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 extracellular loop of pendrin and Prestin modulates their voltage sensing property
Journal of Biological Chemistry, 2018Co-Authors: Makoto Kuwabara, Jing Zheng, Satoe Takahashi, Koichiro Wasano, Justin Bodner, Tomotaka Komori, Sotaro Uemura, Tomohiro Shima, Kazuaki HommaAbstract:Pendrin and Prestin belong to the solute carrier 26 (SLC26) family of anion transporters. Prestin is unique among the SLC26 family members in that it displays voltage-driven motor activity (electromotility) and concurrent gating currents that manifest as nonlinear cell membrane electrical capacitance (nonlinear capacitance (NLC)). Although the anion transport mechanism of the SLC26 proteins has begun to be elucidated, the molecular mechanism of electromotility, which is thought to have evolved from an ancestral ion transport mechanism, still remains largely elusive. Here, we demonstrate that pendrin also exhibits large NLC and that charged residues present in one of the extracellular loops of pendrin and Prestin play significant roles in setting the voltage-operating points of NLC. Our results suggest that the molecular mechanism responsible for sensing voltage is not unique to Prestin among the members of the SLC26 family and that this voltage-sensing mechanism works independently of the anion transport mechanism.
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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.
Peter Dallos - One of the best experts on this subject based on the ideXlab platform.
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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.
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the v499g y501h mutation impairs fast motor kinetics of Prestin and has significance for defining functional independence of individual Prestin subunits
Journal of Biological Chemistry, 2013Co-Authors: Kazuaki Homma, Jing Zheng, Chongwen Duan, Mary Ann Cheatham, Peter DallosAbstract:Outer hair cells (OHCs) are a mammalian innovation for mechanically amplifying sound energy to overcome the viscous damping of the cochlear partition. Although the voltage-dependent OHC membrane motor, Prestin, has been demonstrated to be essential for mammalian cochlear amplification, the molecular mechanism by which Prestin converts electrical energy into mechanical displacement/force remains elusive. Identifying mutations that alter the motor function of Prestin provides vital information for unraveling the energy transduction mechanism of Prestin. We show that the V499G/Y501H mutation does not deprive Prestin of its voltage-induced motor activity, but it does significantly impair the fast motor kinetics and voltage operating range. Furthermore, mutagenesis studies suggest that Val-499 is the primary site responsible for these changes. We also show that V499G/Y501H Prestin forms heteromers with wild-type Prestin and that the fast motor kinetics of wild-type Prestin is not affected by heteromer formation with V499G/Y501H Prestin. These results suggest that Prestin subunits are individually functional within a given multimer. Background: Prestin converts electrical energy into mechanical work. Results: The V499G/Y501H mutation significantly impairs fast motor kinetics of Prestin. Conclusion: Impaired kinetics is attributable to mutation at the Val-499 site that is conserved among SLC26 proteins regardless of their function as motors or transporters. Significance: V499G/Y501H mutated Prestin provides clues to the molecular mechanisms underlying somatic electromotility and thus cochlear amplification.
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dissecting the electromechanical coupling mechanism of the motorprotein Prestin
Communicative & Integrative Biology, 2011Co-Authors: Kazuaki Homma, Peter DallosAbstract:Prestin, which is a member of the solute carrier 26 anion transporter family (SLC26A5), is a voltagedependent membrane-based motor protein that confers electromotility on mammalian cochlear outer hair cells (OHCs).1 OHCs are a mammalian innovation, their presence2 and their endowment with functional Prestin is essential for normal hearing of mammals.3 In order to clarify the molecular mechanism underlying the voltage-dependent motility of Prestin, precise description of the relation between voltage-induced Prestin-associated charge movement and the resulting cell displacement is essential. By simultaneously measuring voltage-dependent charge movement, which is manifested in the nonlinear capacitance (NLC) of the cell membrane, and voltage-induced OHC displacement, we provided compelling experimental evidence that Prestin-associated charge movement and the resulting electromotility are fully coupled, and that Prestin has at least two voltage-dependent conformational transition steps. These findings provide...
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evidence that Prestin has at least two voltage dependent steps
Journal of Biological Chemistry, 2011Co-Authors: Kazuaki Homma, Peter DallosAbstract:Prestin is a voltage-dependent membrane-spanning motor protein that confers electromotility on mammalian cochlear outer hair cells, which is essential for normal hearing of mammals. Voltage-induced charge movement in the Prestin molecule is converted into mechanical work; however, little is known about the molecular mechanism of this process. For understanding the electromechanical coupling mechanism of Prestin, we simultaneously measured voltage-dependent charge movement and electromotility under conditions in which the magnitudes of both charge movement and electromotility are gradually manipulated by the Prestin inhibitor, salicylate. We show that the observed relationships of the charge movement and the physical displacement (q-d relations) are well represented by a three-state Boltzmann model but not by a two-state model or its previously proposed variant. Here, we suggest a molecular mechanism of Prestin with at least two voltage-dependent conformational transition steps having distinct electromechanical coupling efficiencies.
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interaction between the motor protein Prestin and the transporter protein vapa
Biochimica et Biophysica Acta, 2010Co-Authors: Soma Sengupta, Peter Dallos, Kazuaki Homma, Mary Ann Cheatham, Roxanne Edge, Katharine K Miller, Jing ZhengAbstract:Prestin is the motor protein responsible for cochlear outer hair cell (OHC) somatic electromotility. Eliminating this abundant basolateral membrane protein not only causes loss of frequency selectivity and hearing sensitivity, but also leads to OHC death. A membrane-based yeast two-hybrid approach was used to screen an OHC-enriched cDNA (complementary Deoxyribonucleic Acid) library in order to identify Prestin-associated proteins. Several proteins were recognized as potential Prestin partners, including vesicle-associated membrane protein associated protein A (VAPA or VAP-33). VAPA is an integral membrane protein that plays an important role in membrane trafficking, endoplasmic reticulum homeostasis, and the stress-signaling system. The connection between VAPA and Prestin was confirmed through co-immunoprecipitation experiments. This new finding prompted the investigation of the interaction between VAPA and Prestin in outer hair cells. By comparing VAPA expression between wild-type OHCs and OHCs derived from Prestin-knockout mice, we found that VAPA is expressed in OHCs and the quantity of VAPA expressed is related to the presence of Prestin. In other words, less VAPA protein is found in OHCs lacking Prestin. Thus, Prestin appears to modify the expression of VAPA protein in OHCs. Intriguingly, more Prestin protein appears at the plasma membrane when VAPA is co-expressed with Prestin. These data suggest that VAPA could be involved in Prestin's transportation inside OHCs and may facilitate the targeting of this abundant OHC protein to the plasma membrane.
Joseph Santossacchi - One of the best experts on this subject based on the ideXlab platform.
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single particle cryo em structure of the outer hair cell motor protein Prestin
bioRxiv, 2021Co-Authors: Carmen Butan, Jun-ping Bai, Dhasakumar Navaratnam, Qiang Song, Winston Tan, Joseph SantossacchiAbstract:The mammalian outer hair cell (OHC) protein Prestin (Slc26a5), a member of the solute carrier 26 (Slc26) family of membrane proteins, differs from other members of the family owing to its unique piezoelectric-like property that drives OHC electromotility. Prestin is required by OHCs for cochlear amplification, a process that enhances mammalian hearing. Despite substantial biophysical characterization, the mechanistic basis for the Prestins electro-mechanical behavior is not fully understood. To gain insight into such behavior, we have used cryo-electron microscopy at subnanometer resolution (overall resolution of 4.0 A) to investigate the three-dimensional structure of Prestin from gerbil (Meriones unguiculatus). Our studies show that Prestin dimerizes with a 3D architecture strikingly similar to the dimeric conformation observed in the Slc26a9 anion transporter in an inside open/intermediate state, which we infer, based on patch clamp recordings, to reflect the contracted state of Prestin. The structure shows two well separated transmembrane (TM) subunits and two cytoplasmic sulfate transporter and anti-sigma factor antagonist (STAS) domains forming a swapped dimer. The dimerization interface is defined by interactions between the domain-swapped STAS dimer and the transmembrane domains of the opposing half unit, further strengthened by an antiparallel beta strand at its N terminus. The structure also shows that each one of its two transmembrane subunits consists of 14 transmembrane segments organized in two inverted 7-segment repeats with a topology that was first observed in the structure of the bacterial symporter UraA (Lu F, et al., Nature 472, 2011). Finally, the solved anion binding site structural features of Prestin are quite similar to that of Slc26a9 and other family members. Despite this similarity, we find that Slc26a9 lacks the characteristic displacement currents (or NonLinear Capacitance(NLC)) found with Prestin, and we show that mutation of Prestins Cl- binding site removes salicylate competition with anions in the face of normal NLC, thus refuting the yet accepted extrinsic voltage sensor hypothesis and any associated transport-like requirements for voltage-driven electromotility.
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Prestin kinetics and corresponding frequency dependence augment during early development of the outer hair cell within the mouse organ of corti
Scientific Reports, 2019Co-Authors: Jun-ping Bai, Dhasakumar Navaratnam, Joseph SantossacchiAbstract:Several studies have documented the early development of OHC electromechanical behavior. The mechanical response (electromotility, eM) and its electrical correlate (nonlinear capacitance, NLC), resulting from Prestin’s voltage-sensor charge movement, increase over the course of several postnatal days in altricial animals. They increase until about p18, near the time of peripheral auditory maturity. The correspondence of auditory capabilities and Prestin function indicates that mature activity of Prestin occurs at this time. One of the major requirements of eM is its responsiveness across auditory frequencies. Here we evaluate the frequency response of Prestin charge movement in mice over the course of development up to 8 months. We find that in apical turn OHCs Prestin’s frequency response increases during postnatal development and stabilizes when mature hearing is established. The low frequency component of NLC, within in situ explants, agrees with previously reported results on isolated cells. If Prestin activity is independent of cochlear place, as might be expected, then these observations suggest that Prestin activity somehow influences cochlear amplification at high frequencies in spite of its low pass behavior.
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tyrosine motifs are required for Prestin basolateral membrane targeting
Biology Open, 2015Co-Authors: Yifan Zhang, Jun-ping Bai, Joseph Santossacchi, Iman Moeininaghani, Dhasakumar NavaratnamAbstract:Prestin is targeted to the lateral wall of outer hair cells (OHCs) where its electromotility is critical for cochlear amplification. Using MDCK cells as a model system for polarized epithelial sorting, we demonstrate that Prestin uses tyrosine residues, in a YXXΦ motif, to target the basolateral surface. Both Y520 and Y667 are important for basolateral targeting of Prestin. Mutation of these residues to glutamine or alanine resulted in retention within the Golgi and delayed egress from the Golgi in Y667Q. Basolateral targeting is restored upon mutation to phenylalanine suggesting the importance of a phenol ring in the tyrosine side chain. We also demonstrate that Prestin targeting to the basolateral surface is dependent on AP1B (μ1B), and that Prestin uses transferrin containing early endosomes in its passage from the Golgi to the basolateral plasma membrane. The presence of AP1B (μ1B) in OHCs, and parallels between Prestin targeting to the basolateral surface of OHCs and polarized epithelial cells suggest that outer hair cells resemble polarized epithelia rather than neurons in this important phenotypic measure.
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real time measures of Prestin charge and fluorescence during plasma membrane trafficking reveal sub tetrameric activity
PLOS ONE, 2013Co-Authors: Shumin Bian, Dhasakumar Navaratnam, Joseph SantossacchiAbstract:Prestin (SLC26a5) is the outer hair cell integral membrane motor protein that drives cochlear amplification, and has been described as an obligate tetramer. We studied in real time the delivery of YFP-Prestin to the plasma membrane of cells from a tetracycline-inducible cell line. Following the release of temperature block to reinstate trans Golgi network delivery of the integral membrane protein, we measured nonlinear capacitance (NLC) and membrane fluorescence during voltage clamp. Prestin was delivered exponentially to the plasma membrane with a time constant of less than 10 minutes, with both electrical and fluorescence methods showing high temporal correlation. However, based on disparity between estimates of Prestin density derived from either fluorescence or NLC, we conclude that sub-tetrameric forms of Prestin contribute to our electrical and fluorescence measures. Thus, in agreement with previous observations we find that functional Prestin is not an obligate tetramer.
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chloride flux in Prestin expressing cells
Biophysical Journal, 2012Co-Authors: Sheng Zhong, Dhasakumar Navaratnam, Shumin Bian, Joseph SantossacchiAbstract:In Prestin-expressing cells, intracellular Cl- ([Cl-]i) flux plays a preeminent role in promoting Prestin activity, especially since the resulting Vh shift in Prestin's state-probability function (inferred from nonlinear capacitance - NLC) along the Vm axis will effect a motile response. For example, with perforated patch clamp and local perfusion, changing extracellular Cl concentration from 1 mM to 140 mM using Prestin's NLC as a measure of intracellular Cl indicates a several mM increase in intracellular Cl concentration. Nevertheless, the mechanism underlying Cl- flux in Prestin-expressing cells is not clear.To better define Cl- flux, the CFP-YFP-based ratiometric Cl indicator (Cl-sensor, J. Neurosci. Meth., 2008, 170, 67) was modified to reduce pH sensitivity by shifting the pKa away from the physiological pH range. Using excitation ratiometric imaging of fluorescence, [Cl]i was measured in either induced- or non-induced Prestin-expressing HEK cell-lines transfected with our new Cl-sensor construct. Upon changing local extracellular perfusion from 0.2 mM to 140 mM Cl buffer, fluorescence measures indicate a significant difference between induced- and non-induced Prestin cell-lines.We also evaluated [Cl]i flux using a Cl-sensor fusion product of Prestin at the C-terminal to better gauge flux near Prestin's intracellular binding site. Comparison of [Cl]i flux in HEK-293T cells transfected with a normal Prestin-Cl-sensor or a Prestin construct with a C-terminal deletion that eliminated NLC (P709-Cl-sensor) showed no statistical difference in flux. These data suggest that Prestin's NLC is separable from Cl- movement induced by Prestin, as we previously suggested (Bai et al., BJ, 3179, 2009). Further experiments are underway, including voltage dependence of flux by simultaneously monitoring fluorescence ratio signals and NLC using perforated patch.(Supported by NIDCD DC00273 and NIDCD DC 008130)
Fred A. Pereira - One of the best experts on this subject based on the ideXlab platform.
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Diflunisal inhibits Prestin by chloride-dependent mechanism.
PloS one, 2017Co-Authors: Guillaume Duret, Fred A. Pereira, Robert M. RaphaelAbstract:The motor protein Prestin is a member of the SLC26 family of anion antiporters and is essential to the electromotility of cochlear outer hair cells and for hearing. The only direct inhibitor of electromotility and the associated charge transfer is salicylate, possibly through direct interaction with an anion-binding site on Prestin. In a screen to identify other inhibitors of Prestin activity, we explored the effect of the non-steroid anti-inflammatory drug diflunisal, which is a derivative of salicylate. We recorded Prestin activity by whole-cell patch clamping HEK cells transiently expressing Prestin and mouse outer hair cells. We monitored the impact of diflunisal on the Prestin-dependent non-linear capacitance and electromotility. We found that diflunisal triggers two Prestin-associated effects: a chloride independent increase in the surface area and the specific capacitance of the membrane, and a chloride dependent inhibition of the charge transfer and the electromotility in outer hair cells. We conclude that diflunisal affects the cell membrane organization and inhibits Prestin-associated charge transfer and electromotility at physiological chloride concentrations. The inhibitory effects on hair cell function are noteworthy given the proposed use of diflunisal to treat neurodegenerative diseases.
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membrane Prestin expression correlates with the magnitude of Prestin associated charge movement
Hearing Research, 2016Co-Authors: Michelle L Seymour, William E. Brownell, Guillaume Duret, Lavanya Rajagopalan, Matthew J Volk, Haiying Liu, Fred A. PereiraAbstract:Full expression of electromotility, generation of non-linear capacitance (NLC), and high-acuity mammalian hearing require Prestin function in the lateral wall of cochlear outer hair cells (OHCs). Estimates of the number of Prestin molecules in the OHC membrane vary, and a consensus has not emerged about the correlation between Prestin expression and Prestin-associated charge movement in the OHC. Using an inducible Prestin-expressing cell line, we demonstrate that the charge density, but not the voltage at peak capacitance, directly correlates with the amount of Prestin in the plasma membrane. This correlation is evident in studies involving a controlled increase of Prestin expression with time after induction and inducer dose-response. Conversely, membrane Prestin levels and charge density gradually decline together following the reduction of Prestin levels from a steady state by removal of the inducer. Thus, charge density directly correlates with the level of membrane Prestin expression, whereas changing membrane levels of Prestin have no effect on the voltage at peak capacitance in this inducible Prestin-expressing cell line.
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Prestin regulation and function in residual outer hair cells after noise induced hearing loss
PLOS ONE, 2013Co-Authors: Yohan Song, Rosalie Wang, Sung-il Chao, Fred A. Pereira, Andrew K. Groves, William Clifton, Patrick D Raphael, John S. OghalaiAbstract:The outer hair cell (OHC) motor protein Prestin is necessary for electromotility, which drives cochlear amplification and produces exquisitely sharp frequency tuning. TectaC1509G transgenic mice have hearing loss, and surprisingly have increased OHC Prestin levels. We hypothesized, therefore, that Prestin up-regulation may represent a generalized response to compensate for a state of hearing loss. In the present study, we sought to determine the effects of noise-induced hearing loss on Prestin expression. After noise exposure, we performed cytocochleograms and observed OHC loss only in the basal region of the cochlea. Next, we patch clamped OHCs from the apical turn (9–12 kHz region), where no OHCs were lost, in noise-exposed and age-matched control mice. The non-linear capacitance was significantly higher in noise-exposed mice, consistent with higher functional Prestin levels. We then measured Prestin protein and mRNA levels in whole-cochlea specimens. Both Western blot and qPCR studies demonstrated increased Prestin expression after noise exposure. Finally, we examined the effect of the Prestin increase in vivo following noise damage. Immediately after noise exposure, ABR and DPOAE thresholds were elevated by 30–40 dB. While most of the temporary threshold shifts recovered within 3 days, there were additional improvements over the next month. However, DPOAE magnitudes, basilar membrane vibration, and CAP tuning curve measurements from the 9–12 kHz cochlear region demonstrated no differences between noise-exposed mice and control mice. Taken together, these data indicate that Prestin is up-regulated by 32–58% in residual OHCs after noise exposure and that the Prestin is functional. These findings are consistent with the notion that Prestin increases in an attempt to partially compensate for reduced force production because of missing OHCs. However, in regions where there is no OHC loss, the cochlea is able to compensate for the excess Prestin in order to maintain stable auditory thresholds and frequency discrimination.
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Prestin mRNA increases after noise exposure.
2013Co-Authors: Anping Xia, Yohan Song, Rosalie Wang, Simon S. Gao, Will Clifton, Patrick Raphael, Sung-il Chao, Fred A. Pereira, Andrew K. Groves, John S. OghalaiAbstract:(A) The total amount of myosin VIIa mRNA was not significantly different after noise exposure. (B) After normalizing myosin VIIa to the number of residual hair cells at 7 days and one month after noise exposure, there still was no difference in the amount of myosin VIIa per hair cell. (C) The total amount of Prestin mRNA was not significantly different after noise exposure. (D) After normalizing Prestin to the number of residual OHCs at 7 days and one month after noise exposure, there were statistically significant increases in the amount of Prestin mRNA per OHC. (E) The Prestin/myosin VIIa ratio increased after noise exposure. (F) After normalizing the Prestin/myosin VIIa ratio to the number of residual OHCs, the increase after noise exposure persisted.
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Prestin protein level increases after noise exposure.
2013Co-Authors: Anping Xia, Yohan Song, Rosalie Wang, Simon S. Gao, Will Clifton, Patrick Raphael, Sung-il Chao, Fred A. Pereira, Andrew K. Groves, John S. OghalaiAbstract:(A) Representative Western blots of Prestin and myosin VIIa from mice seven days after noise exposure and from control mice. These images come from the same gel. (B) Quantification of the band density revealed that the Prestin/myosin VIIa ratio increased after noise exposure. (C) After normalizing the Prestin/myosin VIIa ratio to the number of residual OHCs, the increase after noise exposure persisted.
Dhasakumar Navaratnam - One of the best experts on this subject based on the ideXlab platform.
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single particle cryo em structure of the outer hair cell motor protein Prestin
bioRxiv, 2021Co-Authors: Carmen Butan, Jun-ping Bai, Dhasakumar Navaratnam, Qiang Song, Winston Tan, Joseph SantossacchiAbstract:The mammalian outer hair cell (OHC) protein Prestin (Slc26a5), a member of the solute carrier 26 (Slc26) family of membrane proteins, differs from other members of the family owing to its unique piezoelectric-like property that drives OHC electromotility. Prestin is required by OHCs for cochlear amplification, a process that enhances mammalian hearing. Despite substantial biophysical characterization, the mechanistic basis for the Prestins electro-mechanical behavior is not fully understood. To gain insight into such behavior, we have used cryo-electron microscopy at subnanometer resolution (overall resolution of 4.0 A) to investigate the three-dimensional structure of Prestin from gerbil (Meriones unguiculatus). Our studies show that Prestin dimerizes with a 3D architecture strikingly similar to the dimeric conformation observed in the Slc26a9 anion transporter in an inside open/intermediate state, which we infer, based on patch clamp recordings, to reflect the contracted state of Prestin. The structure shows two well separated transmembrane (TM) subunits and two cytoplasmic sulfate transporter and anti-sigma factor antagonist (STAS) domains forming a swapped dimer. The dimerization interface is defined by interactions between the domain-swapped STAS dimer and the transmembrane domains of the opposing half unit, further strengthened by an antiparallel beta strand at its N terminus. The structure also shows that each one of its two transmembrane subunits consists of 14 transmembrane segments organized in two inverted 7-segment repeats with a topology that was first observed in the structure of the bacterial symporter UraA (Lu F, et al., Nature 472, 2011). Finally, the solved anion binding site structural features of Prestin are quite similar to that of Slc26a9 and other family members. Despite this similarity, we find that Slc26a9 lacks the characteristic displacement currents (or NonLinear Capacitance(NLC)) found with Prestin, and we show that mutation of Prestins Cl- binding site removes salicylate competition with anions in the face of normal NLC, thus refuting the yet accepted extrinsic voltage sensor hypothesis and any associated transport-like requirements for voltage-driven electromotility.
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Maturation of Voltage-induced Shifts in SLC26a5 (Prestin) Operating Point during Trafficking and Membrane Insertion.
Neuroscience, 2020Co-Authors: Feng Zhai, Lei Song, Jun-ping Bai, Chunfu Dai, Dhasakumar Navaratnam, Joseph Santos-sacchiAbstract:Abstract Prestin (SLC26a5) is an integral membrane motor protein in outer hair cells (OHC) that underlies cochlear amplification. As a voltage-dependent protein, it relies on intrinsic sensor charge to respond to transmembrane voltage (receptor potentials), thereby effecting conformational changes. The protein’s electromechanical actively is experimentally monitored as a bell-shaped nonlinear capacitance (NLC), whose magnitude peaks at a characteristic voltage, Vh. This voltage denotes the midpoint of Prestin’s charge–voltage (Q–V) Boltzmann distribution and region of maximum gain of OHC electromotility. It is an important factor in hearing capabilities for mammals. A variety of biophysical forces can influence the distribution of charge, gauged by shifts in Vh, including prior holding voltage or membrane potential. Here we report that the effectiveness of prior voltage augments during the delivery of Prestin to the membranes in an inducible HEK cell line. The augmentation coincides with an increase in Prestin density, maturing at a characteristic membrane areal density of 870 functional Prestin units per square micrometer, and is likely indicative of Prestin–Prestin cooperative interactions.
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Prestin kinetics and corresponding frequency dependence augment during early development of the outer hair cell within the mouse organ of corti
Scientific Reports, 2019Co-Authors: Jun-ping Bai, Dhasakumar Navaratnam, Joseph SantossacchiAbstract:Several studies have documented the early development of OHC electromechanical behavior. The mechanical response (electromotility, eM) and its electrical correlate (nonlinear capacitance, NLC), resulting from Prestin’s voltage-sensor charge movement, increase over the course of several postnatal days in altricial animals. They increase until about p18, near the time of peripheral auditory maturity. The correspondence of auditory capabilities and Prestin function indicates that mature activity of Prestin occurs at this time. One of the major requirements of eM is its responsiveness across auditory frequencies. Here we evaluate the frequency response of Prestin charge movement in mice over the course of development up to 8 months. We find that in apical turn OHCs Prestin’s frequency response increases during postnatal development and stabilizes when mature hearing is established. The low frequency component of NLC, within in situ explants, agrees with previously reported results on isolated cells. If Prestin activity is independent of cochlear place, as might be expected, then these observations suggest that Prestin activity somehow influences cochlear amplification at high frequencies in spite of its low pass behavior.
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tyrosine motifs are required for Prestin basolateral membrane targeting
Biology Open, 2015Co-Authors: Yifan Zhang, Jun-ping Bai, Joseph Santossacchi, Iman Moeininaghani, Dhasakumar NavaratnamAbstract:Prestin is targeted to the lateral wall of outer hair cells (OHCs) where its electromotility is critical for cochlear amplification. Using MDCK cells as a model system for polarized epithelial sorting, we demonstrate that Prestin uses tyrosine residues, in a YXXΦ motif, to target the basolateral surface. Both Y520 and Y667 are important for basolateral targeting of Prestin. Mutation of these residues to glutamine or alanine resulted in retention within the Golgi and delayed egress from the Golgi in Y667Q. Basolateral targeting is restored upon mutation to phenylalanine suggesting the importance of a phenol ring in the tyrosine side chain. We also demonstrate that Prestin targeting to the basolateral surface is dependent on AP1B (μ1B), and that Prestin uses transferrin containing early endosomes in its passage from the Golgi to the basolateral plasma membrane. The presence of AP1B (μ1B) in OHCs, and parallels between Prestin targeting to the basolateral surface of OHCs and polarized epithelial cells suggest that outer hair cells resemble polarized epithelia rather than neurons in this important phenotypic measure.
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real time measures of Prestin charge and fluorescence during plasma membrane trafficking reveal sub tetrameric activity
PLOS ONE, 2013Co-Authors: Shumin Bian, Dhasakumar Navaratnam, Joseph SantossacchiAbstract:Prestin (SLC26a5) is the outer hair cell integral membrane motor protein that drives cochlear amplification, and has been described as an obligate tetramer. We studied in real time the delivery of YFP-Prestin to the plasma membrane of cells from a tetracycline-inducible cell line. Following the release of temperature block to reinstate trans Golgi network delivery of the integral membrane protein, we measured nonlinear capacitance (NLC) and membrane fluorescence during voltage clamp. Prestin was delivered exponentially to the plasma membrane with a time constant of less than 10 minutes, with both electrical and fluorescence methods showing high temporal correlation. However, based on disparity between estimates of Prestin density derived from either fluorescence or NLC, we conclude that sub-tetrameric forms of Prestin contribute to our electrical and fluorescence measures. Thus, in agreement with previous observations we find that functional Prestin is not an obligate tetramer.