Quantitative FLIM-FRET Measurement of Voltage Dependent Prestin Conformational Changes
Raphael, Robert M.
Master of Science
The transmembrane protein prestin forms an integral part of the mammalian sense of hearing by providing the driving force for the electromotility of the outer hair cell, a specialized cell that resides within the cochlea. This provides the cochlea with an ability to amplify mechanical vibrations, allowing for a high degree of sensitivity and selectivity in auditory transduction. The phenomenon, driven by changes in the transmembrane potential, is thought to be the result of conformational changes in self-associating prestin oligomers. We have previously utilized Forster resonance energy transfer (FRET), by both sensitized emission and acceptor photobleach methods, to detect prestin self -association. While these methods can qualitatively confirm prestin-prestin association, determining nanoscale changes in prestin organization requires greater accuracy than either technique provides. In this thesis, a FRET methodology based on fluorescence lifetime imaging (FLIM), detected by time correlated single photon counting (TCSPC), is implemented and utilized to quantitatively measure conformational changes within prestin-prestin oligomers in response to voltage stimulus.
Prestin; FRET; FLIM; Patch-clamp