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Am J Physiol Cell Physiol 297: C290-C298, 2009. First published June 10, 2009; doi:10.1152/ajpcell.00647.2008
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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Structural rearrangements of the motor protein prestin revealed by fluorescence resonance energy transfer

Kristin Rule Gleitsman,1,2,* Michihiro Tateyama,1,3,* and Yoshihiro Kubo1,3

1Division of Biophysics and Neurobiology, Department of Molecular Physiology, National Institute for Physiological Sciences, Aichi, Japan; 2Department of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California; 3Solution-Orientated Research for Science and Technology, Japan Science and Technology Corporation, Saitama, Japan

Submitted 19 December 2008 ; accepted in final form 5 June 2009

Prestin is a membrane protein expressed in the outer hair cells (OHCs) in the cochlea that is essential for hearing. This unique motor protein transduces a change in membrane potential into a considerable mechanical force, which leads to a cell length change in the OHC. The nonlinear capacitance in cells expressing prestin is recognized to reflect the voltage-dependent conformational change of prestin, of which its precise nature remains unknown. In the present work, we aimed to detect the conformational changes of prestin by a fluorescence resonance energy transfer (FRET)-based technique. We heterologously expressed prestin labeled with fluorophores at the COOH- or NH2-terminus in human embryonic kidney-293T cells, and monitored FRET changes on depolarization-inducing high KCl application. We detected a significant decrease in intersubunit FRET both between the COOH-termini and between the COOH- and NH2-termini. A similar FRET decrease was observed when membrane potential was directly and precisely controlled by simultaneous patch clamp. Changes in FRET were suppressed by either of two treatments known to abolish nonlinear capacitance, V499G/Y501H mutation and sodium salicylate. Our results are consistent with significant movements in the COOH-terminal domain of prestin upon change in membrane potential, providing the first dynamic information on its molecular rearrangements.

cochlear amplifier; outer hair cell; conformational change; total internal reflection fluorescence


Address for reprint requests and other correspondence: Y. Kubo, Div. of Biophysics and Neurobiology, Dept. of Molecular Physiology, National Institute for Physiological Sciences, Nishigoh-naka 38, Myodaiji, Okazaki, Aichi 444-8585, Japan (E-mail: ykubo{at}nips.ac.jp)






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