Biophysical Society Conference | Tahoe 2024

Molecular Biophysics of Membranes

Tuesday Speaker Abstracts

TMC PROTEINS REGULATE COCHLEAR HAIR BUNDLE MEMBRANE VISCOSITY THROUGH LIPID SCRAMBLASE ACTIVITY Shefin Sam George 1 ; Anthony Ricci 1,2 ; 1 Stanford University, OHNS - Head and Neck Surgery, Palo Alto, CA, USA 2 Stanford University, Molecular and Cellular Physiology, Palo Alto, CA, USA Lipid scramblases provide a permeation pathway to translocate phospholipids between membrane leaflets. Therefore, activation of a lipid scramblase results in the externalization of phosphatidylserine (PS), which is usually restricted to the inner leaflet, a hallmark of many relevant biological processes. Transmembrane-like Channel 1 protein (TMC1), which is an integral protein of the cochlear hair cell mechanotransduction (MET) channel, has been implicated to have membrane scramblase activity. However, there has been no direct measures of how the lipid scramblase could affect the cochlear membrane properties. As a first step towards understanding the functional relevance of TMCs in regulating membrane, we used a novel viscosity sensor BODIPY1c based on fluorescence lifetime for precise monitoring of membrane properties within live hair cells. BODIPY1c can also enter hair cells through MET channels and fluorescently label the cytoplasmic membranes, thus allowing identification of hair cells with functional MET channels. We also monitored the membrane scramblase activity using PS-specific binding protein Annexin V (AnV). We find that the membrane viscosity of mammalian cochlear hair bundles decreases during postnatal development and strongly correlates (r 2 =0.75) with the onset of MET. We also see that the lipid scrambling increases during this time frame. This data suggests that MET machinery could reduce the membrane viscosity through lipid scrambling. To test this hypothesis, we tested how genetic deletion of TMC1 and TMC2 would affect the membrane. We find that both the TMC1 mutants and TMC1/TMC2 double mutants have significantly higher membrane viscosity along with absence of lipid scrambling compared to litter mate control. Together this data suggests that TMCs could be directly impacting the membrane through their putative scramblase activity. Membrane is potentially being locally regulated for the MET channel to be more sensitive or faster.

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