Membrane properties of the OHC system

A central concept in mammalian hearing is that outer hair cells (OHCs) generate force through electromotility (eM), driving cochlear amplification and enabling exquisite auditory sensitivity. The motor protein prestin underlies OHC electromotility, expanding and contracting in response to membrane voltage. Prestin’s essential role in human hearing is highlighted by over 16 known (and over a 100 uncharacterized) mutations in the prestin gene causing DFNB61 hearing loss. A key question is how prestin’s voltage sensitivity arises and how its structure compares to other SLC26 family transporters, some of which now appear to have electromotility. Our modeling and electrophysiological data suggest that expansion/ contraction involves core-domain movement relative to the gating domain, similar to the transporter cycle. Moreover the expansion contraction appears to be concentrated at the inner leaflet, similar to the related transporter pendrin. Additionally, our new cryoEM structures identify a hitherto unrecognized phenomenon of coincident reduction in the acuity in the angle between two protomers. Although we were unable to clearly identify structures in lipid vesicles for technical reasons, we believe we have overcome these difficulties with improved algorithms that now allow voltage-dependent structural studies. Specific Aim 1 seeks to define expanded, contracted, and intermediate prestin states and how voltage drives these transitions by: · Determining structures of mutants (V499G/Y501H, R150Q, D485N) with altered voltage sensitivity. · Resolving structures in lipid vesicles under different transmembrane voltages. · Capturing intermediate states using chemical modulation (SCN⁻, salicylate) that varies prestin’s voltage sensitivity and using a mutant V139C that transitions from expanded to contracted state. Specific Aim 2 investigates the molecular mechanism of prestin’s fast voltage-dependent frequency response. A multiscale model will describe conformational transitions as voltage-dependent free energy changes, using enhanced molecular dynamics simulations. The model will predict NLC and frequency responses (1–80 kHz) for wildtype and mutant prestin and generate structural ensembles for comparison with experimental data.