Effects of radiation on the functional expression of voltage-gated potassium channels Kv 10.1

Exposure of biological systems to absorbed radiation doses produces early and late radiogenic responses, including ion channel modulation, oxidative stress, cell migration, and changes in cell metabolism that determine the efficiency of radiotherapy. To study one of these ion channel responses, HEK293 wild-type cells (HEK-WT) and HEK293 cells stably expressing the human ether a-go-go potassium channel (KV 10.1) were used, generously donated by Dr. Walter Stuhmer ), these were irradiated with gamma photons (60-Co, 0.2 Gy/min) in a dose range between 2-10 Gy, the changes produced by radiation in cell proliferation were measured by XTT assay, by analyzing these curves and according to what was reported by [Buch et al., 2012], the duplication rate and the delay time were established to calculate the survival fraction with which the survival curves were constructed. To understand the effects of irradiation on the flow of potassium currents, patch clamp electrophysiological recordings were performed in the voltage clamp mode in the whole cell configuration at room temperature and continuous perfusion with a standard wash solution (37 mM NaCl, 5.4 mM KCl, 2 mM CaCl₂, 1.2 mM MgCl₂, 10 mM HEPES, and 10 mM glucose, 300 m Osm/kg and ph 7.4), borosilicate pipettes (Kwik-Fil, Wpi, USA) with a resistance of 2-3 MΩ were used, filled with an intracellular solution containing 140 mM KCl, 1 mM MgCl₂, 10 mM EGTA, 10 mM HEPES at 300 m Osm/kg and ph 7.2 adjusted with KOH. Macroscopic currents were experimentally recorded on a Multi clamp 700B/Digi data, 1550/pCamp10 system (amplifier/analog-to-digital converter/software, all from Molecular Devices), filtering analog signals at 5 khz and making digital measurements at 10 khz. Kv 10.1 currents were triggered from a holding voltage potential of -70 mV in 250 ms steps from -100 to +50 mV with 10 mV increments. To obtain the I-V curves, the amplitude of the Kv 10.1 currents at the end of the voltage pulses (average of the last 5 ms) was measured and plotted against voltage. The results showed that low doses of radiation (<4 Gy) significantly promote proliferation and clonal capacity of HEK293-Kv 10.1 cells, increasing their radioresistance. Exposure of cells to an absorbed radiation dose of 4 Gy significantly reduced currents through Kv 10.1 evoked by depolarizing pulses between -100 mV and +50 mV. Radiation significantly changed Kv 10.1 currents from 178.9 pA/pF (n = 13) for the control group to 69.3 pA/pF (n = 18) for the group treated with a 4 Gy radiation dose (p = 0.0023), unpaired t-test). (author)