| Among all the ion channels superfamily, the potassium channels are the largest family, and they distribute widely in different type of cell membranes, significantly regulating physiological and pharmacological functions. Leukemia, also known as blood cancer, is a malignant disease of blood or bone marrow. In essence, leukemia is a desease of differentiation disorders, while cell differentiation arrest is the core problem. Induction of leukemia cell differentiation to reduce the malignancy has become the new strategy for the treatment of leukemia nowadays. It was found that the expression of patossium channels increased and further affected the proliferation and migration of leukemia cells. However, the role of patossium channels on differentiation of leukemia cells still remains unclear. Therefore, discovery of potential potassium channel blockers would accelerate the work on leukemia cell differentiation, which is full of theory significance and application potential.During the long term evolution, there are a large number of potassium channel-specific peptide toxins the presence of scorpion venom. It is well-known that these scorpion toxins have played a critical role in the structure-function studies of potassium channels and drug development. In recent years, the value of scorpion toxin peptides as new drugs has become more important while many diseases were found to be related to ion channels. In the thesis, we used scorpion toxin peptides as molecular tools to investigate the role of potassium channels in proliferation and differentiation of leukemia cells.In various types of leukemia, hERG potassium channels are significantly overexpressed, whereas they do not express in normal blood cells such as CD34+cells. In this work, a novel neurotoxic polypeptides named BmKKx2was isolated by screening the cDNA library of scorpion Buthus martensii Karsch, and it could potentially block hERG channel through the structure and function analysis. The pharmacological experiments showed that the recombinated peptide BmKKx2was able to selectively inhibit the currents of hERG channel with an IC50value of6.7±1.7nM in HEK293cells. Furthermore, toxin BmKKx2could also inhibit the currents of native hERG channels in leukemic cell line K562cells. In the presence of BmKKx2, the proliferation of K562cells were suppressed; and cell cycle analysis showed K562cell were arrested in the Gl/S phrase after the blockage of hERG channels. Subsequently, we constructed an erythroid differentiation model of K562cells by treatment with Ara-C; the GPA expression were tested as a marker for the level of differentiation. We found the the blockage of hERG channel by BmKKx2could enhance the Ara-C induced erythroid differentiation of K562cells; the toxin dose-dependent effect were also detected. To futher confirm this novel function of toxin BmKKx2, we conducted a hERG-deficent cell line by recombinant lentiviral particles containing hERG shRNAs. In the hERG-knockdown cell lines, the enhancement of erythroid differentiation by BmKKx2remarkably decreased, which proved this unique role of toxin BmKKx2depended on the blockage of hERG channels. Interestingly, toxin BmKKx2treatment could suppress the expression of hERG channel at both mRNA and protein levels during the erythroid differentiation of K562cells. Besides the function of toxin BmKKx2to enhance the erythroid differentiation, it was also found to enhance the differentiation dependent apoptosis (DDA) during erythroid differentiation of K562cells. In addition, the blockage of hERG potassium channel by toxin BmKKx2was able to decrease the intracellular Ca2+concentration during the erythroid differentiation of K562cells, providing an insight into the mechanism of the hERG potassium channel regulating this cellular process.In addition to the over expression of hERG channels in leukemia, other types of potassium channels such as Kvl.3channels are also expressed on leukemic cells or cell lines. Since the patossium channels can regualate membrane potential and Ca2+entry, they might also modulate blood cell differentiation similar with hERG channels in K562cells.Based on this hypothesis, we chosed the human promyelocytic leukemia cell line HL60and human monocytic leukemia cell line THP-1, with the potential for granulocyte differentiation and monocyte differentiation respectively. Both of these cell lines selectively express Kvl.3potassium channels instead of hERG channels. By using Kvl.3channel-specific scorpion toxin BmKTX with IC50value of90.5+33.5pM, the differentiation of HL60and THP-1were enhanced after the Kvl.3channel blockage. The express of Kvl.3channels was found to decrease, which was similar to that of hERG channels in K562cells. These findings lay the foundation for the in-depth research of Kvl.3channel role in the proliferation, differation and mechanism of HL60and THP-1cells in the future.In smmary, we isolated a novel hERG channel-specific peptide toxin named BmKKx2from scorpion Buthus martensii Karsch. This toxin could effectively blocked the hERG channel current in K562cells, thereby inhibiting the cell proliferation and enhancing erythroid differentiation.Meanwhile, toxin BmKKx2suppressed the hERG channel expression during erythroid differentiation, and strengthened the differentiation-dependent apoptosis (DDA) of K562cells. We also proved that another scorpion toxin BmKTX could promote the differentiation of HL60cells and THP-1cells by the blockage of Kvl.3, an overexpressed potassium channels expressed on leukemic cells. The blockage via BmKTX could also suppress the expression of Kvl.3channels during the differentiatin process. Therefore, this thesis revealed the the important role of potassium channels in regulating leukemic cell differentiation, which indicated the potential application of scorpion toxins regulating the differentiation of leukemia as the blockers for the potassium channels. |
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