Project summary. Some proteins have the unique ability to sense and respond to mechanical force, a process called mechanotransduction, and can confer mechanosensitivity to cells, tissue, or organelles that express them. Sound waves in the ear, caress of a feather on the skin, or blood flow in arterial vessels are some instances where a force-inducing stimulus such as vibration, pressure, or stretch activates mechanically activated (MA) ion channels that initiate a cascade of events allowing the body to hear, sense touch, or regulate blood pressure. In the past decade, identification of novel MA ion channels like PIEZOs, K2Ps, TMCs, and OSCA/TMEM63s has revealed their importance in many physiological processes, but mechanistic details of how these channels sense force is, strikingly, incomplete. A major challenge impeding the field in comprehending MA channel gating mechanisms is the lack of a channel activation method that faithfully replicates the transduction of force, within a physiological environment. This proposal aims to apply photoswitchable lipids as a new and innovative method to assay mechanically activated (MA) ion channels, which will facilitate the study of these channels with greater ease, precision, and detail. In vivo, mechanical stimulation exerts force, which alters tension within the cell membrane where mechanosensitive proteins reside. MA ion channels detect this change in membrane tension leading to channel activation. Traditional in vitro techniques to activate MA channels are rather crude including pushing on membrane with a blunt glass probe or stretching the membrane by applying pressure. Although these techniques to alter membrane tension have enabled measurement of MA channel activity, they are indirect, low-throughput, and poorly mimic physiological stimuli. Here I propose to modulate membrane tension by directly targeting lipids that encompass the channel using photoswitchable lipids (or photolipids). Incorporation of the photochromic molecule azobenzene into fatty acyl chains provides optical control over lipids as they undergo cis-trans isomerization when irradiated with UV-A and blue light. Therefore, azobenzene-modified lipids can be used to reversibly manipulate membrane structure with light, which can either directly activate or modulate MA ion channel activity. Using the bona-fide MA ion channel family OSCAs we will screen and optimize photolipids to selectively change membrane properties on a cellular scale and assay MA channel activity with electrophysiology and/or calcium imaging. This unique strategy will be combined with structural, functional, and pharmacological studies to gain a better perspective on the cellular and molecular underpinnings of MA ion channel functions. Ultimately, this work will also lead to a deeper mechanistic understanding of mechanotransduction processes that drive vital physiological and pathological states in humans.

Project Narrative Mechanotransduction, the conversion of physiological force into biological signals, is crucial for many physiological processes such as touch or pain sensation, blood pressure regulation, and muscle contraction. The importance of mechanotransduction is underscored by its role in disorders such as chronic pain, atherosclerosis, and muscular dystrophies. Mechanotransduction is driven by ion channels activated when force is applied to cell membranes, but the lack of a highly precise method for MA channel activation in vitro has hampered progress in the field. This proposal uses an innovative and tractable technique to activate and assay MA ion channels that will enable their deep biophysical and pharmacological characterization, which will be vital to probe their in vivo consequences.