Identification of the binding pockets for peptide ligands in DEG/ENaC ion channels

DEG/ENaC 离子通道中肽配体结合袋的鉴定

• 批准号: 405378766
• 负责人: Professor Dr. Stefan Gründer
• 金额: --
• 依托单位: Institut für Physiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/405378766?language=en
• 关键词: Identification; binding; pockets; peptide; ligands; Identification; binding; pockets; peptide; ligands

Acid-Sensing Ion Channels (ASICs) are ligand-gated Na+ channels with important physiological functions and great pathophysiological impact. They are activated by transient acidification during synaptic transmission. Longer lasting acidosis, which is typical for inflammatory pain or ischemia, also activates ASICs. In animal models of ischemic stroke, the activation of ASICs exacerbates neuronal degeneration. Activation of ASICs in inflammatory diseases of the nervous system appears to contribute also to axonal degeneration. ASIC-inhibitors are already used in clinical trials of multiple sclerosis patients. During longer activation (> 5 sec) ASICs desensitize completely. Therefore, it is believed that different modulators change the ASIC activity in such a way, that they can still contribute to signal transduction during a long-lasting acidosis. An especially important group of ASC-modulators are neuropeptides. RFamide neuropeptides, for example, slow desensitization of ASICs, in particular of ASIC3, an ASIC with important function for detection of painful acidosis, and induce a sustained current that does not desensitize. Dynorphins, endogenous opioid peptides, on the other hand shift the steady-state inactivation curves of ASIC1a in such a way, that this important ASIC of the central nervous system is not completely inactivated even at slight acidosis (pH 7.0). The binding site on ASICs is neither known for RFamide nor for dynorphins. Identification of the binding site is, however, a prerequisite for the pharmacological intervention with the modulation by peptides, for example by competitive drugs. ASICs are close relatives of peptide-gated ion channels of the freshwater polyp Hydra, which are probably involved in neuromuscular transmission. These so-called Hydra Na+ channels (HyNaCs) are directly gated by their ligand, the Hydra-RFamides. It is conceivable that binding of peptides, for modulation of activity or for direct activation, is a conserved feature of this group of ion channels. This would predict that also the peptide binding pocket is conserved.In this grant application, we propose to molecularly characterize the binding pocket for RFamides on ASIC3, for dynorphins on ASIC1a, and for Hydra-RFamides on HyNaCs. To achieve this goal, we propose a combination of different methods: 1) site-directed mutagenesis and functional analysis of the mutants, 2) systematic modifications of the peptide ligands, 3) in silico prediction of the binding site, and 4) photo-crosslinking of the ligands to their receptors and subsequent purification of the complexes and mass spectrometric identification of the binding site. The combination of these methods will thoroughly characterize the binding pockets on the three channels and will deepen our understanding of gating and modulation of ASICs by neuropeptides.

酸性离子通道（ASIC）是配体门控的Na+通道，具有重要的生理功能和巨大的病理生理影响。它们在突触传播过程中通过瞬时酸化激活。持续的酸中毒较长，这是炎症性疼痛或缺血的典型特征，也会激活ASIC。在缺血性中风的动物模型中，ASIC的激活加剧了神经元变性。神经系统炎症性疾病中ASIC的激活似乎也导致了轴突变性。 ASIC抑制剂已经用于多发性硬化症患者的临床试验中。在更长的激活（> 5秒）期间，ASIC完全脱敏。因此，据信，不同的调节剂以这种方式改变了ASIC活性，以至于它们仍然可以在持久的酸中毒过程中有助于信号转导。一个特别重要的ASC调节剂是神经肽。例如，rFamide神经肽，例如，ASIC3的ASIC脱敏缓慢脱敏，这是一种具有重要功能可检测疼痛酸中毒的ASIC，并诱导不脱敏的持续电流。另一方面，内源性阿片类肽的动源是ASIC1A的稳态灭活曲线，即使在轻微的酸中毒时，这种重要的中枢神经系统的重要ASIC也不会完全失活（pH 7.0）。 ASIC上的结合位点既不以RFamide或Dynorphins的形式闻名。但是，结合位点的鉴定是对肽调节的药理干预措施的先决条件，例如竞争药物。 ASIC是淡水息肉的肽门控离子通道的近亲，它们可能与神经肌肉传播有关。这些所谓的Hydra Na+通道（HYNACS）由它们的配体Hydra-rfamide直接门控。可以想象，用于调制活性或直接激活的肽的结合是这组离子通道的保守特征。这将预测肽结合口袋也是保存的。在此赠款应用中，我们建议分子表征ASIC3上的rfamides的结合袋，ASIC1A上的Dynorphins和Hynacs上的hydra-rfamide。为了实现这一目标，我们提出了不同方法的组合：1）突变体的位置定向诱变和功能分析，2）肽配体的系统修饰，3）在结合位点的硅硅预测中，以及4）4）将配体对其受体和后续质量识别的构成和质量识别的相互联系。这些方法的结合将彻底表征三个通道上的结合口袋，并将加深我们对神经肽对ASIC的门控和调节的理解。