Giffard Rona G, Lee Yong-Sun, Ouyang Yi-Bing, Murphy Sherry L, Monyer Hannah
Department of Anesthesia, S272, Stanford University, Stanford, CA 94305-5117, USA.
Eur J Neurosci. 2003 Dec;18(11):2935-45. doi: 10.1046/j.1460-9568.2003.03053.x.
Regulation of pH in the central nervous system is critical to normal brain function and response to pathophysiological conditions. Here we identify two novel variants of the sodium-driven chloride bicarbonate exchanger (NCBE) from brain. The developmental expression pattern seen by in situ hybridization for the 90-bp containing insert (insert A) reveals strong expression in spinal cord and brain beginning in embryonic development. High-level expression is seen in cerebellar Purkinje cells and principal cells in hippocampus. The variant missing a 39-bp insert at the 3' end (insert B) encodes a protein in which the deduced carboxyterminal three amino acids are replaced with a unique 21 amino acid stretch terminating in a PDZ motif. rb2NCBE, the PDZ motif-encoding variant, is more highly expressed in astrocytes than is rb1NCBE. Both variants are expressed at similar levels in neurons. Expression varies with age and cell type. The FLAG epitope was fused in-frame at the amino terminus and each variant was expressed using a retroviral vector to study subcellular localization. Both variants were associated with the plasma membrane, but rb2NCBE colocalized with actin filaments to a greater extent, suggesting the PDZ form may interact with the cytoskeleton, whereas rb1NCBE was more often seen in intracellular vesicles. The PDZ motif-containing variant was much more active in pH regulation, with the expected ionic dependence on Na+, HCO3- and Cl- when expressed in 3T3 cells. These results are a first step towards understanding the regulation of expression and activity of this transporter in the brain.
中枢神经系统中pH值的调节对于正常脑功能及对病理生理状况的反应至关重要。在此，我们鉴定出大脑中钠驱动的氯碳酸氢根交换体（NCBE）的两种新型变体。通过原位杂交观察到的含90个碱基对插入片段（插入片段A）的发育表达模式显示，从胚胎发育开始，脊髓和大脑中就有强烈表达。在小脑浦肯野细胞和海马体主细胞中可见高水平表达。在3'端缺失39个碱基对插入片段的变体（插入片段B）编码一种蛋白质，其中推导的羧基末端三个氨基酸被一个独特的21个氨基酸延伸所取代，该延伸以一个PDZ基序结尾。rb2NCBE，即编码PDZ基序的变体，在星形胶质细胞中的表达比rb1NCBE更高。两种变体在神经元中的表达水平相似。表达随年龄和细胞类型而变化。FLAG表位在氨基末端框内融合，每个变体使用逆转录病毒载体表达以研究亚细胞定位。两种变体都与质膜相关，但rb2NCBE与肌动蛋白丝的共定位程度更高，这表明PDZ形式可能与细胞骨架相互作用，而rb1NCBE更常见于细胞内囊泡中。含PDZ基序的变体在pH调节方面活性更高，在3T3细胞中表达时，对Na+、HCO3-和Cl-具有预期的离子依赖性。这些结果是迈向理解该转运体在大脑中表达和活性调节的第一步。
