Bjerregaard Henriette, Severinsen Kasper, Said Saida, Wiborg Ove, Sinning Steffen
From the Translational Neuropsychiatry Unit, Department of Clinical Medicine, Aarhus University, Aarhus University Hospital, Skovagervej 2, DK-8240 Risskov, Denmark.
From the Translational Neuropsychiatry Unit, Department of Clinical Medicine, Aarhus University, Aarhus University Hospital, Skovagervej 2, DK-8240 Risskov, Denmark
J Biol Chem. 2015 Mar 20;290(12):7747-55. doi: 10.1074/jbc.M114.573477. Epub 2015 Jan 22.
Serotonergic neurotransmission is modulated by the membrane-embedded serotonin transporter (SERT). SERT mediates the reuptake of serotonin into the presynaptic neurons. Conformational changes in SERT occur upon binding of ions and substrate and are crucial for translocation of serotonin across the membrane. Our understanding of these conformational changes is mainly based on crystal structures of a bacterial homolog in various conformations, derived homology models of eukaryotic neurotransmitter transporters, and substituted cysteine accessibility method of SERT. However, the dynamic changes that occur in the human SERT upon binding of ions, the translocation of substrate, and the role of cholesterol in this interplay are not fully elucidated. Here we show that serotonin induces a dualistic conformational response in SERT. We exploited the substituted cysteine scanning method under conditions that were sensitized to detect a more outward-facing conformation of SERT. We found a novel high affinity outward-facing conformational state of the human SERT induced by serotonin. The ionic requirements for this new conformational response to serotonin mirror the ionic requirements for translocation. Furthermore, we found that membrane cholesterol plays a role in the dualistic conformational response in SERT induced by serotonin. Our results indicate the existence of a subpopulation of SERT responding differently to serotonin binding than hitherto believed and that membrane cholesterol plays a role in this subpopulation of SERT.
血清素能神经传递由膜嵌入的血清素转运体（SERT）调节。SERT介导血清素重新摄取到突触前神经元中。离子和底物结合后，SERT会发生构象变化，这对于血清素跨膜转运至关重要。我们对这些构象变化的理解主要基于细菌同源物在各种构象下的晶体结构、真核神经递质转运体的推导同源模型以及SERT的取代半胱氨酸可及性方法。然而，人类SERT在离子结合、底物转运时发生的动态变化以及胆固醇在这种相互作用中的作用尚未完全阐明。在这里，我们表明血清素在SERT中诱导二元构象反应。我们在对检测SERT更外向构象敏感的条件下利用取代半胱氨酸扫描方法。我们发现血清素诱导了人类SERT一种新的高亲和力外向构象状态。这种对血清素新构象反应的离子需求反映了转运的离子需求。此外，我们发现膜胆固醇在血清素诱导的SERT二元构象反应中起作用。我们的结果表明存在一部分SERT对血清素结合的反应与迄今所认为的不同，并且膜胆固醇在这部分SERT中起作用。
