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.

酸敏离子通道(ASICs)是一种配体门控的钠离子通道，具有重要的生理功能和重要的病理生理影响。它们在突触传递过程中被瞬间酸化激活。持续时间较长的酸中毒是炎症性疼痛或缺血的典型表现，也会激活ASICs。在缺血性中风的动物模型中，ASICs的激活加剧了神经元的退化。神经系统炎症性疾病中ASICs的激活似乎也是轴突变性的原因。ASIC抑制剂已经用于多发性硬化症患者的临床试验。在较长时间的激活过程中(&gt；5秒)，ASIC会完全失去敏感性。因此，人们认为，不同的调节剂以这种方式改变ASIC的活性，在长期酸中毒期间，它们仍然可以参与信号转导。一类特别重要的ASC调节剂是神经肽。例如，雷米特神经肽对ASICs，特别是对检测痛性酸中毒具有重要功能的ASIC3，具有缓慢的脱敏作用，并诱导不脱敏的持续电流。另一方面，强啡肽，内源性阿片肽，以这种方式移动ASIC1a的稳态失活曲线，使中枢神经系统的这一重要的ASIC即使在轻微的酸中毒(pH 7.0)下也不会完全失活。ASIC上的结合位点既不是RFamide的已知结合位点，也不是强啡肽的已知结合位点。然而，识别结合部位是通过肽的调节进行药理干预的先决条件，例如通过竞争性药物的调节。ASICs是淡水息肉Hydra多肽门控离子通道的近亲，可能参与神经肌肉传递。这些所谓的Hydra Na+通道(HyNaCs)直接由它们的配体Hydra-R家族控制。可以想象，为了调节活性或为了直接激活，多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守的。在这项拨款申请中，我们建议从分子上表征ASIC3上RFamide的结合口袋，ASIC1a上的强啡肽结合口袋，以及HyNaCs上的Hydra-RFamide结合口袋。为了实现这一目标，我们提出了一种不同方法的组合：1)突变的定点突变和功能分析，2)肽配体的系统修饰，3)结合位点的电子预测，以及4)配体与其受体的光交联和随后的络合物的纯化和结合位点的质谱学鉴定。这些方法的结合将彻底表征这三个通道上的结合口袋，并将加深我们对神经肽对ASICs的门控和调制的理解。