Determining the Topology of the Acid-Sensing Ion Channel Intracellular Domains

Abstract

Acid-sensing ion channels (ASICs) are trimeric cation-selective channels activated by decreases in extracellular pH. The intracellular N and C terminal tails of ASIC1 variously influence channel gating, ion selectivity, channel trafficking, and signaling in ischemic cell death. While there are numerous x-ray and cryo-EM structures of the extracellular and transmembrane segments of ASIC1, these important intracellular tails remain unresolved. Here we set out to map the coarse topology of these intracellular domains using the voltage-sensitive dipicrylamine as a dark acceptor for fluorescent proteins inserted into various positions of chicken ASIC1 tails. With this patch clamp FRET approach, we find that the N-terminus of the channel undergoes an axial motion between the resting and desensitized states of the channel, while no movement of the C-tail is apparent. Further, when labeling both tails with pH-insensitive FRET donor-acceptor pairs, we find that the tails of the channel to not appear to separate as the channel transitions between the resting and active/desensitized states. Together, these data allow us to build a topological model of the intracellular tails of ASIC1 that will be a foundation for working hypotheses in future experiments.