Browsing by Subject "Ionenkanal"

Now showing 1 - 2 of 2

Charakterisierung der lichtinduzierten Internalisierung des Ionenkanals TRPL aus Drosophila melanogaster
(2012) Oberegelsbacher, Claudia; Huber, Armin
The light-dependent isomerization of rhodopsin (Rh1), which takes place in the compound eyes of Drosophila, leads to the activation of the visual signaling cascade. The result is a depolarizing receptor potential caused by the Ca2+-influx through the two cation channels TRP and TRPL. This Ca2+ influx subsequently mediates a change in the subcellular localization of the TRPL channel by inducing its translocation. TRPL of dark-adapted flies is located inside the rhabdomeres, whereas upon illumination, TRPL translocates to a yet unidentified storage compartment in the cell body of the photoreceptor cell. The translocation is reversible; however, the underlying mechanism remains largely unclear. Based on the observation of TRPL-containing vesicles on immunocytochemical sections of illuminated flies a vesicular transport mechanism has been proposed for TRPL translocation. In the present work, the mechanism underlying light-dependent TRPL internalization was studied. Using immunocytochemical techniques, a co-localization of rhodopsin and TRPL was observed in endocytic vesicles. Like many other G-protein coupled receptors, Rhodopsin undergoes endocytosis following activation. The rate of Rh1 internalization depends on the amount of metarhodopsin and, therefore, on the light quality used for illumination. The internalization rate was determined by counting Rh1- and TRPL-positive vesicles observed upon illumination with different light qualities. Surprisingly, the light quality that induced the highest number of Rh1-positive vesicles (i.e. blue light) caused the lowest number of TRPL-positive vesicles, while illumination with orange light induced strong TRPL internalization, but poor Rh1 endocytosis. Likewise, time courses of TRPL internalization were significantly faster in orange light compared to blue light. These findings may indicate a competition between TRPL and Rh1 for a common internalization factor. Analysis of endocytosis in different mutants showed that the internalization of TRPL required Ca2+ influx mediated by the activation of the phototransduction cascade, whereas internalization of Rhodopsin was Ca2+-independent. Therefore, the trigger for activating TRPL and Rh1 endocytosis seems to be different, although both types of internalization were mechanistically similar and depended on dynamin function. The internalization of Rhodopsin is mediated by Rab5. A screen of dominant negative Rab mutants revealed that the light-induced internalization of TRPL is mediated by Rab5 and RabX4. Accordingly, the involvement of Rab5 constitutes another common feature in the endocytosis of TRPL and Rh1. Arrestins play an important role in regulating the endocytosis of rhodopsin. Whereas arrestin2 mediates the inactivation of metarhodopsin, arrestin1 is responsible for subsequent rhodopsin endocytosis. The endocytosis of TRPL is independent of arrestins, but arrestin2 fulfills an important function regarding the stability of the TRPL protein in the rhabdomere. In the present work, the analysis of different arr2 alleles revealed a complete degradation of the TRPL protein after ten days in darkness, but not in light. This finding suggests that arrestin2 has a possible function as a scaffolding protein in the rhabdomer of dark-adapted flies, but not of light-adapted flies, when TRPL is located in a storage compartment in the cell body. There is another fundamental difference between the two transport mechanisms regarding the fate of the protein after it has been internalized. Rhodopsin undergoes rapid lysosomal degradation whereas the trafficking of TRPL is described as a recycling mechanism. In this work, it was possible to show colocalization of TRPL with recycling endosomes indicating an involvement of these compartments in TRPL trafficking. Furthermore, rhodopsin but not TRPL showed colocalization with a lysosomal marker in light-adapted flies, providing additional evidence for the recycling of the TRPL channel.
Patch clamp experiments with human neuron-like cells under different gravity conditions
(2010) Kohn, Florian Peter Michael; Hanke, Wolfgang
Gravitation influences many physical, chemical and biological processes. Cells and their behaviour are no exception. The gravitational impact on the activity of neuronal cells is very important with the perspective of manned space missions. Previous experiments with vertebrates showed that the velocity of neuronal impulses (action potentials) in nerve fibres is decreasing under zero gravity and is increasing at high gravity. There are many theories about the changed properties of the nervous systems under zero gravity, but the molecular principles are mostly unexplored. For this dissertation hardware for patch clamp experiments under microgravity was developed. The patch clamp technique is a common used tool in investigating the electrophysiological properties of cells and single ion channels. Since the conditions during parabolic flights render classic patch clamp nearly impossible, advanced chip-based planar patch-clamp hardware, the Port-a-Patch from Nanion Technologies was integrated into a setup. This setup had to comply with the mandatory design and safety regulations to be used in parabolic flights. Two parabolic flight campaigns were used to validate the hardware, adapt the patch clamp procedures to the special conditions during a parabolic flight (as time pressure and vibrations) and to choose a suitable cell line from the available cell lines. As the laboratory conditions on ground were insufficient at the beginning of the project, the main focus had to lie on robust cells. The SH-SY5Y cell line was chosen for their robustness (they already have been used successfully by other teams) and their origin from the human brain (glioblastoma). During two subsequent parabolic flight campaigns patch clamp experiments were performed and whole cell currents of SH-SY5Y cells were recorded with increasing success rate. Pulse protocols were used to create current-voltage (I-V) characteristics. To obtain 20 seconds of microgravity, two phases of hypergravity, each lasting 20 seconds, had to be endured by the passengers. This fact allowed the subsequent recording of whole cell currents of the same cell during normal Gravity (1g), hypergravity (1.8g) and microgravity (approx. 10-3g) to compare the I-V characteristics of the different gravity conditions. For SH-SY5Y cells it was shown that a gravity dependence of the whole cell currents exists. The micro- and hypergravity whole cell currents were changed compared to 1g flight controls. At -20 and -10mV, the hypergravity current was significantly decreased compared to 1g, by 13.5% at -20mV and by 7.4% at -10mV. A significant 1.8g current < 0g current relation could be observed at potentials between -20 and +10mV. At -20mV the microgravity whole cell currents were increased by 13.5%, at -10mV by 7.4%, at 0mV by 6.2% and at +10mV by 3.9%. The duration and complexity of the used pulse protocols were limited by the time of each gravity phase (20-22 seconds). In a last parabolic flight campaign, therefore the passive electrophysiological properties of the cell membrane were investigated. As the laboratory conditions at the site were greatly improved in the meantime, especially for cell culture, new cell lines could be tested for their usability. SNB19 cells, also originating from the human brain (astrocytoma) were chosen due to their good sealing quality and stability compared to SH-SY5Y cells and constant pulse protocols near the estimated resting potential (-80 and -60mV) were performed. At constant -80mV and -60mV, it was shown that the whole cell currents during the variable gravity conditions are significantly increased compared to the 1g in-flight controls. The hypergravity current of SNB19 was increased by 2.2% at -80V and by 8.2% at -60mV. The microgravity current was increased by 4.1% at -80mV and 3.7% at -60mV. The acquired I-V characteristic of SNB19 differs from the I-V characteristic of SH-SY5Y. These findings show that the planar patch clamp technique can be used in parabolic flights to investigate the electrophysiological properties of single. Furthermore the findings suggest that the electrophysiological properties of single cells originating from the human brain exist are gravity dependent.