Wang J B, Moriwaki A, Uhl G R
Division of Intramural Research, National Institute on Drug Abuse, Baltimore, MD 21224.
J Neurochem. 1995 Mar;64(3):1416-9. doi: 10.1046/j.1471-4159.1995.64031416.x.
Studies with thiol-modifying reagents have suggested that cysteines might play important roles in the function of the dopamine transporter (DAT). To identify DAT cysteines with important thiol groups, we have studied six mutant dopamine transporters in which cysteines were replaced by alanines. Substitutions of cysteines assigned to the DAT's second putative extracellular loop--positions 180 and 189--dramatically decreased the expression of the mutant transporters. Substitutions at positions 90, 242, 305, and 345 had no significant effect in decreasing dopamine uptake, MPP+ uptake, or cocaine analogue binding. Immunostaining COS cells transfected with Cys180 and Cys189 to Ala mutants revealed reduced membrane staining and prominent staining in perinuclear regions consistent with Golgi apparatus. These results suggest that cysteines i the DAT second extracellular loop may provide sulfide residues crucial to full transporter expression, at least in part, through interference with membrane insertion. Conceivably, they might also provide the targets for the influences of thiol-modifying reagents in modifying the function of the wild-type DAT expressed in striatal membranes.
使用硫醇修饰试剂进行的研究表明，半胱氨酸可能在多巴胺转运体（DAT）的功能中发挥重要作用。为了鉴定具有重要硫醇基团的DAT半胱氨酸，我们研究了六个半胱氨酸被丙氨酸取代的突变型多巴胺转运体。位于DAT第二个假定细胞外环的半胱氨酸（第180和189位）被取代后，显著降低了突变型转运体的表达。第90、242、305和345位的取代对多巴胺摄取、MPP+摄取或可卡因类似物结合的降低没有显著影响。对用Cys180和Cys189突变为Ala的突变体转染的COS细胞进行免疫染色，结果显示膜染色减少，且在与高尔基体一致的核周区域有明显染色。这些结果表明，DAT第二个细胞外环中的半胱氨酸可能至少部分地通过干扰膜插入来提供对转运体完整表达至关重要的硫化物残基。可以想象，它们也可能为硫醇修饰试剂影响纹状体膜中野生型DAT功能提供靶点。
