Application of fluorescence recovery after photobleaching to study prestin lateral mobility in the human embryonic kidney cell

The transmembrane protein prestin is crucial to outer hair cell (OHC) electromotility and contributes to the sensitivity and frequency selectivity of mammalian hearing. The molecular mechanisms of electromotility remain unclear, but prestin is purported to function as both a voltage sensor and a molecular motor. Understanding the role of prestin requires characterizing its organization and behavior in the plasma membrane. Fluorescence recovery after photobleaching (FRAP) provides a powerful means to quantitatively study molecular diffusion. However, OHCs are inherently fragile ex vivo, and dynamic studies of prestin require model systems, such as human embryonic kidney (HEK) cells, expressing fluorescently labeled prestin. Utilizing this system, we provide the first direct, quantitative measurement of prestin lateral mobility. The results show remarkably different diffusion behavior for prestin-green fluorescent protein (GFP) as compared to a control protein, human somatostatin receptor 5 (SSTR5). Prestin-GFP FRAP experiments reveal immobile fractions approaching 50%, low effective diffusion coefficients, and recovery times slower than those of SSTR5. Secondary bleaching of a region reveals distinctly different diffusion parameters, which we propose reflect the transient confinement of prestin in the HEK cell. Although uncharacterized, intermolecular interactions between prestin and the membrane and/ or cytoskeleton may be important for the unique properties of prestin in electromotile OHCs. (C) 2007 Society of Photo-Optical Instrumentation Engineers.