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

左旋多巴（L-DOPA，DOPA）被认为是中枢神经系统中的一种神经递质和/或神经调质，然而，与DOPA能传递有关的分子，如DOPA受体、DOPA转运体等，仍有待于鉴定。在这项研究中，我们试图通过遗传学的方法，通过使用秀丽隐杆线虫（C.elegans）来鉴定这些分子。在含有DOPA（1-10 mM）的琼脂平板上，该蠕虫表现出觅食行为、前后运动、咽部泵血加快等特征性行为，我们用甲磺酸乙酯处理该蠕虫以分离缺乏DOPA诱导的这些行为反应的突变体。通过对F_2代的筛选，我们分离到9个突变体，命名为dpa 1-9（dopa response abnormalities），目前正在进行STS定位，以确定连锁群。我们还发现，非洲爪蟾卵母细胞注射从兔肠上皮细胞提取的mRNA显示5倍增加多巴摄取活性相比，非注射卵母细胞。DOPA的摄取呈Na+依赖性，而非Cl-依赖性。酪氨酸、苯丙氨酸、亮氨酸和赖氨酸在1 mM时抑制多巴的摄取，而D-多巴、DA和谷氨酸则不抑制多巴的摄取。共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是注射兔肠上皮mRNA的卵母细胞Na+依赖性DOPA摄取的必需成分。我们现在正试图做表达克隆的功能多巴转运蛋白分子，这可能是与rBAT。

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.