FLIM-FRET Measurements of Voltage Dependent Conformational Changes in the Outer Hair Cell Motor Protein Prestin

Abstract

Cochlear sensory cells, outer hairs cells (OHCs), elongate and contract in response to changes in transmembrane potential. This electromotility is important for auditory frequency selectivity and sensitivity and is believed to be driven by a motor protein, prestin, which is highly expressed within the membrane of OHCs. Experiments have shown that prestin undergoes self-association leading to the observation that oligomerization might be important for proper function. We have previously utilized acceptor photobleach fluorescence resonance energy transfer (FRET) to study pretin-prestin interactions in living cells. However, traditional FRET, based upon detection of fluorescence intensity, is inherently subjected to artifacts that arise from variations in excitation intensity, photobleaching, and fluorophore concentration. We have recently implemented fluorescence lifetime imaging microscopy (FLIM) to perform robust FLIM-FRET measurements in HEK cells cotransfected with prestin-TFP and prestin-YFP. FRET efficiency was calculated by fitting the lifetime decay of prestin-TFP in cells clamped at various holding potentials. High FRET efficiencies were measured at hyperpolarized potentials, and low FRET efficiencies were measured at depolarized. The results indicated that changes in the transmembrane potential induce a conformational change in prestin that can be detected by FLIM-FRET. Thus, FLIM-FRET can provide a sensitive assay for the mechanical correlate of prestin function.