From the Translational Neuropsychiatry Unit, Department of Clinical Medicine, Aarhus University, Aarhus University Hospital, Skovagervej 2, DK-8240 Risskov, Denmark and.
the Department of Chemistry and Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Langelandsgade 140, DK-8000 Aarhus C, Denmark.
J Biol Chem. 2018 Mar 9;293(10):3510-3523. doi: 10.1074/jbc.M117.809046. Epub 2018 Jan 19.
The serotonin transporter (SERT) is important for reuptake of the neurotransmitter serotonin from the synaptic cleft and is also the target of most antidepressants. It has previously been shown that cholesterol in the membrane bilayer affects the conformation of SERT. Although recent crystal structures have identified several potential cholesterol-binding sites, it is unclear whether any of these potential cholesterol sites are occupied by cholesterol and functionally relevant. In the present study, we focus on the conserved cholesterol site 1 (CHOL1) located in a hydrophobic groove between TM1a, TM5, and TM7. By molecular dynamics simulations, we demonstrate a strong binding of cholesterol to CHOL1 in a membrane bilayer environment. In biochemical experiments, we find that cholesterol depletion induces a more inward-facing conformation favoring substrate analog binding. Consistent with this, we find that mutations in CHOL1 with a negative impact on cholesterol binding induce a more inward-facing conformation, and, vice versa, mutations with a positive impact on cholesterol binding induce a more outward-facing conformation. This shift in transporter conformation dictated by the ability to bind cholesterol in CHOL1 affects the apparent substrate affinity, maximum transport velocity, and turnover rates. Taken together, we show that occupation of CHOL1 by cholesterol is of major importance in the transporter conformational equilibrium, which in turn dictates ligand potency and serotonin transport activity. Based on our findings, we propose a mechanistic model that incorporates the role of cholesterol binding to CHOL1 in the function of SERT.
血清素转运体（SERT）对于从突触间隙中重新摄取神经递质血清素非常重要，也是大多数抗抑郁药的作用靶点。先前已经表明，膜双层中的胆固醇会影响 SERT 的构象。尽管最近的晶体结构已经确定了几个潜在的胆固醇结合位点，但尚不清楚这些潜在的胆固醇结合位点中是否有任何被胆固醇占据并且具有功能相关性。在本研究中，我们专注于位于 TM1a、TM5 和 TM7 之间的疏水性凹槽中的保守胆固醇结合位点 1（CHOL1）。通过分子动力学模拟，我们证明了胆固醇在膜双层环境中与 CHOL1 具有很强的结合能力。在生化实验中，我们发现胆固醇耗竭会诱导更倾向于底物类似物结合的内向构象。与此一致，我们发现对胆固醇结合具有负面影响的 CHOL1 突变会诱导更内向的构象，反之亦然，对胆固醇结合具有积极影响的突变会诱导更外向的构象。这种由 CHOL1 中胆固醇结合能力决定的转运体构象的转变会影响表观底物亲和力、最大转运速度和周转率。总之，我们表明胆固醇占据 CHOL1 对转运体构象平衡具有重要意义，而转运体构象平衡又决定了配体效力和血清素转运活性。基于我们的发现，我们提出了一个机制模型，该模型将胆固醇结合到 CHOL1 中的作用纳入 SERT 的功能中。
