The Motor Protein Prestin Is a Bullet-shaped Molecule with Inner Cavities

Abstract

Prestin is a transmembrane motor protein localized at the outer hair cells (OHCs) of the mammalian inner ear. Voltage-dependent conformational changes in prestin generate changes in the length of OHCs. A loss of prestin function is reported to induce severe auditory deficiencies, suggesting prestin-dependent changes of OHC length may be at least a part of cochlear amplification. Here we expressed the recombinant FLAG-fused prestin proteins in Sf9 cells and purified to particles of a uniform size in EM. The square-shaped top view of purified prestin, the binding of multiple anti-FLAG antibodies to each prestin particle, the native-PAGE analysis, and the much larger molecular weight obtained from size exclusion chromatography than the estimation for the monomer all support that prestin is a tetramer (Zheng, J., Du, G. G., Anderson, C. T., Keller, J. P., Orem, A., Dallos, P., and Cheatham, M. (2006) J. Biol. Chem. 281, 19916 -19924). From negatively stained prestin particles, the three-dimensional structure was reconstructed at 2 nm resolution assuming 4-fold symmetry. Prestin is shown to be a bulletshaped particle with a large cytoplasmic domain. The surface representation demonstrates indentations on the molecule, and the slice images indicate the inner cavities of sparse densities. The dimensions, 77 ؋ 77 ؋ 115 Å , are consistent with the previously reported sizes of motor proteins on the surface of OHCs.The mammalian ear has specialized function in collecting sound signals and transmitting them to the nerve. Several amplifying systems are developed for sound collection. The pinna and ear canal funnel have suitable shapes for effective sound collection and condensation. The bones of the middle ear (malleus, incus, and stapes) convert the sound energy in the eardrum to the pressure waves in the fluid of the cochlea with 20 -40-fold amplification.In the inner ear, a positive feedback loop generates synchronous force by sensing the vibrations within the organ of Corti, which amplifies gained sound signals more than 100 times, although the mechanisms are not clearly understood (1, 2). One of the hypotheses is that voltage-dependent changes in the length of the outer hair cells (OHCs) 2 generate the lateral membrane motility and amplify the sound (3-5). These changes are proposed to be caused by prestin (SLC26A5) (6, 7), which is a membrane integral protein and is categorized to the solute linked carrier (SLC) 26 family of anion transporters. It has been also reported that prestin responds to the membrane potential by harboring cytoplasmic anions (Cl Ϫ or HCO 3 Ϫ ), as extrinsic voltage sensors, and changes its structure (8). Because prestin is densely embedded in the plasma membrane of the OHCs, the total length of the cell also changes as much as 5% as follows: decreases by depolarization and increases by hyperpolarization (9, 10). A voltage-dependent structural change was also observed in heterologously expressed cells, which strongly supports that prestin is a motor protein (6).The prestin gene (81.4 kDa, 744 a...

Structure of TRPV1 channel revealed by electron cryomicroscopy

“…Surprisingly, the structure described is a very open wireframelike structure, with overall volume exceeding that of much larger proteins, such as the InsP 3 receptor (1,252 kDa, vs. 383 kDa for TRPC3), and with insufficient continuous density to form an obvious transmembrane channel domain. It bears little resemblance to a structure derived from electron microscopy of TRPC3 in negative stain by the same group (38), although the latter is strikingly similar to their bullet-shaped negative-stain structures of TRPM2 (689 kDa) (39), and the unrelated membrane protein, prestin (326 kDa) (40). Based on our preliminary results with TRPV2 and TRPY1, a yeast channel related most closely to the TRPC family among mammalian channels, it seems likely that the structures of TRP channels in general follow the basic structural organization described here for TRPV1.…”

Purification of the Motor Protein Prestin from Chinese Hamster Ovary Cells Stably Expressing Prestin

“…However, although the baculovirus/Sf9 insect cell system is generally used as a high-expression system due to its well-organized and easy-to-use method, the post-translational modification pattern of prestin expressed in the Sf9 cells is considered to be different from that of native prestin. In fact, the glycosylation pattern of prestin in Sf9 cells and that in native prestin are found to differ from each other (11) (16) (17) . Since the glycosylation pattern is related to the protein stability, there is a possibility that the stability of prestin expressed in Sf9 cells is lower than that expressed in mammalian OHCs.…”

“…Investigation using purified prestin, which would enable us to obtain signals from only prestin, is a promising approach to gain new information on prestin. Recently, it has been reported that recombinant prestin molecules were purified from Sf9 insect cells using a baculovirus expression system, and employing such molecules, it was revealed by electron microscopy that prestin is bullet-shaped (16) . However, although the baculovirus/Sf9 insect cell system is generally used as a high-expression system due to its well-organized and easy-to-use method, the post-translational modification pattern of prestin expressed in the Sf9 cells is considered to be different from that of native prestin.…”

High-Resolution AFM Imaging of Prestin Purified and Reconstituted into an Artificial Lipid Bilayer

“…Recently, two significant studies on such structure have been reported. Mio et al [7] purified prestin from insect cells which were engineered to express prestin and observed purified prestin by EM. Results indicated that prestin is a bullet-shaped molecule.…”