Molecular mechanisms of bacterial homologs of neurotransmitter:sodium symporters

描述(申请人提供)：神经递质：钠转运体(NSS)将底物的积累与钠离子沿其浓度梯度沿质膜向下移动相结合，因此构成细胞信号和动态平衡的关键要素。神经递质包括多巴胺、5-羟色胺和去甲肾上腺素的转运体-苯丙胺、可卡因和抗抑郁药物的靶标-以及GABA和甘氨酸的转运体，它们是治疗癫痫和精神分裂症的靶标。2005年，Gouaux小组在1.65？解决了Leut的结构，Leut是一种细菌NSS同系物，在封闭的结合口袋中与1个Leu和2个Na+结合结晶(称为初级底物结合(S1)位)。该结构不容易提供底物从细胞外或细胞内侧到S1位点的路径的线索。在前一个项目期间，在细胞外前庭发现了一个意想不到的第二底物结合(S2)位点；结合和通量实验表明，这两个结合位点可以同时占据。S2位点的底物变构地触发细胞内Na+和S1位点底物的释放，从而起到“符号效应器”的作用。因为三环抗抑郁剂(TCA)与这个S2位点结合的方式不同，它们不会促进底物从S1位点释放，因此起到联合解偶联剂的作用来抑制转运。确定与转运相关的构象变化和在转运体内形成的渗透途径是该项目的长期目标，对于理解人类神经递质转运体的功能机制以及药物如何作用于这些机制至关重要。为了实现这一目标，在与研究人员积极合作的基础上开发了一种综合方法，这些研究人员在计算建模(Harel Weinstein)、膜蛋白结晶学(Pul Nissen)和单分子荧光光谱学(Scott Blanchard)方面的专业知识使本申请中描述的多学科组合方法成为可能。提出了以下具体目标：1)利用我们关于洗涤剂、突变和离子取代对S2结合的特异性和调节的新发现，开发条件，使我们能够理解S2结合位点对Leut性质的调节，并解决底物与S2结合的Leut的结构。这将提供原子分辨率数据，为我们关于底物结合到这个位点的运输中的关键作用的机制假说提供信息。2)根据底物转运体的特定构象变化来描述底物转运的机制，这些构象变化将底物结合到S2-位点所触发的变构信号传播到转运蛋白的胞内门，并允许底物向内释放。3)建立我们在细菌转运体的结构和功能发现的相关性，以了解SERT和DAT的功能。我们将：a)证明S2位点在这些人类转运蛋白中的重要功能作用，以及b)使用依赖于氯的Leut突变体来确定Cl-结合位点的结构，从而阐明Cl-在SERT和DAT中的功能作用。与公共卫生相关：神经递质转运体是可卡因和安非他明等精神刺激性药物的靶标，也是抗抑郁药以及治疗癫痫和精神分裂症的新药开发的靶标。识别转运的构象动力学、转运体内的渗透途径以及底物和抑制物结合的作用对于理解人类神经递质转运体的作用机制以及药物如何作用于这些机制至关重要。我们已经建立的强大的方法将使我们能够达成这一理解，并解决药物作用和以坚实的结构和功能信息为基础的治疗设计指南。

DESCRIPTION (provided by applicant): Neurotransmitter:sodium symporters (NSS) couple the accumulation of substrate to the movement of sodium ions down their concentration gradient across the plasma membrane, and as such constitute key elements in cellular signaling and homeostasis. NSS include the transporters for dopamine, serotonin and norepinephrine-targets for amphetamine, cocaine, and antidepressant drugs-as well as the transporters for GABA and glycine, which are targeted for treatment of epilepsy and schizophrenia. In 2005 the Gouaux group solved at 1.65 ¿ the structure of LeuT, a bacterial NSS homolog, crystallized with 1 Leu and 2 Na+ bound in an occluded binding pocket (referred to as primary substrate binding (S1) site). The structure provided no easy clues to the pathway of substrate to the S1 site from the extracellular or the intracellular side. An unexpected second substrate binding (S2) site located in the extracellular vestibule was identified during the previous project period; binding and flux experiments showed that the two binding sites can be occupied simultaneously. Substrate in the S2 site allosterically triggers intracellular release of Na+ and substrate from the S1 site, thereby functioning as a "symport effector." Because tricyclic antidepressants (TCA) bind differently to this S2 site, they do not promote substrate release from the S1 site and thus act as symport uncouplers to inhibit transport. Identifying the conformational changes associated with transport and the permeation pathways that are formed within the transporter are long term goals of this project critical to understanding the functional mechanisms of the human neurotransmitter transporters and how drugs act upon these mechanisms. To achieve this goal, an integrated approach has been developed based on active collaborations with investigators whose expertise in computational modeling (Harel Weinstein), membrane protein crystallography (Poul Nissen), and single-molecule fluorescence spectroscopy (Scott Blanchard) enables the combined multidisciplinary approach described in this application. The following specific aims are proposed: 1) To use our novel discoveries regarding the specificity and modulation of S2 binding, by detergents, mutations, and ionic substitution, to develop conditions that enable us to understand the regulation of LeuT properties by the S2 binding site and to solve a structure of LeuT with substrate bound to the S2 site. This will provide atomic resolution data to inform our mechanistic hypothesis as to the essential role in transport of substrate binding to this site. 2) To characterize the mechanism of substrate transport in terms of specific conformational changes in the transporter that propagate the allosteric signal triggered by substrate binding to the S2-site towards the intracellular gate of the transporter and allow inward release of substrate. 3) To establish the relevance of our structural and functional findings in bacterial transporters to understanding the function of SERT and DAT. We will: a) demonstrate the essential functional role of the S2 site in these human transporters, and b) use a Cl-- dependent LeuT mutant to determine the structure of the Cl- binding site and thus to explicate the functional role of Cl- in SERT and DAT. PUBLIC HEALTH RELEVANCE: Neurotransmitter transporters are the target of psychostimulant drugs such as cocaine and amphetamine and are targets for antidepressants as well as for new drugs in development for the treatment of epilepsy and schizophrenia. Identifying the conformational dynamics of transport, the permeation pathways within the transporter, and the role of substrate and inhibitor binding are critical for understanding the functional mechanisms of the human neurotransmitter transporters and how drugs act upon these mechanisms. The powerful approaches we have established will allow us to reach this understanding and address drug action and guidelines for therapy design anchored in solid structural and functional information.