Structure-based analysis of the molecular mechanisms of prestin-dependent cochlear amplification

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Active mechanical amplification in the mammalian cochlea requires a process termed electromotility, i.e. voltage-driven ultrafast length changes of sensory outer hair cells (OHC). The elementary motor that generates motility is prestin (SLC26A5), an anion transporter-related, OHC-specific membrane protein. It is thought that electromotility arises from conformational changes of prestin that are directly triggered by changes in membrane potential and involve the binding of anions to the protein. In the previous funding period, we have established a 3D structural model of prestin and non-mammalian homologs, based on homology modeling, molecular dynamics simulations and - experimentally - extensive mutagenesis. The properties of the structure converge nicely with many of the functional features of prestin including anion dependence and the functional impact of previously identified domains. Based on this structural information we now want to examine the conformational dynamics that give rise to the generation of movement/or force. We will analyze voltage and anion dependent conformational changes by state-dependence of modification of introduced cysteine residues and by environmentally sensitive dyes. Beyond the molecular mode of action, it is also still unclear how prestin contributes to mechanical amplification: in a slow manner by setting the set-point of the active process or ultrafast by producing force in a cycle by cycle manner at high acoustic frequencies. To distinguish between these mechanisms, we will introduce kinetically altered prestin mutants into mice by cutting-edge genome engineering methods and analyze the peripheral hearing in terms of cochlear amplification.