DOPAMINE TRANSPORTER SITE-DIRECTED MUTATIONS DIFFERENTIALLY ALTER SUBSTRATE TRANSPORT AND COCAINE BINDING
DOPAMINE TRANSPORTER SITE-DIRECTED MUTATIONS DIFFERENTIALLY ALTER SUBSTRATE TRANSPORT AND COCAINE BINDING
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DOI：
10.1073/pnas.89.16.7782
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发表时间：
1992-08-15
影响因子：
11.1
通讯作者：
UHL, GR
中科院分区：
文献类型：
--
作者：
KITAYAMA, S;SHIMADA, S;UHL, GR
位于多巴胺转运蛋白（DAT）的三个疏水区域内的极性氨基酸类似于对儿茶酚胺受体的配体识别重要的那些氨基酸。通过表达在这些极性残基中突变的DAT cDNA来检查这些氨基酸的可能的功能意义。用丙氨酸、甘氨酸或谷氨酸替代79位的天冬氨酸显著降低了[H-3]多巴胺和氚标记的帕金森病诱导神经毒素1-甲基-4-苯基吡啶鎓（MPP+）的摄取，并降低了突变体对氚标记的可卡因类似物（-）-2-β-甲氧羰基-3-β-（4-氟苯基）托烷（CFT）的亲和力，而不影响B（max）。在第七疏水区的位置356和359处的丝氨酸残基被丙氨酸或甘氨酸取代引起[H-3]多巴胺和[H-3]MPP+摄取减少，而[H-3]CFT结合受影响较小。在第八疏水区中两个丝氨酸的取代产生了[H-3]多巴胺和[H-3]MPP+摄取和[H-3]CFT结合的野生型值。这些结果表明，天冬氨酸和丝氨酸残基躺在第一和第七疏水推定跨膜区域内的DAT功能是至关重要的，并提供鉴定的残基差异重要的可卡因结合和多巴胺摄取。
Polar amino acids lying within three hydrophobic regions of the dopamine transporter (DAT) are analogous to those important for ligand recognition by catecholamine receptors. Possible functional significance of these amino acids was examined by expressing DAT cDNAs mutated in these polar residues. Replacement of aspartate at position 79 with alanine, glycine, or glutamate dramatically reduced uptake of [H-3]dopamine and the tritium-labeled Parkinsonism-inducing neurotoxin 1-methyl-4-phenylpyridinium (MPP+) and reduced the mutants' affinity for the tritium-labeled cocaine analog (-)-2-beta-carbomethoxy-3-beta-(4-fluorophenyl)tropane (CFT) without affecting B(max). Replacement of the serine residues at positions 356 and 359 in the seventh hydrophobic region by alanine or glycine caused reductions in [H-3]dopamine and [H-3]MPP+ uptake, whereas [H-3]CFT binding was less affected. Substitution of two serines in the eighth hydrophobic region yielded wild-type values for [H-3]dopamine and [H-3]MPP+ uptake and [H-3]CFT binding. These results demonstrate that aspartate and serine residues lying within the first and seventh hydrophobic putative transmembrane regions are crucial for DAT function and provide identification of residues differentially important for cocaine binding and for dopamine uptake.