Koji Iida

Tohoku University, Miyagi, Japan

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Publications (31)32.31 Total impact

  • Nano-Biomedical Engineering 2012 - The Tohoku University Global Centre of Excellence Programme; 01/2012
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    ABSTRACT: In the present study, purified prestin was observed by atomic force microscopy (AFM). First, the lipid bilayer was formed on a freshly cleaved mica disk by the deposition of small unilamellar lipid vesicles. Such bilayer was destabilized by incubation with a detergent. Afterward, purified prestin was added to the destabilized lipid bilayer. Finally, a high resolution image of the prestin-reconstituted lipid bilayer was acquired by AFM. As a result, densely embedded prestin with a diameter of 11.0±1.3 nm was visualized. From the obtained image, cytoplasmic side of prestin was found to form a ring-like structure with four peaks and one valley at its center.
    11/2011; 1403. DOI:10.1063/1.3658084
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    ABSTRACT: The SLC26A4 gene encodes the transmembrane protein pendrin, which is involved in the homeostasis of the ion concentration of the endolymph of the inner ear, most likely by acting as a chloride/bicarbonate transporter. Mutations in the SLC26A4 gene cause sensorineuronal hearing loss. However, the mechanisms responsible for such loss have remained unknown. Therefore, in this study, we focused on the function of ten missense pendrin mutations (p.P123S (Pendred syndrome), p.M147V (NSEVA), p.K369E (NSEVA), p.A372V (Pendred syndrome/NSEVA), p.N392Y (Pendred syndrome), p.C565Y (NSEVA), p.S657N (NSEVA), p.S666F (NSEVA), p.T721M (NSEVA) and p.H723R (Pendred syndrome/NSEVA)) reported in Japanese patients, and analyzed their cellular localization and anion exchanger activity using HEK293 cells transfected with each mutant gene. Immunofluorescent staining of the cellular localization of the pendrin mutants revealed that p.K369E and p.C565Y, as well as wild-type pendrin, were transported to the plasma membrane, while 8 other mutants were retained in the cytoplasm. Furthermore, we analyzed whether salicylate, as a pharmacological chaperone, restores normal plasma membrane localization of 8 pendrin mutants retained in the cytoplasm to the plasma membrane. Incubation with 10 mM of salicylate of the cells transfected with the mutants induced the transport of 4 pendrin mutants (p.P123S, p.M147V, p.S657Y and p.H723R) from the cytoplasm to the plasma membrane and restored the anion exchanger activity. These findings suggest that salicylate might contribute to development of a new method of medical treatment for sensorineuronal hearing loss caused by the mutation of the deafness-related proteins, including pendrin.
    Hearing research 12/2010; 270(1-2):110-8. DOI:10.1016/j.heares.2010.08.015 · 2.97 Impact Factor
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    ABSTRACT: Prestin is the motor protein of cochlear outer hair cells and is essential for mammalian hearing. The present study aimed to clarify the structure of prestin by atomic force microscopy (AFM). Prestin was purified from Chinese hamster ovary cells which had been modified to stably express prestin, and then reconstituted into an artificial lipid bilayer. Immunofluorescence staining with anti-prestin antibody showed that the cytoplasmic side of prestin was possibly face up in the reconstituted lipid bilayer. AFM observation indicated that the cytoplasmic surface of prestin was ring-like with a diameter of about 11 nm.
    FEBS letters 07/2010; 584(13):2872-6. DOI:10.1016/j.febslet.2010.04.076 · 3.17 Impact Factor
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    ABSTRACT: Prestin is a key molecule for mammalian hearing. The present study investigated changes in characteristics of prestin by culturing prestin-transfected cells with salicylate, an antagonist of prestin. As a result, the plasma membrane localization of prestin bearing a mutation in the GTSRH sequence, which normally accumulates in the cytoplasm, was recovered. Moreover, the nonlinear capacitance of the majority of the mutants, which is a signature of prestin activity, was also recovered. Thus, the present study discovered a new effect of salicylate on prestin, namely, the promotion of the plasma membrane expression of prestin mutants in an active state.
    FEBS letters 04/2010; 584(11):2327-32. DOI:10.1016/j.febslet.2010.04.010 · 3.17 Impact Factor
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    ABSTRACT: The motor protein prestin in the plasma membrane of cochlear outer hair cells is believed to be the origin of their electromotility. Several characteristics of prestin have been clarified by introduction of mutations into prestin. In the present study, the aim was to investigate whether or not salicylate has the ability to promote the plasma membrane expression of prestin mutants accumulated in the cytoplasm. Six prestin mutants created in our previous study, namely, G127A, T128A, S129A, R130A, H131A and S129T, were used. These mutants were engineered to be expressed in HEK293 cells by transfection and effects of salicylate on prestin were then investigated by immunofluorescence staining and the whole-cell patch-clamp. When the cells were cultured without salicylate, immunofluorescence staining showed that all prestin mutants were accumulated in the cytoplasm. The patch-clamp recording indicated that H131A and S129T did not show nonlinear capacitance (NLC), which reflects the amount of functional prestin in the plasma membrane, and that the other four mutants showed NLC significantly smaller than that of wild-type (WT) prestin. On the other hand, when 10 mM salicylate was used, immunofluorescence staining suggested that the plasma membrane expression of all prestin mutants was recovered. Especially, the plasma membrane expression of G127A and R130A was recovered to the WT prestin level. By the patch-clamp method, NLC in G127A, T127A, S129A and R130A were shown to statistically increase, although H131A and S129T did not exhibit NLC. As in the plasma membrane expression, NLC of G127A and that of R130A recovered to the WT prestin level by salicylate. The results suggest that the prestin mutants were misfolded in the cytoplasm and that salicylate has the ability to induce mutants’ correct folding, promoting their transport to the plasma membrane, which led to the recovery of NLC.
    IFMBE proceedings 01/2010; 31. DOI:10.1007/978-3-642-14515-5_415
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    ABSTRACT: The motor protein prestin in cochlear outer hair cells is a member of the solute carrier 26 family, but among the proteins of that family, only prestin can confer the cells with nonlinear capacitance (NLC) and motility. In the present study, to clarify contributions of unique amino acids of prestin, namely, Met-122, Met-225 and Thr-428, to the characteristics of prestin, mutations were introduced into those amino acids. As a result, NLC remained unchanged by both replacement of Met-122 by isoleucine and that of Thr-428 by leucine, suggesting that those amino acids were not important for the generation of NLC. Surprisingly, the replacement of Met-225 by glutamine statistically increased NLC as well as the motility of prestin-expressing cells without an increase in the amount of prestin expression in the plasma membrane. This indicates that Met-225 in prestin somehow adjusts NLC and the motility of prestin-expressing cells.
    Biochemical and Biophysical Research Communications 10/2009; 389(4):569-74. DOI:10.1016/j.bbrc.2009.09.016 · 2.30 Impact Factor

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    ABSTRACT: Prestin is the motor protein of cochlear outer hair cells, which exhibit elongation and contraction in response to acoustic stimuli. In this study, the effects of mutations in unique amino acids, which among members of the SLC26 family were only present in prestin, on the characteristics of prestin were investigated. As a result, a mutation in Met-225 of prestin was found to increase nonlinear capacitance, which is a signature of prestin activity.
  • Hiroshi Wada · Michio Murakoshi · Koji Iida · Shun Kumano ·

  • Conference Paper: INNER EAR BIOMECHANICS

    Nano-Biomedical Engineering 2009 - The Tohoku University Global Centre of Excellence Programme; 01/2009
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    ABSTRACT: Prestin is believed to be the motor protein expressed in the plasma membrane of outer hair cells (OHCs). In this study, characterization of prestin was carried out by sitedirected mutagenesis focusing on the unique amino acids in prestin. Five prestin mutants, namely, M122I, C192A, M225Q, C415A and T428L, were engineered to be separately expressed in HEK293 cells. The characteristics of those mutants were then investigated. As a result, it was found that the charge density of M225Q, which reflects the charge transfer of prestin in the plasma membrane, was 1.7 times larger than that of wild-type prestin (WT), although the charge density of the other mutants were similar to that of WT. On the other hand, the amount of M225Q molecules in the plasma membrane was 1.2 times greater than that of WT molecules. The increase in the amount of prestin molecules due to the mutation of M225 probably indicates that prestin became more stable, but such increase does not correspond to the increase of the charge density. Hence, the mutation of M225 was considered to increase the stability of prestin, leading to an increase in the ratio of active prestin molecules to the total amount of prestin molecules in the plasma membrane.
    IFMBE proceedings 01/2009; 23. DOI:10.1007/978-3-540-92841-6_560
  • Michio Murakoshi · Koji Iida · Shun Kumano · Hiroshi Wada ·
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    ABSTRACT: Prestin, a membrane protein of the outer hair cells (OHCs), is known to be the motor which drives OHC somatic electromotility. Electron microscopic studies showed the lateral membrane of the OHCs to be densely covered with 10-nm particles, they being believed to be a motor protein. Imaging by atomic force microscopy (AFM) of prestin-transfected Chinese hamster ovary (CHO) cells revealed 8- to 12-nm particle-like structures to possibly be prestin. However, since there are many kinds of intrinsic membrane proteins other than prestin in the plasma membranes of OHCs and CHO cells, it was impossible to clarify which structures observed in such membranes were prestin. In the present study, an experimental approach combining AFM with quantum dots (Qdots), used as topographic surface markers, was carried out to detect individual prestin molecules. The inside-out plasma membranes were isolated from the prestin-transfected and untransfected CHO cells. Such membranes were then incubated with antiprestin primary antibodies and Qdot-conjugated secondary antibodies. Fluorescence labeling of the prestin-transfected CHO cells but not of the untransfected CHO cells was confirmed. The membranes were subsequently scanned by AFM, and Qdots were clearly seen in the prestin-transfected CHO cells. Ring-like structures, each with four peaks and one valley at its center, were observed in the vicinity of the Qdots, suggesting that these structures are prestin expressed in the plasma membranes of the prestin-transfected CHO cells.
    Pflügers Archiv - European Journal of Physiology 09/2008; 457(4):885-98. DOI:10.1007/s00424-008-0560-z · 4.10 Impact Factor
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    ABSTRACT: Acetylation and deacetylation of proteins occur in cells in response to various stimuli, and are reversibly catalyzed by histone acetyltransferase and histone deacetylase (HDAC), respectively. EoL-1 cells have an FIP1L1-PDGFRA fusion gene that causes transformation of eosinophilic precursor cells into leukemia cells. The HDAC inhibitors apicidin and n-butyrate suppress the proliferation of EoL-1 cells and induce differentiation into eosinophils by a decrease in the protein level of FIP1L1-PDGFRalpha without affecting the mRNA level for FIP1L1-PDGFRA. In this study, we analyzed the mechanism by which the protein level of FIP1L1-PDGFRalpha is decreased by apicidin and n-butyrate. EoL-1 cells were incubated in the presence of the HDAC inhibitors apicidin, trichostatin A or n-butyrate. The protein levels of FIP1L1-PDGFRalpha and phosphorylated eIF-2alpha were determined by Western blotting. Actinomycin D and cycloheximide were used to block RNA synthesis and protein synthesis, respectively, in the chasing experiment of the amount of FIP1L1-PDGFRalpha protein. When apicidin- and n-butyrate-treated EoL-1 cells were incubated in the presence of actinomycin D, the decrease in the protein level of FIP1L1-PDGFRalpha was significantly enhanced when compared with controls. In contrast, the protein levels were not changed by cycloheximide among these groups. Apicidin and n-butyrate induced the continuous phosphorylation of eIF-2alpha for up to 8 days. The decrease in the level of FIP1L1-PDGFRalpha protein by continuous inhibition of HDAC may be due to the decrease in the translation rate of FIP1L1-PDGFRA.
    International Archives of Allergy and Immunology 02/2008; 146 Suppl 1(Suppl 1):7-10. DOI:10.1159/000126053 · 2.67 Impact Factor

  • Journal of Biomechanical Science and Engineering 01/2008; 3(2):221-234. DOI:10.1299/jbse.3.221

  • Journal of Biomechanical Science and Engineering 01/2008; 3(2):287-298. DOI:10.1299/jbse.3.287
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    ABSTRACT: When the ear is exposed to traumatic loud noise, outer hair cells (OHCs) are damaged and thus permanent hearing loss occurs. Recently, prior conditioning with heat stress has been reported to protect OHCs from traumatic noise exposure by increasing the stiffness of the OHC soma and has also been reported to enhance distortion product otoacoustic emissions [DPOAEs; Murakoshi, M., Yoshida, N., Kitsunai, Y., Iida, K., Kumano, S., Suzuki, T., Kobayashi, T., Wada, H., 2006. Effects of heat stress on Young's modulus of outer hair cells in mice. Brain Res. 1107, 121-130]. In the present study, to further investigate the heat stress-induced protective mechanism of hearing and such stress-induced DPOAE enhancement mechanism, the amount of filamentous actin (F-actin), which is concerned with cell stiffness, and the amount of prestin, which is concerned with the generation of DPOAEs, were examined in OHCs, with and without heat stress. Heat stress was found to increase the amount of F-actin 6-24 h after heat stress. The greatest increase in the amount of F-actin was observed at the cuticular plate where F-actin anchors the roots of the stereocilia to the cell body. Based on this result, the part of the stereocilia most reinforced and protected by heat stress was concluded to be the roots of the stereocilia. In contrast with F-actin, heat stress did not affect the amount of prestin.
    Brain Research 11/2007; 1177(1):47-58. DOI:10.1016/j.brainres.2007.08.019 · 2.84 Impact Factor
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    ABSTRACT: The high sensitivity of mammalian hearing is achieved by amplification of the motion of the cochlear partition. This cochlear amplification is thought to be generated by the elongation and contraction of outer hair cells (OHCs) in response to acoustical stimulation. This motility is made possible by a membrane protein embedded in the lateral membrane of OHCs. Although a fructose transporter, GLUT-5, was initially proposed to be this protein, a later study identified the gene of the motor protein distributed throughout the OHC plasma membrane. This protein has been named "prestin." However, although previous morphological studies by electron microscopy and atomic force microscopy (AFM) found the lateral wall of OHCs to be covered with 10-nm particles, believed to be motor proteins, it is unknown whether such particles consist only of prestin or are a complex of GLUT-5 and prestin molecules. To determine if the 10-nm particles are indeed constituted only of prestin, plasma membranes of prestin-transfected and untransfected Chinese hamster ovary (CHO) cells, which do not express GLUT-5, were observed by AFM. First, the cells attached to a substrate were sonicated so that only the plasma membrane remained on the substrate. The cytoplasmic face of the cell was observed by the tapping mode of the AFM in liquid. As a result, particle-like structures were recognized on the plasma membranes of both the prestin-transfected and untransfected CHO cells. Comparison of the difference in the frequency distribution of these structures between those two cells showed approximately 75% of the particle-like structures with a diameter of 8-12 nm in the prestin-transfected CHO cells to be possibly constituted only by prestin molecules. Our data suggest that the densely packed 10-nm particles observed on the OHC lateral wall are likely to be constituted only of prestin molecules.
    Journal of the Association for Research in Otolaryngology 10/2006; 7(3):267-78. DOI:10.1007/s10162-006-0041-z · 2.60 Impact Factor
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    ABSTRACT: The motion of the inner hair cell (IHC) stereocilia, which results in tension in the tip links connected to mechanically gated ion channels, mediates the auditory transduction process. However, it is difficult to directly observe the motion of the stereocilia because of their minute dimensions and complex structure. In this study, to investigate such motion, a finite element method model of the tall, middle and short IHC stereocilia, including the tip and lateral links extending between the stereocilia, was constructed. By applying an analytically estimated fluid force caused by a stimulus of 60dB SPL at 500Hz to the model, the dynamic behavior of the stereocilia was analyzed. Numerical results showed that the stereocilia moved in phase and that the maximum tensions of 2.5fN and 2.1fN occurred in the tip link connecting the tall and middle stereocilia and in the tip link connecting the middle and short stereocilia, respectively. By contrast, under the condition in which the lateral links were removed, maximum tension in the former increased to 11.6fN, while that in the latter only increased to 2.3fN. It was therefore suggested that the lateral links protect the MET channels located at taller stereocilia against large stimuli and subject the channels located in the same IHC to forces of similar size.
    JSME International Journal Series C 09/2006; 49(3):828-836. DOI:10.1299/jsmec.49.828 · 0.39 Impact Factor

Publication Stats

100 Citations
32.31 Total Impact Points


  • 2003-2011
    • Tohoku University
      • Department of Bioengineering and Robotics
      Miyagi, Japan
  • 2004-2005
    • Juntendo University
      • Department of Otorhinolaryngology
      Edo, Tōkyō, Japan