RESUMEN
Charged moieties in the outer hair cell (OHC) membrane motor protein, prestin, are driven by transmembrane voltage to power OHC electromotility (eM) and cochlear amplification (CA), an enhancement of mammalian hearing. Consequently, the speed of prestin's conformational switching constrains its dynamic influence on micromechanics of the cell and the organ of Corti. Corresponding voltage-sensor charge movements in prestin, classically assessed as a voltage-dependent, nonlinear membrane capacitance (NLC), have been used to gauge its frequency response, but have been validly measured only out to 30 kHz. Thus, controversy exists concerning the effectiveness of eM in supporting CA at ultrasonic frequencies where some mammals can hear. Using megahertz sampling of guinea pig (either sex) prestin charge movements, we extend interrogations of NLC into the ultrasonic range (up to 120 kHz) and find an order of magnitude larger response at 80 kHz than previously predicted, indicating that an influence of eM at ultrasonic frequencies is likely, in line with recent in vivo results (Levic et al., 2022). Given wider bandwidth interrogations, we also validate kinetic model predictions of prestin by directly observing its characteristic cut-off frequency under voltage-clamp as the intersection frequency (Fis), near 19 kHz, of the real and imaginary components of complex NLC (cNLC). The frequency response of prestin displacement current noise determined from either the Nyquist relation or stationary measures aligns with this cut-off. We conclude that voltage stimulation accurately assesses the spectral limits of prestin activity, and that voltage-dependent conformational switching is physiologically significant in the ultrasonic range.SIGNIFICANCE STATEMENT The motor protein prestin powers outer hair cell (OHC) electromotility (eM) and cochlear amplification (CA), an enhancement of high-frequency mammalian hearing. The ability of prestin to work at very high frequencies depends on its membrane voltage-driven conformation switching. Using megahertz sampling, we extend measures of prestin charge movement into the ultrasonic range and find response magnitude at 80 kHz an order of magnitude larger than previously estimated, despite confirmation of previous low pass characteristic frequency cut-offs. The frequency response of prestin noise garnered by the admittance-based Nyquist relation or stationary noise measures confirms this characteristic cut-off frequency. Our data indicate that voltage perturbation provides accurate assessment of prestin performance indicating that it can support cochlear amplification into a higher frequency range than previously thought.
Asunto(s)
Células Ciliadas Auditivas Externas , Ultrasonido , Animales , Cobayas , Células Ciliadas Auditivas Externas/fisiología , Cóclea , Audición , Membrana Celular/metabolismo , MamíferosRESUMEN
Zebrafish are popular models for biological discovery. For investigators of the auditory and vestibular periphery, manipulations of hair cell and synaptic mechanisms have relied on inferences from extracellular recordings of physiological activity. We now provide data showing that hair cells and supporting cells of the lateral line can be directly patch-clamped, providing the first recordings of ionic channel activity, synaptic vesicle release, and gap junctional coupling in the neuromasts of living fish. Such capabilities will allow more detailed understanding of mechano-sensation of the zebrafish.
Asunto(s)
Células Ciliadas Auditivas/fisiología , Sistema de la Línea Lateral/inervación , Sistema de la Línea Lateral/fisiología , Neuronas/fisiología , Técnicas de Placa-Clamp , Animales , Fenómenos Electrofisiológicos , Femenino , Lateralidad Funcional/fisiología , Uniones Comunicantes/fisiología , Larva , Masculino , Canales de Potasio/fisiología , Vesículas Sinápticas/fisiología , Pez CebraRESUMEN
The function of prestin (SLC26a5), an anion transport family member, has evolved to enhance auditory sensitivity and frequency selectivity by providing mechanical feedback via outer hair cells (OHC) into the organ of Corti. The frequency extent of this boost is governed by the voltage-dependent kinetics of the protein's charge movements, otherwise known as nonlinear capacitance (NLC) that we measure in membrane patches under voltage clamp. Here we extend our previous studies on guinea pig OHCs by studying the frequency response of NLC in the mouse OHC, a species with higher frequency auditory needs. We find that the characteristic frequency cut-off (F is ) for the mouse surpasses that of the guinea pig, being 27 kHz vs. 19 kHz, respectively; nevertheless, each shows significant activity in the ultrasonic range. We also evaluate the influence of anion binding on prestin frequency response. Several single point mutations within the chloride binding pocket of prestin (e.g., S396E, S398E) lack anion influence. In agreement, we show absence of anion binding through molecular dynamics (MD) simulations. NLC F is in the S396E knock-in mouse remains the same as controls, indicating that high frequency activity is likely governed by viscoelastic loads within the membrane characterized by stretched-exponential frequency roll-off. Accordingly, treatment with MßCD, which removes membrane cholesterol, possibly from prestin itself, and can alter membrane fluidity, augments NLC F is out to 39 kHz. Although interactions between membrane lipid and prestin have been suggested from structural studies to arise at their interfacial boundaries within the membrane, our MD simulations suggest that phospholipids can insert within transmembrane domains of prestin during voltage perturbation. Such novel lipid-protein interactions could account for our observed changes in the phase of prestin's voltage-sensor charge movements across frequency. We hypothesize that because prestin tertiary structures of all species studied to-date are indistinguishable, it is likely that any special auditory requirements of individual species for cochlear amplification have evolved to capitalize on prestin performance by modifying, not the protein itself, but the external loads on the protein, including those within the membrane and organ of Corti. Significance: Prestin is believed to provide cochlear amplification in mammals that possess a wide range of frequency sensitivities, yet its tertiary structure is indistinguishable among those species studied. We find that prestin kinetics is faster in mice than in guinea pigs, mice showing higher frequency auditory capabilities. Chloride binding is not influential, but membrane lipids/viscosity is. We suggest that the evolution of prestin's species performance involves modifications of impinging loads, not the protein itself.
RESUMEN
Large conductance (BK) calcium activated potassium channels (Slo) are ubiquitous and implicated in a number of human diseases including hypertension and epilepsy. BK channels consist of a pore forming α-subunit (Slo) and a number of accessory subunits. In hair cells of nonmammalian vertebrates these channels play a critical role in electrical resonance, a mechanism of frequency selectivity. Hair cell BK channel clusters on the surface and currents increase along the tonotopic axis and contribute significantly to the responsiveness of these hair cells to sounds of high frequency. In contrast, messenger RNA levels encoding the Slo gene show an opposite decrease in high frequency hair cells. To understand the molecular events underlying this paradox, we used a yeast two-hybrid screen to isolate binding partners of Slo. We identified Rack1 as a Slo binding partner and demonstrate that PKC activation increases Slo surface expression. We also establish that increased Slo recycling of endocytosed Slo is at least partially responsible for the increased surface expression of Slo. Moreover, analysis of several PKC phosphorylation site mutants confirms that the effects of PKC on Slo surface expression are likely indirect. Finally, we show that Slo clusters on the surface of hair cells are also increased by increased PKC activity and may contribute to the increasing amounts of channel clusters on the surface of high-frequency hair cells.
Asunto(s)
Membrana Celular/metabolismo , Proteínas de Unión al GTP/metabolismo , Células Ciliadas Auditivas/metabolismo , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína Quinasa C/fisiología , Receptores de Superficie Celular/metabolismo , Animales , Membrana Celular/genética , Pollos , Cóclea/metabolismo , Proteínas de Unión al GTP/biosíntesis , Proteínas de Unión al GTP/genética , Regulación de la Expresión Génica , Células HEK293 , Células Ciliadas Auditivas/fisiología , Humanos , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Canales de Potasio de Gran Conductancia Activados por el Calcio/genética , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Fosforilación/genética , Proteína Quinasa C/biosíntesis , Proteína Quinasa C/genética , Receptores de Cinasa C Activada , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/genética , Regulación hacia Arriba/genéticaRESUMEN
Large-conductance calcium-activated potassium (BK) channels are ubiquitous and play an important role in a number of diseases. In hair cells of the ear, they play a critical role in electrical tuning, a mechanism of frequency discrimination. These channels show variable kinetics and expression along the tonotopic axis. Although the molecular underpinnings to its function in hair cells are poorly understood, it is established that BK channels consist of a pore-forming α-subunit (Slo) and a number of accessory subunits. Here we identify CDK5, a member of the cyclin-dependent kinase family, as an interacting partner of Slo. We show CDK5 to be present in hair cells and expressed in high concentrations in the cuticular plate and in the circumferential zone. In human embryonic kidney cells, we show that CDK5 inhibits surface expression of Slo by direct phosphorylation of Slo. Similarly, we note that CDK5 affects Slo voltage activation and deactivation kinetics, by a direct phosphorylation of T847. Taken together with its increasing expression along the tonotopic axis, these data suggest that CDK5 likely plays a critical role in electrical tuning and surface expression of Slo in hair cells.
Asunto(s)
Cóclea/metabolismo , Quinasa 5 Dependiente de la Ciclina/metabolismo , Células Ciliadas Auditivas/metabolismo , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Animales , Señalización del Calcio/fisiología , Membrana Celular/genética , Membrana Celular/metabolismo , Pollos , Transferencia Resonante de Energía de Fluorescencia , Biblioteca de Genes , Células HEK293 , Humanos , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Potenciales de la Membrana/fisiología , Técnicas de Placa-Clamp , Fosforilación , Técnicas del Sistema de Dos Híbridos , Xenopus laevisRESUMEN
The mammalian outer hair cell (OHC) protein prestin (Slc26a5) differs from other Slc26 family members due to its unique piezoelectric-like property that drives OHC electromotility, the putative mechanism for cochlear amplification. Here, we use cryo-electron microscopy to determine prestin's structure at 3.6 Å resolution. Prestin is structurally similar to the anion transporter Slc26a9. It is captured in an inward-open state which may reflect prestin's contracted state. Two well-separated transmembrane (TM) domains and two cytoplasmic sulfate transporter and anti-sigma factor antagonist (STAS) domains form a swapped dimer. The transmembrane domains consist of 14 transmembrane segments organized in two 7+7 inverted repeats, an architecture first observed in the bacterial symporter UraA. Mutation of prestin's chloride binding site removes salicylate competition with anions while retaining the prestin characteristic displacement currents (Nonlinear Capacitance), undermining the extrinsic voltage sensor hypothesis for prestin function.
Asunto(s)
Microscopía por Crioelectrón , Células Ciliadas Auditivas Externas/metabolismo , Transportadores de Sulfato/química , Animales , Proteínas de Transporte de Anión , Sitios de Unión , Células CHO , Cricetulus , Gerbillinae , Células Ciliadas Vestibulares/metabolismo , Transporte Iónico , Proteínas de la Membrana/química , Proteínas Motoras Moleculares/química , Proteínas Motoras Moleculares/metabolismo , Dominios ProteicosRESUMEN
Reports have proposed a putative role for ßV spectrin in outer hair cells (OHCs) of the cochlea. In an ongoing investigation of the role of the cytoskeleton in electromotility, we tested mice with a targeted exon deletion of ßV spectrin (Spnb5), and unexpectedly find that Spnb5(-/-) animals' auditory thresholds are unaffected. Similarly, these mice have normal OHC electromechanical activity (otoacoustic emissions) and non-linear capacitance. In contrast, magnitudes of auditory brainstem response (ABR) wave 1-amplitudes are significantly reduced. Evidence of a synaptopathy was absent with normal hair cell CtBP2 counts. In Spnb5(-/-) mice, the number of afferent and efferent nerve fibers is decreased. Consistent with this data, Spnb5 mRNA is present in Type I and II spiral ganglion neurons, but undetectable in OHCs. Together, these data establish that ßV spectrin is important for hearing, affecting neuronal structure and function. Significantly, these data support that ßV spectrin as is not functionally important to OHCs as has been previously suggested.
Asunto(s)
Células Ciliadas Auditivas Externas , Espectrina , Animales , Cóclea/fisiología , Potenciales Evocados Auditivos del Tronco Encefálico , Células Ciliadas Auditivas Externas/fisiología , Ratones , Ratones Noqueados , Emisiones Otoacústicas Espontáneas , Espectrina/genética , Espectrina/metabolismoRESUMEN
Widespread soil acidification due to atmospheric acid deposition and agricultural fertilization may greatly accelerate soil carbonate dissolution and CO2 release. However, to date, few studies have addressed these processes. Here, we use meta-analysis and nationwide-survey datasets to investigate changes in soil inorganic carbon (SIC) stocks in China. We observe an overall decrease in SIC stocks in topsoil (0-30 cm) (11.33 g C m-2 yr-1) from the 1980s to the 2010s. Total SIC stocks have decreased by â¼8.99 ± 2.24% (1.37 ± 0.37 Pg C). The average SIC losses across China (0.046 Pg C yr-1) and in cropland (0.016 Pg C yr-1) account for â¼17.6%-24.0% of the terrestrial C sink and 57.1% of the soil organic carbon sink in cropland, respectively. Nitrogen deposition and climate change have profound influences on SIC cycling. We estimate that â¼19.12%-19.47% of SIC stocks will be further lost by 2100. The consumption of SIC may offset a large portion of global efforts aimed at ecosystem carbon sequestration, which emphasizes the importance of achieving a better understanding of the indirect coupling mechanisms of nitrogen and carbon cycling and of effective countermeasures to minimize SIC loss.
RESUMEN
Changing kinetics of large-conductance potassium (BK) channels in hair cells of nonmammalian vertebrates, including the chick, plays a critical role in electrical tuning, a mechanism used by these cells to discriminate between different frequencies of sound. BK currents are less abundant in low-frequency hair cells and show large openings in response to a rise in intracellular Ca(2+) at a hair cell's operating voltage range (spanning -40 to -60 mV). Although the molecular underpinnings of its function in hair cells are poorly understood, it is established that BK channels consist of a pore-forming α-subunit (Slo) and a number of accessory subunits. Currents from the α (Slo)-subunit alone do not show dramatic increases in response to changes in Ca(2+) concentrations at -50 mV. We have cloned the chick ß(4)- and ß(1)-subunits and show that these subunits are preferentially expressed in low-frequency hair cells, where they decrease Slo surface expression. The ß(4)-subunit in particular is responsible for the BK channel's increased responsiveness to Ca(2+) at a hair cell's operating voltage. In contrast, however, the increases in relaxation times induced by both ß-subunits suggest additional mechanisms responsible for BK channel function in hair cells.
Asunto(s)
Calcio/fisiología , Células Ciliadas Auditivas/metabolismo , Activación del Canal Iónico/fisiología , Subunidades beta de los Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Animales , Señalización del Calcio/fisiología , Membrana Celular/genética , Membrana Celular/metabolismo , Pollos , Cóclea/metabolismo , Regulación hacia Abajo/genética , Subunidades beta de los Canales de Potasio de Gran Conductancia Activados por el Calcio/genética , Canales de Potasio de Gran Conductancia Activados por el Calcio/química , Mecanotransducción Celular/fisiología , Potenciales de la Membrana/fisiología , Oocitos/fisiología , Xenopus laevisRESUMEN
Prestin is a member of the SLC26 family of anion transporters that is responsible for outer hair cell (OHC) electromotility. Measures of voltage-evoked charge density (Q(sp)) of prestin indicated that the protein is highly expressed in OHCs, with single cells expressing up to 10 million molecules within the lateral membrane. In contrast, charge density measures in transfected cells indicated that they express, at best, only a fifth as many proteins on their surface. We sought to determine whether associations with other OHC-specific proteins could account for this difference. Using a yeast two-hybrid technique, we found microtubule-associated protein 1S (MAP1S) bound to prestin. The interaction was limited to the STAS domain of prestin and the region connecting the heavy and light chain of MAP1S. Using reciprocal immunoprecipitation and Forster resonance energy transfer, we confirmed these interactions. Furthermore, co-expression of prestin with MAP1S resulted in a 2.7-fold increase in Q(sp) in single cells that was paralleled by a 2.8-fold increase in protein surface expression, indicating that the interactions are physiological. Quantitative PCR data showed gradients in the expression of prestin and MAP1S across the tonotopic axis that may partially contribute to a previously observed 6-fold increase in Q(sp) in high frequency hair cells. These data highlight the importance of protein partner effects on prestin.
Asunto(s)
Proteínas de Transporte de Anión/genética , Células Ciliadas Auditivas Externas/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Animales , Proteínas de Transporte de Anión/metabolismo , Encéfalo/metabolismo , Cóclea/anatomía & histología , Cóclea/efectos de los fármacos , Cóclea/fisiología , Colchicina/farmacología , Cartilla de ADN , Transferencia Resonante de Energía de Fluorescencia , Gerbillinae , Cobayas , Células Ciliadas Auditivas Externas/citología , Células Ciliadas Auditivas Externas/efectos de los fármacos , Especificidad de Órganos , Órgano Espiral/fisiología , Reacción en Cadena de la Polimerasa , Proteínas/genética , Proteínas/metabolismo , Ratas , Transportadores de SulfatoRESUMEN
Prestin is a member of the SLC26 family of anion transporters and is responsible for electromotility in outer hair cells, the basis of cochlear amplification in mammals. It is an anion transporting transmembrane protein, possessing nine cysteine residues, which generates voltage-dependent charge movement. We determine the role these cysteine residues play in the voltage sensing capabilities of prestin. Mutations of any single cysteine residue had little or no effect on charge movement. However, using combinatorial substitution mutants, we identified a cysteine residue pair (C415 and either C192 or C196) whose mutation reduced or eliminated charge movement. Furthermore, we show biochemically that surface expression of mutants with markedly reduced functionality can be near normal; however, we identify two monomers of the protein on the surface of the cell, the larger of which correlates with surface charge movement. Because we showed previously by Förster resonance energy transfer that monomer interactions are required for charge movement, we tested whether disulfide interactions were required for dimerization. Using Western blots to detect oligomerization of the protein in which variable numbers of cysteines up to and including all nine cysteine residues were mutated, we show that disulfide bond formation is not essential for dimer formation. Taken together, we believe these data indicate that intramembranous cysteines are constrained, possibly via disulfide bond formation, to ensure structural features of prestin required for normal voltage sensing and mechanical activity.
Asunto(s)
Sustitución de Aminoácidos , Proteínas de Transporte de Anión/química , Proteínas de Transporte de Anión/metabolismo , Cisteína/metabolismo , Conductividad Eléctrica , Animales , Proteínas de Transporte de Anión/genética , Células CHO , Cricetinae , Cricetulus , Cisteína/genética , Disulfuros/química , Capacidad Eléctrica , Gerbillinae , Mutación , Pliegue de Proteína , Multimerización de Proteína , Estructura Cuaternaria de ProteínaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Large-conductance calcium-activated potassium (BK) channels play a critical role in electrical resonance, a mechanism of frequency selectivity in chicken hair cells. We determine that BK currents are dependent on inward flow of Ca2+ , and intracellular buffering of Ca2+ . Entry of Ca2+ is further amplified locally by calcium-induced Ca2+ release (CICR) in close proximity to plasma membrane BK channels. Ca2+ imaging reveals peripheral clusters of high concentrations of Ca2+ that are suprathreshold to that needed to activate BK channels. Protein kinase A (PKA) activation increases the size of BK currents likely by recruiting more BK channels due to spatial spread of high Ca2+ concentrations in turn from increasing CICR. STORM imaging confirms the presence of nanodomains with ryanodine and IP3 receptors in close proximity to the Slo subunit of BK channels. Together, these data require a rethinking of how electrical resonance is brought about and suggest effects of CICR in synaptic release. Both genders were included in this study.
Asunto(s)
Proteínas Aviares/metabolismo , Señalización del Calcio , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células Ciliadas Auditivas/metabolismo , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Animales , Células Cultivadas , Pollos , Femenino , Células Ciliadas Auditivas/fisiología , Masculino , Potenciales de la MembranaRESUMEN
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.
Asunto(s)
Células Ciliadas Auditivas Externas , Proteínas , Animales , Proteínas de Transporte de Anión , Membrana Celular/metabolismo , Capacidad Eléctrica , Células Ciliadas Auditivas Externas/metabolismo , Potenciales de la Membrana , Técnicas de Placa-Clamp , Proteínas/metabolismoRESUMEN
Naked mole-rats are highly vocal, eusocial, subterranean rodents with, counterintuitively, poor hearing. The causes underlying their altered hearing are unknown. Moreover, whether altered hearing is degenerate or adaptive to their unique lifestyles is controversial. We used various methods to identify the factors contributing to altered hearing in naked and the related Damaraland mole-rats and to examine whether these alterations result from relaxed or adaptive selection. Remarkably, we found that cochlear amplification was absent from both species despite normal prestin function in outer hair cells isolated from naked mole-rats. Instead, loss of cochlear amplification appears to result from abnormal hair bundle morphologies observed in both species. By exploiting a well-curated deafness phenotype-genotype database, we identified amino acid substitutions consistent with abnormal hair bundle morphology and reduced hearing sensitivity. Amino acid substitutions were found in unique groups of six hair bundle link proteins. Molecular evolutionary analyses revealed shifts in selection pressure at both the gene and the codon level for five of these six hair bundle link proteins. Substitutions in three of these proteins are associated exclusively with altered hearing. Altogether, our findings identify the likely mechanism of altered hearing in African mole-rats, making them the only identified mammals naturally lacking cochlear amplification. Moreover, our findings suggest that altered hearing in African mole-rats is adaptive, perhaps tailoring hearing to eusocial and subterranean lifestyles. Finally, our work reveals multiple, unique evolutionary trajectories in African mole-rat hearing and establishes species members as naturally occurring disease models to investigate human hearing loss.
Asunto(s)
Adaptación Fisiológica/genética , Sordera/genética , Evolución Molecular , Audición/genética , Ratas Topo/fisiología , África , Sustitución de Aminoácidos , Animales , Células Ciliadas Auditivas/fisiología , Células Ciliadas Auditivas/ultraestructura , Microscopía Electrónica de Rastreo , Selección GenéticaRESUMEN
The integral membrane protein prestin, a member of the SLC26 anion transporter family, is responsible for the voltage-driven electromotility of mammalian outer hair cells. It was argued that the evolution of prestin's motor function required a loss of the protein's transport capabilities. Instead, it was proposed that prestin manages only an abortive hemicycle that results in the trapped anion acting as a voltage sensor, to generate the motor's signature gating charge movement or nonlinear capacitance. We demonstrate, using classical radioactive anion ([(14)C]formate and [(14)C]oxalate) uptake studies, that in contrast to previous observations, prestin is able to transport anions. The prestin-dependent uptake of both these anions was twofold that of cells transfected with vector alone, and comparable to SLC26a6, prestin's closest phylogenetic relative. Furthermore, we identify a potential chloride-binding site in which the mutations of two residues (P328A and L326A) preserve nonlinear capacitance, yet negate anion transport. Finally, we distinguish 12 charged residues out of 22, residing within prestin's transmembrane regions, that contribute to unitary charge movement, i.e., voltage sensing. These data redefine our mechanistic concept of prestin.
Asunto(s)
Proteínas de Transporte de Anión/metabolismo , Transporte Iónico/fisiología , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Análisis de Varianza , Animales , Proteínas de Transporte de Anión/genética , Antiportadores/metabolismo , Células CHO , Radioisótopos de Carbono , Cloruros/metabolismo , Cricetinae , Cricetulus , Capacidad Eléctrica , Formiatos/metabolismo , Gerbillinae , Transporte Iónico/efectos de los fármacos , Ratones , Mutación Missense , Oxalatos/metabolismo , Técnicas de Placa-Clamp , Salicilatos/farmacología , Transportadores de SulfatoRESUMEN
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.
Asunto(s)
Capacidad Eléctrica , Regulación del Desarrollo de la Expresión Génica , Células Ciliadas Auditivas Externas/metabolismo , Audición/fisiología , Mecanotransducción Celular , Proteínas Motoras Moleculares/metabolismo , Órgano Espiral/metabolismo , Animales , Animales Recién Nacidos , Vías Auditivas , Células Ciliadas Auditivas Externas/citología , Cinética , Ratones , Ratones Endogámicos C57BL , Órgano Espiral/citologíaRESUMEN
Paradiplozoon yunnanensis n. sp. (Monogenea, Diplozoidae) is described from the gills of Sikukia gudgeri Smith, 1931 (Cyprinidae) collected from Jinghong Basin, a tributary of the international Lancang-Mekong River. This is the first diplozoid species from S. gudgeri and its description increases the number of Paradiplozoon species recorded in China to 25. The new species is distinguished from congeners by a combination of morphological and molecular features. The anterior end of the median plate is thickened in the marginal area and a narrow rectangular trapeze spur connects to the anterior jaw through two separate anterior joining sclerites. The posterior end of the median plate sclerite is invaginated with a smooth strip-shaped posterior joining sclerite. Comparison of a newly obtained sequence of rRNA ITS2 with 18 other congeneric sequences from GenBank provides support for separation of the new species.
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Cyprinidae/parasitología , Enfermedades de los Peces/epidemiología , Trematodos/anatomía & histología , Trematodos/aislamiento & purificación , Infecciones por Trematodos/epidemiología , Animales , China/epidemiología , ADN de Helmintos/genética , Enfermedades de los Peces/parasitología , Branquias/parasitología , Filogenia , ARN Ribosómico/genética , Ríos/parasitología , Trematodos/clasificación , Trematodos/genéticaRESUMEN
Prestin in the lateral membrane of outer hair cells, is responsible for electromotility (EM) and a corresponding nonlinear capacitance (NLC). Prestin's voltage sensitivity is influenced by intracellular chloride. A regulator of intracellular chloride is a stretch-sensitive, non-selective conductance within the lateral membrane, GmetL. We determine that prestin itself possesses a stretch-sensitive, non-selective conductance that is largest in the presence of thiocyanate ions. This conductance is independent of the anion transporter mechanism. Prestin has been modeled, based on structural data from related anion transporters (SLC26Dg and UraA), to have a 7 + 7 inverted repeat structure with anion transport initiated by chloride binding at the intracellular cleft. Mutation of residues that bind intracellular chloride, and salicylate treatment which prevents chloride binding, have no effect on thiocyanate conductance. In contrast, other mutations reduce the conductance while preserving NLC. When superimposed on prestin's structure, the location of these mutations indicates that the ion permeation pathway lies between the core and gate ring of helices, distinct from the transporter pathway. The uncoupled current is reminiscent of an omega current in voltage-gated ion channels. We suggest that prestin itself is the main regulator of intracellular chloride concentration via a route distinct from its transporter pathway.
Asunto(s)
Activación del Canal Iónico/genética , Transportadores de Sulfato , Animales , Células CHO , Cricetulus , Células HEK293 , Humanos , Transporte Iónico/genética , Mutación , Estructura Secundaria de Proteína , Transportadores de Sulfato/química , Transportadores de Sulfato/genética , Transportadores de Sulfato/metabolismoRESUMEN
Prestin, the transmembrane motor protein is a novel protein underlying the motility of the outer hair cells. Nonlinear capacitance (NLC) or gating charge current, which can be observed in both auditory and transfected non-auditory cells, is the electrical signature of prestin's electromechanical activity. To test the functional role of the C-terminus of prestin, several charged residue clusters were reversed en-block by site-directed mutagenesis. They are D/E to K at 516, 518, 522, 524, 527, 528 and 531 (cluster a); R/K to D at 571, 572, 573, 576, 577 and 580 (cluster b); R to D at 571; and E/D to K at 608, 609, 610, 611, 612 and 613 (cluster c). These constructs were transfected into Chinese hamster ovary cells (CHO) and NLC recordings were performed to evaluate the effects of these charge substitutions. All of the mutants showed NLC. Charge cluster a reversal significantly reduced the maximum charge movement (Qmax). All but one mutation (charge cluster c reversal) shifted V(h), indicative of the operating voltage range, in the depolarizing direction. None of the mutations affected unitary charge movement (z). These data suggest that the C-terminus of prestin lies outside the membrane voltage field, and may play an important role in controlling the operating voltage range through control of the protein's conformational energy profile via allosteric means.