Uptake inhibitors but not substrates induce protease resistance in extracellular loop two of the dopamine transporter
Uptake inhibitors but not substrates induce protease resistance in extracellular loop two of the dopamine transporter
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DOI：
10.1124/mol.65.3.692
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发表时间：
2004-03-01
影响因子：
3.6
通讯作者：
Vaughan, RA
中科院分区：
文献类型：
--
作者：
Gaffaney, JD;Vaughan, RA
使用胰蛋白酶蛋白水解和表位特异性免疫印迹检查抑制剂和底物结合期间多巴胺转运蛋白 (DAT) 的细胞外环二 (EL2) 的蛋白酶敏感性的变化。在对照大鼠纹状体膜中，EL2 限制区域中的 DAT 的蛋白水解是由 0.001 至 10 μg/ml 胰蛋白酶产生的。然而，在存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)、马吲哚、2β-甲氧甲氧基-3β-(4-氟苯基)托烷(β-CFT)、诺米芬辛、苯托品或(-)-可卡因的情况下，100至1000倍需要更高浓度的胰蛋白酶才能产生相当水平的蛋白水解作用。配体诱导的蛋白酶抗性与其对 DAT 结合的亲和力相关，在 Zn2+、(-)- 可卡因或去甲肾上腺素或血清素转运蛋白抑制剂中未观察到，并且不是由胰蛋白酶催化活性改变引起的。总之，这些结果支持了这样的假设：摄取抑制剂与 DAT 的相互作用诱导 EL2 中的蛋白酶抗性构象。相反，底物的结合不会诱导 EL2 中的蛋白酶抗性，这表明底物和抑制剂在结合过程中与 DAT 的相互作用不同。为了评估 EL2 蛋白水解对 DAT 功能的影响，用 [H-3] CFT 和 [H-3] 多巴胺测定了胰蛋白酶消化的 DAT 的结合和转运特性。消化降低了结合的 B-max 和摄取的 V-max，其量与蛋白水解程度成正比，表明 EL2 的结构完整性是维持 DAT 结合和转运功能所必需的。这些数据共同提供了有关 EL2 抑制剂和底物相互作用的新信息，可能将蛋白酶抗性 DAT 构象与转运抑制机制联系起来。
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 mug/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 [H-3] CFT and [H-3] dopamine. Digestion decreased the B-max for binding and the V-max 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.