研究鋅離子轉運蛋白1 在 PC12 細胞中調節鋅離子恆定和代謝活性的功能

Abstract

Zn2+與多種蛋白質交互作用，調節多樣生理活性，其在不同亞細胞區域的濃度決定相關蛋白質的活性。多項研究表明，細胞質 Zn2+ 濃度 ([Zn2+]i) 的失調與神經退化性疾病的進展息息相關。細胞膜和細胞器上的轉運蛋白透過促進 Zn2+ 跨膜通量，對於維持 Zn2+ 恆定極為重要。 Zn2+ 轉運蛋白 1 (ZnT1) 主要位於細胞膜上，對於將 Zn2+ 輸出到細胞外以降低 [Zn2+]i 至關重要。我們先前的報告表明，多巴胺透過 cAMP-NO 訊號通路升高 [Zn2+]i，激活 PC12 細胞和培養神經元的自噬和發炎。為了表徵 ZnT1 在調節 Zn2+ 恆定和細胞活動中的影響，此研究在 PC12 細胞中過度表現 ZnT1 並檢查多巴胺處理下的代謝活動。以多巴胺和ZnCl2 處理PC12 細胞可升高 [Zn2+]i，降低透過Zn2+ 敏感螢光染料（FluoZin-3）和MTT [3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴化物] 監測的代謝活性測定。ZnT1 過度表現下調了多巴胺處理的 PC12 細胞中 Zn2+ 反應並增強了代謝活性。此外，ZnT1 過度表現略微升高了用多巴胺處理的 PC12 細胞中的粒線體電位。這些發現強調了 ZnT1 在維持 Zn2+ 恆定以保護粒線體代謝活性方面的重要影響。因此，了解 ZnT1 和其他 Zn2+ 轉運蛋白如何調節神經系統中 Zn2+ 恆定對於闡明其在病理條件下神經退化性疾病進展中的作用至關重要。

Zn2+ interacts with numerous proteins to regulate diverse physiological activities and its concentration at different subcellular regions determine the activities of associated proteins. Deficits in regulating cytosolic Zn2+ concentration ([Zn2+]i) has been associated with the progression of neurodegenerative diseases in various studies. Transporter proteins at the cell membrane and organelles can maintain Zn2+ homeostasis by facilitating Zn2+ flux across membranes. Zn2+ transporter 1 (ZnT1), mainly located at the cell membrane to export Zn2+ out of cells to lower [Zn2+]i. Our previous report has shown that dopamine can elevates [Zn2+]i via activating the cAMP-NO signaling pathway, activating autophagy and inflammation in PC12 cells and cultured neurons. To investigate the effects of ZnT1 in regulating the Zn2+ homeostasis and cellular activities, we overexpressed ZnT1 in PC12 cells and examined the metabolic activities under dopamine treatment. Treating PC12 cells with dopamine and ZnCl2 elevated the [Zn2+]i and decreased the metabolic activities monitored by a Zn2+ sensitive fluorescence dye, FluoZin-3, and MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] assay, respectively. ZnT1 overexpression down-regulated the Zn2+ response and enhanced metabolic activity in PC12 cells treated with dopamine. In addition, ZnT1 overexpression slightly elevated the mitochondria potential in PC12 cells under dopamine treatment. These findings underscore ZnT1's critical role in maintaining Zn2+ homeostasis to preserve mitochondrial metabolic activity. Henceforth, understanding how ZnT1 and other Zn2+ transporters regulate Zn2+ homeostasis in the nervous system is crucial for elucidating their roles in the progression of neurodegenerative diseases under pathological conditions.