NON-PROTON LIGAND ACTIVATION OF ASIC3 IS MEDIATED BY CALCIUM DEPLETION

Purpose: Acid-sensing ion channels (ASICs) are trimeric, sodium-selective channels located throughout the central and peripheral nervous systems. These ion channels are activated by extracellular protons and involved in ischemia, neurotransmission, and pain nociception. Thus, ASICs are intriguing molecular targets for the development of novel pharmaceutical treatments. However, there is a lack of selective compounds that can differentiate specific ASIC subtypes. One ASIC subtype, the peripherally located ASIC3, is sensitive to extracellular calcium, which acts to stabilize the closed, inactive state. Recently, non-proton ligands, such as 2-guanidine-4-methylquinazoline (GMQ), have been identified to selectively activate ASIC3. Controversy remains over what constitutes the ASIC3 GMQ binding site and how other mediators of ASIC3 activity, such as calcium, influence non-proton ligand activity. We hypothesize that non-proton ligand activation of ASIC3 requires the depletion, or removal, of calcium. Methods: Chimeric receptors combining the extracellular (ETC), transmembrane (TM), and intracellular (ITC) domains of rat ASIC3 and chicken ASIC1 were generated and subcloned into a mammalian expression vector with an amino-terminal GFP tag. Each of these vectors were transfected into Chinese hamster ovarian (CHO) cells where successful transfection was confirmed using fluorescence microscopy. Each chimeric receptor was assessed for function using whole-cell patch clamp electrophysiology. The non-proton ligand GMQ was assessed in each ASIC in the presence and absence of calcium. Results: Our preliminary data confirm that ASIC3 is activated and held open by a lowering of extracellular calcium concentration at high pH (pH 8.0), thus minimizing proton influence. This activation is enhanced by the addition of GMQ to the extracellular solution at the same pH in a concentration dependent manner. Our data suggest that when calcium is removed from the extracellular solution at high pH, GMQ stimulates ASIC3 activity. Conclusions: The non-proton ligand activity, mediated by GMQ, is dependent on the removal of calcium from the ASIC3 calcium block site, which appears to be void in ASIC1. Future studies will focus on elucidating the GMQ activation kinetics and key residues in both the extracellular and transmembrane domains critical for GMQ binding and activity. The understanding of ASIC3 activation via this novel mechanism may allow for the development of novel compounds specifically targeting ASIC3.