Tyr-95 and Ile-172 in transmembrane segments 1 and 3 of human serotonin transporters interact to establish high affinity recognition of antidepressants
Tyr-95 and Ile-172 in transmembrane segments 1 and 3 of human serotonin transporters interact to establish high affinity recognition of antidepressants
复制标题
DOI：
10.1074/jbc.m505055200
复制
发表时间：
2006-01-27
影响因子：
4.8
通讯作者：
Blakely, RD
中科院分区：
文献类型：
--
作者：
Henry, LK;Field, JR;Blakely, RD
在之前的研究中，我们研究了血清素转运体（SERT）对抗抑郁药和底物识别的结构决定因素，我们发现人类SERT跨膜段1 （TM1）中的tyr95是几种拮抗剂结合的主要决定因素，包括外消旋西酞普兰（RS -CIT）。在这里，我们描述了hSERT TM3 （Ile-172）中的一个单独的位点，当切换到相应的果蝇SERT残基（I172M）时，它会影响(RS)-CIT识别。hSERT I172M突变体对多种抑制剂如(RS)-CIT、氯丙咪嗪、RTI-55、氟西汀、可卡因、尼索西汀、马辛多尔和诺非芬的抑制作用明显丧失，而对底物（包括5 -羟色胺和3,4-亚甲基二氧甲基苯丙胺）的识别则不受影响。拮抗剂相互作用的选择性与这种替代是明显的，因为抗抑郁药天奈汀和帕罗西汀的效力不变。使用[I-125] mfz2 -24的光亲和标记研究证实了可卡因模拟物识别的降低。与I172M取代相反，该位置的其他取代显著影响底物识别和/或运输活性。此外，小鼠突变（mSERT I172M）在抑制剂效力方面也表现出类似的选择性变化。与hSERT或mSERT不同，小鼠多巴胺转运蛋白（V152M）或人去甲肾上腺素转运蛋白（V148M）的类似替换导致转运蛋白结合底物，但在底物随后的易位中缺乏。在正常血清素识别的情况下，双突变体hSERT Y95F/ I172M对(RS)-CIT识别具有协同影响（类似于(RS)-CIT效力降低10,000倍）。活性较低的对映体(R)-CIT与(S)-CIT一样对I172M取代有反应，但对Y95F取代相对不敏感，并且在Y95F/ I172M处没有表现出协同损失。一个在TM1和TM3中具有单一半胱氨酸取代的hSERT突变体导致形成一个高亲和力的镉金属配位位点，表明这些结构域在SERT的三级结构中接近。这些研究为SERT拮抗剂的不同结合位点提供了证据，并揭示了CIT立体异构体差异靶向TM1和TM3之间的密切相互作用。
In previous studies examining the structural determinants of antidepressant and substrate recognition by serotonin transporters (SERTs), we identified Tyr-95 in transmembrane segment 1 (TM1) of human SERT as a major determinant of binding for several antagonists, including racemic citalopram ((RS)-CIT). Here we described a separate site in hSERT TM3 (Ile-172) that impacts (RS)-CIT recognition when switched to the corresponding Drosophila SERT residue (I172M). The hSERT I172M mutant displays a marked loss of inhibitor potency for multiple inhibitors such as (RS)-CIT, clomipramine, RTI-55, fluoxetine, cocaine, nisoxetine, mazindol, and nomifensine, whereas recognition of substrates, including serotonin and 3,4-methylenedioxymethamphetamine, is unaffected. Selectivity for antagonist interactions is evident with this substitution because the potencies of the antidepressants tianeptine and paroxetine are unchanged. Reduced cocaine analog recognition was verified in photoaffinity labeling studies using [I-125] MFZ 2-24. In contrast to the I172M substitution, other substitutions at this position significantly affected substrate recognition and/or transport activity. Additionally, the mouse mutation (mSERT I172M) exhibits similar selective changes in inhibitor potency. Unlike hSERT or mSERT, analogous substitutions in mouse dopamine transporter (V152M) or human norepinephrine transporter (V148M) result in transporters that bind substrate but are deficient in the subsequent translocation of the substrate. A double mutant hSERT Y95F/ I172M had a synergistic impact on ( RS)-CIT recognition (similar to 10,000-fold decrease in ( RS)-CIT potency) in the context of normal serotonin recognition. The less active enantiomer (R)-CIT responded to the I172M substitution like (S)-CIT but was relatively insensitive to the Y95F substitution and did not display a synergistic loss at Y95F/ I172M. An hSERT mutant with single cysteine substitutions in TM1 and TM3 resulted in formation of a high affinity cadmium metal coordination site, suggesting proximity of these domains in the tertiary structure of SERT. These studies provided evidence for distinct binding sites coordinating SERT antagonists and revealed a close interaction between TM1 and TM3 differentially targeted by stereoisomers of CIT.