Structure and Function of a Serotonin Transporter

描述（由申请人提供）：血浆5 -羟色胺转运蛋白（SERT）终止5 -羟色胺能神经传递，因此在形成突触信号的持续时间和强度方面起着关键作用。它的工作原理是将预先存在的梯度耦合到从突触到神经元和胶质细胞质的5 -羟色胺的热力学不利运动。这个分子机器已经引起了临床的极大关注，因为它的功能障碍与多种衰弱的神经精神疾病有关，如抑郁症、自闭症、强迫症和广泛性焦虑症。此外，它是许多精神活性药物的目标，如抗抑郁药、可卡因和安非他明衍生物“摇头丸”。尽管具有这样的临床和药理学意义，但底物易位、抑制和调节机制的原子水平细节仍然难以捉摸。本提案的两个目标包括：1)解决SERT与底物、离子和抑制剂配合物的结构；2)通过氢-氘交换质谱（HDX-MS）探测这些相互作用的构象动力学。基于结构的假设随后将通过位点定向诱变、半胱氨酸交联、瞬态和稳态通量动力学、解离/结合动力学、x射线晶体学和HDX-MS的组合进行测试。成功将允许进一步研究特定拮抗剂结合位点，甚至可能研究耐药性的分子基础，从而为合理的药物设计工作铺平道路。当最终与超出本提案范围的体内工作相结合时，SERT结构/功能研究也可能揭示疾病相关多态性的分子基础。实现这些目标中的任何一个都可能对实验室和临床产生重大影响。

DESCRIPTION (provided by applicant): The plasmalemmal serotonin transporter (SERT) terminates serotonergic neurotransmission and thus plays a critical role in shaping the duration and magnitude of synaptic signaling. It works by coupling preexisting ion gradients to the thermodynamically unfavorable movement of serotonin from the synapse to neuronal and glial cytoplasms. This molecular machine has garnered significant clinical attention because its dysfunction has been implicated in multiple debilitating neuropsychiatric diseases such as depression, autism, obsessive- compulsive disorder, and generalized anxiety. Furthermore, it is the target of numerous psychoactive agents such as antidepressants, cocaine, and the amphetamine derivative "ecstasy". Despite such clinical and pharmacological significance, atomic-level detail into the mechanism of substrate translocation, inhibition, and regulation has remained elusive. Two goals of this proposal include 1) solving the structure of a SERT in complex with substrates, ions, and inhibitors; and 2) probing the conformational dynamics of these interactions via hydrogen-deuterium exchange mass spectrometry (HDX-MS). Structure-based hypotheses will subsequently be tested with a combination of site-directed mutagenesis, cysteine crosslinking, transient and steady-state flux kinetics, dissociation/association binding kinetics, X-ray crystallography, and HDX-MS. Success will permit further investigation into specific antagonist binding sites and perhaps into the molecular basis for drug resistance, thereby paving the way for rational drug design efforts. When eventually coupled with in vivo work beyond the scope of this proposal, SERT structure/function studies may also shed light on the molecular underpinnings of disease-associated polymorphisms. Achieving any of one of these objectives would likely have significant impact in both the laboratory and the clinic.