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

L-多巴（DOPA）被认为是中枢神经系统中的神经递质和/或神经调节剂。然而，被认为参与多巴能传递的分子，例如多巴受体、多巴转运蛋白仍有待鉴定。在这项研究中，我们尝试利用秀丽隐杆线虫（C.elegans）通过遗传方法鉴定此类分子。在含有DOPA（1-10 mM）的琼脂平板上，蠕虫表现出觅食行为、前后运动、加速咽抽吸等特征行为。我们用甲磺酸乙酯处理蠕虫，以分离出缺乏这种多巴诱导的行为反应的突变体。通过筛选F2后代，我们分离出9个突变体，并将这些突变体命名为dpa 1-9（多巴反应异常）。我们现在正在对确定的连锁群进行STS作图。我们还发现，与未注射的卵母细胞相比，注射从兔肠上皮提取的 mRNA 的非洲爪蟾卵母细胞的多巴摄取活性增加了 5 倍。多巴的摄取是Na依赖性的，而不是Cl依赖性的。 1 mM 的酪氨酸、苯丙氨酸、亮氨酸和赖氨酸抑制多巴的摄取，而 D-多巴、DA 和谷氨酸不抑制多巴的摄取。共注射大中性氨基酸摄取系统激活剂rBAT的反义RNA，显着抑制多巴摄取活性，从而表明rBAT是注射兔肠上皮mRNA的卵母细胞中Na依赖性多巴摄取的重要组成部分。我们现在正尝试对可能与 rBAT 相关的功能性多巴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.