Analysis of intracellular translocation of choline acetyltransferase by bioimaging

胆碱乙酰转移酶（Choline acetyltransferase, ChAT）是一种合成乙酰胆碱的酶，在许多动物中，ChAT mRNA的多种亚型都与之有关。由于这些异构体大多是非编码剪接变异体，在体内只产生了约68 kDa的同源ChAT蛋白。最近的证据表明，存在一种编码ChAT mRNA剪接的蛋白质变体，该变体缺乏大鼠ChAT基因的外显子6-9。经Western blot和免疫组织化学证实，该编码蛋白优先在外周神经系统表达，因此被命名为外周型ChAT （pChAT）。然而，pChAT的功能意义尚不清楚。为了获得这个问题的线索，我们在活的人胚胎肾细胞（HEK293细胞）和PC-12细胞中使用绿色荧光蛋白（GFP）检测了pChAT和常见类型ChAT （cChAT）之间细胞内运输的可能差异。共聚焦激光扫描显微镜显示，pChAT-GFP在细胞质中检测到，但在细胞核中未检测到，而cChAT-GFP在细胞质和细胞核中均存在。在用leptomycin B（一种核输出途径抑制剂）治疗后，pChAT- gfp在细胞质和细胞核中都可以检测到，这表明pChAT可以易位到细胞核。相比之下，leptomycin B治疗似乎不影响核内chchat - gfp的含量。与蛋白激酶C抑制剂孵育后，细胞核中pChAT-GFP的积累增加，而cChAT-GFP的积累没有增加。这些结果清楚地表明，pChAT与cChAT在细胞内运输方面存在差异，这可能反映了pChAT与cChAT在生理作用上的差异。

Choline acetyltransferase(ChAT), the synthesizing enzyme for acetylcholine, has been implicated to involve multiple isoforms of ChAT mRNA in several animals. Since these isoforms are mostly non-coding splice variants, only a homologous ChAT protein of about 68 kDa has been shown to be produced in vivo. Recent evidence indicates the existence of a protein coding splice variant of ChAT mRNA, which lacks exons 6-9 of the rat ChAT gene. The encoded protein was designated ChAT of a peripheral type(pChAT), because of its preferential expression in the peripheral nervous system as confirmed by Western blot and immunohistochemistry. However, functional significance of pChAT is unknown. To obtain a clue to this question, we examined a possible difference in intracellular trafficking between pChAT and the well-known ChAT of the common type(cChAT) using green fluorescent protein(GFP) in living human embryonic kidney cells (HEK293 cells) and PC-12 cells. Confocal laser scanning microscopy revealed that pChAT-GFP was detectable in the cytoplasm but not in the nucleus, whereas cChAT-GFP was found in both cytoplasm and nucleus. Following treatment with leptomycin B, a nuclear export pathway inhibitor, pChAT-GFP became detectable in both cytoplasm and nucleus, indicating that pChAT can be translocated to the nucleus. In contrast, the leptomycin B treatment did not seem to affect the content of intranuclear cChAT-GFP. After incubation with protein kinase C inhibitors, enhanced accumulation of pChAT-GFP but not cChAT-GFP occurred in the nucleus. These results clearly indicate that pChAT varies from cChAT in intracellular transportation, probably reflecting the difference in physiological roles between pChAT and cChAT.