Gaffaney Jon D, Vaughan Roxanne A
Department of Biochemistry and Molecular Biology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58203, USA.
Mol Pharmacol. 2004 Mar;65(3):692-701. doi: 10.1124/mol.65.3.692.
Changes in protease sensitivity of extracellular loop two (EL2) of the dopamine transporter (DAT) during inhibitor and substrate binding were examined using trypsin proteolysis and epitope-specific immunoblotting. In control rat striatal membranes, proteolysis of DAT in a restricted region of EL2 was produced by 0.001 to 10 microg/ml trypsin. However, in the presence of the dopamine uptake blockers [2-(diphenylmethoxyl) ethyl]-4-(3phenylpropyl) piperazine (GBR 12909), mazindol, 2beta-carbomethoxy-3beta-(4-flourophenyl)tropane (beta-CFT), nomifensine, benztropine, or (-)-cocaine, 100- to 1000-fold higher concentrations of trypsin were required to produce comparable levels of proteolysis. Protease resistance induced by ligands was correlated with their affinity for DAT binding, was not observed with Zn2+, (+)-cocaine, or inhibitors of norepinephrine or serotonin transporters, and was not caused by altered catalytic activity of trypsin. Together, these results support the hypothesis that the interaction of uptake inhibitors with DAT induces a protease-resistant conformation in EL2. In contrast, binding of substrates did not induce protease resistance in EL2, suggesting that substrates and inhibitors interact with DAT differently during binding. To assess the effects of EL2 proteolysis on DAT function, the binding and transport properties of trypsin-digested DAT were assayed with [3H]CFT and [3H]dopamine. Digestion decreased the Bmax for binding and the Vmax for uptake in amounts that were proportional to the extent of proteolysis, indicating that the structural integrity of EL2 is required for maintenance of both DAT binding and transport functions. Together this data provides novel information about inhibitor and substrate interactions at EL2, possibly relating the protease resistant DAT conformation to a mechanism of transport inhibition.
利用胰蛋白酶消化和表位特异性免疫印迹法，研究了多巴胺转运体（DAT）细胞外环二（EL2）在抑制剂和底物结合过程中蛋白酶敏感性的变化。在对照大鼠纹状体膜中，0.001至10微克/毫升的胰蛋白酶可使EL2的一个受限区域内的DAT发生蛋白水解。然而，在多巴胺摄取阻滞剂[2 - （二苯基甲氧基）乙基]-4 - （3 - 苯基丙基）哌嗪（GBR 12909）、吗茚酮、2β - 甲氧羰基 - 3β - （4 - 氟苯基）托烷（β - CFT）、诺米芬辛、苯海索或（ - ） - 可卡因存在的情况下，需要高100至1000倍的胰蛋白酶浓度才能产生相当程度的蛋白水解。配体诱导的蛋白酶抗性与其对DAT结合的亲和力相关，在锌离子、（ + ） - 可卡因或去甲肾上腺素或5 - 羟色胺转运体抑制剂存在时未观察到这种抗性，且不是由胰蛋白酶催化活性改变引起的。总之，这些结果支持以下假说：摄取抑制剂与DAT的相互作用在EL2中诱导了一种抗蛋白酶构象。相比之下，底物的结合并未在EL2中诱导蛋白酶抗性，这表明底物和抑制剂在结合过程中与DAT的相互作用方式不同。为了评估EL2蛋白水解对DAT功能的影响，用[³H]CFT和[³H]多巴胺测定了胰蛋白酶消化后的DAT的结合和转运特性。消化降低了结合的最大结合容量（Bmax）和摄取的最大转运速率（Vmax），其降低程度与蛋白水解程度成正比，表明EL2的结构完整性对于维持DAT的结合和转运功能都是必需的。这些数据共同提供了关于EL2处抑制剂和底物相互作用的新信息，可能将抗蛋白酶的DAT构象与转运抑制机制联系起来。
