This study was conducted to elucidate the interaction and biotic effect of indigenous microorganisms, especially micro animals and bacteria on prosperity and decay of GEMs (genetically engineered microorganisms). Bacterial interaction means competition for substrate to grow between GEMs and indigenous bacteria, and prey-predator interaction means effect of micro animals on survival of GEMs. So, it is considered that basic part of environmental effects of GEMs will be made clear with analysi of these phenomena. Escherichia coli HB101/pBR325 as GEM, its host strain E. coli HB101, and Bacillus cereus MC as one of dominant species of indigenous bacteria were supplied. As predator the micro animals,Tetrahymena thermophila (ciliata, filter feeder) and Aeolosoma hemprichi (oligochaeta, detritus feeder) were supplied. The results obtained from this study can be concluded as follows: 1) GEMs showed few potentials to beat their host strains in their growth competition; 2) predation of micro animals influenced equally GEMs and their host strains, and these microorganisms, such as bacteria, were expected to decrease in environment; 3) fluculation enabled GEMs to escape from predation of micro animals, but GEMs were not expected to survive long-term because various predator species of micro animals exist in environment; 4) effect of micro animals, especially their predation, on survival of GEMs was so important that more practical research should be conducted; 5) it suggests that the results obtained from this study can be used to establish regular guidelines for environmental release of GEMs such as bioremediation technologies.

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Hypokalemic periodic paralysis (HypoPP) is a rare genetic disease associated with mutations in CACNA1S or SCN4A, encoding Cav1.1 or Nav1.4, respectively. Most HypoPP-associated missense changes occur at the arginine residues within the voltage-sensing domain (VSD) of these channels. It is established that such mutations destroy the hydrophobic seal separating the external water and the internal cytosolic crevices, resulting in the generation of aberrant leak currents called gating pore currents. Presently, the gating pore currents are thought to underlie HypoPP. Here, we generated HEK293T-based HypoPP-model cell lines with the Sleeping Beauty transposon system that co-express mouse inward-rectifier potassium channel (mKir2.1) and HypoPP2-associated Nav1.4 variants. Whole cell patch-clamp measurements confirmed that mKir2.1 successfully hyperpolarized the membrane potential to comparable levels to myofibers, and that some Nav1.4 variants induced notable proton-based gating pore currents. Importantly, we succeeded in fluorometrically measuring the gating pore currents in these variants using a ratiometric pH indicator, SNARF-4F. Our optical method provides a potential in vitro platform for high-throughput drug screen, not only for HypoPP but also for other channelopathies caused by VSD mutations.

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