Prasad Harish C, Zhu Chong-Bin, McCauley Jacob L, Samuvel Devadoss J, Ramamoorthy Sammanda, Shelton Richard C, Hewlett William A, Sutcliffe James S, Blakely Randy D
Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232-8548, USA.
Proc Natl Acad Sci U S A. 2005 Aug 9;102(32):11545-50. doi: 10.1073/pnas.0501432102. Epub 2005 Jul 29.
Human serotonin [5-hydroxytryptamine (5-HT)] transporters (hSERT, 5HTT, and SLC6A4) inactivate 5-HT after release and are prominent targets for therapeutic intervention in mood, anxiety, and obsessive-compulsive disorders. Multiple hSERT coding variants have been identified, although to date no comprehensive functional analysis of these variants has been reported. We transfected hSERT or 10 hSERT coding variants and examined total and surface protein expression, antagonist recognition, and transporter modulation by posttranslational, regulatory pathways. Two variants, Pro339Leu and Ile425Val, demonstrated significant changes in surface expression supporting alterations in 5-HT transport capacity (V(max)). Regardless of basal transport activity, all SERT variants displayed a capacity for rapid, phorbol ester-triggered down-regulation. Remarkably, five variants (Thr4Ala, Gly56Ala, Glu215Lys, Lys605Asn, and Pro612Ser) demonstrated no capacity for 5-HT uptake stimulation after acute protein kinase G (PKG)/p38 mitogen-activated protein kinase (MAPK) activation. Epstein-Barr virus (EBV)-transformed lymphocytes natively expressing the most common of these variants (Gly56Ala) exhibited a similar loss of 5-HT uptake stimulation by PKG/p38 MAPK activators. HeLa cells transfected with the Gly56Ala variant demonstrated elevated basal phosphorylation and, unlike hSERT, could not be further phosphorylated after 8-bromo cGMP (8BrcGMP) treatments. These studies reveal cellular phenotypes associated with naturally occurring human SERT coding variants and suggest that altered transporter regulation by means of PKG/p38 MAPK-linked pathways may influence risk for disorders attributed to compromised 5-HT signaling.
人类血清素[5-羟色胺（5-HT）]转运体（hSERT、5HTT和SLC6A4）在5-HT释放后使其失活，是治疗情绪、焦虑和强迫症的重要干预靶点。已鉴定出多个hSERT编码变体，尽管迄今为止尚未有对这些变体进行全面功能分析的报道。我们转染了hSERT或10个hSERT编码变体，并通过翻译后调控途径检查了总蛋白和表面蛋白表达、拮抗剂识别以及转运体调节。两个变体Pro339Leu和Ile425Val在表面表达上有显著变化，支持5-HT转运能力（Vmax）的改变。无论基础转运活性如何，所有SERT变体都表现出对佛波酯触发的快速下调的能力。值得注意的是，五个变体（Thr4Ala、Gly56Ala、Glu215Lys、Lys605Asn和Pro612Ser）在急性蛋白激酶G（PKG）/p38丝裂原活化蛋白激酶（MAPK）激活后没有5-HT摄取刺激能力。天然表达这些变体中最常见的一种（Gly56Ala）的爱泼斯坦-巴尔病毒（EBV）转化淋巴细胞对PKG/p38 MAPK激活剂也表现出类似的5-HT摄取刺激丧失。用Gly56Ala变体转染的HeLa细胞显示基础磷酸化升高，并且与hSERT不同，在8-溴环鸟苷（8BrcGMP）处理后不能进一步磷酸化。这些研究揭示了与天然存在的人类SERT编码变体相关的细胞表型，并表明通过PKG/p38 MAPK相关途径改变转运体调节可能影响因5-HT信号受损而导致的疾病风险。
