ABSTRACT Piezo2 is a non-selective Ca2+-permeable cation channel activated by mechanical force. It is expressed in peripheral sensory neurons of the dorsal root ganglia (DRG) and it plays important roles in gentle touch and injury-induced mechanical pain. Piezos are large proteins, with 38 transmembrane domains and their membrane footprint is larger than that of any other known ion channels. This suggests that membrane lipids could significantly impact the behavior of these channels, but exploration of the effects of membrane lipids on Piezo function is in its early stages. The goal of this proposal is to gain insights into how lipids regulate Piezo channels, building upon our recent identification of membrane lipids that specifically inhibit Piezo2 activity while not affecting Piezo1. The proposal is based on our recent finding that the TMEM120A protein robustly inhibits Piezo2, but not Piezo1 channels. Structural studies of TMEM120A indicate that this protein shows similarity to a lipid modifying enzyme ELOVL7, with a CoA molecule bound to each monomer. We hypothesized that TMEM120A inhibits Piezo2 by modifying cellular lipid composition. Our preliminary data show that expressing TMEM120A increases levels of lipids in the Kennedy pathway of triglyceride synthesis, with the most robust changes in phosphatidic acid (PA) and lysophosphatidic acid (LPA) with saturated acyl chains. Intracellular PA delivery through the whole cell patch pipette inhibited Piezo2, but not Piezo1 activity. Generating endogenous PA in the plasma membrane by optogenetic activation of phospholipase D inhibited Piezo2, but not Piezo1 activity. Intracellular delivery of LPA, as well as incubation with carbacyclic PA (ccPA), a non-metabolizable analogue of PA and LPA inhibited Piezo2 channels. Our data identify PA and LPA as endogenous inhibitors of Piezo2 channels. In our proposal we aim to identify the molecular determinants of the effects of PA and LPA (Aim1), investigate their roles in regulation of Piezo2 activity by signaling pathways (Aim 2), and tests in vivo effectiveness of this lipid regulation on Piezo2 mediated sensory processes (Aim 3). Overall, we identified novel lipid regulators of Piezo2 channels, that do not affect Piezo1. Our data will provide important insight into the regulation of Piezo2 by membrane lipids, and the knowledge gained from this study has the potential to be utilized in the future development of Piezo2 inhibitors for treating various pain conditions.

PROJECT NARRATIVE Piezo2 is a non-selective Ca2+ permeable mechanically activated ion channel, that is indispensable for gentle touch and it is involved in injury-induced mechanical pain. We identify a novel lipid regulator of this channel, that inhibits Piezo2, but not Piezo1 activity. Our data will provide insight not how these channels are regulated, and may help identifying selective pharmacological inhibitors of Piezo2 in the future.