Annual International Conference of the IEEE Engineering in Medicine and Biology - Proceedings, 2007
The electromotility of cochlear outer hair cells (OHCs) is a required process for normal hearing,... more The electromotility of cochlear outer hair cells (OHCs) is a required process for normal hearing, and involves a membrane-based mechanism in which the transmembrane protein, prestin, plays a central role. We have investigated the contribution of prestin to the mechanics and electromechanics of the cell membrane using membrane tethers formed from human embryonic kidney cells. Our results suggest that prestin appears to change membrane tension and amplify electrically-evoked force generation, while a single point mutation of alanine to tryptophan in prestin reduces electrically-evoked force generation without affecting the membrane tension. We propose that prestin and membrane work in synergy to produce the electrical and mechanical changes that are required during OHC electromotility.
Background: Identification of bona fide direct nuclear receptor gene targets has been challenging... more Background: Identification of bona fide direct nuclear receptor gene targets has been challenging but essential for understanding regulation of organismal physiological processes.
Background: Prestin, encoded by the gene SLC26A5, is a transmembrane protein of the cochlear oute... more Background: Prestin, encoded by the gene SLC26A5, is a transmembrane protein of the cochlear outer hair cell (OHC). Prestin is required for the somatic electromotile activity of OHCs, which is absent in OHCs and causes severe hearing impairment in mice lacking prestin. In humans, the role of sequence variations in SLC26A5 in hearing loss is less clear. Although prestin is expected to be required for functional human OHCs, the clinical significance of reported putative mutant alleles in humans is uncertain.
Prestin is an essential component of the molecular motor of cochlear outer hair cells that contri... more Prestin is an essential component of the molecular motor of cochlear outer hair cells that contribute to frequency selectivity and sensitivity of mammalian hearing. A model system to study prestin employs its transfection into cultured HEK 293 cells. Our goal was to characterize prestin's trafficking pathway and localization in the plasma membrane. We used immuno-colocalization of prestin with intracellular and plasma membrane markers and sucrose density fractionation to analyze prestin in membrane compartments. Voltage clamping was used to measure nonlinear capacitance (NLC), prestin's electrical signature. Prestin targets to the membrane by 24 hours post-transfection when NLC is measurable. Prestin then concentrates into membrane foci that colocalize and fractionate with membrane microdomains. Depleting membrane cholesterol content altered prestin localization and NLC. Prestin activity in HEK 293 cells results from expression in the plasma membrane and altering membrane lipid content affects prestin localization and activity.
We have recently described in genital skin fibroblasts (GSF) a relatively abundant 56 kDa protein... more We have recently described in genital skin fibroblasts (GSF) a relatively abundant 56 kDa protein with androgen-binding activity. This protein is missing in GSF of most patients with complete androgen insensitivity syndrome (CAI). The protein has many characteristics compatible with the androgen receptor; it has in fact been tentatively considered as a precursor or degradation form of the prototypic (approximately 100 kDa) human androgen receptor. We have prepared an antiserum to this protein, which allowed us to detect it as a direct product by in vitro translation of mRNA from GSF. It is thus very unlikely to be a degradation product of a larger precursor. Furthermore, covalent photolytic labeling of this protein with the androgen analogue [3H]mibolerone revealed a much lower affinity for this protein than is known for the androgen receptor. Finally, the GSF of two exceptional patients with complete androgen insensitivity syndrome due to negligible androgen receptor-binding activity express this protein normally, as determined on two-dimensional gels by Western blot analysis with the antiserum and by photolytic covalent labeling with androgen analogues. These data indicate that the protein is not a precursor or a degradation product of the receptor; nor is it androgen-induced. They are more compatible with the idea that the protein is another member of the steroid/thyroid/retinoic acid receptor supergene family, perhaps as an unorthodox product of the human androgen receptor gene.
Nor-1 belongs to the nur subfamily of nuclear receptor transcription factors. The precise role of... more Nor-1 belongs to the nur subfamily of nuclear receptor transcription factors. The precise role of Nor-1 in mammalian development has not been established. However, recent studies indicate a function for this transcription factor in oncogenesis and apoptosis. To examine the spatiotemporal expression pattern of Nor-1 and the developmental and physiological consequences of Nor-1 ablation, Nor-1-null mice were generated by insertion of the lacZ gene into the Nor-1 genomic locus. Disruption of the Nor-1 gene results in inner ear defects and partial bidirectional circling behavior. During early otic development, Nor-1 is expressed exclusively in the semicircular canal forming fusion plates. After formation of the membranous labyrinth, Nor-1 expression in the vestibule is limited to nonsensory epithelial cells localized at the inner edge of the semicircular canals and to the ampullary and utricular walls. In the absence of Nor-1, the vestibular walls fuse together as normal; however, the endolymphatic fluid space in the semicircular canals is diminished and the roof of the ampulla appears flattened due to defective continual proliferative growth of the semicircular canals.
We report on the linear and nonlinear dielectric properties of budding yeast (S. cerevisiae) cell... more We report on the linear and nonlinear dielectric properties of budding yeast (S. cerevisiae) cells, one strain of which has been genetically modified to express prestin. This motor protein plays a crucial role in the large electromotility exhibited by the outer hair cells of mammalian inner ears. Live cell suspensions exhibit enormous dielectric responses, which can be used to probe metabolic activity, membrane potential, and other properties. The aims of this study are: (1) to compare the dielectric responses of organisms expressing prestin from those of control specimens, and (2) ultimately to further develop dielectric response as a tool to study live cells, proteins, and lipids.
An active process within the cochlea is necessary to obtain the sensitivity and frequency selecti... more An active process within the cochlea is necessary to obtain the sensitivity and frequency selectivity characteristic of mammalian hearing. This process is realized, at least in part, through the electromotile response of outer hair cells (OHCs). Electromotility requires the presence of prestin, a transmembrane protein highly expressed in the OHC lateral wall. Very little is known about how prestin functions at the molecular level to elicit electromotility, but theoretical models and recent experiments suggest that prestin-prestin interactions are required. To explore the extent of proposed prestin interactions, we employ fluorescence resonance energy transfer (FRET). FRET is a powerful optical technique capable of measuring inter-fluorophore distances less than 10 nm. Using human embryonic kidney cells (HEKs) as a model cell system and the standard FRET pair, cyan fluorescent protein (CFP) as the donor and yellow fluorescent protein (YFP) as the acceptor, we assay for the self-association of prestin under steady-state conditions using acceptor photobleach FRET (apFRET) and sensitized emission FRET (seFRET). Our findings from apFRET indicate the presence of prestin self-association when HEKs express both prestin-CFP and prestin-YFP in the membrane. The average FRET efficiency was ∼9%, but values as high as 20% were measured.
The voltage-dependent movement, or electromotility, of cochlear outer hair cells contributes to c... more The voltage-dependent movement, or electromotility, of cochlear outer hair cells contributes to cochlear amplification in mammalian hearing. Outer hair-cell electromotility involves a membrane-based motor in which the membrane protein prestin plays a central role. We have investigated the contribution of prestin to the mechanics and electromechanical force (EMF) generation of the membrane using membrane tethers formed from human embryonic kidney (HEK) cells. Several measures of membrane tether mechanics are greater in tethers pulled from HEK cells transfected with prestin when compared to control untransfected HEK cells. A single point mutation of alanine to tryptophan (A100W) in prestin eliminates prestinassociated charge movement and diminishes EMF but does not alter passive membrane mechanics. These results suggest that prestin-associated charge transfer is necessary for maximal EMF generation by the membrane.
Biochemical and Biophysical Research Communications, 1988
We have found in genital skin fibroblasts an abundant 56 kD protein which appears related to the ... more We have found in genital skin fibroblasts an abundant 56 kD protein which appears related to the androgen receptor. The protein is detected in the soluble fraction by two-dimensional polyacrylamide gel electrophoresis as two spots with isoelectric points of 6.7 and 6.5 respectively. Photoaffinity labelling of GSF with 8 nM or 50 nM [3H]-methyltrienolone selectively labels the two protein spots but with an apparently lower affinity than is known for the androgen receptor. The two protein spots stem from one protein as judged from peptide patterns of partial proteolytic digests and the equal labelling of both spots with methyltrienolone. Cells from subjects with mutant androgen receptors generally lack the 56 kD protein and labelling with methyltrienolone fails, but the protein is not the androgen receptor itself. We propose the hypothesis that the 56 kD protein is synthesized from the same gene as the androgen receptor and that androgen may not be its natural ligand.
The dynamic embryonic expression of germ cell nuclear factor (GCNF), an orphan nuclear receptor, ... more The dynamic embryonic expression of germ cell nuclear factor (GCNF), an orphan nuclear receptor, suggests that it may play an important role during early development. To determine the physiological role of GCNF, we have generated a targeted mutation of the GCNF gene in mice. Germ line mutation of the GCNF gene proves that the orphan nuclear receptor is essential for embryonic survival and normal development. GCNF ؊/؊ embryos cannot survive beyond 10.5 days postcoitum (dpc), probably due to cardiovascular failure. Prior to death, GCNF ؊/؊ embryos suffer significant defects in posterior development. Unlike GCNF ؉/؉ embryos, GCNF ؊/؊ embryos do not turn and remain in a lordotic position, the majority of the neural tube remains open, and the hindgut fails to close. GCNF ؊/؊ embryos also suffer serious defects in trunk development, specifically in somitogenesis, which terminates by 8.75 dpc. The maximum number of somites in GCNF ؊/؊ embryos is 13 instead of 25 as in the GCNF ؉/؉ embryos. Interestingly, the tailbud of GCNF ؊/؊ embryos develops ectopically outside the yolk sac. Indeed, alterations in expression of multiple marker genes were identified in the posterior of GCNF ؊/؊ embryos, including the primitive streak, the node, and the presomitic mesoderm. These results suggest that GCNF is required for maintenance of somitogenesis and posterior development and is essential for embryonic survival. These results suggest that GCNF regulates a novel and critical developmental pathway involved in normal anteroposterior development.
Problem: COUP-TFI (Chicken Ovalbumin Upstream Promoter-Transcription Factor I) is an orphan membe... more Problem: COUP-TFI (Chicken Ovalbumin Upstream Promoter-Transcription Factor I) is an orphan member of the steroid/thyroid nuclear receptor superfamily. It is highly expressed in the developing nervous system. Null mutant COUP-TFI mice have glossopharyngeal nerve dysfunction that compromises feeding ability and usually results in perinatal death. Surviving null mutant mice demonstrate neuronal differentiation defects, altered thalamocortical axon guidance, and apoptosis of cerebral cortical layer IV. COUP-TFI expression is also important for inner ear organogenesis. Null mutants have a shortened cochlear duct, abnormal cochlear innervation, and malformed vestibular chambers. Additionally, by P20 the organ of Corti in the basal turn has degenerated. Interestingly, the organ of Corti at the mid-modiolar region has only subtle anatomic irregularities and, in particular, contains normal-appearing inner and outer hair cells. We sought to determine the effect of this mutation on cochlear function.
Recent advances in the developmental biology, genetics and cell biology of the inner ear are guid... more Recent advances in the developmental biology, genetics and cell biology of the inner ear are guiding research to novel therapeutic modalities -a market currently estimated to be at least US$10 billion. This article highlights prospects to manipulate the mammalian hearing organ with gene and stem cell delivery to the inner ear to protect, repair or regenerate the hair cells, supporting cells and associated nerves. IUBMB Life, 58: 525-530, 2006
Glycosylation is a common post-translational modification of proteins and is implicated in a vari... more Glycosylation is a common post-translational modification of proteins and is implicated in a variety of cellular functions including protein folding, degradation, sorting and trafficking, and membrane protein recycling. The membrane protein prestin is an essential component of the membrane-based motor driving electromotility changes (electromotility) in the outer hair cell (OHC), a central process in auditory transduction. Prestin was earlier identified to possess two N-glycosylation sites (N163, N166) that, when mutated, marginally affect prestin nonlinear capacitance (NLC) function in cultured cells. Here, we show that the double mutant prestin NN163/166AA is not glycosylated and shows the expected NLC properties in the untreated and cholesterol-depleted HEK 293 cell model. In addition, unlike WT prestin that readily forms oligomers, prestin NN163/166AA is enriched as monomers and more mobile in the plasma membrane, suggesting that oligomerization of prestin is dependent on glycosylation but is not essential for the generation of NLC in HEK 293 cells. However, in the presence of increased membrane cholesterol, unlike the hyperpolarizing shift in NLC seen with WT prestin, cells expressing prestin NN163/166AA exhibit a linear capacitance function. In an attempt to explain this finding, we discovered that both WT prestin and prestin NN163/166AA participate in cholesterol-dependent cellular trafficking. In contrast to WT prestin, prestin NN163/166AA shows a significant cholesterol-dependent decrease in cellsurface expression, which may explain the loss of NLC function. Based on our observations, we conclude that glycosylation regulates self-association and cellular trafficking of prestin NN163/166AA . These observations are the first to implicate a regulatory role for cellular trafficking and sorting in prestin function. We speculate that the cholesterol regulation of prestin occurs through localization to and internalization from membrane microdomains by clathrin-and caveolin-dependent mechanisms.
Journal of Steroid Biochemistry and Molecular Biology, 1996
COUP-TFs are orphan members of the steroid/thyroid hormone receptor superfamily. COUP-TF homologu... more COUP-TFs are orphan members of the steroid/thyroid hormone receptor superfamily. COUP-TF homologues have been cloned in several species, from Drosophila to man. The vertebrate COUP-TFs can be classified into four subgroups according to sequence homology in their ligand-binding domain. COUP-TFs bind to AGGTCA direct repeats or palindromes with various spacings. These include the response elements of several other members of
The outer hair cell (OHC) motor protein prestin is necessary for electromotility, which drives co... more The outer hair cell (OHC) motor protein prestin is necessary for electromotility, which drives cochlear amplification and produces exquisitely sharp frequency tuning. Tecta(C1509G) 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 ...
Annual International Conference of the IEEE Engineering in Medicine and Biology - Proceedings, 2007
The electromotility of cochlear outer hair cells (OHCs) is a required process for normal hearing,... more The electromotility of cochlear outer hair cells (OHCs) is a required process for normal hearing, and involves a membrane-based mechanism in which the transmembrane protein, prestin, plays a central role. We have investigated the contribution of prestin to the mechanics and electromechanics of the cell membrane using membrane tethers formed from human embryonic kidney cells. Our results suggest that prestin appears to change membrane tension and amplify electrically-evoked force generation, while a single point mutation of alanine to tryptophan in prestin reduces electrically-evoked force generation without affecting the membrane tension. We propose that prestin and membrane work in synergy to produce the electrical and mechanical changes that are required during OHC electromotility.
Background: Identification of bona fide direct nuclear receptor gene targets has been challenging... more Background: Identification of bona fide direct nuclear receptor gene targets has been challenging but essential for understanding regulation of organismal physiological processes.
Background: Prestin, encoded by the gene SLC26A5, is a transmembrane protein of the cochlear oute... more Background: Prestin, encoded by the gene SLC26A5, is a transmembrane protein of the cochlear outer hair cell (OHC). Prestin is required for the somatic electromotile activity of OHCs, which is absent in OHCs and causes severe hearing impairment in mice lacking prestin. In humans, the role of sequence variations in SLC26A5 in hearing loss is less clear. Although prestin is expected to be required for functional human OHCs, the clinical significance of reported putative mutant alleles in humans is uncertain.
Prestin is an essential component of the molecular motor of cochlear outer hair cells that contri... more Prestin is an essential component of the molecular motor of cochlear outer hair cells that contribute to frequency selectivity and sensitivity of mammalian hearing. A model system to study prestin employs its transfection into cultured HEK 293 cells. Our goal was to characterize prestin's trafficking pathway and localization in the plasma membrane. We used immuno-colocalization of prestin with intracellular and plasma membrane markers and sucrose density fractionation to analyze prestin in membrane compartments. Voltage clamping was used to measure nonlinear capacitance (NLC), prestin's electrical signature. Prestin targets to the membrane by 24 hours post-transfection when NLC is measurable. Prestin then concentrates into membrane foci that colocalize and fractionate with membrane microdomains. Depleting membrane cholesterol content altered prestin localization and NLC. Prestin activity in HEK 293 cells results from expression in the plasma membrane and altering membrane lipid content affects prestin localization and activity.
We have recently described in genital skin fibroblasts (GSF) a relatively abundant 56 kDa protein... more We have recently described in genital skin fibroblasts (GSF) a relatively abundant 56 kDa protein with androgen-binding activity. This protein is missing in GSF of most patients with complete androgen insensitivity syndrome (CAI). The protein has many characteristics compatible with the androgen receptor; it has in fact been tentatively considered as a precursor or degradation form of the prototypic (approximately 100 kDa) human androgen receptor. We have prepared an antiserum to this protein, which allowed us to detect it as a direct product by in vitro translation of mRNA from GSF. It is thus very unlikely to be a degradation product of a larger precursor. Furthermore, covalent photolytic labeling of this protein with the androgen analogue [3H]mibolerone revealed a much lower affinity for this protein than is known for the androgen receptor. Finally, the GSF of two exceptional patients with complete androgen insensitivity syndrome due to negligible androgen receptor-binding activity express this protein normally, as determined on two-dimensional gels by Western blot analysis with the antiserum and by photolytic covalent labeling with androgen analogues. These data indicate that the protein is not a precursor or a degradation product of the receptor; nor is it androgen-induced. They are more compatible with the idea that the protein is another member of the steroid/thyroid/retinoic acid receptor supergene family, perhaps as an unorthodox product of the human androgen receptor gene.
Nor-1 belongs to the nur subfamily of nuclear receptor transcription factors. The precise role of... more Nor-1 belongs to the nur subfamily of nuclear receptor transcription factors. The precise role of Nor-1 in mammalian development has not been established. However, recent studies indicate a function for this transcription factor in oncogenesis and apoptosis. To examine the spatiotemporal expression pattern of Nor-1 and the developmental and physiological consequences of Nor-1 ablation, Nor-1-null mice were generated by insertion of the lacZ gene into the Nor-1 genomic locus. Disruption of the Nor-1 gene results in inner ear defects and partial bidirectional circling behavior. During early otic development, Nor-1 is expressed exclusively in the semicircular canal forming fusion plates. After formation of the membranous labyrinth, Nor-1 expression in the vestibule is limited to nonsensory epithelial cells localized at the inner edge of the semicircular canals and to the ampullary and utricular walls. In the absence of Nor-1, the vestibular walls fuse together as normal; however, the endolymphatic fluid space in the semicircular canals is diminished and the roof of the ampulla appears flattened due to defective continual proliferative growth of the semicircular canals.
We report on the linear and nonlinear dielectric properties of budding yeast (S. cerevisiae) cell... more We report on the linear and nonlinear dielectric properties of budding yeast (S. cerevisiae) cells, one strain of which has been genetically modified to express prestin. This motor protein plays a crucial role in the large electromotility exhibited by the outer hair cells of mammalian inner ears. Live cell suspensions exhibit enormous dielectric responses, which can be used to probe metabolic activity, membrane potential, and other properties. The aims of this study are: (1) to compare the dielectric responses of organisms expressing prestin from those of control specimens, and (2) ultimately to further develop dielectric response as a tool to study live cells, proteins, and lipids.
An active process within the cochlea is necessary to obtain the sensitivity and frequency selecti... more An active process within the cochlea is necessary to obtain the sensitivity and frequency selectivity characteristic of mammalian hearing. This process is realized, at least in part, through the electromotile response of outer hair cells (OHCs). Electromotility requires the presence of prestin, a transmembrane protein highly expressed in the OHC lateral wall. Very little is known about how prestin functions at the molecular level to elicit electromotility, but theoretical models and recent experiments suggest that prestin-prestin interactions are required. To explore the extent of proposed prestin interactions, we employ fluorescence resonance energy transfer (FRET). FRET is a powerful optical technique capable of measuring inter-fluorophore distances less than 10 nm. Using human embryonic kidney cells (HEKs) as a model cell system and the standard FRET pair, cyan fluorescent protein (CFP) as the donor and yellow fluorescent protein (YFP) as the acceptor, we assay for the self-association of prestin under steady-state conditions using acceptor photobleach FRET (apFRET) and sensitized emission FRET (seFRET). Our findings from apFRET indicate the presence of prestin self-association when HEKs express both prestin-CFP and prestin-YFP in the membrane. The average FRET efficiency was ∼9%, but values as high as 20% were measured.
The voltage-dependent movement, or electromotility, of cochlear outer hair cells contributes to c... more The voltage-dependent movement, or electromotility, of cochlear outer hair cells contributes to cochlear amplification in mammalian hearing. Outer hair-cell electromotility involves a membrane-based motor in which the membrane protein prestin plays a central role. We have investigated the contribution of prestin to the mechanics and electromechanical force (EMF) generation of the membrane using membrane tethers formed from human embryonic kidney (HEK) cells. Several measures of membrane tether mechanics are greater in tethers pulled from HEK cells transfected with prestin when compared to control untransfected HEK cells. A single point mutation of alanine to tryptophan (A100W) in prestin eliminates prestinassociated charge movement and diminishes EMF but does not alter passive membrane mechanics. These results suggest that prestin-associated charge transfer is necessary for maximal EMF generation by the membrane.
Biochemical and Biophysical Research Communications, 1988
We have found in genital skin fibroblasts an abundant 56 kD protein which appears related to the ... more We have found in genital skin fibroblasts an abundant 56 kD protein which appears related to the androgen receptor. The protein is detected in the soluble fraction by two-dimensional polyacrylamide gel electrophoresis as two spots with isoelectric points of 6.7 and 6.5 respectively. Photoaffinity labelling of GSF with 8 nM or 50 nM [3H]-methyltrienolone selectively labels the two protein spots but with an apparently lower affinity than is known for the androgen receptor. The two protein spots stem from one protein as judged from peptide patterns of partial proteolytic digests and the equal labelling of both spots with methyltrienolone. Cells from subjects with mutant androgen receptors generally lack the 56 kD protein and labelling with methyltrienolone fails, but the protein is not the androgen receptor itself. We propose the hypothesis that the 56 kD protein is synthesized from the same gene as the androgen receptor and that androgen may not be its natural ligand.
The dynamic embryonic expression of germ cell nuclear factor (GCNF), an orphan nuclear receptor, ... more The dynamic embryonic expression of germ cell nuclear factor (GCNF), an orphan nuclear receptor, suggests that it may play an important role during early development. To determine the physiological role of GCNF, we have generated a targeted mutation of the GCNF gene in mice. Germ line mutation of the GCNF gene proves that the orphan nuclear receptor is essential for embryonic survival and normal development. GCNF ؊/؊ embryos cannot survive beyond 10.5 days postcoitum (dpc), probably due to cardiovascular failure. Prior to death, GCNF ؊/؊ embryos suffer significant defects in posterior development. Unlike GCNF ؉/؉ embryos, GCNF ؊/؊ embryos do not turn and remain in a lordotic position, the majority of the neural tube remains open, and the hindgut fails to close. GCNF ؊/؊ embryos also suffer serious defects in trunk development, specifically in somitogenesis, which terminates by 8.75 dpc. The maximum number of somites in GCNF ؊/؊ embryos is 13 instead of 25 as in the GCNF ؉/؉ embryos. Interestingly, the tailbud of GCNF ؊/؊ embryos develops ectopically outside the yolk sac. Indeed, alterations in expression of multiple marker genes were identified in the posterior of GCNF ؊/؊ embryos, including the primitive streak, the node, and the presomitic mesoderm. These results suggest that GCNF is required for maintenance of somitogenesis and posterior development and is essential for embryonic survival. These results suggest that GCNF regulates a novel and critical developmental pathway involved in normal anteroposterior development.
Problem: COUP-TFI (Chicken Ovalbumin Upstream Promoter-Transcription Factor I) is an orphan membe... more Problem: COUP-TFI (Chicken Ovalbumin Upstream Promoter-Transcription Factor I) is an orphan member of the steroid/thyroid nuclear receptor superfamily. It is highly expressed in the developing nervous system. Null mutant COUP-TFI mice have glossopharyngeal nerve dysfunction that compromises feeding ability and usually results in perinatal death. Surviving null mutant mice demonstrate neuronal differentiation defects, altered thalamocortical axon guidance, and apoptosis of cerebral cortical layer IV. COUP-TFI expression is also important for inner ear organogenesis. Null mutants have a shortened cochlear duct, abnormal cochlear innervation, and malformed vestibular chambers. Additionally, by P20 the organ of Corti in the basal turn has degenerated. Interestingly, the organ of Corti at the mid-modiolar region has only subtle anatomic irregularities and, in particular, contains normal-appearing inner and outer hair cells. We sought to determine the effect of this mutation on cochlear function.
Recent advances in the developmental biology, genetics and cell biology of the inner ear are guid... more Recent advances in the developmental biology, genetics and cell biology of the inner ear are guiding research to novel therapeutic modalities -a market currently estimated to be at least US$10 billion. This article highlights prospects to manipulate the mammalian hearing organ with gene and stem cell delivery to the inner ear to protect, repair or regenerate the hair cells, supporting cells and associated nerves. IUBMB Life, 58: 525-530, 2006
Glycosylation is a common post-translational modification of proteins and is implicated in a vari... more Glycosylation is a common post-translational modification of proteins and is implicated in a variety of cellular functions including protein folding, degradation, sorting and trafficking, and membrane protein recycling. The membrane protein prestin is an essential component of the membrane-based motor driving electromotility changes (electromotility) in the outer hair cell (OHC), a central process in auditory transduction. Prestin was earlier identified to possess two N-glycosylation sites (N163, N166) that, when mutated, marginally affect prestin nonlinear capacitance (NLC) function in cultured cells. Here, we show that the double mutant prestin NN163/166AA is not glycosylated and shows the expected NLC properties in the untreated and cholesterol-depleted HEK 293 cell model. In addition, unlike WT prestin that readily forms oligomers, prestin NN163/166AA is enriched as monomers and more mobile in the plasma membrane, suggesting that oligomerization of prestin is dependent on glycosylation but is not essential for the generation of NLC in HEK 293 cells. However, in the presence of increased membrane cholesterol, unlike the hyperpolarizing shift in NLC seen with WT prestin, cells expressing prestin NN163/166AA exhibit a linear capacitance function. In an attempt to explain this finding, we discovered that both WT prestin and prestin NN163/166AA participate in cholesterol-dependent cellular trafficking. In contrast to WT prestin, prestin NN163/166AA shows a significant cholesterol-dependent decrease in cellsurface expression, which may explain the loss of NLC function. Based on our observations, we conclude that glycosylation regulates self-association and cellular trafficking of prestin NN163/166AA . These observations are the first to implicate a regulatory role for cellular trafficking and sorting in prestin function. We speculate that the cholesterol regulation of prestin occurs through localization to and internalization from membrane microdomains by clathrin-and caveolin-dependent mechanisms.
Journal of Steroid Biochemistry and Molecular Biology, 1996
COUP-TFs are orphan members of the steroid/thyroid hormone receptor superfamily. COUP-TF homologu... more COUP-TFs are orphan members of the steroid/thyroid hormone receptor superfamily. COUP-TF homologues have been cloned in several species, from Drosophila to man. The vertebrate COUP-TFs can be classified into four subgroups according to sequence homology in their ligand-binding domain. COUP-TFs bind to AGGTCA direct repeats or palindromes with various spacings. These include the response elements of several other members of
The outer hair cell (OHC) motor protein prestin is necessary for electromotility, which drives co... more The outer hair cell (OHC) motor protein prestin is necessary for electromotility, which drives cochlear amplification and produces exquisitely sharp frequency tuning. Tecta(C1509G) 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 ...
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