Optical interrogation of acid-sensing ion channel activation and desensitization through genetic code expansion

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Project Summary This proposal will paint a clear picture of the molecular function of a unique family of proton- sensitive ion channels, known as Acid-sensing ion channels (ASICs). Located primarily in the central and peripheral nervous system, ASICs play a wide range of roles from synaptic plasticity to pain. In acidosis-related CNS pathologies like cerebral ischemia, ASICs have been implicated in causing neurodegeneration. These channels are activated by acidic shifts in extracellular pH below 7, where excess protons in solution bind the large extracellular domain and allow for the pore to open. ASICs primarily reside in three states: 1. At physiological or alkaline pH, the channel resides in an inactive resting state. 2. During mild acidification, the channel reverts to a proton-bound, inactive conformation known as the desensitized state. 3. Upon initial heavy acidification, the channel is in an open state, but over prolonged exposures, the channel desensitizes. While the crystal structures of these states help illuminate potential changes in the channel between these state transitions, experimental testing is required to functionally assess whether they are pertinent to channel gating. To elucidate this, the two following specific aims will be employed. Aim 1 will determine conformational changes that are necessary and sufficient for the mechanisms of activation using noncanonical amino acid incorporation to induce optogenetic crosslinking and sidechain isomerization at regions that undergo large conformational changes and test the functional consequences. Specifically, this aim will determine the contributions of the acidic pocket and palm domain to activation. Aim 2 will evaluate the mechanism of β11-12 linker regulation of desensitization by first understanding the role of Asp415, a residue within the linker, and its conformational consequences in desensitization through the use site-directed mutagenesis and photocrosslinking. Further, we will investigate the surrounding environment of the β11-12 linker by a phylogenetic comparison of ASIC isoforms and how this environment can control the movement of the linker, which we have previously shown to control desensitization. The outcomes of these two aims will fill the gaps in our knowledge for ASIC gating processes to allow for the development of ASIC-targeted therapeutics. Furthermore, this study will lay the groundwork for optogenetic modulation of ASICs through genetic code expansion. These tools will provide more precise control over the channels activity in in vitro and in vivo model systems. The fellowship training plan that accompanies this proposal will support my growth in reading, writing and communication skills to become a successful scientist. Technically, the training plan acts as a guide to further enhance my electrophysiological skills, i.e. patch clamp, and bolster my knowledge and skill in optogenetics, genetic code expansion and molecular modelling. The University of Rochester and the Department of Pharmacology and Physiology is renowned for its ion channel research, which provides the necessary facilities and mentoring required to complete my fellowship training plan.