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

Subject Area Molecular Biology and Physiology of Neurons and Glial Cells
Pharmacology
Term from 2018 to 2022
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 405378766
 
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.
DFG Programme Research Grants
 
 

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