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Published August 2009 | public
Journal Article

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

Abstract

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 NH_2-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 NH_2-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.

Additional Information

Copyright © 2009 by the American Physiological Society. Submitted 19 December 2008 ; accepted in final form 5 June 2009. This work was supported in part by research grants from the Ministry of Education, Science, Sports, Culture to Yoshihiro Kubo. K. R. Gleitsman was supported by the East Asia and Pacific Summer Institute program of the National Science Foundation and the Japan Society for Promotion of Science. The authors are grateful to Dr. B. Fakler (University of Freiburg, Germany) for providing us with rat prestin cDNA, Dr. A. Miyawaki for Venus YFP cDNA, and Dr. J. Miyazaki for pcXN_2 expression vector. We also thank members of the Kubo laboratory for helpful discussions and T. Yamamoto and Y. Asai for technical assistance. K. R. Gleitsman especially thanks Professors Dennis Dougherty and Henry Lester (Caltech) for support for the undertaking of this research and Dr. M. Torrice (Caltech) for helpful comments on early drafts of this manuscript. K. R. Gleitsman and M. Tateyama made equal contribution to this study.

Additional details

Created:
August 21, 2023
Modified:
October 18, 2023