Wang Wenfei, Sonders Mark S, Ukairo Okechukwu T, Scott Helen, Kloetzel Megan K, Surratt Christopher K
Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York, USA.
Mol Pharmacol. 2003 Aug;64(2):430-9. doi: 10.1124/mol.64.2.430.
Cocaine initiates its euphoric effects by binding to the dopamine transporter (DAT), blocking uptake of synaptic dopamine. It has been hypothesized that the DAT transmembrane aspartic acid residue D79 forms an ionic interaction with charged nitrogen atoms in both dopamine and cocaine. We examined the consequences of novel and previously studied mutations of the D79 residue on DAT uptake of [3H]dopamine, DAT binding of the cocaine analog [3H]WIN 35,428, and drug inhibition of each process, all under identical conditions. The rat D79E DAT mutation decreased dopamine uptake Vmax by 7-fold and decreased dopamine turnover by 4-fold. Wild-type DAT displayed near-perfect agreement in the uptake and binding inhibition potencies for substrates, but cocaine and other nonsubstrate inhibitor drugs were approximately 3-fold less potent in uptake than in binding assays. Apparent affinities for substrates were unaffected by the D79E mutation unless the catechol moiety was modified. Strikingly, potencies for nonsubstrate inhibitors in uptake and binding assays matched for D79E DAT, because of a 3-fold lowering of binding affinities relative to WT DAT. The present findings reveal a complex role for D79 in determining substrate specificity and high-affinity binding of DAT inhibitors. We propose that at least two discrete inhibitor-binding DAT conformations or populations exist and that the DAT conformation/population responsible for inhibitor high-affinity binding is less responsible for dopamine uptake. The findings may be extensible to other psychostimulants and antidepressants that display discrepancies between binding affinity and monoamine uptake inhibition potency and may be relevant to development of a long-sought "cocaine antagonist".
可卡因通过与多巴胺转运体（DAT）结合，阻断突触多巴胺的摄取，从而产生欣快效应。据推测，DAT跨膜天冬氨酸残基D79与多巴胺和可卡因中的带电荷氮原子形成离子相互作用。我们在相同条件下，研究了D79残基新的和先前已研究的突变对[3H]多巴胺的DAT摄取、可卡因类似物[3H]WIN 35,428的DAT结合以及每个过程的药物抑制的影响。大鼠D79E DAT突变使多巴胺摄取Vmax降低7倍，多巴胺周转降低4倍。野生型DAT在底物摄取和结合抑制效力方面显示出近乎完美的一致性，但可卡因和其他非底物抑制剂药物在摄取试验中的效力比结合试验中低约3倍。除非儿茶酚部分被修饰，否则底物的表观亲和力不受D79E突变的影响。引人注目的是，对于D79E DAT，非底物抑制剂在摄取和结合试验中的效力相匹配，因为相对于野生型DAT，其结合亲和力降低了3倍。目前的研究结果揭示了D79在确定DAT抑制剂的底物特异性和高亲和力结合方面的复杂作用。我们提出，至少存在两种离散的抑制剂结合DAT构象或群体，并且负责抑制剂高亲和力结合 的DAT构象/群体对多巴胺摄取的作用较小。这些发现可能适用于其他在结合亲和力和单胺摄取抑制效力之间存在差异的精神兴奋剂和抗抑郁药，并且可能与长期寻求的“可卡因拮抗剂”的开发相关。
