Studies On The Relationship Of Drosophila Selenoprotein DSelK And The Cell Ca²⁺ Concentrations

Selenoprotein is a specific protein which is encoding by one of the termination codon, UGA, in organisms. All of the selenoproteins whose functions have been elucidated have important physiological function. Selenoprotein dSelK is one of the three selenoproteins in Drosophila melanogaster and is the only one that locates on endoplasmic reticulum. Elucidating the biological function has important roles for the researches on the animal physiological metabolism.In our earlier microarray studies it was found that the expression of inositol 1,4,5,-tris-phosphate receptor （IP3 receptor）, locating on endoplasmic reticulum （ER） and controlling the releasing of Ca²⁺ from ER to cytosol, was apparently reduced after the knockdown of dSelK in Schneider 2（S2）by dsRNAi. On the other hand, the expression of inositol 1,4,5, -tris- phosphate Kinase1 （ IP3 Kinase1）, locates in cytosol and having the opposite functions, was apparently increased. And it was concluded that the expression of dSelK is related to the releasing of Ca²⁺ from the endoplasmic reticulum to the cytosol. In the paper the real-time PCR was used to confirm the accuracy of the microarray results. Then, the dSelK in S2 cells was knockdown by dsRNAi, the changes of the cytosolic free Ca²⁺ after the knockdown of dSelK were detected by fluorescent probe Fluo-3/AM and laser confocal microscope. The expression plasmid of fusion protein of dSelK and dsRed, pAC-dSelK-dsRed-N1, was also constructed, and the changes of the cytosolic free Ca²⁺ after the over expression of dSelK were detected by the same method as the above. The apoptosis of S2 cells after the over expression of dSelK was detected by Hoechest staining.The real-time PCR results verified the microarray results that when the expression of Drosophila selenoprotein dSelK was knocked down in Drosophila S2 cells, the expression of the ER-located IP3 receptor, which increase the releasing of Ca²⁺ from ER to cytosol was apparently reduced, on the other hand the expression of IP3 Kinase1, which has an opposite functions was apparently increased. The cytosolic free Ca²⁺ concentrations in S2 cells were significantly reduced after the knockdown of dSelK by dsRNAi. On the other hand, the cytosolic free Ca²⁺ concentrations in S2 cells were significantly increased after the over expression of dSelK. And the cytosolic free Ca²⁺ concentrations were not significantly changed after the over expression of the negative control protein, the ER structure protein PDI. There was no apoptosis phenomenon appeared in the S2 cells over expressed dSelK proteins stained by Hoechest.To sum up, by real-time PCR experiment and the detecting of the changes of cytosolic free Ca²⁺ after the knockdown and the over expression of Drosophila selenoprotein dSelK it was found that Drosophila selenoprotein dSelK can control the releasing of Ca²⁺ from ER to the cytosol, and the releasing of Ca²⁺ is realized by increasing the expression of IP3 receptor located on ER. The above studies can play important roles on revealing the biological functions of its Homo homologue, SelK and other selenoproteins in organism.