Targeting Specific ASIC Subunits and Heteromers Using Protein Engineering

PROJECT SUMMARY/ABSTRACT Acid-sensing ion channels (ASICs) are a family of low pH-activated ion channels found throughout the central and peripheral nervous systems. ASICs are important players in several neurological conditions such as cell death following ischemic stroke, neurodegeneration, fear and anxiety and synaptic plasticity. ASICs are particularly important in various forms of pain like cutaneous nociception and inflammatory hyperalgesia. Consistent with this role, the subunits ASIC1a, ASIC1b and ASIC 3 are all found in dorsal root ganglia and trigeminal ganglia neurons from both human and rodent. Moreover, pain sensation can be reduced by pharmacological blockade of ASIC1, using the selective toxins mambalgin or psalmotoxin1, or inhibition of ASIC3, using the toxin APETx2. These effects are attenuated in mice where ASIC1 or ASIC3 have been deleted, further implicating these channels as important mediators of peripheral pain. Thus, ASICs – and in particular ASIC1a, 1b and 3 – are attractive targets for analgesic treatments. However, translational efforts have been complicated by several factors. In particular, the binding site for APETx2 on ASIC3 is unknown, preventing drug design efforts that exploit this toxin. Also, the ASIC1 inhibitor mambalgin acts differently in rodent versus human ASICs. While mambalgin inhibits rodent ASIC1b, thereby producing peripheral analgesia, it actually potentiates human ASIC1b. And finally, the physiological AISC targets are likely heteromers of these subunits that would be less sensitive to individual subunit specific toxins. Here we propose to overcome these challenges through two specific aims. First, we have identified a novel binding site for APETx2 based on computational docking and supported by experimental evidence. We will further test this novel site through a combination of patch clamp electrophysiology and surface plasmon resonance assays. Second, we will generate and test a series of novel bivalent toxins for inhibitory effect on heteromeric ASICs of defined stoichiometry using a concatemer system. Taken together, these proposed experiments may identify the long sought APETx2 binding site, generate a library of valuable heteromeric ASIC concatemers and test an array of novel bivalent toxins at these physiologically relevant heteromers. These toxins may serve as useful research compounds, either as functional modulators or specific markers when conjugated to fluorophores. Most importantly, we may identify toxin pairings to act as a peripheral analgesic leads for further development.