEM-81 e por ET-1 em B16 e quais os mecanismos de sinalização intracelular envolvidos nessa modulação; 2) os níveis protéicos de rodopsina também poderiam ser modulados por SRTX S6c em GEM-81 e por ET-1 em B16. Através de PCR em tempo real (quantitativo), demonstrou-se que SRTX S6c e ET-1 modulam os níveis do RNAm para rodopsina em GEM-81 e B16, respectivamente, de forma temporal e dose-dependente. Em GEM-81, essa modulação envolve a ativação de uma PKC e da cascata das MAPKs. Já em B16, há o envolvimento de PLC, cálcio como mensageiro intracelular, calmodulina, quinase dependente de cálcio/calmodulina e PKC. Através de ensaios de Western blotting, foi demostrado que na linhagem GEM-81 os níveis protéicos de rodopsina não são significativamente alterados por 24 horas de tratamento com SRTX 10-9M S6c, sugerindo o envolvimento de mecanismos de controle pós-transcricional na modulação da expressão protéica de rodopsina. Nas células B16 cuja extração de proteína total ocorreu 0 ou 6h após o fim do tratamento de 24h com ET-1 10-10M, os níveis protéicos de rodopsina não são significativamente alterados. Já nas células cuja proteína total foi extraída 3h após o fim do tratamento com ET-1, observou-se uma diminuição significativa dos níveis da proteína rodopsina. Esses resultados sugerem o envolvimento de mecanismos de controle pós-transcricional na modulação da expressão protéica de rodopsina, mecanismos estes exacerbados nas células B16 cuja extração de proteína ocorreu 3h após o fim do tratamento. / Endothelins (ETs) and sarafotoxins (SRTXs) belong to a family of vasoconstrictor peptides, which can regulate pigment migration and/or production in vertebrate pigment cells (chromatophores). In teleostean fish, ETs/SRTXs induce pigment migration. In human melanocytes, ETs promote melanogenesis and mitogenesis. ETs also regulate the transcription of several genes. These effects are mediated by different intracellular signaling pathways, such as the phospholipase C (PLC), protein kinase C (PKC) and the mitogen-activated protein kinase (MAPK) cascade. Rhodopsin is a photopigment responsible for photon detection, found in vertebrate rod cells. Rhodopsin gene transcription regulation in teleostean fish and mammals seems to occur through conserved elements. Chromatophores can respond directly to light, promoting the migration of pigment granules along the cells dedritic processes. These light-evoked responses are probably mediated by photoreceptive molecules expressed by these cells. The teleost Carassius auratus erythrophoroma cell line, GEM-81 and Mus musculus B16 melanocytes express rhodopsin, as well as the ET receptors, ETB and ETA, respectively. The aim of this study was to determine whether 1) rhodopsin mRNA levels could be modulated by SRTX S6c in GEM-81 cells and ET-1 in B16 cells and the intracellular signaling mechanisms involved; 2) rhodopsin protein levels could also be modulated by SRTX S6c in GEM-81 and ET-1 in B16 cells. Using real time (quantitative) PCR, we demonstrated that SRTX S6c and ET-1 modulate rhodopsin mRNA levels in GEM-81 and B16, respectively, in a time and dose-dependent way. In GEM-81, this modulation involves the activation of a PKC and the MAPK cascade. In B16, it involves PLC, calcium as a second messenger, calmodulin, a calcium/calmodulin dependent kinase and PKC. The Western blotting assays demonstrated that in GEM-81 cells rhodopsin protein levels are not significantly altered by a 24-hour treatment with 10-9M SRTX S6c, suggesting the involvement of post-transcriptional mechanisms in the modulation of rhodopsin expression. In B16 cells, whose total protein was extracted 0 or 6 hours after the 24-hour treatment with 10-10M ET-1, rhodopsin protein levels were not significantly altered. When the cells total protein was extracted 3 hours after the 24-hour treatment with ET-1, a significant reduction in rhodopsin protein levels was observed. These results also suggest the involvement of post-transcriptional mechanisms in the modulation of rhodopsin expression in this cell line. These mechanisms could be somehow exacerbated in B16 cells whose protein was extracted 3 hours after the treatment.

Das haloalkaphile Archaeon Natronomas pharaonis nutzt den Lichtrezeptor, das Sensorische Rhodopsin II (NpSRII), im Komplex mit dem halobakteriellen Transducer (NpHtrII) zur photophoben Antwort auf schädliches grün-blaues Licht und entsprechender Steuerung des Flagellenmotors um optimale Umgebungen zum Überleben aufzusuchen. In einer Membran rekonstituiert bildet der Rezeptor/Transducer Komplex eine 2:2 Stöchiometrie aus, wobei ein Transducer-Dimer von zwei Rezeptor-Molekülen flankiert wird. Durch die Lichtanregung wird ein Photozyklus initiiert, dessen Intermediate sich aufgrund ihrer optischen und/oder strukturellen Eigenschaften unterscheiden. In dieser Studie sind die strukturellen Änderungen des Rezeptors und des Transducers während des Photozyklus mit Hilfe der Elektronenspinresonanz (ESR)-Spektroskopie in Kombination mit der ortsspezifischen Spinmarkierung aufgeklärt worden. Als Methoden wurden dabei die zeitaufgelöste ESR-Spektroskopie und Abstandsmessungen in unterschiedlichen Intermediaten mit Hilfe von cw- und Puls-ESR-Techniken genutzt. Der Signaltransfer nach Initiierung des Photozyklus im Rezeptor, die Weiterleitung des Signals zum Transducer durch die Auswärtsbewegung der Helix F und die damit verbundene Verschiebung des thermodynamischen Gleichgewichts in der HAMP-Domäne des Transducers konnten beobachtet und analysiert werden. Die Methode der ESR-Spektroskopie erweist sich als mächtige biophysikalische Technik, die eine direkte und zeitaufgelöste Analyse von strukturellen Konformationsänderungen in Membranproteinen und die strukturelle Aufklärung unterschiedlicher Intermediate erlaubt.

Mikrobielle Rhodopsine sind lichtsensitive Proteine, die von Mikroorganismen exprimiert werden um Licht wahrzunehmen oder dessen Energie zu nutzen. Ionen-transportierende mikrobielle Rhodopsine begründeten das Feld der Optogenetik. Hier erlauben sie transmembrane Ionenflüsse lichtsensitiv zu machen und neuronale Aktivität mit Licht zu steuern. Eine zielführende Nutzung beruht auf ihrer molekularen Charakterisierung, um sie dem Experiment anzupassen und es sinnvoll zu entwerfen. Teil I der Arbeit beschäftigt sich mit dem rotverschobenen Kanalrhodopsin ReaChR. Obwohl es mit breitem, nicht gaussförmigen Aktionsspektrum mit maximalen Strömen um 600 nm publiziert wurde, zeigte das Blitzlichtspektrum hier maximale Aktivität bei 535 nm ohne Besonderheiten. Mit steigender Intensität und längeren Pulsen verbreiterte sich das Spektrum; sehr ähnlich zum publizierten Spektrum. Dieses einzigartige Verhalten wird durch sekundäre Photochemie erklärt, welche zu einem komplexen Photozyklus mit lichtinduzierten Übergangen führt. Mutationen an Schlüsselpositionen wurden genutzt, um ReaChR über die publizierten Daten hinaus zu charakterisieren und neue Eigenschaften zu generieren. In Teil II wurde die auswärtsgerichtete Natriumpumpe KR2 elektrophysiologisch charakterisiert, was zuvor von schlechter Membranständigkeit in Säugetierzellen verhindert wurde. Ein verbessertes KR2 mit höherer Membranständigkeit und 60-fach größeren Photoströmen erlaubte Selektivitätsmessungen, welche zeigten, dass der Strom von Natriumionen getragen wird, wohingegen nichts auf Protonentransport hindeutete. Bei ausreichender Substratkonzentration war der Strom anders als bei Chlorid- oder Protonenpumpen von der Membranspannung unabhängig. Die Expression in Mausneuronen ermöglichte die reversible Unterdrückung von Aktionspotentialen mit Licht, wobei der Ausstrom von Kationen einen komplementären Weg zur neuronale Aktivitätsunterdrückung bietet, wenn etablierte Werkzeuge schlecht oder nicht funktionieren. / Microbial rhodopsins are photosensitive proteins utilized by fungi, algae, and prokaryotes to sense light or harness its' energy. Ion transporting microbial rhodopsins initiated the field of optogenetics, where they are applied to render transmembrane ion fluxes light sensitive and control neuronal activity with light. Part I of the thesis focused on the electrophysiological characterization of the red-shifted channelrhodopsin ReaChR. Although published with a broad, non-Gaussian shaped action spectrum peaking around 600 nm, the flash action spectra of ReaChR recorded here had a maximum at 535 nm without peculiarities. Increasing intensities and prolonging illumination broadened the spectrum, which finally peaked around 600 nm. This unique behavior stems from pronounced secondary photochemistry leading to a complex photocycle with various light-induced transitions especially under constant illumination. Mutations at key positions like the central gate, DC-pair or counter ions were employed to characterize the properties of ReaChR beyond published data and engineer new features. In part II an electrophysiological characterization of the outward Na+ pump KR2 was pursued, which was hindered by poor membrane targeting in mammalian cells before. Engineering of eKR2 improved membrane targeting and lead to 60-fold larger photocurrents than in the wild type. Selectivity measurements revealed that the stationary photocurrent is primarily carried by sodium with no evidence for proton transport. At sufficient substrate concentration stationary photocurrents were independent of the membrane voltage distinguishing eKR2 from proton and chloride pumps. Finally, eKR2 reliably and reversibly inhibited action potential firing already at 0.5 mW/mm2 green illumination in cultured hippocampal mouse neurons. Inhibiting action potential firing through cation extrusion poses a complementary way of neuronal silencing in contexts where established tools are unfavorable or even impossible to use.

No description available.