Molecular origin of plasma membrane citrate transporter in human prostate epithelial cells
Molecular origin of plasma membrane citrate transporter in human prostate epithelial cells
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人前列腺上皮细胞质膜柠檬酸转运蛋白的分子起源
作者：
Maciej P. Mazurek;P. Prasad;E. Gopal;S. P. Fraser;L. Bolt;Nahit Rizaner;C. Palmer;C. Foster;F. Palmieri;V. Ganapathy;W. Stühmer;M. Djamgoz;M. Mycielska
前列腺是高度特化的哺乳动物器官，可产生和释放大量柠檬酸盐。然而，柠檬酸盐释放机制尚不清楚。在这里，我们展示了从人正常前列腺上皮 PNT2-C2 细胞中分子克隆柠檬酸转运蛋白的结果，先前已证明该细胞表达这种机制。通过使用 cDNA 末端 PCR 快速扩增，我们确定前列腺载体是线粒体转运蛋白 SLC25A1 的亚型，具有不同的第一个外显子。我们通过在人胚胎肾细胞中表达该克隆并进行放射性示踪剂实验和全细胞膜片钳记录来确认该克隆的功能。通过使用针对序列不同部分的短干扰RNA，我们证实克隆蛋白是负责柠檬酸盐释放的主要前列腺转运蛋白。我们还生产了一种特异性抗体，并将克隆的转运蛋白定位到细胞的质膜上。通过使用相同的抗体，我们已经证明克隆的转运蛋白在非恶性人体组织中表达。
The prostate is a highly specialized mammalian organ that produces and releases large amounts of citrate. However, the citrate release mechanism is not known. Here, we present the results of molecular cloning of a citrate transporter from human normal prostate epithelial PNT2‐C2 cells shown previously to express such a mechanism. By using rapid amplification of cDNA ends PCR, we determined that the prostatic carrier is an isoform of the mitochondrial transporter SLC25A1 with a different first exon. We confirmed the functionality of the clone by expressing it in human embryonic kidney cells and performing radiotracer experiments and whole‐cell patch‐clamp recordings. By using short interfering RNAs targeting different parts of the sequence, we confirmed that the cloned protein is the main prostatic transporter responsible for citrate release. We also produced a specific antibody and localized the cloned transporter protein to the plasma membrane of the cells. By using the same antibody, we have shown that the cloned transporter is expressed in non‐malignant human tissues.