Structural rearrangements of the motor protein prestin revealed by fluorescence resonance energy transfer.
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.
Gleitsman
Kristin Rule
KR
Division of Biophysics and Neurobiology, Department of Molecular Physiology, National Institute for Physiological Sciences, Aichi, Japan.
Tateyama
Michihiro
M
Kubo
Yoshihiro
Y
D027321:Anion Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000378:metabolism; D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D018072:Hair Cells, Auditory, Outer / Q000378:metabolism; D006801:Humans; D008564:Membrane Potentials / Q000502:physiology; D018408:Patch-Clamp Techniques; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012980:Sodium Salicylate / Q000378:metabolism; D000076988:Sulfate Transporters
D027321:Anion Transport Proteins; D000894:Anti-Inflammatory Agents, Non-Steroidal; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; C498719:SLC26A5 protein, human; D000076988:Sulfate Transporters; D012980:Sodium Salicylate