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Real-time characterisation of neuropeptide binding to a membrane receptor involved in pain and ischemic stroke

Project description

Ligand binding assay protocol sets the stage for new treatment options

Brain tissue acidosis is a result of either an increase in carbon dioxide tension or an accumulation of acids produced by metabolism. Normally, local acidification of brain tissue serves as a neuronal signal, transduced via acid-sensing ion channels (ASIC1a). Local acidosis has been linked to conditions such as chronic pain, ischaemic stroke and psychiatric disorders. Recently, the potentially therapeutic role of ASIC1a has been pointed out, but no drugs are currently available to target the channels under pathological conditions. The EU-funded hsPCF-FRET project aims to characterise the binding of the neuropeptide big dynorphin to ASIC1a in real time and identify binding sites and interactions. The results will aid the future design of ASIC1a inhibitors with the potential to treat chronic pain and ischaemia.

Objective

Under physiological conditions, localized acidification of brain tissue serves as neuronal signal that get synaptically transduced via acid-sensing ion channels (ASIC1a). Local acidosis has, however, also been linked to some of the most prevalent neurological disorders such as chronic pain, ischemic stroke and psychiatric diseases. ASIC1a has thus emerged as drug target with great potential, but no drugs are currently available that specifically target the channels under pathological conditions. A few known neuropeptides modulate ASIC1a and could thus serve as scaffolds for a new generation of ASIC1a-selective drugs to, for example, treat pain without the typical downsides of opioids. Advances have, however, been hampered by the limited understanding of detailed protein-peptide interactions. Thus, the aim of the proposed project is to directly characterize the binding of the neuropeptide Big Dynorphin to ASIC1a in real time. Here, I will use a unique in-house developed high-sensitivity fluorescence patch-clamp electrophysiology setup and establish a protocol for a FRET-based ligand-binding assay. Together with site-directed mutagenesis, this approach will be able to identify state-dependent binding sites and key interactions, and allow direct analysis of binding affinity and kinetics under pathological conditions; all in intact membranes and with unprecedented (microsecond) temporal resolution. This information will aid future design of ASIC inhibitors with the potential to treat chronic pain and ischemia. The technology developed for this work will also enable ligand-binding studies of other membrane proteins in living cells and with high temporal resolution and will thus be of great potential value for a broad field. The project will expand my existing electrophysiology skills and add highly versatile expertise in fluorescent measurements. I thus anticipate my project to have significant personal and scientific impact beyond the scope of this proposal.

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Coordinator

KOBENHAVNS UNIVERSITET
Net EU contribution
€ 207 312,00
Address
Norregade 10
1165 Kobenhavn
Denmark

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Region
Danmark Hovedstaden Byen København
Activity type
Higher or Secondary Education Establishments
Links
Other funding
€ 0,00