The human dopamine transporter forms a tetramer in the plasma membrane - Cross-linking of a cysteine in the fourth transmembrane segment is sensitive to cocaine analogs
The human dopamine transporter forms a tetramer in the plasma membrane - Cross-linking of a cysteine in the fourth transmembrane segment is sensitive to cocaine analogs
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DOI：
10.1074/jbc.c300349200
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发表时间：
2003-11-14
影响因子：
4.8
通讯作者：
Javitch, JA
中科院分区：
文献类型：
--
作者：
Hastrup, H;Sen, N;Javitch, JA
使用半胱氨酸交联，我们之前证明多巴胺转运蛋白（DAT）至少是同型二聚体，跨膜片段（TM）6的细胞外末端位于对称二聚体界面。我们现在已经探索了 DAT 作为质膜中的高级低聚物存在的可能性。野生型 DAT 的半胱氨酸交联导致 SDS-PAGE 上的条带与二聚体、三聚体和四聚体一致，表明 DAT 在质膜中形成四聚体。仅重新引入 Cys(243) 或 Cys(306) 的半胱氨酸耗尽的 DAT (CD-DAT) 与二聚体交联，表明 TM4 和 TM6 中的这些内源半胱氨酸分别在对称二聚体界面处交联。将 Cys(243) 和 Cys(306) 重新引入 CD-DAT 会产生与野生型无法区分的交联模式，具有二聚体、三聚体和四聚体带。这表明TM4界面和TM6界面是不同的，并进一步表明DAT可能作为二聚体的二聚体存在于质膜中，具有两个对称的同二聚体界面。可卡因类似物 MFZ 2-12 和其他 DAT 抑制剂（包括苯托品和马吲哚）可保护 Cys(243) 免于交联。相比之下，DAT 的两种底物，多巴胺和酪胺，并没有显着影响交联。我们认为抑制剂产生的交联受损是由于 TM4 界面的构象变化造成的，进一步证明这些化合物不是中性阻断剂，而是本身对转运蛋白的结构有影响。
Using cysteine cross-linking, we demonstrated previously that the dopamine transporter (DAT) is at least a homodimer, with the extracellular end of transmembrane segment (TM) 6 at a symmetrical dimer interface. We have now explored the possibility that DAT exists as a higher order oligomer in the plasma membrane. Cysteine cross-linking of wild type DAT resulted in bands on SDS-PAGE consistent with dimer, trimer, and tetramer, suggesting that DAT forms a tetramer in the plasma membrane. A cysteine-depleted DAT (CD-DAT) into which only Cys(243) or Cys(306) was reintroduced was cross-linked to dimer, suggesting that these endogenous cysteines in TM4 and TM6, respectively, were crosslinked at a symmetrical dimer interface. Reintroduction of both Cys(243) and Cys(306) into CD-DAT led to a pattern of cross-linking indistinguishable from that of wild type, with dimer, trimer, and tetramer bands. This indicated that the TM4 interface and the TM6 interface are distinct and further suggested that DAT may exist in the plasma membrane as a dimer of dimers, with two symmetrical homodimer interfaces. The cocaine analog MFZ 2-12 and other DAT inhibitors, including benztropine and mazindol, protected Cys(243) against cross-linking. In contrast, two substrates of DAT, dopamine and tyramine, did not significantly impact cross-linking. We propose that the impairment of cross-linking produced by the inhibitors results from a conformational change at the TM4 interface, further demonstrating that these compounds are not neutral blockers but by themselves have effects on the structure of the transporter.