RESUMEN
Mitochondrial dysfunction causes poorly understood tissue-specific pathology stemming from primary defects in respiration, coupled with altered reactive oxygen species (ROS), metabolic signaling, and apoptosis. The A1555G mtDNA mutation that causes maternally inherited deafness disrupts mitochondrial ribosome function, in part, via increased methylation of the mitochondrial 12S rRNA by the methyltransferase mtTFB1. In patient-derived A1555G cells, we show that 12S rRNA hypermethylation causes ROS-dependent activation of AMP kinase and the proapoptotic nuclear transcription factor E2F1. This retrograde mitochondrial-stress relay is operative in vivo, as transgenic-mtTFB1 mice exhibit enhanced 12S rRNA methylation in multiple tissues, increased E2F1 and apoptosis in the stria vascularis and spiral ganglion neurons of the inner ear, and progressive E2F1-dependent hearing loss. This mouse mitochondrial disease model provides a robust platform for deciphering the complex tissue specificity of human mitochondrial-based disorders, as well as the precise pathogenic mechanism of maternally inherited deafness and its exacerbation by environmental factors.
Asunto(s)
Sordera/metabolismo , Modelos Animales de Enfermedad , Factor de Transcripción E2F1/metabolismo , Animales , ADN Mitocondrial/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Oído Interno/patología , Ganglión/patología , Humanos , Ratones , Ratones Transgénicos , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Mutación , Neuronas/patología , ARN Ribosómico/metabolismo , Especies Reactivas de Oxígeno , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
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
Outer hair cell (OHC) nonlinear membrane capacitance derives from voltage-dependent sensor charge movements within the membrane protein prestin (SLC26a5) that drive OHC electromotility. The ability of the protein to influence hearing depends on its reaction to membrane receptor potentials across auditory frequency. Estimates of prestin's frequency response have been evaluated by several groups out to tens of kHz in voltage-clamped macro-patches of OHC membrane. The response is a power function of frequency that is down 40 dB at 77 kHz. Despite these observations, concerns remain that the macro-patch approach is flawed due to mechanical constraints of pipette solution column load or patch size itself. In the absence of these influences, prestin's frequency response is posited by some to be ultrasonic in nature. Here we evaluate the influence of these putative confounding factors on prestin's frequency response. We show that neither pipette column height nor negative or positive pipette pressure substantially influence total sensor charge frequency response. Additionally, patch surface area has negligible influence. We conclude that the speed of voltage-driven conformational changes in prestin within the plasma membrane is accurately assessed with the macro-patch technique, permitting investigations of membrane characteristics that can substantially alter prestin's performance bandwidth. We illustrate significant alterations in bandwidth by perturbation of membrane fluidity and chloride anion concentration. Finally, we speculate that OHC membrane characteristics may differ along the tonotopic axis of the cochlea to tune nonlinear membrane capacitance frequency cutoffs.
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Células Ciliadas Auditivas Externas , Proteínas , Membrana Celular/metabolismo , Capacidad Eléctrica , Células Ciliadas Auditivas Externas/metabolismo , Potenciales de la Membrana/fisiología , Técnicas de Placa-Clamp , Proteínas/metabolismoRESUMEN
Fragile X syndrome (FXS) is characterized by hypersensitivity to sensory stimuli, including environmental sounds. We compared the auditory brainstem response (ABR) recorded in vivo in mice lacking the gene (Fmr1-/y ) for fragile X mental retardation protein (FMRP) with that in wild-type animals. We found that ABR wave I, which represents input from the auditory nerve, is reduced in Fmr1-/y animals, but only at high sound levels. In contrast, wave IV, which represents the activity of auditory brainstem nuclei is enhanced at all sound levels, suggesting that loss of FMRP alters the central processing of auditory signals. Current-clamp recordings of neurons in the medial nucleus of the trapezoid body in the auditory brainstem revealed that, in contrast to neurons from wild-type animals, sustained depolarization triggers repetitive firing rather than a single action potential. In voltage-clamp recordings, K+ currents that activate at positive potentials ("high-threshold" K+ currents), which are required for high-frequency firing and are carried primarily by Kv3.1 channels, are elevated in Fmr1-/y mice, while K+ currents that activate near the resting potential and inhibit repetitive firing are reduced. We therefore tested the effects of AUT2 [((4-({5-[(4R)-4-ethyl-2,5-dioxo-1-imidazolidinyl]-2-pyridinyl}oxy)-2-(1-methylethyl) benzonitrile], a compound that modulates Kv3.1 channels. AUT2 reduced the high-threshold K+ current and increased the low-threshold K+ currents in neurons from Fmr1-/y animals by shifting the activation of the high-threshold current to more negative potentials. This reduced the firing rate and, in vivo, restored wave IV of the ABR. Our results from animals of both sexes suggest that the modulation of the Kv3.1 channel may have potential for the treatment of sensory hypersensitivity in patients with FXS.SIGNIFICANCE STATEMENT mRNA encoding the Kv3.1 potassium channel was one of the first described targets of the fragile X mental retardation protein (FMRP). Fragile X syndrome is caused by loss of FMRP and, in humans and mice, causes hypersensitivity to auditory stimuli. We found that components of the auditory brain response (ABR) corresponding to auditory brainstem activity are enhanced in mice lacking FMRP. This is accompanied by hyperexcitability and altered potassium currents in auditory brainstem neurons. Treatment with a drug that alters the voltage dependence of Kv3.1 channels normalizes the imbalance of potassium currents, as well as ABR responses in vivo, suggesting that such compounds may be effective in treating some symptoms of fragile X syndrome.
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Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/metabolismo , Canales de Potasio Shaw/metabolismo , Animales , Vías Auditivas , Percepción Auditiva , Tronco Encefálico/efectos de los fármacos , Núcleo Coclear/fisiología , Fenómenos Electrofisiológicos , Potenciales Evocados Auditivos del Tronco Encefálico/efectos de los fármacos , Potenciales Evocados Auditivos del Tronco Encefálico/genética , Femenino , Síndrome del Cromosoma X Frágil/tratamiento farmacológico , Síndrome del Cromosoma X Frágil/genética , Hidantoínas/farmacología , Técnicas In Vitro , Masculino , Ratones , Ratones Noqueados , Técnicas de Placa-Clamp , Piridinas/farmacologíaRESUMEN
The voltage-dependent protein SLC26a5 (prestin) underlies outer hair cell electromotility (eM), which is responsible for cochlear amplification in mammals. The electrical signature of eM is a bell-shaped nonlinear capacitance (NLC), deriving from prestin sensor-charge (Qp) movements, which peaks at the membrane voltage, Vh, where charge is distributed equally on either side of the membrane. Voltage dependencies of NLC and eM differ depending on interrogation frequency and intracellular chloride, revealing slow intermediate conformational transitions between anion binding and voltage-driven Qp movements. Consequently, NLC exhibits low-pass characteristics, substantially below prevailing estimates of eM frequency response. Here we study in guinea pig and mouse of either sex synchronous prestin electrical (NLC, Qp) and mechanical (eM) activity across frequencies under voltage clamp (whole cell and microchamber). We find that eM and Qp magnitude and phase correspond, indicating tight piezoelectric coupling. Electromechanical measures (both NLC and eM) show dual-Lorentzian, low-pass behavior, with a limiting (τ2) time constant at Vh of 32.6 and 24.8 µs, respectively. As expected for voltage-dependent kinetics, voltage excitation away from Vh has a faster, flatter frequency response, with our fastest measured τ2 for eM of 18.2 µs. Previous observations of ultrafast eM (τ ≈ 2 µs) were obtained at offsets far removed from Vh We hypothesize that trade-offs in eM gain-bandwith arising from voltage excitation at membrane potentials offset from Vh influence the effectiveness of cochlear amplification across frequencies.SIGNIFICANCE STATEMENT Of two types of hair cells within the organ of Corti, inner hair cells and outer hair cells, the latter evolved to boost sensitivity to sounds. Damage results in hearing loss of 40-60 dB, revealing amplification gains of 100-1000× that arise from voltage-dependent mechanical responses [electromotility (eM)]. eM, driven by the membrane protein prestin, may work beyond 70 kHz. However, this speed exceeds, by over an order of magnitude, kinetics of typical voltage-dependent membrane proteins. We find eM is actually low pass in nature, indicating that prestin bears kinetics typical of other membrane proteins. These observations highlight potential difficulties in providing sufficient amplification beyond a cutoff frequency near 20 kHz. Nevertheless, observed trade-offs in eM gain-bandwith may sustain cochlear amplification across frequency.
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Percepción Auditiva/fisiología , Células Ciliadas Auditivas Externas/fisiología , Mecanotransducción Celular/fisiología , Proteínas Motoras Moleculares/fisiología , Proteínas/fisiología , Animales , Femenino , Cobayas , Cinética , Masculino , RatonesRESUMEN
In general, SLC26 solute carriers serve to transport a variety of anions across biological membranes. However, prestin (SLC26a5) has evolved, now serving as a motor protein in outer hair cells (OHCs) of the mammalian inner ear and is required for cochlear amplification, a mechanical feedback mechanism to boost auditory performance. The mechanical activity of the OHC imparted by prestin is driven by voltage and controlled by anions, chiefly intracellular chloride. Current opinion is that chloride anions control the Boltzmann characteristics of the voltage sensor responsible for prestin activity, including Qmax, the total sensor charge moved within the membrane, and Vh, a measure of prestin's operating voltage range. Here, we show that standard narrow-band, high-frequency admittance measures of nonlinear capacitance (NLC), an alternate representation of the sensor's charge-voltage (Q-V) relationship, is inadequate for assessment of Qmax, an estimate of the sum of unitary charges contributed by all voltage sensors within the membrane. Prestin's slow transition rates and chloride-binding kinetics adversely influence these estimates, contributing to the prevalent concept that intracellular chloride level controls the quantity of sensor charge moved. By monitoring charge movement across frequency, using measures of multifrequency admittance, expanded displacement current integration, and OHC electromotility, we find that chloride influences prestin kinetics, thereby controlling charge magnitude at any particular frequency of interrogation. Importantly, however, this chloride dependence vanishes as frequency decreases, with Qmax asymptoting at a level irrespective of the chloride level. These data indicate that prestin activity is significantly low-pass in the frequency domain, with important implications for cochlear amplification. We also note that the occurrence of voltage-dependent charge movements in other SLC26 family members may be hidden by inadequate interrogation timescales, and that revelation of such activity could highlight an evolutionary means for kinetic modifications within the family to address hearing requirements in mammals.
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Proteínas de Transporte de Anión/metabolismo , Cloruros/metabolismo , Células Ciliadas Auditivas Externas/fisiología , Mecanotransducción Celular/fisiología , Potenciales de la Membrana/fisiología , Animales , Aniones/metabolismo , Células Cultivadas , Capacidad Eléctrica , Cobayas , Cinética , Modelos Lineales , Modelos Moleculares , Modelos Neurológicos , Dinámicas no Lineales , Técnicas de Placa-ClampRESUMEN
The A1555G mutation in the 12S rRNA gene of human mitochondrial DNA causes maternally inherited, nonsyndromic deafness, an extreme case of tissue-specific mitochondrial pathology. A transgenic mouse strain that robustly overexpresses the mitochondrial 12S ribosomal RNA methyltransferase TFB1M (Tg-mtTFB1 mice) exhibits progressive hearing loss that we proposed models aspects of A1555G-related pathology in humans. Although our previous studies of Tg-mtTFB1 mice implicated apoptosis in the spiral ganglion and stria vascularis because of mitochondrial reactive oxygen species-mediated activation of AMP kinase (AMPK) and the nuclear transcription factor E2F1, detailed auditory pathology was not delineated. Herein, we show that Tg-mtTFB1 mice have reduced endocochlear potential, indicative of significant stria vascularis dysfunction, but without obvious signs of strial atrophy. We also observed decreased auditory brainstem response peak 1 amplitude and prolonged wave I latency, consistent with apoptosis of spiral ganglion neurons. Although no major loss of hair cells was observed, there was a mild impairment of voltage-dependent electromotility of outer hair cells. On the basis of these results, we propose that these events conspire to produce the progressive hearing loss phenotype in Tg-mtTFB1 mice. Finally, genetically reducing AMPK α1 rescues hearing loss in Tg-mtTFB1 mice, confirming that aberrant up-regulation of AMPK signaling promotes the observed auditory pathology. The relevance of these findings to human A1555G patients and the potential therapeutic value of reducing AMPK activity are discussed.
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Sordera/patología , Enfermedades Mitocondriales/patología , Proteínas Quinasas Activadas por AMP/deficiencia , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/fisiología , Animales , Apoptosis/fisiología , ADN Mitocondrial/genética , Sordera/genética , Sordera/fisiopatología , Modelos Animales de Enfermedad , Potenciales Evocados Auditivos del Tronco Encefálico/fisiología , Células Ciliadas Auditivas Internas/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/fisiopatología , Mutación , Órgano Espiral/patología , Tiempo de Reacción/fisiología , Transducción de Señal/fisiología , Ganglio Espiral de la Cóclea/patología , Ganglio Espiral de la Cóclea/fisiopatología , Estría Vascular/patología , Factores de Transcripción/genéticaRESUMEN
Outer hair cells (OHCs) drive cochlear amplification that enhances our ability to detect and discriminate sounds. The motor protein, prestin, which evolved from the SLC26 anion transporter family, underlies the OHC's voltage-dependent mechanical activity (eM). Here we report on simultaneous measures of prestin's voltage-sensor charge movement (nonlinear capacitance, NLC) and eM that evidence disparities in their voltage dependence and magnitude as a function of intracellular chloride, challenging decades' old dogma that NLC reports on eM steady-state behavior. A very simple kinetic model, possessing fast anion-binding transitions and fast voltage-dependent transitions, coupled together by a much slower transition recapitulates these disparities and other biophysical observations on the OHC. The intermediary slow transition probably relates to the transporter legacy of prestin, and this intermediary gateway, which shuttles anion-bound molecules into the voltage-enabled pool of motors, provides molecular delays that present as phase lags between membrane voltage and eM. Such phase lags may help to effectively inject energy at the appropriate moment to enhance basilar membrane motion.
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Proteínas de Transporte de Anión/química , Proteínas de Transporte de Anión/metabolismo , Proteínas/química , Proteínas/metabolismo , Animales , Fenómenos Biomecánicos , Fenómenos Biofísicos , Cloruros/metabolismo , Capacidad Eléctrica , Fenómenos Electrofisiológicos , Cobayas , Células Ciliadas Auditivas Externas/metabolismo , Cinética , Mecanotransducción Celular , Modelos Biológicos , Proteínas Motoras Moleculares/química , Proteínas Motoras Moleculares/metabolismoRESUMEN
The cylindrical outer hair cell (OHC) of Corti's organ drives cochlear amplification by a voltage-dependent activation of the molecular motor, prestin (SLC26a5), in the cell's lateral membrane. The voltage-dependent nature of this process leads to the troublesome observation that the membrane resistor-capacitor filter could limit high-frequency acoustic activation of the motor. Based on cable theory, the unique 30 nm width compartment (the extracisternal space, ECS) formed between the cell's lateral membrane and adjacent subsurface cisternae (SSC) could further limit the influence of receptor currents on lateral membrane voltage. Here, we use dual perforated/whole-cell and loose patch clamp on isolated OHCs to sequentially record currents resulting from excitation at apical, middle, and basal loose patch sites before and after perforated patch rupture. We find that timing of currents is fast and uniform before whole-cell pipette washout, suggesting little voltage attenuation along the length of the lateral membrane. Prior treatment with salicylate, a disrupter of the SSC, confirms the influence of the SSC on current spread. Finally, a cable model of the OHC, which can match our data, indicates that the SSC poses a minimal barrier to current flow across it, thereby facilitating rapid delivery of voltage excitation to the prestin-embedded lateral membrane.
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Fenómenos Electrofisiológicos , Células Ciliadas Auditivas Externas/fisiología , Animales , Electrodos , Fenómenos Electrofisiológicos/efectos de los fármacos , Cobayas , Células Ciliadas Auditivas Externas/efectos de los fármacos , Modelos Biológicos , Técnicas de Placa-Clamp , Factores de TiempoRESUMEN
The outer hair cell is electromotile, its membrane motor identified as the protein SLC26a5 (prestin). An area motor model, based on two-state Boltzmann statistics, was developed about two decades ago and derives from the observation that outer hair cell surface area is voltage-dependent. Indeed, aside from the nonlinear capacitance imparted by the voltage sensor charge movement of prestin, linear capacitance (Clin) also displays voltage dependence as motors move between expanded and compact states. Naturally, motor surface area changes alter membrane capacitance. Unit linear motor capacitance fluctuation (δCsa) is on the order of 140 zeptofarads. A recent three-state model of prestin provides an alternative view, suggesting that voltage-dependent linear capacitance changes are not real but only apparent because the two component Boltzmann functions shift their midpoint voltages (Vh) in opposite directions during treatment with salicylate, a known competitor of required chloride binding. We show here using manipulations of nonlinear capacitance with both salicylate and chloride that an enhanced area motor model, including augmented δCsa by salicylate, can accurately account for our novel findings. We also show that although the three-state model implicitly avoids measuring voltage-dependent motor capacitance, it registers δCsa effects as a byproduct of its assessment of Clin, which increases during salicylate treatment as motors are locked in the expanded state. The area motor model, in contrast, captures the characteristics of the voltage dependence of δCsa, leading to a better understanding of prestin.
Asunto(s)
Proteínas de Transporte de Anión/fisiología , Membrana Celular/metabolismo , Cloruros/química , Proteínas/metabolismo , Salicilatos/química , Animales , Proteínas de Transporte de Anión/genética , Capacidad Eléctrica , Electrofisiología , Cobayas , Células Ciliadas Auditivas Externas/metabolismo , Concentración 50 Inhibidora , Modelos QuímicosRESUMEN
Outer hair cells (OHC) possess voltage-dependent membrane bound molecular motors, identified as the solute carrier protein SLC26a5, that drive somatic motility at acoustic frequencies. The electromotility (eM) of OHCs provides for cochlear amplification, a process that enhances auditory sensitivity by up to three orders of magnitude. In this study, using whole cell voltage clamp and mechanical measurement techniques, we identify disparities between voltage sensing and eM that result from stretched exponential electromechanical behavior of SLC26a5, also known as prestin, for its fast responsiveness. This stretched exponential behavior, which we accurately recapitulate with a new kinetic model, the meno presto model of prestin, influences the protein's responsiveness to chloride binding and provides for delays in eM relative to membrane voltage driving force. The model predicts that in the frequency domain, these delays would result in eM phase lags that we confirm by measuring OHC eM at acoustic frequencies. These lags may contribute to canceling viscous drag, a requirement for many models of cochlear amplification.
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Movimiento Celular , Células Ciliadas Auditivas Externas/metabolismo , Potenciales de la Membrana , Proteínas/metabolismo , Animales , Cloruros/metabolismo , Cobayas , Células Ciliadas Auditivas Externas/fisiología , Unión Proteica , Estructura Terciaria de Proteína , Proteínas/química , Tiempo de Reacción , SonidoRESUMEN
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.
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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
Alterations in membrane capacitance can arise from linear and nonlinear sources. For example, changes in membrane surface area or dielectric properties can modify capacitance linearly, whereas sensor residues of voltage-dependent proteins can modify capacitance nonlinearly. Here, we examined the effects of fast temperature jumps induced by an infrared (IR) laser in control and prestin (SLC26a5)-transfected human embryonic kidney (HEK) cells under whole-cell voltage clamp. Prestin's voltage sensor imparts a characteristic bell-shaped, voltage-dependent nonlinear capacitance (NLC). Temperature jumps in control HEK cells cause a monophasic increase in membrane capacitance (Cm) regardless of holding voltage due to double-layer effects. Prestin-transfected HEK cells, however, additionally show a biphasic increase/decrease in Cm with a reversal potential corresponding to the voltage at peak NLC of prestin (Vh), attributable to a rapid temperature-following shift in Vh, with shift rates up to 14 V/s over the course of a 5 ms IR pulse. Treatment with salicylate, a known inhibitor of NLC, reestablishes control cell behavior. A simple kinetic model recapitulates our biophysical observations. These results verify a voltage-dependent protein's ability to respond to fast temperature perturbations on a par with double-layer susceptibility. This likely arises from prestin's unique ability to move sensor charge at kilohertz rates, which is required for the outer hair cells' role as a cochlear amplifier.
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Proteínas de Transporte de Anión/metabolismo , Rayos Infrarrojos , Rayos Láser , Capacidad Eléctrica , Células HEK293 , Humanos , Cinética , Modelos Biológicos , Transportadores de Sulfato , TemperaturaRESUMEN
Tightly regulated and robust mitochondrial activities are critical for normal hearing. Previously, we demonstrated that Fus1/Tusc2 KO mice with mitochondrial dysfunction exhibit premature hearing loss. Molecular analysis of the cochlea revealed hyperactivation of the mTOR pathway, oxidative stress, and altered mitochondrial morphology and quantity, suggesting compromised energy sensing and production. Here, we investigated whether the pharmacological modulation of metabolic pathways using rapamycin (RAPA) or 2-deoxy-D-glucose (2-DG) supplementation can protect against hearing loss in female Fus1 KO mice. Additionally, we aimed to identify mitochondria- and Fus1/Tusc2-dependent molecular pathways and processes critical for hearing. We found that inhibiting mTOR or activating alternative mitochondrial energetic pathways to glycolysis protected hearing in the mice. Comparative gene expression analysis revealed the dysregulation of critical biological processes in the KO cochlea, including mitochondrial metabolism, neural and immune responses, and the cochlear hypothalamic-pituitary-adrenal axis signaling system. RAPA and 2-DG mostly normalized these processes, although some genes showed a drug-specific response or no response at all. Interestingly, both drugs resulted in a pronounced upregulation of critical hearing-related genes not altered in the non-treated KO cochlea, including cytoskeletal and motor proteins and calcium-linked transporters and voltage-gated channels. These findings suggest that the pharmacological modulation of mitochondrial metabolism and bioenergetics may restore and activate processes critical for hearing, thereby protecting against hearing loss.
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Non-spiking sensory hair cells of the auditory and vestibular systems encode a dynamic range of graded signals with high fidelity by vesicle exocytosis at ribbon synapses. Ribeye, the most abundant protein in the synaptic ribbon, is composed of a unique A domain specific for ribbons and a B-domain nearly identical to the transcriptional corepressor CtBP2. CTBP2 and the B-domain of Ribeye contain a surface cleft that binds to proteins harboring a PXDLS/T peptide motif. Little is known about the importance of this binding site in synaptic function. Piccolo has a well-conserved PVDLT motif and we find that overexpressed Ribeye exhibits striking co-localization with Piccolo in INS-cells, while two separate mutants containing mutations in PXDLS/T-binding region, fail to co-localize with Piccolo. Similarly, co-transfected Ribeye and a piccolo fragment containing the PVDLT region co-localize in HEK cells. Expression of wild-type Ribeye-YFP in zebrafish neuromast hair cells returns electron densities to ribbon structures and mostly rescued normal synaptic transmission and morphological phenotypes in a mutant zebrafish lacking most Ribeye. By contrast, Ribeye-YFP harboring a mutation in the PXDLS/T-binding cleft resulted in ectopic electron dense aggregates that did not collect vesicles and the persistence of ribbons lacking electron densities. Furthermore, overexpression failed to return capacitance responses to normal levels. These results point toward a role for the PXDLS/T-binding cleft in the recruitment of Ribeye to ribbons and in normal synaptic function.
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Outer hair cells (OHC) function as both receptors and effectors in providing a boost to auditory reception. Amplification is driven by the motor protein prestin, which is under anionic control. Interestingly, we now find that the major, 4-AP-sensitive, outward K(+) current of the OHC (I(K)) is also sensitive to Cl(-), although, in contrast to prestin, extracellularly. I(K) is inhibited by reducing extracellular Cl(-) levels, with a linear dependence of 0.4%/mM. Other voltage-dependent K(+) (Kv) channel conductances in supporting cells, such as Hensen and Deiters' cells, are not affected by reduced extracellular Cl(-). To elucidate the molecular basis of this Cl(-)-sensitive I(K), we looked at potential molecular candidates based on Cl(-) sensitivity and/or similarities in kinetics. For I(K), we identified three different Ca(2+)-independent components of I(K) based on the time constant of inactivation: a fast, transient outward current, a rapidly activating, slowly inactivating current (Ik(1)), and a slowly inactivating current (Ik(2)). Extracellular Cl(-) differentially affects these components. Because the inactivation time constants of Ik(1) and Ik(2) are similar to those of Kv1.5 and Kv2.1, we transiently transfected these constructs into CHO cells and found that low extracellular Cl(-) inhibited both channels with linear current reductions of 0.38%/mM and 0.49%/mM, respectively. We also tested heterologously expressed Slick and Slack conductances, two intracellularly Cl(-)-sensitive K(+) channels, but found no extracellular Cl(-) sensitivity. The Cl(-) sensitivity of Kv2.1 and its robust expression within OHCs verified by single-cell RT-PCR indicate that these channels underlie the OHC's extracellular Cl(-) sensitivity.
Asunto(s)
Cloruros/fisiología , Líquido Extracelular/fisiología , Células Ciliadas Auditivas Externas/fisiología , Canales de Potasio Shab/fisiología , Animales , Células CHO , Cricetinae , Cricetulus , CobayasRESUMEN
The outer hair cell (OHC) is celebrated on the 21st birthday of prestin's identification and the year of this molecular motor's sub-nanometer structural solution. Dogmatic conceptions of OHC performance have been challenged by decades of biophysical interrogations that must be influential on hearing, but which have received little attention by cochlear modelers. Here we point to these interrogations and present a compilation of articles in a Special Issue of Hearing Research that reconsiders the OHC's role in cochlear amplification, as well as the cell's basic physiology. We are getting closer to understanding these special cells of Dallos.
Asunto(s)
Células Ciliadas Auditivas Externas , Células Ciliadas Vestibulares , Biofisica , Cóclea/fisiología , Células Ciliadas Auditivas Externas/fisiología , Audición/fisiologíaRESUMEN
The OHC drives cochlear amplification, and prestin activity is the basis. The frequency response of nonlinear capacitance (NLC), which is a ratiometric measure of prestin's voltage-sensor charge movement (dQp/dVm), depends on the location of AC voltage excitation along prestin's operating voltage range, being slowest at the voltage (Vh) where NLC peaks. Here we directly investigate the coupling between prestin charge movement (Qp) and electromotility (eM) at frequencies up to 6.25 kHz, and find tight correspondence between the two at operating voltages displaced from Vh. Near Vh, however, eM shows a slower frequency response than Qp. We reason that coupling is more susceptible to molecular/cellular loads at Vh, where prestin compliance is expected to be maximal. Recent cryo-EM studies have begun to shed light on structural features of prestin that impact its performance against loads. This article is part of the Special Issue Outer hair cell Edited by Joseph Santos-Sacchi and Kumar Navaratnam.
Asunto(s)
Células Ciliadas Auditivas Externas , Células Ciliadas Vestibulares , Cóclea , Capacidad Eléctrica , Células Ciliadas Auditivas Externas/fisiología , Técnicas de Placa-ClampRESUMEN
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.