Radiation induced activation of potassium-channels: The role of ROS and calcium

Ionizing radiation (IR), in particular photons, is a quasi-universal tool in medical diagnostics and in tumor therapy. The negative side effects of this high-energy photon irradiation, which often cause secondary cancers or cell invasiveness, are well documented. The classical paradigm still is that all these effects can be traced back to irradiation induced DNA damage. Damage to other cellular compartments has been neglected for a long time. Recent research, however, has demonstrated that a calcium-activated K+-channel (hIK-channel) is activated by different types of ionizing radiation, e.g. γ-irradiation (Kuo et al., 1993), X-ray, α-particles and heavy-ion irradiation (Roth, 2013). The elevated K+ conductance results in a membrane hyperpolarization; the latter is a known signal for cell cycle progression. In the present thesis I elucidate the signal cascade, which is activated by IR and which finally activates hIK channels. In order to examine whether excursion in the concentration of cellular hydrogen peroxide (H2O2), or of the free concentration of Ca2+ ([Ca2+]cyt) are involved in signaling after IR, I employed several genetically encoded fluorescence sensors. The generation of reactive oxygen species (ROS), especially H2O2, was measured before and immediately after cells were challenged with either 405 nm UV laser micro-irradiation, X-rays or heavy-ion irradiation with a sensor for H2O2 (HyPer) and a sensor for the glutathione redox-buffer (Grx1-roGFP2). The latter is a sink for all ROS, which are eliminated in a cell by the oxidation of glutathione. These measurements provide for the first time robust quantitative data on the generation of ROS directly after irradiation in single living cells with a high temporal and spatial resolution. The data show that ROS molecules are generated immediately after the irradiation stress. They are rapidly buffered by an efficient redox-buffer system, which involves glutathione. When the buffer is exhausted the concentration of ROS is increasing throughout the cell; the latter could be monitored directly by an increase in the concentration of H2O2, a known second messenger in the cell. This general pattern is observed with some variations after exposing cells to X-ray stress (1-10 Gy) and 405 nm UV-irradiation (0.5-4.5 mJ/µm2). The latter micro-irradiation experiments of the cells with laser light provide the additional information that the ROS response is maximal in the compartment, which is directly irradiated and that an irradiation of the nucleus generates about 2 to 3 times more H2O2 than the equivalent irradiation of the cytosol. Also an irradiation of cells with heavy-ions causes an increase in H2O2 concentration, but the response is more variable and not all cells reveal an increase in H2O2. Further experiments suggest that the rise in H2O2, which is generated in cells as a responds to irradiation stress, is sufficient to trigger a signal cascade, involving an increase in [Ca2+]cyt. The latter hypothesis is supported by the finding that an incubation of A549 cells and HEK293 cells in a buffer with H2O2 is triggering an elevation in [Ca2+]cyt. This was measured with a FRET based Ca2+ sensor (YC3.60). The fact that challenging the same cells with the identical amount of H2O2 is sufficient to stimulate the Ca2+-activated hIK channel suggests that channel activation is mediated via a H2O2 induced increase in [Ca2+]cyt. This upstream part of the signaling cascade is independent of the cell type and found in HEK293 cells and A549 cells. The increase in membrane conductance, which is downstream of these events, is only elevated in cells like A549 cells, which express the hIK channel. When hIK channels are transiently expressed in HEK293 cells, also these cells, which are in their native form insensitive to IR, respond to the radiation stress with an increase in membrane conductance. Collectively the data show that cells, which functionally express hIK channels, are sensitive to ionizing irradiation. The activation of these Ca2+ sensitive channels, which can have severe impacts on the differentiation of cells, is based on an elevation in [Ca2+]cyt in these cells; the latter gain is the result of a rapid elevation of ROS molecules in the nucleus but also in the cytosol of cells, which under went an exposure to ionizing irradiation.