Identification of molecules mediating DOPAergic neurotransmission-Genetic analysis of DOPA-resistant mutants of C.elegans

L多巴被认为是中枢神经系统中的神经递质和/或神经调节剂，但参与多巴能传递的分子，如多巴受体、多巴转运体等尚不清楚。在这项研究中，我们试图利用秀丽隐杆线虫(C.elegans)的遗传方法来鉴定这些分子。在含有DOPA(1-10 mm)的琼脂平板上，蠕虫表现出提前老化、前后移动、加速咽抽吸等特征行为。我们用甲烷磺酸乙酯处理这些蠕虫，以分离缺乏这种DOPA诱导的行为反应的突变体。通过对F2代的筛选，我们分离到9个突变体，命名为dpa 1-9(多巴反应异常)。我们正在对确定的连锁群进行STS定位。我们还发现，注射了从兔肠上皮中提取的mRNA的非洲爪哇卵母细胞，摄取DOPA的活性比未注射的卵母细胞高5倍。DOPA的摄取依赖于钠离子，而不依赖于氯离子。酪氨酸、苯丙氨酸、亮氨酸和赖氨酸在1 mM处对DOPA的摄取有抑制作用，而D-DOPA、DA和谷氨酸对DOPA的摄取无抑制作用。联合注射大分子中性氨基酸摄取系统激活剂rBAT的反义RNA可显著抑制DOPA摄取活性，表明rBAT是注射了兔肠上皮信使RNA的卵母细胞摄取多巴胺钠的必需成分。我们现在正在尝试对可能与rBAT相关的功能DOPA转运蛋白分子进行表达克隆。

L-DOPA (DOPA) is proposed to be a neurotransmitter and/or neuromodulator in CNS.However, molecules which are supposed to be involved in DOPAergic transmission, for example, DOPA receptors, DOPA transpoters, remain to be identified. In this study, we have attempted to identify such molecules by genetic approach by use of Caenorhabditis elegans (C.elegans). On agar plates containing DOPA (1-10 mM), the worms showed characteristic behaviors such as foreaging behavior, back and forward movement, accelerated pharyngeal pumping and so on. We treated the worms with ethyl methanesulfonate to isolate mutants which lack such DOPA-induced behavioral responses. By screening F2 progeny, we isolated 9 mutants, and called these mutants dpa 1-9 (dopa response abnormality).We are now doing STS mapping to determined linkage group. We also found that Xenopus laevis oocytes injected with mRNA extracted from rabbit intestinal epithelium showed 5-fold increase in DOPA-uptake activity compared to non-injected oocytes. The uptake of DOPA were Na+-dependent but not Cl--dependent. The uptake of DOPA was inhibited by tyrosine, phenylalanine, leucine and lysine at 1 mM, whereas was not inhibited by D- DOPA, DA and glutamate. Co-injection of antisense-RNA of rBAT, an activator for uptake system of large neutral amino acid, markedly depressed the DOPA uptake activity, thereby indicating that rBAT is an essential component for Na+-dependent DOPA uptake in the oocyte injected with mRNA from rabbit intestinal epithelium. We are now attempting to do expression cloning for functional DOPA transporter molecules which is probably associated with rBAT.