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

酸敏感离子通道（Acid-Sensing Ion Channels，ASICs）是一类具有重要生理功能和病理生理影响的配体门控Na+通道。它们在突触传递过程中被瞬时酸化激活。较长时间的酸中毒，这是典型的炎性疼痛或缺血，也激活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。神经系统炎性疾病中ASIC的激活似乎也有助于轴突变性。ASIC抑制剂已经用于多发性硬化症患者的临床试验。在较长的激活过程中（> 5秒），ASIC完全脱敏。因此，认为不同的调节剂以这样的方式改变ASIC活性，使得它们在长期酸中毒期间仍然可以有助于信号转导。一组特别重要的ASC调节剂是神经肽。RF酰胺神经肽，例如，减缓ASIC，特别是ASIC 3（一种具有检测疼痛性酸中毒的重要功能的ASIC）的脱敏，并诱导不脱敏的持续电流。另一方面，强啡肽，内源性阿片肽，以这样的方式移动ASIC 1a的稳态失活曲线，即使在轻微的酸中毒（pH 7.0）下，中枢神经系统的这种重要ASIC也不会完全失活。ASIC上的结合位点既不知道RFamide也不知道强啡肽。然而，结合位点的鉴定是通过肽（例如通过竞争性药物）进行调节的药理学干预的先决条件。ASICs是淡水水螅的肽门控离子通道的近亲，其可能参与神经肌肉传递。这些所谓的Hydra Na+通道（HyNaCs）直接由其配体Hydra-RFamides门控。可以想象，肽的结合，用于调节活性或用于直接活化，是这组离子通道的保守特征。这将预示着肽结合口袋也是保守的。在本授权申请中，我们建议对ASIC 3上的RFamides、ASIC 1a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）配体与其受体的光交联，以及随后的复合物的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口袋，并将加深我们对神经肽对ASIC的门控和调节的理解。

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.