Investigating the role of lipid membrane in the cochlear hair cell mechanotransduction - Project Summary/Abstract The mechano-electrical transduction (MET) process allows the transduction of mechanical information from sound into electrical signals, and it is a fundamental step in cochlear system function. Failures in this process lead to hearing loss and deafness. Understanding the basic properties of MET will lead to a better understanding of deafness, leading to targeted treatments and therapies. MET takes place at the level of the hair bundle and is mediated by tip links, extracellular proteins connecting shorter stereocilia to adjacent taller stereocilia. Deflections of the hair bundle towards the tallest stereocilia row increase tip-link tension and open MET channels that reside at the top of the shorter stereocilia. Although there is a large body of work regarding lipid membrane modulation of mechanosensitive ion channels, there is a limited but growing body of data on lipid modulation of cochlear hair cell MET. The lipid environment can affect channels indirectly through changes in membrane mechanics, or directly through individual lipid/protein interactions. PIP2, an endogenous phospholipid, modulates MET channel properties, potentially through a direct interaction or indirectly by altering membrane mechanics. A stretch activated channel modifier, GsMTx4 reduces the resting open probability (Po) of MET channel while also blocking the increase in Po induced by lowering external calcium or depolarizing the hair cell, suggesting the lipid membrane may be involved in modulating MET channel Po. The effect of voltage and calcium could be mediated through changes in lipid packing due to multivalent ions interacting between adjacent lipids. Our recent direct assessment of membrane diffusivity of individual stereocilium at a time using two-photon Fluorescent Recovery after Photobleaching (FRAP) demonstrated that stereocilia membrane is sensitive to calcium and voltage but not the soma, and MET channel Po co-varies with membrane diffusivity, supporting the hypothesis that the MET channel can be modulated by membrane mechanics. However, due to spatial and temporal limitations of FRAP, we were unable to monitor stereocilia membrane locally and dynamically. To further test this hypothesis and overcome current technological limitations, I will combine electrophysiology with live-cell fluorescence lifetime imaging (FLIM) of a novel viscosity sensor to examine the membrane viscosity with improved spatio-temporal resolution for the first time in mammalian cochlea. I will assess local and temporal changes in the stereociliary membrane viscosity with voltage, calcium, and membrane components like cholesterol and PIP2 and correlate these effects to changes in MET channel Po. These studies will enhance our basic understanding of the importance of lipid membrane in hair cell mechanotransduction. Understanding the crucial components in the mechanical underpinnings of the stereocilia are both biophysically and biologically relevant. The development and use of these new technologies will greatly advance my career as an independent investigator and likely have broader applications in the auditory field and beyond.
