Ferrer J V, Javitch J A
Center for Molecular Recognition and Departments of Pharmacology, College of Physicians and Surgeons, Columbia University, 630 W. 168th Street, New York, NY 10032, USA.
Proc Natl Acad Sci U S A. 1998 Aug 4;95(16):9238-43. doi: 10.1073/pnas.95.16.9238.
Cocaine and other psychostimulants act by blocking the dopamine transporter. Binding of the cocaine analog, [3H]2-beta-carbomethoxy-3-beta-(4-fluorophenyl) tropane (CFT) to the dopamine transporter is sensitive to polar sulfhydryl-specific derivatives of methanethiosulfonate (MTS). These reagents preferentially react with water-accessible, reduced cysteines. The human dopamine transporter has 13 cysteines. Their topology is not completely determined. We sought to identify those cysteine residues the modification of which affects CFT binding and to determine the topology of these reactive cysteines. We mutated each of the cysteines, one at a time and in various combinations, to residues that preserved binding and transport, and we tested the sensitivity of each of the mutant transporters to the reagents. One construct, X5C, had five mutated cysteines (C90A, C135A, C306A, C319F, and C342A). Using a membrane preparation in which both extracellular and intracellular cysteines could be accessible, we found that CFT binding in X5C, as compared with wild-type transporter, was two orders of magnitude less sensitive to MTS ethylammonium (MTSEA). The wild-type cysteines were substituted back into X5C, one at a time, and these constructs were tested in cells and in membranes. Cys-90 and Cys-306 appear to be extracellular, and Cys-135 and Cys-342 appear to be intracellular. Each of these residues is predicted to be in extramembranous loops. The binding of cocaine increases the rate of reaction of MTSEA and MTS ethyltrimethylammonium with the extracellular Cys-90 and therefore acts by inducing a conformational change. Cocaine decreases the rate of reaction of MTSEA with Cys-135 and Cys-342, acting either directly or indirectly on these intracellular residues.
可卡因和其他精神兴奋剂通过阻断多巴胺转运体发挥作用。可卡因类似物[3H]2-β-甲氧羰基-3-β-(4-氟苯基)托烷（CFT）与多巴胺转运体的结合对甲硫基磺酸酯（MTS）的极性巯基特异性衍生物敏感。这些试剂优先与可接近水的还原型半胱氨酸反应。人类多巴胺转运体有13个半胱氨酸。它们的拓扑结构尚未完全确定。我们试图确定那些修饰后会影响CFT结合的半胱氨酸残基，并确定这些反应性半胱氨酸的拓扑结构。我们将每个半胱氨酸一次一个或多种组合地突变为保留结合和转运功能的残基，并测试每个突变转运体对试剂的敏感性。一种构建体X5C有五个突变的半胱氨酸（C90A、C135A、C306A、C319F和C342A）。使用一种细胞外和细胞内半胱氨酸均可接近的膜制剂，我们发现与野生型转运体相比，X5C中的CFT结合对MTS乙铵（MTSEA）的敏感性低两个数量级。将野生型半胱氨酸一次一个地重新引入X5C中，并在细胞和膜中测试这些构建体。半胱氨酸-90和半胱氨酸-306似乎位于细胞外，而半胱氨酸-135和半胱氨酸-342似乎位于细胞内。预计这些残基中的每一个都位于膜外环中。可卡因增加了MTSEA和MTS乙三甲基铵与细胞外半胱氨酸-90的反应速率，因此通过诱导构象变化起作用。可卡因降低了MTSEA与半胱氨酸-135和半胱氨酸-342的反应速率，直接或间接作用于这些细胞内残基。
