Asic Activation Mechanisms Delineated Through Genetic Code Expansion

Acid-sensing Ion Channels (ASICs) are a unique class of ion channels that are activated through acidification of the extracellular space. These trimeric sodium-selective ion channels are found in the central and peripheral nervous system, where they are critical players in a myriad of conditions including ischemic cell death, synaptic plasticity, and fear conditioning. As such, determining how these channels function on a molecular level will aid the development of useful pharmacological tools. Currently, ASIC activation is thought to require protonation of acidic side chains in two distinct domains: the acidic pocket and the palm domain. Both of these regions show the largest differences in α-carbon position between the resting and toxin-stabilized open state structures. Single or multiple point mutations to these domains shift ASIC pH-response curves, further supporting their importance. However, it is unclear if these domains function independently or if one is the dominant driver of activation. To explore this issue, we incorporated photomodulatory noncanonical amino acids into these domains in fluorescently-tagged chicken ASIC1. Fast perfusion patch clamp experiments demonstrate multiple insertion positions are functional. We find several positions in the acidic pocket exhibit state-dependent UV modulation. In general, sites that can be modulated in the resting state decrease pH sensitivity while positions modulated in the desensitized conformation increase sensitivity. Additionally, induced steric hindrance in the acidic pocket likely prevents closure during activation, decreasing overall sensitivity of the channel to acidification. However, in all cases thus far examined, modulated channels can still be activated by strongly acidic stimuli. These findings indicate that the acidic pocket is an important, but not essential, allosteric site for channel activation.