Cationic interactions at the human dopamine transporter reveal binding conformations for dopamine distinguishable from those for the cocaine analog 2α-carbomethoxy-3α-(4-fluorophenyl)tropane
Cationic interactions at the human dopamine transporter reveal binding conformations for dopamine distinguishable from those for the cocaine analog 2α-carbomethoxy-3α-(4-fluorophenyl)tropane
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DOI：
10.1046/j.1471-4159.2002.00941.x
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发表时间：
2002-06-01
影响因子：
4.7
通讯作者：
Reith, MEA
中科院分区：
文献类型：
--
作者：
Chen, NH;Sun, LL;Reith, MEA
在膜制备中，CFT，苯托烷类可卡因类似物，和多巴胺（DA）相互作用的重组人多巴胺转运蛋白（hDAT）在Na+ -无介质。Na+显着增加转运体的亲和力CFT，但有很少或没有影响DA抑制CFT结合的效力。将[Na+ ]从20提高到155 mm可减少Li+诱导的DA K（i）的增加，但对CFT K（d）无影响。155 mm Na+的存在增强了CFT K（d）对低pH的耐受性，但对DA K（i）的耐受性没有影响。在跨膜结构域（TM）1中用亮氨酸取代色氨酸84（W84 L）或在TM 6中用天冬酰胺取代天冬氨酸313（D313 N）没有或仅适度地增强Na+非依赖性CFT结合的亲和力，并且保留了DA、Li+、K+或H+抑制这种结合的接近正常的能力。然而，突变显著增强了CFT结合的Na+刺激以及Na+拮抗Li+和H+抑制CFT结合。相反，突变既不改变DAK（i）的Na+不敏感特性，也不增强DAK-1对Li+抑制作用的Na+保护，但它们引起DAK（i）对H+抑制作用的Na+保护。这些结果是一致的DA的结合构象的存在是可区分的CFT，并与DA和CFT结合的阳离子相互作用的差分关联。这些突变可能改变hDAT的Na+结合状态，优先加强Na+和CFT结合之间的正变构偶联，并降低Li+或H+对CFT结合的影响。
In membrane preparations, CFT, a phenyltropane cocaine analog, and dopamine (DA) interact with the recombinant human dopamine transporter (hDAT) in Na+ -free medium. Na+ markedly increased the transporter's affinity for CFT, but had little or no effect on DA potency for inhibiting CFT binding. Raising [Na+ ] from 20 to 155 mm reduced Li+ -induced increase in DA K (i) , but not CFT K (d) . The presence of 155 mm Na+ enhanced the tolerance to low pH of CFT K (d) but not DA K (i) . Leucine substitution for tryptophan 84 (W84L) in transmembrane domain (TM) 1 or asparagine substitution for aspartate 313 (D313N) in TM 6 did not or only modestly enhance the affinity of Na+ -independent CFT binding, and retained the near normal ability of DA, Li+ , K+ , or H+ to inhibit this binding. However, the mutations significantly enhanced the Na+ stimulation of CFT binding as well as the Na+ antagonism against Li+ and H+ inhibition of CFT binding. In contrast, the mutations neither changed the Na+ -insensitive feature of DA K (i) nor enhanced the Na+ protection of DA K-i against Li+ 's inhibitory effect, though they caused Na+ protection of DA K (i) against H+ 's inhibitory action. These results are consistent with the existence of binding conformations for DA that are distinguishable from those for CFT, and with a differential association of cation interactions with DA and CFT binding. The mutations likely alter Na+ -bound state(s) of hDAT, preferentially strengthening the positive allosteric coupling between Na+ and CFT binding, and reducing the impact of Li+ or H+ on the CFT binding.