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

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.

酸敏感离子通道（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。它们在突触传递过程中被短暂的酸化激活。长期持续的酸中毒（典型的炎性疼痛或缺血症状）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 的激活似乎也会导致轴突变性。 ASIC 抑制剂已用于多发性硬化症患者的临床试验。在较长的激活时间（> 5 秒）期间，ASIC 完全不敏感。因此，人们相信不同的调节剂以这样的方式改变 ASIC 活性，即它们在长期酸中毒期间仍然有助于信号转导。一组特别重要的 ASC 调节剂是神经肽。例如，RFamide 神经肽可减缓 ASIC 的脱敏，特别是 ASIC3（一种对检测疼痛性酸中毒具有重要功能的 ASIC），并诱导不会脱敏的持续电流。另一方面，强啡肽（内源性阿片肽）会改变 ASIC1a 的稳态失活曲线，即使在轻微酸中毒（pH 7.0）下，这种重要的中枢神经系统 ASIC 也不会完全失活。 RFamide 和强啡肽在 ASIC 上的结合位点均未知。然而，结合位点的识别是通过肽调节的药理学干预的先决条件，例如通过竞争性药物。 ASIC 是淡水息肉水螅的肽门控离子通道的近亲，后者可能参与神经肌肉传递。这些所谓的 Hydra Na+ 通道 (HyNaC) 直接由其配体 Hydra-RFamides 门控。可以想象，用于调节活性或直接激活的肽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守的。在本次资助申请中，我们建议对 ASIC3 上的 RFamides、ASIC1a 上的强啡肽和 HyNaCs 上的 Hydra-RFamides 的结合口袋进行分子表征。为了实现这一目标，我们提出了不同方法的组合：1）突变体的定点诱变和功能分析，2）肽配体的系统修饰，3）结合位点的计算机预测，以及4）光-配体与其受体的交联以及随后的复合物纯化和结合位点的质谱鉴定。这些方法的结合将彻底表征三个通道上的结合袋，并将加深我们对神经肽对 ASIC 的门控和调节的理解。