Assessment of prestin self-association using fluorescence resonance energy transfer

An active process within the cochlea is necessary to obtain the sensitivity and frequency selectivity characteristic of mammalian hearing. This process is realized, at least in part, through the electromotile response of outer hair cells (OHCs). Electromotility requires the presence of prestin, a transmembrane protein highly expressed in the OHC lateral wall. Very little is known about how prestin functions at the molecular level to elicit electromotility, but theoretical models and recent experiments suggest that prestin-prestin interactions are required. To explore the extent of proposed prestin interactions, we employ fluorescence resonance energy transfer (FRET). FRET is a powerful optical technique capable of measuring inter-fluorophore distances less than 10 nm. Using human embryonic kidney cells (HEKs) as a model cell system and the standard FRET pair, cyan fluorescent protein (CFP) as the donor and yellow fluorescent protein (YFP) as the acceptor, we assay for the self-association of prestin under steady-state conditions using acceptor photobleach FRET (apFRET) and sensitized emission FRET (seFRET). Our findings from apFRET indicate the presence of prestin self-association when HEKs express both prestin-CFP and prestin-YFP in the membrane. The average FRET efficiency was approximately 9%, but values as high as 20% were measured. Notably, a higher efficiency of energy transfer ranging from 10-30% was obtained with seFRET. Additionally, we report an apFRET efficiency of approximately 10% for cells expressing a CFP-prestin-YFP double fusion protein. We discuss the significance of these measurements for establishing the presence of prestin-prestin interactions in transfected HEK cells.