The third transmembrane domain of the serotonin transporter contains residues associated with substrate and cocaine binding
The third transmembrane domain of the serotonin transporter contains residues associated with substrate and cocaine binding
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DOI：
10.1074/jbc.272.45.28321
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发表时间：
1997-11-07
影响因子：
4.8
通讯作者：
Rudnick, G
中科院分区：
文献类型：
--
作者：
Chen, JG;Sachpatzidis, A;Rudnick, G
血清素转运蛋白 (SERT) 第三跨膜结构域中的 20 个残基一次突变为半胱氨酸，几乎所有这些突变体都对血清素 (5-HT) 转运具有完全活性，并且对带正电荷的半胱氨酸试剂 [2-(三甲基铵)乙基]甲硫磺酸盐 (MTSET) 的灭活不敏感，两个活性突变体 I172C 和I179C 对 MTSET 的快速失活敏感，但对带负电荷的试剂（2-磺基乙基）甲硫磺酸盐 (MTSES) 相对不敏感。 I172C 的失活被 5-HT 和可卡因阻断，但 I179C 没有类似的保护，用半胱氨酸替换 Tyr-175 导致转运活性低的突变体，并且在邻近的 Tyr-176 处，半胱氨酸替换完全阻断转运，Y175C 和 Y176C 突变体在细胞表面的表达水平分别为野生型的 84% 和 69% (C109A) SERT，突变体 Y175C 和 Y176C 的可卡因亲和力低于 C109A，这是通过高亲和力可卡因类似物 2β-甲甲氧基-3β-(4-[I-125]碘苯基)托烷 (beta-CIT) 的置换来测量的。对于 Y176C，5-HT 亲和力也降低，MTSET 灭活了 β-CIT 与 I172C 和 Y176C 的结合，但仅轻微抑制与 I179C 和 C109A 的结合，当这些位置被丙氨酸、丝氨酸或蛋氨酸取代时，未观察到 172、176 和 179 位半胱氨酸取代的 MTSET 敏感性。 Ile-172、Tyr-176 和 Ile-179 位于 α-螺旋跨膜元件的一个面上，并且 Ile-172 和 Tyr-176 靠近 5-HT 和可卡因的结合位点。
Twenty residues in the third transmembrane domain of the serotonin transporter (SERT) were mutated, one at a time, to cysteine, Almost all of these mutants were fully active for serotonin (5-HT) transport and insensitive to inactivation by the positively charged cysteine reagent [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET), Two active mutants, I172C and I179C, were sensitive to rapid inactivation by MTSET but were relatively insensitive to the negatively charged reagent (2-sulfonatoethyl)methanethiosulfonate (MTSES). Inactivation of I172C was blocked by 5-HT and cocaine, but I179C was not similarly protected, Replacement of Tyr-175 with cysteine resulted in a mutant with low transport activity, and, at the neighboring Tyr-176, cysteine replacement completely blocked transport, The Y175C and Y176C mutants were expressed on the cell surface at levels 84% and 69%, respectively, that of wild type (C109A) SERT, Mutants Y175C and Y176C had lower cocaine affinity than C109A, as measured by displacement of the high affinity cocaine analog 2 beta-carbomethoxy-3 beta-(4-[I-125]iodophenyl)tropane (beta-CIT). For Y176C, 5-HT affinity also was decreased, MTSET inactivated beta-CIT binding to I172C and Y176C, but only slightly inhibited binding to I179C and C109A, The MTSET sensitivity of cysteine replacements at positions 172, 176, and 179 was not observed when these positions were replaced with alanine, serine, or methionine, The results suggest that Ile-172, Tyr-176 and Ile-l79 are on one face of an alpha-helical transmembrane element, and that Ile-172 and Tyr-176 are in proximity to the binding site for 5-HT and cocaine.