Acid-sensing ion channel modulation by nonproton ligands: the influence of divalent cations

dc.creatorSmith, Rachel N.
dc.creatorGonzales, Eric B.
dc.description.abstract1. The acid-sensing ion channel (ASIC) is emerging as a potential mediator for a variety of pathologies, such as stroke, pain, and mental health diseases. Despite their involvement in multiple age-related pathologies, the ability to selectively target the ASIC subtypes remains unidentified. As their name suggests, ASICs are activated by an increase in extracellular protons, however other ASIC ligands include natural venom toxins, guanidine containing compounds, and calcium. Nonproton ligands, like 2-guanidine-4-methylquinazoline (GMQ), have been identified to selectively activate the peripheral ASIC3 via the nonproton ligand sensor domain (NPLSD). A pair of glutamates in rat ASIC3 (E79 and E423) responsible for GMQ activation is present in the structural determinant chicken ASIC1, despite having no direct modulation effect on the channel. We previously showed that cASIC1 could be activated by GMQ following a based on a partially activated channel state. Interestingly, low calcium concentrations cause the peripherally located ASIC3 subtype to be partially activated. We proposed that direct nonproton ligand activation of ASIC3 is possible due to the actions of the transmembrane domains (TMD) where calcium sensitivity resides. Additionally, the introduction of ASIC3 TMDs into a GMQ insensitive ASIC subtype (cASIC1) will reveal nonproton ligand sensitivity. 2. Chimeric receptors combining the extracellular, transmembrane, and intracellular domains of rat ASIC3 and chicken ASIC1 were generated to individually isolate the calcium and nonproton ligand effects on channel activation. Each chimeric receptor was assessed for function using whole cell patch clamp electrophysiology. 3. We confirmed that rASIC3 is activated and held open when extracellular calcium concentrations are reduced with minimal proton influence (pH 8.0). Low-calcium-activation of rASIC3 is further enhanced by the addition of GMQ in a concentration dependent manner. These effects are absent in cASIC1. The chimera termed cASIC1 (rASIC3-TM/ITC) is comprised of the extracellular domain of cASIC1 and the transmembrane/intracellular domains of rASIC3, and can be activated by GMQ in the absence of calcium, although its sensitivity to GMQ is reduced. Thus, GMQ activation was introduced in cASIC1 by replacing the transmembrane domains with those of ASIC3. 4. This data suggests that the ASIC3 TM domains dictate NPLSD influence on channel activity. Fully understanding how we can interrupt or enhance channel activation will allow us to preferentially target these ion channels, potentially leading to the promotion of developing novel therapeutics to interact with ASICs.
dc.titleAcid-sensing ion channel modulation by nonproton ligands: the influence of divalent cations