Electrophysiological investigation of prestin function: Impact of cysteine replacement and characterization of self-association during voltage stimulation
McGuire, Ryan M.
Doctor of Philosophy thesis
Almost 25 years ago unique piezoelectric activity was discovered in neuroepithelial cells of the mammalian cochlea. These cells, outer hair cells (OHCs), are mechanoelectrical transducers capable of converting changes in membrane potential into whole cell axial deformations. This so called reverse transduction allows OHCs to impart force upon surrounding structures that comprise the cochlear sensory organ, the organ of Corti. Electromotility has been suggested to serve as the cochlear amplifier since it provides cycle by cycle positive feedback that enhances basilar membrane motion. Without this active process, hearing sensitivity and frequency discrimination are profoundly diminished. The motor protein prestin has recently been identified as a critical component of OHC electromotility and thought to populate the 11 nm particles observed in freeze-fractured OHC lateral membranes as either oligomers or complexes with accessory proteins. The mechanism for prestin activity is unknown and a common method of study has involved mutation of the protein, expression in a surrogate system, and characterization of function using electrophysiology. Our mutational study of prestin, involving replacement of cysteine residues, demonstrates that disulfide bonding is not required for oligomerization or function. Cysteine residues 196 and 415 are however critical to prestin activity, and might be important structural determinants of the protein's putative chloride binding pocket. We provide the first experimental evidence linking prestin conformational changes to function and report the discovery of voltage-dependent self-association. Voltage-dependent interactions provide a fluorescence signature for prestin activity and a molecular basis for the mechanism of electromotility. Finally, we have developed a biotinylated prestin reporter construct that allows extracellular binding of prestin, identification of the membrane localized fraction, and potentially provides a platform for forced oligomerization.