The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Jing Zheng - One of the best experts on this subject based on the ideXlab platform.
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deletion of exons 17 and 18 in prestin s stas domain results in loss of function
Scientific Reports, 2019Co-Authors: Satoe Takahashi, Jing Zheng, Tetsuji Yamashita, Jian Zuo, Kazuaki Homma, Yingjie Zhou, Mary Ann CheathamAbstract:Cochlear outer hair cells (OHC) express the motor protein, prestin, which is required for sensitivity and frequency selectivity. Because our previous work showed that a calmodulin binding site (CBS) was located in prestin’s C-terminal, specifically within the intrinsically disordered region, we sought to delete the IDR to study the functional significance of calcium-dependent, calmodulin binding on OHC function. Although the construct lacking the IDR (∆IDR prestin) demonstrated wildtype-like nonlinear capacitance (NLC) in HEK293T cells, the phenotype in ∆IDR prestin knockins (KI) was similar to that in prestin knockouts: thresholds were elevated, NLC was absent and OHCs were missing from basal regions of the cochlea. Although ∆IDR prestin mRNA was measured, no prestin protein was detected. At the mRNA level, both of prestin’s exons 17 and 18 were entirely removed, rather than the smaller region encoding the IDR. Our hybrid exon that contained the targeted deletion (17–18 ∆IDR) failed to splice in vitro and prestin protein lacking exons 17 and 18 aggregated and failed to target the cell membrane. Hence, the absence of prestin protein in ∆IDR KI OHCs may be due to the unexpected splicing of the hybrid 17–18 ∆IDR exon followed by rapid degradation of nonfunctional prestin protein.
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prestin contributes to membrane compartmentalization and is required for normal innervation of outer hair cells
Frontiers in Cellular Neuroscience, 2018Co-Authors: Satoe Takahashi, Kazuaki Homma, Mary Ann Cheatham, Bechara Kachar, Willy Sun, Yingjie Zhou, Jing ZhengAbstract:Outer hair cells (OHC) act as amplifiers and their function is modified by medial olivocochlear (MOC) efferents. The unique OHC motor protein, prestin, provides the molecular basis for somatic electromotility, which is required for sensitivity and frequency selectivity, the hallmarks of mammalian hearing. Prestin proteins are the major component of the lateral membrane of mature OHCs, which separates apical and basal domains. To investigate the contribution of prestin to this unique arrangement, we compared the distribution of membrane proteins in OHCs of wildtype (WT) and prestin-knockout (KO) mice. In WT, the apical protein PMCA2 was exclusively localized to the hair bundles, while it was also found at the lateral membrane in KOs. Similarly, a basal protein KCNQ4 did not coalesce at the base of OHCs but was widely dispersed in mice lacking prestin. Since the expression levels of PMCA2 and KCNQ4 remained unchanged in KOs, the data indicate that prestin is required for the normal distribution of apical and basal membrane proteins in OHCs. Since OHC synapses predominate in the basal subnuclear region, we also examined the synaptic architecture in prestin-KO mice. Although neurite densities were not affected, MOC efferent terminals in prestin-KO mice were no longer constrained to the basal pole as in WT. This trend was evident as early as at postnatal day 12. Furthermore, terminals were often enlarged and frequently appeared as singlets when compared to the multiple clusters of individual terminals in WT. This abnormality in MOC synaptic morphology in prestin-KO mice is similar to defects in mice lacking MOC pathway proteins such as α9/α10 nicotinic acetylcholine receptors and BK channels, indicating a role for prestin in the proper establishment of MOC synapses. To investigate the contribution of prestin’s electromotility, we also examined OHCs from a mouse model that expresses non-functional prestin (499-prestin). We found no changes in PMCA2 localization and MOC synaptic morphology in OHCs from 499-prestin mice. Taken together, these results indicate that prestin, independent of its motile function, plays an important structural role in membrane compartmentalization, which is required for the formation of normal efferent-OHC synapses in mature OHCs.
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the r130s mutation significantly affects the function of prestin the outer hair cell motor protein
Journal of Molecular Medicine, 2016Co-Authors: Satoe Takahashi, Jing Zheng, Mary Ann Cheatham, Kazuaki HommaAbstract:A missense mutation, R130S, was recently found in the prestin gene, SLC26A5, of patients with moderate to severe hearing loss (DFNB61). In order to define the pathology of hearing loss associated with this missense mutation, a recombinant prestin construct harboring the R130S mutation (R130S-prestin) was generated, and its functional consequences examined in a heterologous expression system. We found that R130S-prestin targets the plasma membrane but less efficiently compared to wild-type. The voltage operating point and voltage sensitivity of the motor function of R130S-prestin were similar to wild-type prestin. However, the motor activity of R130S-prestin is greatly reduced at higher voltage stimulus frequencies, indicating a reduction in motor kinetics. Our study thus provides experimental evidence that supports a causal relationship between the R130S mutation in the prestin gene and hearing loss found in patients with this missense mutation.
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susceptibility of outer hair cells to cholesterol chelator 2 hydroxypropyl β cyclodextrine is prestin dependent
Scientific Reports, 2016Co-Authors: Satoe Takahashi, Kazuaki Homma, Chongwen Duan, Mary Ann Cheatham, Yingjie Zhou, Shinichi Nishimura, Jessie Chen, Aisha Ahmad, Jing ZhengAbstract:Niemann-Pick type C1 disease (NPC1) is a fatal genetic disorder caused by impaired intracellular cholesterol trafficking. Recent studies reported ototoxicity of 2-hydroxypropyl- β-cyclodextrin (HPβCD), a cholesterol chelator and the only promising treatment for NPC1. Because outer hair cells (OHCs) are the only cochlear cells affected by HPβCD, we investigated whether prestin, an OHC-specific motor protein, might be involved. Single, high-dose administration of HPβCD resulted in OHC death in prestin wildtype (WT) mice whereas OHCs were largely spared in prestin knockout (KO) mice in the basal region, implicating prestin’s involvement in ototoxicity of HPβCD. We found that prestin can interact with cholesterol in vitro, suggesting that HPβCD-induced ototoxicity may involve disruption of this interaction. Time-lapse analysis revealed that OHCs isolated from WT animals rapidly deteriorated upon HPβCD treatment while those from prestin-KOs tolerated the same regimen. These results suggest that a prestin-dependent mechanism contributes to HPβCD ototoxicity.
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prestin dependence of outer hair cell survival and partial rescue of outer hair cell loss in prestinv499g y501h knockin mice
PLOS ONE, 2015Co-Authors: Mary Ann Cheatham, Peter Dallos, Kazuaki Homma, Roxanne Edge, Emily L Leserman, Jing ZhengAbstract:A knockin (KI) mouse expressing mutated prestinV499G/Y501H (499 prestin) was created to study cochlear amplification. Recordings from isolated outer hair cells (OHC) in this mutant showed vastly reduced electromotility and, as a consequence, reduced hearing sensitivity. Although 499 prestin OHCs were normal in stiffness and longer than OHCs lacking prestin, accelerated OHC death was unexpectedly observed relative to that documented in prestin knockout (KO) mice. These observations imply an additional role of prestin in OHC maintenance besides its known requirement for mammalian cochlear amplification. In order to gain mechanistic insights into prestin-associated OHC loss, we implemented several interventions to improve survival. First, 499 prestin KI’s were backcrossed to Bak KO mice, which lack the mitochondrial pro-apoptotic gene Bak. Because oxidative stress is implicated in OHC death, another group of 499 prestin KI mice was fed the antioxidant diet, Protandim. 499 KI mice were also backcrossed onto the FVB murine strain, which retains excellent high-frequency hearing well into adulthood, to reduce the compounding effect of age-related hearing loss associated with the original 499 prestin KIs. Finally, a compound heterozygous (chet) mouse expressing one copy of 499 prestin and one copy of KO prestin was also created to reduce quantities of 499 prestin protein. Results show reduction in OHC death in chets, and in 499 prestin KIs on the FVB background, but only a slight improvement in OHC survival for mice receiving Protandim. We also report that improved OHC survival in 499 prestin KIs had little effect on hearing phenotype, reaffirming the original contention about the essential role of prestin’s motor function in cochlear amplification.
Hiroshi Wada - One of the best experts on this subject based on the ideXlab platform.
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prestin binding peptides as ligands for targeted polymersome mediated drug delivery to outer hair cells in the inner ear
International Journal of Pharmaceutics, 2012Co-Authors: Elena V Surovtseva, Michio Murakoshi, Hiroshi Wada, Alexander H Johnston, Weikai Zhang, Ya Zhang, Tracey A Newman, Ilmari PyykkoAbstract:Targeted delivery of treatment agents to the inner ear using nanoparticles is an advanced therapeutic approach to cure or alleviate hearing loss. Designed to target the outer hair cells of the cochlea, two 12-mer peptides (A665 and A666) with affinity to prestin were identified following 3 rounds of sequential phage display. Two-round display with immobilized prestin protein was used to enrich the library for full-length prestin. The last round was performed using Cos-7 cells transiently transfected with a cCFP-prestin plasmid to display phages expressing peptides restrictive to the extracellular loops of prestin. The binding properties of A665 and A666 shown by flow cytometry demonstrated selectivity to prestin-expressing Chinese hamster ovary cells. PEG6K-b-PCL19K polymersomes covalently labelled with these peptides demonstrated effective targeting to outer hair cells in a rat cochlear explant study.
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salicylate induced translocation of prestin having mutation in the gtsrh sequence to the plasma membrane
FEBS Letters, 2010Co-Authors: Shun Kumano, Koji Iida, Michio Murakoshi, Kouhei Tsumoto, Katsuhisa Ikeda, Toshimitsu Kobayashi, Izumi Kumagai, Kenji Ishihara, Hiroshi WadaAbstract: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.
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mutation induced reinforcement of prestin expressing cells
Biochemical and Biophysical Research Communications, 2009Co-Authors: Shun Kumano, Xiaodong Tan, Koji Iida, Michio Murakoshi, Hiroshi WadaAbstract: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.
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immune atomic force microscopy of prestin transfected cho cells using quantum dots
Pflügers Archiv: European Journal of Physiology, 2009Co-Authors: Michio Murakoshi, Koji Iida, Shun Kumano, Hiroshi WadaAbstract: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.
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purification of the motor protein prestin from chinese hamster ovary cells stably expressing prestin
Journal of Biomechanical Science and Engineering, 2008Co-Authors: Koji Iida, Michio Murakoshi, Shun Kumano, Kouhei Tsumoto, Katsuhisa Ikeda, Toshimitsu Kobayashi, Izumi Kumagai, Hiroshi WadaAbstract:Prestin is regarded as the motor protein of cochlear outer hair cells (OHCs). Due to the conformational change of prestin, OHCs are believed to contract and elongate, this OHC motility realizing the high sensitivity, wide dynamic range and sharp tuning of the auditory system of mammals. Since its identification in 2000, prestin has been intensively investigated. As a result, knowledge about the structure and function of prestin has been gradually accumulated by studies using prestin-expressing cells. Purification of prestin would allow further analysis, e.g., crystal structure analysis, to obtain knowledge about prestin at the molecular level. Recently, it has been reported that recombinant prestin was purified from Sf9 insect cells and that structural analysis was carried out by electron microscopy. In the present study, an attempt was made to purify prestin from another expression system, i.e., mammalian Chinese hamster ovary (CHO) cells stably transfected with gerbil prestin. First, since it is unclear which detergents are suitable for solubilization of prestin, the best detergent for solubilization was selected from among 8 kinds of detergent commonly used for membrane protein isolation. The optimum concentration of the detergent was also determined. As a result, it was clarified that 10 mM n-nonyl-β-D-thiomaltopyranoside efficiently solubilizes prestin. Next, using this detergent, purification of prestin by anti-FLAG affinity chromatography was performed, and 84 ± 23 μg of purified prestin was obtained from 2×109 3×FLAG-tagged prestin-expressing CHO cells.
Kirk W. Beisel - One of the best experts on this subject based on the ideXlab platform.
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Lizard and Frog Prestin: Evolutionary Insight into Functional Changes
2016Co-Authors: Jie Tang, Jason L. Pecka, Bernd Fritzsch, Kirk W. BeiselAbstract:The plasma membrane of mammalian cochlear outer hair cells contains prestin, a unique motor protein. Prestin is the fifth member of the solute carrier protein 26A family. Orthologs of prestin are also found in the ear of non-mammalian vertebrates such as zebrafish and chicken. However, these orthologs are electrogenic anion exchangers/transporters with no motor function. Amphibian and reptilian lineages represent phylogenic branches in the evolution of tetrapods and subsequent amniotes. Comparison of the peptide sequences and functional properties of these prestin orthologs offer new insights into prestin evolution. With the recent availability of the lizard and frog genome sequences, we examined amino acid sequence and function of lizard and frog prestins to determine how they are functionally and structurally different from prestins of mammals and other non-mammals. Somatic motility, voltage-dependent nonlinear capacitance (NLC), the two hallmarks of prestin function, and transport capability were measured in transfected human embryonic kidney cells using voltage-clamp and radioisotope techniques. We demonstrated that while the transport capability of lizard and frog prestin was compatible to that of chicken prestin, the NLC of lizard prestin was more robust than that of chicken’s and was close to that of platypus. However, unlike platypus prestin which has acquired motor capability, lizard or frog prestin did not demonstrate motor capability. Lizard and frog prestins do not possess the same 11-amino-acid motif that is likely the structural adaptation for motor function in mammals. Thus, lizard and frog prestins appear to be functionally mor
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prestin at year 14 progress and prospect
Hearing Research, 2014Co-Authors: Sandor Lovas, Kirk W. BeiselAbstract:Prestin, the motor protein of cochlear outer hair cells, was identified 14 years ago. Prestin-based outer hair cell motility is responsible for the exquisite sensitivity and frequency selectivity seen in the mammalian cochlea. Prestin is the 5th member of an eleven-member membrane transporter superfamily of SLC26A proteins. Unlike its paralogs, which are capable of transporting anions across the cell membrane, prestin primarily functions as a motor protein with unique capability of performing direct and reciprocal electromechanical conversion on microsecond time scale. Significant progress in the understanding of its structure and the molecular mechanism has been made in recent years using electrophysiological, biochemical, comparative genomics, structural bioinformatics, molecular dynamics simulation, site-directed mutagenesis and domain-swapping techniques. This article reviews recent advances of the structural and functional properties of prestin with focus on the areas that are critical but still controversial in understanding the molecular mechanism of how prestin works: The structural domains for voltage sensing and interaction with anions and for conformational change. Future research directions and potential application of prestin are also discussed. This article is part of a Special Issue entitled .
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lizard and frog prestin evolutionary insight into functional changes
PLOS ONE, 2013Co-Authors: Jie Tang, Jason L. Pecka, Bernd Fritzsch, Kirk W. BeiselAbstract:The plasma membrane of mammalian cochlear outer hair cells contains prestin, a unique motor protein. Prestin is the fifth member of the solute carrier protein 26A family. Orthologs of prestin are also found in the ear of non-mammalian vertebrates such as zebrafish and chicken. However, these orthologs are electrogenic anion exchangers/transporters with no motor function. Amphibian and reptilian lineages represent phylogenic branches in the evolution of tetrapods and subsequent amniotes. Comparison of the peptide sequences and functional properties of these prestin orthologs offer new insights into prestin evolution. With the recent availability of the lizard and frog genome sequences, we examined amino acid sequence and function of lizard and frog prestins to determine how they are functionally and structurally different from prestins of mammals and other non-mammals. Somatic motility, voltage-dependent nonlinear capacitance (NLC), the two hallmarks of prestin function, and transport capability were measured in transfected human embryonic kidney cells using voltage-clamp and radioisotope techniques. We demonstrated that while the transport capability of lizard and frog prestin was compatible to that of chicken prestin, the NLC of lizard prestin was more robust than that of chicken’s and was close to that of platypus. However, unlike platypus prestin which has acquired motor capability, lizard or frog prestin did not demonstrate motor capability. Lizard and frog prestins do not possess the same 11-amino-acid motif that is likely the structural adaptation for motor function in mammals. Thus, lizard and frog prestins appear to be functionally more advanced than that of chicken prestin, although motor capability is not yet acquired.
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engineered pendrin protein an anion transporter and molecular motor
Journal of Biological Chemistry, 2011Co-Authors: Jie Tang, Xiaodong Tan, Jason L. Pecka, Kirk W. BeiselAbstract:Pendrin and prestin both belong to a distinct anion transporter family called solute carrier protein 26A, or SLC26A. Pendrin (SLC26A4) is a chloride-iodide transporter that is found at the luminal membrane of follicular cells in the thyroid gland as well as in the endolymphatic duct and sac of the inner ear, whereas prestin (SLC26A5) is expressed in the plasma membrane of cochlear outer hair cells and functions as a unique voltage-dependent motor. We recently identified a motif that is critical for the motor function of prestin. We questioned whether it was possible to create a chimeric pendrin protein with motor capability by integrating this motility motif from prestin. The chimeric pendrin was constructed by substituting residues 160–179 in human pendrin with residues 156–169 from gerbil prestin. Non-linear capacitance and somatic motility, two hallmarks representing prestin function, were measured from chimeric pendrin-transfected human embryonic kidney 293 cells using the voltage clamp technique and photodiode-based displacement measurement system. We showed that this 14-amino acid substitution from prestin was able to confer pendrin with voltage-dependent motor capability despite the amino acid sequence disparity between pendrin and prestin. The molecular mechanism that facilitates motor function appeared to be the same as prestin because the motor activity depended on the concentration of intracellular chloride and was blocked by salicylate treatment. Radioisotope-labeled formate uptake measurements showed that the chimeric pendrin protein retained the capability to transport formate, suggesting that the gain of motor function was not at the expense of its inherent transport capability. Thus, the engineered pendrin was capable of both transporting anions and generating force.
Mary Ann Cheatham - One of the best experts on this subject based on the ideXlab platform.
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deletion of exons 17 and 18 in prestin s stas domain results in loss of function
Scientific Reports, 2019Co-Authors: Satoe Takahashi, Jing Zheng, Tetsuji Yamashita, Jian Zuo, Kazuaki Homma, Yingjie Zhou, Mary Ann CheathamAbstract:Cochlear outer hair cells (OHC) express the motor protein, prestin, which is required for sensitivity and frequency selectivity. Because our previous work showed that a calmodulin binding site (CBS) was located in prestin’s C-terminal, specifically within the intrinsically disordered region, we sought to delete the IDR to study the functional significance of calcium-dependent, calmodulin binding on OHC function. Although the construct lacking the IDR (∆IDR prestin) demonstrated wildtype-like nonlinear capacitance (NLC) in HEK293T cells, the phenotype in ∆IDR prestin knockins (KI) was similar to that in prestin knockouts: thresholds were elevated, NLC was absent and OHCs were missing from basal regions of the cochlea. Although ∆IDR prestin mRNA was measured, no prestin protein was detected. At the mRNA level, both of prestin’s exons 17 and 18 were entirely removed, rather than the smaller region encoding the IDR. Our hybrid exon that contained the targeted deletion (17–18 ∆IDR) failed to splice in vitro and prestin protein lacking exons 17 and 18 aggregated and failed to target the cell membrane. Hence, the absence of prestin protein in ∆IDR KI OHCs may be due to the unexpected splicing of the hybrid 17–18 ∆IDR exon followed by rapid degradation of nonfunctional prestin protein.
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prestin contributes to membrane compartmentalization and is required for normal innervation of outer hair cells
Frontiers in Cellular Neuroscience, 2018Co-Authors: Satoe Takahashi, Kazuaki Homma, Mary Ann Cheatham, Bechara Kachar, Willy Sun, Yingjie Zhou, Jing ZhengAbstract:Outer hair cells (OHC) act as amplifiers and their function is modified by medial olivocochlear (MOC) efferents. The unique OHC motor protein, prestin, provides the molecular basis for somatic electromotility, which is required for sensitivity and frequency selectivity, the hallmarks of mammalian hearing. Prestin proteins are the major component of the lateral membrane of mature OHCs, which separates apical and basal domains. To investigate the contribution of prestin to this unique arrangement, we compared the distribution of membrane proteins in OHCs of wildtype (WT) and prestin-knockout (KO) mice. In WT, the apical protein PMCA2 was exclusively localized to the hair bundles, while it was also found at the lateral membrane in KOs. Similarly, a basal protein KCNQ4 did not coalesce at the base of OHCs but was widely dispersed in mice lacking prestin. Since the expression levels of PMCA2 and KCNQ4 remained unchanged in KOs, the data indicate that prestin is required for the normal distribution of apical and basal membrane proteins in OHCs. Since OHC synapses predominate in the basal subnuclear region, we also examined the synaptic architecture in prestin-KO mice. Although neurite densities were not affected, MOC efferent terminals in prestin-KO mice were no longer constrained to the basal pole as in WT. This trend was evident as early as at postnatal day 12. Furthermore, terminals were often enlarged and frequently appeared as singlets when compared to the multiple clusters of individual terminals in WT. This abnormality in MOC synaptic morphology in prestin-KO mice is similar to defects in mice lacking MOC pathway proteins such as α9/α10 nicotinic acetylcholine receptors and BK channels, indicating a role for prestin in the proper establishment of MOC synapses. To investigate the contribution of prestin’s electromotility, we also examined OHCs from a mouse model that expresses non-functional prestin (499-prestin). We found no changes in PMCA2 localization and MOC synaptic morphology in OHCs from 499-prestin mice. Taken together, these results indicate that prestin, independent of its motile function, plays an important structural role in membrane compartmentalization, which is required for the formation of normal efferent-OHC synapses in mature OHCs.
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the r130s mutation significantly affects the function of prestin the outer hair cell motor protein
Journal of Molecular Medicine, 2016Co-Authors: Satoe Takahashi, Jing Zheng, Mary Ann Cheatham, Kazuaki HommaAbstract:A missense mutation, R130S, was recently found in the prestin gene, SLC26A5, of patients with moderate to severe hearing loss (DFNB61). In order to define the pathology of hearing loss associated with this missense mutation, a recombinant prestin construct harboring the R130S mutation (R130S-prestin) was generated, and its functional consequences examined in a heterologous expression system. We found that R130S-prestin targets the plasma membrane but less efficiently compared to wild-type. The voltage operating point and voltage sensitivity of the motor function of R130S-prestin were similar to wild-type prestin. However, the motor activity of R130S-prestin is greatly reduced at higher voltage stimulus frequencies, indicating a reduction in motor kinetics. Our study thus provides experimental evidence that supports a causal relationship between the R130S mutation in the prestin gene and hearing loss found in patients with this missense mutation.
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susceptibility of outer hair cells to cholesterol chelator 2 hydroxypropyl β cyclodextrine is prestin dependent
Scientific Reports, 2016Co-Authors: Satoe Takahashi, Kazuaki Homma, Chongwen Duan, Mary Ann Cheatham, Yingjie Zhou, Shinichi Nishimura, Jessie Chen, Aisha Ahmad, Jing ZhengAbstract:Niemann-Pick type C1 disease (NPC1) is a fatal genetic disorder caused by impaired intracellular cholesterol trafficking. Recent studies reported ototoxicity of 2-hydroxypropyl- β-cyclodextrin (HPβCD), a cholesterol chelator and the only promising treatment for NPC1. Because outer hair cells (OHCs) are the only cochlear cells affected by HPβCD, we investigated whether prestin, an OHC-specific motor protein, might be involved. Single, high-dose administration of HPβCD resulted in OHC death in prestin wildtype (WT) mice whereas OHCs were largely spared in prestin knockout (KO) mice in the basal region, implicating prestin’s involvement in ototoxicity of HPβCD. We found that prestin can interact with cholesterol in vitro, suggesting that HPβCD-induced ototoxicity may involve disruption of this interaction. Time-lapse analysis revealed that OHCs isolated from WT animals rapidly deteriorated upon HPβCD treatment while those from prestin-KOs tolerated the same regimen. These results suggest that a prestin-dependent mechanism contributes to HPβCD ototoxicity.
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prestin dependence of outer hair cell survival and partial rescue of outer hair cell loss in prestinv499g y501h knockin mice
PLOS ONE, 2015Co-Authors: Mary Ann Cheatham, Peter Dallos, Kazuaki Homma, Roxanne Edge, Emily L Leserman, Jing ZhengAbstract:A knockin (KI) mouse expressing mutated prestinV499G/Y501H (499 prestin) was created to study cochlear amplification. Recordings from isolated outer hair cells (OHC) in this mutant showed vastly reduced electromotility and, as a consequence, reduced hearing sensitivity. Although 499 prestin OHCs were normal in stiffness and longer than OHCs lacking prestin, accelerated OHC death was unexpectedly observed relative to that documented in prestin knockout (KO) mice. These observations imply an additional role of prestin in OHC maintenance besides its known requirement for mammalian cochlear amplification. In order to gain mechanistic insights into prestin-associated OHC loss, we implemented several interventions to improve survival. First, 499 prestin KI’s were backcrossed to Bak KO mice, which lack the mitochondrial pro-apoptotic gene Bak. Because oxidative stress is implicated in OHC death, another group of 499 prestin KI mice was fed the antioxidant diet, Protandim. 499 KI mice were also backcrossed onto the FVB murine strain, which retains excellent high-frequency hearing well into adulthood, to reduce the compounding effect of age-related hearing loss associated with the original 499 prestin KIs. Finally, a compound heterozygous (chet) mouse expressing one copy of 499 prestin and one copy of KO prestin was also created to reduce quantities of 499 prestin protein. Results show reduction in OHC death in chets, and in 499 prestin KIs on the FVB background, but only a slight improvement in OHC survival for mice receiving Protandim. We also report that improved OHC survival in 499 prestin KIs had little effect on hearing phenotype, reaffirming the original contention about the essential role of prestin’s motor function in cochlear amplification.
Kazuaki Homma - One of the best experts on this subject based on the ideXlab platform.
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deletion of exons 17 and 18 in prestin s stas domain results in loss of function
Scientific Reports, 2019Co-Authors: Satoe Takahashi, Jing Zheng, Tetsuji Yamashita, Jian Zuo, Kazuaki Homma, Yingjie Zhou, Mary Ann CheathamAbstract:Cochlear outer hair cells (OHC) express the motor protein, prestin, which is required for sensitivity and frequency selectivity. Because our previous work showed that a calmodulin binding site (CBS) was located in prestin’s C-terminal, specifically within the intrinsically disordered region, we sought to delete the IDR to study the functional significance of calcium-dependent, calmodulin binding on OHC function. Although the construct lacking the IDR (∆IDR prestin) demonstrated wildtype-like nonlinear capacitance (NLC) in HEK293T cells, the phenotype in ∆IDR prestin knockins (KI) was similar to that in prestin knockouts: thresholds were elevated, NLC was absent and OHCs were missing from basal regions of the cochlea. Although ∆IDR prestin mRNA was measured, no prestin protein was detected. At the mRNA level, both of prestin’s exons 17 and 18 were entirely removed, rather than the smaller region encoding the IDR. Our hybrid exon that contained the targeted deletion (17–18 ∆IDR) failed to splice in vitro and prestin protein lacking exons 17 and 18 aggregated and failed to target the cell membrane. Hence, the absence of prestin protein in ∆IDR KI OHCs may be due to the unexpected splicing of the hybrid 17–18 ∆IDR exon followed by rapid degradation of nonfunctional prestin protein.
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prestin contributes to membrane compartmentalization and is required for normal innervation of outer hair cells
Frontiers in Cellular Neuroscience, 2018Co-Authors: Satoe Takahashi, Kazuaki Homma, Mary Ann Cheatham, Bechara Kachar, Willy Sun, Yingjie Zhou, Jing ZhengAbstract:Outer hair cells (OHC) act as amplifiers and their function is modified by medial olivocochlear (MOC) efferents. The unique OHC motor protein, prestin, provides the molecular basis for somatic electromotility, which is required for sensitivity and frequency selectivity, the hallmarks of mammalian hearing. Prestin proteins are the major component of the lateral membrane of mature OHCs, which separates apical and basal domains. To investigate the contribution of prestin to this unique arrangement, we compared the distribution of membrane proteins in OHCs of wildtype (WT) and prestin-knockout (KO) mice. In WT, the apical protein PMCA2 was exclusively localized to the hair bundles, while it was also found at the lateral membrane in KOs. Similarly, a basal protein KCNQ4 did not coalesce at the base of OHCs but was widely dispersed in mice lacking prestin. Since the expression levels of PMCA2 and KCNQ4 remained unchanged in KOs, the data indicate that prestin is required for the normal distribution of apical and basal membrane proteins in OHCs. Since OHC synapses predominate in the basal subnuclear region, we also examined the synaptic architecture in prestin-KO mice. Although neurite densities were not affected, MOC efferent terminals in prestin-KO mice were no longer constrained to the basal pole as in WT. This trend was evident as early as at postnatal day 12. Furthermore, terminals were often enlarged and frequently appeared as singlets when compared to the multiple clusters of individual terminals in WT. This abnormality in MOC synaptic morphology in prestin-KO mice is similar to defects in mice lacking MOC pathway proteins such as α9/α10 nicotinic acetylcholine receptors and BK channels, indicating a role for prestin in the proper establishment of MOC synapses. To investigate the contribution of prestin’s electromotility, we also examined OHCs from a mouse model that expresses non-functional prestin (499-prestin). We found no changes in PMCA2 localization and MOC synaptic morphology in OHCs from 499-prestin mice. Taken together, these results indicate that prestin, independent of its motile function, plays an important structural role in membrane compartmentalization, which is required for the formation of normal efferent-OHC synapses in mature OHCs.
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the r130s mutation significantly affects the function of prestin the outer hair cell motor protein
Journal of Molecular Medicine, 2016Co-Authors: Satoe Takahashi, Jing Zheng, Mary Ann Cheatham, Kazuaki HommaAbstract:A missense mutation, R130S, was recently found in the prestin gene, SLC26A5, of patients with moderate to severe hearing loss (DFNB61). In order to define the pathology of hearing loss associated with this missense mutation, a recombinant prestin construct harboring the R130S mutation (R130S-prestin) was generated, and its functional consequences examined in a heterologous expression system. We found that R130S-prestin targets the plasma membrane but less efficiently compared to wild-type. The voltage operating point and voltage sensitivity of the motor function of R130S-prestin were similar to wild-type prestin. However, the motor activity of R130S-prestin is greatly reduced at higher voltage stimulus frequencies, indicating a reduction in motor kinetics. Our study thus provides experimental evidence that supports a causal relationship between the R130S mutation in the prestin gene and hearing loss found in patients with this missense mutation.
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susceptibility of outer hair cells to cholesterol chelator 2 hydroxypropyl β cyclodextrine is prestin dependent
Scientific Reports, 2016Co-Authors: Satoe Takahashi, Kazuaki Homma, Chongwen Duan, Mary Ann Cheatham, Yingjie Zhou, Shinichi Nishimura, Jessie Chen, Aisha Ahmad, Jing ZhengAbstract:Niemann-Pick type C1 disease (NPC1) is a fatal genetic disorder caused by impaired intracellular cholesterol trafficking. Recent studies reported ototoxicity of 2-hydroxypropyl- β-cyclodextrin (HPβCD), a cholesterol chelator and the only promising treatment for NPC1. Because outer hair cells (OHCs) are the only cochlear cells affected by HPβCD, we investigated whether prestin, an OHC-specific motor protein, might be involved. Single, high-dose administration of HPβCD resulted in OHC death in prestin wildtype (WT) mice whereas OHCs were largely spared in prestin knockout (KO) mice in the basal region, implicating prestin’s involvement in ototoxicity of HPβCD. We found that prestin can interact with cholesterol in vitro, suggesting that HPβCD-induced ototoxicity may involve disruption of this interaction. Time-lapse analysis revealed that OHCs isolated from WT animals rapidly deteriorated upon HPβCD treatment while those from prestin-KOs tolerated the same regimen. These results suggest that a prestin-dependent mechanism contributes to HPβCD ototoxicity.
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prestin dependence of outer hair cell survival and partial rescue of outer hair cell loss in prestinv499g y501h knockin mice
PLOS ONE, 2015Co-Authors: Mary Ann Cheatham, Peter Dallos, Kazuaki Homma, Roxanne Edge, Emily L Leserman, Jing ZhengAbstract:A knockin (KI) mouse expressing mutated prestinV499G/Y501H (499 prestin) was created to study cochlear amplification. Recordings from isolated outer hair cells (OHC) in this mutant showed vastly reduced electromotility and, as a consequence, reduced hearing sensitivity. Although 499 prestin OHCs were normal in stiffness and longer than OHCs lacking prestin, accelerated OHC death was unexpectedly observed relative to that documented in prestin knockout (KO) mice. These observations imply an additional role of prestin in OHC maintenance besides its known requirement for mammalian cochlear amplification. In order to gain mechanistic insights into prestin-associated OHC loss, we implemented several interventions to improve survival. First, 499 prestin KI’s were backcrossed to Bak KO mice, which lack the mitochondrial pro-apoptotic gene Bak. Because oxidative stress is implicated in OHC death, another group of 499 prestin KI mice was fed the antioxidant diet, Protandim. 499 KI mice were also backcrossed onto the FVB murine strain, which retains excellent high-frequency hearing well into adulthood, to reduce the compounding effect of age-related hearing loss associated with the original 499 prestin KIs. Finally, a compound heterozygous (chet) mouse expressing one copy of 499 prestin and one copy of KO prestin was also created to reduce quantities of 499 prestin protein. Results show reduction in OHC death in chets, and in 499 prestin KIs on the FVB background, but only a slight improvement in OHC survival for mice receiving Protandim. We also report that improved OHC survival in 499 prestin KIs had little effect on hearing phenotype, reaffirming the original contention about the essential role of prestin’s motor function in cochlear amplification.
