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

酸感离子通道（asic）是一种配体门控的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瞬时酸化激活。持续时间较长的酸中毒，是典型的炎症性疼痛或缺血，也会激活asic。在缺血性脑卒中的动物模型中，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。在较长的激活期间（bbb50秒），asic完全脱敏。因此，我们认为不同的调节剂改变ASIC活性的方式，它们仍然可以在长期酸中毒期间促进信号转导。一组特别重要的asc调节剂是神经肽。例如，RFamide神经肽可以减缓ASIC的脱敏，特别是ASIC3， ASIC3是一种对检测疼痛性酸中毒具有重要功能的ASIC，并且可以诱导持续电流而不会脱敏。另一方面，内源性阿片肽Dynorphins以这样一种方式改变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。asic上的结合位点既不知道RFamide也不知道dynorphins。然而，结合位点的鉴定是通过肽（例如竞争性药物）调节进行药理学干预的先决条件。asic是淡水水螅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（hynac）是由它们的配体Hydra- rfamides直接控制的。可以想象，肽的结合，以调节活性或直接激活，是这组离子通道的保守特征。这也可以预测肽结合袋是保守的。在本申请中，我们建议对ASIC3上的RFamides、ASIC1a上的dynorphins和HyNaCs上的Hydra-RFamides的结合袋进行分子表征。为了实现这一目标，我们提出了不同方法的组合：1)位点定向诱变和突变体的功能分析，2)肽配体的系统修饰，3)结合位点的硅预测，以及4)配体与其受体的光交联，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.