| Mineral elements are necessary for organisms to survive. They are components of cells and play important roles in regulating physiological functions. However, when the balance of essential metal ions in the body is destroyed or toxic metal ions intrude into the body, it will lead to the occurrence of various diseases, such as cardiovascular disease, neuropathic degenerative diseases, etc. Chloride iodine quinoline(Clioquinol, CQ) first was used in the treatment of malaria, due to its some side effects, such as retinopathy, heart disease, etc, it was banned to use for a long time in some areas. Recent studies have shown that the drug can lower the metal ion concentration in the central nervous system and has beneficial effect on neurodegenerative disease. Therefore, CQ becomes attractive again. However, its cellular and molecular mechanism is unclear. In this study, we investigated the probable mechanism of CQ on the intracellular metal ions homeostasis in Saccharomyces cerevisiae.Firstly, we measured the contents of some cations, including Zn2+, Fe3+, Cu2+, K+, Mg2+ and Ca2+ in different tissues of mouse after the treatment of CQ using ICP-AES. The data showed that the levels of Zn2+and Fe3+ were decreased, while those of Ca2+ were increased in the treated tissues compared to the untreated controls. The data were further confirmed in yeast. It is well known that calcium ion is essential for signal transduction, takes part in synthesis and release of neurotransmitter. Therefore, it is necessary to explore the regulating mechanism of CQ on intracellular calcium ion. In different calcium ion transporters defect strains including pmc1△、pmr1△、vcx1△、cch1△, we found that CQ inhibited cells to uptake Zn2+and Fe3+, and promoted the Ca2+ enrichment, which was concentration-dependent. The data was different from those treated with EDTA. However, the intracellular calcium ion content was not affected by CQ in pmr1△, we speculated that PMR1 p is the target protein of CQ or CQ inhibits the expression of PMR1.Secondly, we detected the influence of CQ on the expression of PMR1 by using the RT-PCR and fluorescence microscopy. The data showed that CQ inhibited the expression of PMR1. Furthermore, we found that CQ inhibited the expression of transcription factor CRZ1 of PMR1. PMR1 promoter regions(-1000 ~0bp) containing three pieces of different transcription factor binding domains, were inserted into the front of initiation codon of the luciferase marker genes(Ycplac33- Rluc), respectively. The luciferase detection showed that CQ regulated transcription factor CRZ1 p to combine with- 918 bp area of PMR1 promoter. At the same time, by using the RT-PCR and fluorescence microscopy, we found that CQ inhibited the expression of CRZ1.PMR1p is the calcium pump ATPase of endoplasmic reticulum and golgi membrane. Endoplasmic reticulum is the main place to store intracellular calcium ion, which is closely related to the endoplasmic reticulum stress. Therefore, we investigated the role of CQ in endoplasmic reticulum stress. The results showed that the mechanism is different from DTT, CQ changed the calcium homeostasis by inhibiting the expression of PMR1 to regulate the endoplasmic reticulum stress reaction.In summary, we conclude that CQ inhibits the expression of PMR1 by suppressing the expression of transcription factor CRZ1, which results in the decrease of the capacity of endoplasmic reticulum and golgi apparatus to use and excrete intracellular calcium ion, and promoting the absorption of calcium of the vacuole. That is the reason why the level of intracellular calcium ion is increased in CQ treated cells. |
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