Stephan M M, Chen M A, Penado K M, Rudnick G
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
Biochemistry. 1997 Feb 11;36(6):1322-8. doi: 10.1021/bi962150l.
The serotonin transporter (SERT) is a member of a highly homologous family of proteins responsible for the reuptake of biogenic amines from the synaptic cleft. We took advantage of native restriction sites in SERT to construct a chimeric transporter containing a small (34 amino acid) region of the norepinephrine transporter. The substituted region corresponds to about half of the largest extracellular loop. This chimera transports serotonin very slowly compared to wild type SERT. However, it binds serotonin and the cocaine analog 2beta-carbomethoxy-3beta-(4-[125I]iodophenyl)tropane with a high affinity indistinguishable from wild type. It has the same specificity as wild type SERT for the antidepressants paroxetine and desipramine. The low rate of transport does not appear to be due to poor expression, since the chimeric transporter is expressed at the membrane surface at close to wild type levels as measured by cell surface biotinylation. These observations lead us to conclude that, rather than playing a role in substrate or drug binding, this region of the large extracellular loop may be involved in the conformational changes associated with substrate translocation into the cell.
血清素转运体（SERT）是一个高度同源蛋白家族的成员，负责从突触间隙重新摄取生物胺。我们利用SERT中的天然限制性酶切位点构建了一个嵌合转运体，其中包含去甲肾上腺素转运体的一个小区域（34个氨基酸）。被替换的区域大约相当于最大的细胞外环的一半。与野生型SERT相比，这种嵌合体转运血清素的速度非常慢。然而，它与血清素以及可卡因类似物2β-甲氧羰基-3β-（4-[125I]碘苯基）托烷具有与野生型无法区分的高亲和力结合。它对抗抑郁药帕罗西汀和地昔帕明具有与野生型SERT相同的特异性。转运速率低似乎并非由于表达不佳，因为通过细胞表面生物素化测量，嵌合转运体在膜表面的表达水平接近野生型。这些观察结果使我们得出结论，大细胞外环的这个区域可能不是在底物或药物结合中起作用，而是可能参与了与底物转运到细胞内相关的构象变化。
