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

左旋多巴（DOPA）被认为是中枢神经系统中的一种神经递质和/或神经调节剂。然而，参与多巴能传递的分子，如DOPA受体、DOPA转座等仍有待确定。在这项研究中，我们试图利用秀丽隐杆线虫（秀丽隐杆线虫）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，虫体表现出觅食行为、前后运动、咽泵加速等特征行为。我们用甲磺酸乙酯处理蠕虫，分离出缺乏多巴诱导的行为反应的突变体。通过筛选F2后代，我们分离到9个突变体，并将这些突变体命名为dpa 1-9（多巴反应异常）。我们现在正在做STS映射到确定的连锁群。我们还发现，与未注射的卵母细胞相比，注射了兔肠上皮提取的mRNA的非洲爪蟾卵母细胞的多巴摄取活性增加了5倍。多巴的摄取依赖于Na+，而不依赖于Cl。1 mM时酪氨酸、苯丙氨酸、亮氨酸和赖氨酸对多巴摄取有抑制作用，而D-多巴、DA和谷氨酸对多巴摄取无抑制作用。rBAT的反义rna是大中性氨基酸摄取系统的激活剂，共注射rBAT可显著降低DOPA摄取活性，从而表明rBAT是兔肠上皮mRNA注入卵母细胞Na+依赖性DOPA摄取的重要成分。我们现在正尝试对可能与rBAT相关的功能性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.