Atomic force microscopy imaging of the structure of the motor protein prestin reconstituted into an artificial lipid bilayer

Prestin is the motor protein of cochlear outer hair cells and is essential for mammalian hearing. The present study aimed to clarify the structure of prestin by atomic force microscopy (AFM). Prestin was purified from Chinese hamster ovary cells which had been modified to stably express prestin, and then reconstituted into an artificial lipid bilayer. Immunofluorescence staining with anti-prestin antibody showed that the cytoplasmic side of prestin was possibly face up in the reconstituted lipid bilayer. AFM observation indicated that the cytoplasmic surface of prestin was ring-like with a diameter of about 11 nm.     

Reduction in Noise-Induced Functional Loss of the Cochleae in Mice with Pre-Existing Cochlear Dysfunction Due to Genetic Interference of Prestin

Various cochlear pathologies, such as acoustic trauma, ototoxicity and age-related degeneration, cause hearing loss. These pre-existing hearing losses can alter cochlear responses to subsequent acoustic overstimulation. So far, the knowledge on the impacts of pre-existing hearing loss caused by genetic alteration of cochlear genes is limited. Prestin is the motor protein expressed exclusively in outer hair cells in the mammalian cochlea. This motor protein contributes to outer hair cell motility. At present, it is not clear how the interference of prestin function affects cochlear responses to acoustic overstimulation. To address this question, a genetic model of prestin dysfunction in mice was created by inserting an internal ribosome entry site (IRES)-CreER^T2-FRT-Neo-FRT cassette into the prestin locus after the stop codon. Homozygous mice exhibit a threshold elevation of auditory brainstem responses with large individual variation. These mice also display a threshold elevation and a shift of the input/output function of the distortion product otoacoustic emission, suggesting a reduction in outer hair cell function. The disruption of prestin function reduces the threshold shifts caused by exposure to a loud noise at 120 dB (sound pressure level) for 1 h. This reduction is positively correlated with the level of pre-noise cochlear dysfunction and is accompanied by a reduced change in Cdh1 expression, suggesting a reduction in molecular responses to the acoustic overstimulation. Together, these results suggest that prestin interference reduces cochlear stress responses to acoustic overstimulation.     

IR Laser-Induced Perturbations of the Voltage-Dependent Solute Carrier Protein SLC26a5

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 (C_m) regardless of holding voltage due to double-layer effects. Prestin-transfected HEK cells, however, additionally show a biphasic increase/decrease in C_m with a reversal potential corresponding to the voltage at peak NLC of prestin (V_h), attributable to a rapid temperature-following shift in V_h, 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.     

Lizard and Frog Prestin: Evolutionary Insight into Functional Changes

The plasma membrane of mammalian cochlear outer hair cells contains prestin, a unique motor protein. Prestin is the fifth member of the solute carrier protein 26A family. Orthologs of prestin are also found in the ear of non-mammalian vertebrates such as zebrafish and chicken. However, these orthologs are electrogenic anion exchangers/transporters with no motor function. Amphibian and reptilian lineages represent phylogenic branches in the evolution of tetrapods and subsequent amniotes. Comparison of the peptide sequences and functional properties of these prestin orthologs offer new insights into prestin evolution. With the recent availability of the lizard and frog genome sequences, we examined amino acid sequence and function of lizard and frog prestins to determine how they are functionally and structurally different from prestins of mammals and other non-mammals. Somatic motility, voltage-dependent nonlinear capacitance (NLC), the two hallmarks of prestin function, and transport capability were measured in transfected human embryonic kidney cells using voltage-clamp and radioisotope techniques. We demonstrated that while the transport capability of lizard and frog prestin was compatible to that of chicken prestin, the NLC of lizard prestin was more robust than that of chicken’s and was close to that of platypus. However, unlike platypus prestin which has acquired motor capability, lizard or frog prestin did not demonstrate motor capability. Lizard and frog prestins do not possess the same 11-amino-acid motif that is likely the structural adaptation for motor function in mammals. Thus, lizard and frog prestins appear to be functionally more advanced than that of chicken prestin, although motor capability is not yet acquired.     

Expression, purification and characterisation of the C-terminal STAS domain of the SLC26 anion transporter prestin

The membrane protein prestin is the voltage-sensitive molecular motor underlying somatic electromotility of outer hair cells. In order to produce adequate quantities to perform structural and functional studies, we cloned and expressed in bacterial systems three variants of the cytosolic C-terminal STAS domain of prestin from Rattus norvegicus. While the expression level of the longer form of the C-terminal domain (fragment [505–744]) was very low or absent, we succeeded in the overexpression of two shorter fragment of the STAS domain (fragments [529–744], PreCD_L, and [529–720], PreCD_S). These two polypeptides were purified to homogeneity and characterised by circular dichroism, fluorescence spectroscopy and dynamic light scattering. The two proteins possess a three-dimensional structure and show a great tendency to aggregate. In particular, PreCD_L is present in solution mainly as dimers and tetramers. These data correlate with that of full-length prestin that forms stable tetramers, suggesting that the C-terminal domain play an important role in modulating the properties of the entire prestin.     

IR Laser-Induced Perturbations of the Voltage-Dependent Solute Carrier Protein SLC26a5

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 (C_m) regardless of holding voltage due to double-layer effects. Prestin-transfected HEK cells, however, additionally show a biphasic increase/decrease in C_m with a reversal potential corresponding to the voltage at peak NLC of prestin (V_h), attributable to a rapid temperature-following shift in V_h, 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.     

Prestin's role in cochlear frequency tuning and transmission of mechanical responses to neural excitation

The remarkable power amplifier [1] of the cochlea boosts low-level and compresses high-level vibrations of the basilar membrane (BM) [2]. By contributing maximally at the characteristic frequency (CF) of each point along its length, the amplifier ensures the exquisite sensitivity, narrow frequency tuning, and enormous dynamic range of the mammalian cochlea. The motor protein prestin in the outer hair cell (OHC) lateral membrane is a prime candidate for the cochlear power amplifier [3]. The other contender for this role is the ubiquitous calcium-mediated motility of the hair cell stereocilia, which has been demonstrated in vitro and is based on fast adaptation of the mechanoelectrical transduction channels [4, 5]. Absence of prestin [6] from OHCs results in a 40-60 dB reduction in cochlear neural sensitivity [7]. Here we show that sound-evoked BM vibrations in the high-frequency region of prestin(-/-) mice cochleae are, surprisingly, as sensitive as those of their prestin(+/+) siblings. The BM vibrations of prestin(-/-) mice are, however, broadly tuned to a frequency approximately a half octave below the CF of prestin(+/+) mice at similar BM locations. The peak sensitivity of prestin(+/+) BM tuning curves matches the neural thresholds. In contrast, prestin(-/-) BM tuning curves at their best frequency are >50 dB more sensitive than the neural responses. We propose that the absence of prestin from OHCs, and consequent reduction in stiffness of the cochlea partition, changes the passive impedance of the BM at high frequencies, including the CF. We conclude that prestin influences the cochlear partition's dynamic properties that permit transmission of its vibrations into neural excitation. Prestin is crucial for defining sharp and sensitive cochlear frequency tuning by reducing the sensitivity of the low-frequency tail of the tuning curve, although this necessitates a cochlear amplifier to determine the narrowly tuned tip.     

Salicylate-induced translocation of prestin having mutation in the GTSRH sequence to the plasma membrane

Prestin is a key molecule for mammalian hearing. The present study investigated changes in characteristics of prestin by culturing prestin-transfected cells with salicylate, an antagonist of prestin. As a result, the plasma membrane localization of prestin bearing a mutation in the GTSRH sequence, which normally accumulates in the cytoplasm, was recovered. Moreover, the nonlinear capacitance of the majority of the mutants, which is a signature of prestin activity, was also recovered. Thus, the present study discovered a new effect of salicylate on prestin, namely, the promotion of the plasma membrane expression of prestin mutants in an active state.     

Generation of somatic electromechanical force by outer hair cells may be influenced by prestin–CASK interaction at the basal junction with the Deiter’s cell

The motor protein, prestin, situated in the basolateral plasma membrane of cochlear outer hair cells (OHCs), underlies the generation of somatic, voltage-driven mechanical force, the basis for the exquisite sensitivity, frequency selectivity and dynamic range of mammalian hearing. The molecular and structural basis of the ontogenetic development of this electromechanical force has remained elusive. The present study demonstrates that this force is significantly reduced when the immature subcellular distribution of prestin found along the entire plasma membrane persists into maturity, as has been described in previous studies under hypothyroidism. This observation suggests that cochlear amplification is critically dependent on the surface expression and distribution of prestin. Searching for proteins involved in organizing the subcellular localization of prestin to the basolateral plasma membrane, we identified cochlear expression of a novel truncated prestin splice isoform named prestin 9b (Slc26A5d) that contains a putative PDZ domain-binding motif. Using prestin 9b as the bait in a yeast two-hybrid assay, we identified a calcium/calmodulin-dependent serine protein kinase (CASK) as an interaction partner of prestin. Co-immunoprecipitation assays showed that CASK and prestin 9b can interact with full-length prestin. CASK was co-localized with prestin in a membrane domain where prestin-expressing OHC membrane abuts prestin-free OHC membrane, but was absent from this area for thyroid hormone deficiency. These findings suggest that CASK and the truncated prestin splice isoform contribute to confinement of prestin to the basolateral region of the plasma membrane. By means of such an interaction, the basal junction region between the OHC and its Deiter’s cell may contribute to efficient generation of somatic electromechanical force.     

Membrane thickness sensitivity of prestin orthologs: the evolution of a piezoelectric protein

How proteins evolve new functionality is an important question in biology; prestin (SLC26A5) is a case in point. Prestin drives outer hair cell somatic motility and amplifies mechanical vibrations in the mammalian cochlea. The motility of mammalian prestin is analogous to piezoelectricity, in which charge transfer is coupled to changes in membrane area occupied by the protein. Intriguingly, nonmammalian prestin orthologs function as anion exchangers but are apparently nonmotile. We previously found that mammalian prestin is sensitive to membrane thickness, suggesting that prestin's extended conformation has a thinner hydrophobic height in the lipid bilayer. Because prestin-based motility is a mammalian specialization, we initially hypothesized that nonmotile prestin orthologs, while functioning as anion transporters, should be much less sensitive to membrane thickness. We found the exact opposite to be true. Chicken prestin was the most sensitive to thickness changes, displaying the largest shift in voltage dependence. Platypus prestin displayed an intermediate response to membrane thickness and gerbil prestin was the least sensitive. To explain these observations, we present a theory where force production, rather than displacement, was selected for the evolution of prestin as a piezoelectric membrane motor.     

Complex Dielectric Properties of Sulfate-Reducing Bacteria Suspensions

Sulfate-reducing bacteria (SRB) can potentially enhance the remediation of heavy metals in the subsurface. Previous geophysical research has demonstrated the sensitivity of electrical measurements to SRB-mediated mineral transformation in porous media. However, the inherent dielectric properties of SRB and their direct contribution to the electrical properties of porous media are poorly understood. We studied the complex dielectric properties of SRB (Desulfovibrio vulgaris) suspensions at different concentrations and at different growth stages using a two-electrode dielectric spectroscopy measurement over the frequency range of 20 Hz to 1 MHz. Our results show higher dielectric responses (relative dielectric permittivity, real and imaginary conductivity) occurred with higher bacteria concentration at frequencies <10 kHz. Additionally, permittivity and conductivity both decreased as cells aged from mid-log phase to late stationary phase. Our results suggest that dielectric spectroscopy measurements can be used to noninvasively monitor biomass and various growth stages of SRB. Our work advances the interpretation of electrical signals associated with SRB observed in the subsurface.     

Mutation-induced reinforcement of prestin-expressing cells

The motor protein prestin in cochlear outer hair cells is a member of the solute carrier 26 family, but among the proteins of that family, only prestin can confer the cells with nonlinear capacitance (NLC) and motility. In the present study, to clarify contributions of unique amino acids of prestin, namely, Met-122, Met-225 and Thr-428, to the characteristics of prestin, mutations were introduced into those amino acids. As a result, NLC remained unchanged by both replacement of Met-122 by isoleucine and that of Thr-428 by leucine, suggesting that those amino acids were not important for the generation of NLC. Surprisingly, the replacement of Met-225 by glutamine statistically increased NLC as well as the motility of prestin-expressing cells without an increase in the amount of prestin expression in the plasma membrane. This indicates that Met-225 in prestin somehow adjusts NLC and the motility of prestin-expressing cells.     

Cysteine Mutagenesis Reveals Transmembrane Residues Associated with Charge Translocation in Prestin

The solute carrier transmembrane protein prestin (SLC26A5) drives an active electromechanical transduction process in cochlear outer hair cells that increases hearing sensitivity and frequency discrimination in mammals. A large intramembraneous charge movement, the nonlinear capacitance (NLC), is the electrical signature of prestin function. The transmembrane domain (TMD) helices and residues involved in the intramembrane charge displacement remain unknown. We have performed cysteine-scanning mutagenesis with serine or valine replacement to investigate the importance of cysteine residues to prestin structure and function. The distribution of oligomeric states and membrane abundance of prestin was also probed to investigate whether cysteine residues participate in prestin oligomerization and/or NLC. Our results reveal that 1) Cys-196 (TMD 4) and Cys-415 (TMD 10) do not tolerate serine replacement, and thus maintaining hydrophobicity at these locations is important for the mechanism of charge movement; 2) Cys-260 (TMD 6) and Cys-381 (TMD 9) tolerate serine replacement and are probably water-exposed; and 3) if disulfide bonds are present, they do not serve a functional role as measured via NLC. These novel findings are consistent with a recent structural model, which proposes that prestin contains an occluded aqueous pore, and we posit that the orientations of transmembrane domain helices 4 and 10 are essential for proper prestin function.     

Prestin-based outer hair cell motility is necessary for mammalian cochlear amplification

It is a central tenet of cochlear neurobiology that mammalian ears rely on a local, mechanical amplification process for their high sensitivity and sharp frequency selectivity. While it is generally agreed that outer hair cells provide the amplification, two mechanisms have been proposed: stereociliary motility and somatic motility. The latter is driven by the motor protein prestin. Electrophysiological phenotyping of a prestin knockout mouse intimated that somatic motility is the amplifier. However, outer hair cells of knockout mice have significantly altered mechanical properties, making this mouse model unsatisfactory. Here, we study a mouse model without alteration to outer hair cell and organ of Corti mechanics or to mechanoelectric transduction, but with diminished prestin function. These animals have knockout-like behavior, demonstrating that prestin-based electromotility is required for cochlear amplification.     

Voltage and frequency dependence of prestin-associated charge transfer

Membrane protein prestin is a critical component of the motor complex that generates forces and dimensional changes in cells in response to changes in the cell membrane potential. In its native cochlear outer hair cell, prestin is crucial to the amplification and frequency selectivity of the mammalian ear up to frequencies of tens of kHz. Other cells transfected with prestin acquire voltage-dependent properties similar to those of the native cell. The protein performance is critically dependent on chloride ions, and intrinsic protein charges also play a role. We propose an electro-diffusion model to reveal the frequency and voltage dependence of electric charge transfer by prestin. The movement of the combined charge (i.e., anion and protein charges) across the membrane is described with a Fokker–Planck equation coupled to a kinetic equation that describes the binding of chloride ions to prestin. We found a voltage- and frequency-dependent phase shift between the transferred charge and the applied electric field that determines capacitive and resistive components of the transferred charge. The phase shift monotonically decreases from zero to −90° as a function of frequency. The capacitive component as a function of voltage is bell-shaped, and decreases with frequency. The resistive component is bell-shaped for both voltage and frequency. The capacitive and resistive components are similar to experimental measurements of charge transfer at high frequencies. The revealed nature of the transferred charge can help reconcile the high-frequency electrical and mechanical observations associated with prestin, and it is important for further analysis of the structure and function of this protein.     

Tuning of the outer hair cell motor by membrane cholesterol

Cholesterol affects diverse biological processes, in many cases by modulating the function of integral membrane proteins. We observed that alterations of cochlear cholesterol modulate hearing in mice. Mammalian hearing is powered by outer hair cell (OHC) electromotility, a membrane-based motor mechanism that resides in the OHC lateral wall. We show that membrane cholesterol decreases during maturation of OHCs. To study the effects of cholesterol on hearing at the molecular level, we altered cholesterol levels in the OHC wall, which contains the membrane protein prestin. We show a dynamic and reversible relationship between membrane cholesterol levels and voltage dependence of prestin-associated charge movement in both OHCs and prestin-transfected HEK 293 cells. Cholesterol levels also modulate the distribution of prestin within plasma membrane microdomains and affect prestin self-association in HEK 293 cells. These findings indicate that alterations in membrane cholesterol affect prestin function and functionally tune the outer hair cell.     

Effect of membrane mechanics on charge transfer by the membrane protein prestin

Prestin was found in the membrane of outer hair cells (OHCs) located in the cochlea of the mammalian inner ear. These cells convert changes in the membrane potential into dimensional changes and (if constrained) to an active electromechanical force. The OHCs provide the ear with the mechanism of amplification and frequency selectivity that is effective up to tens of kHz. Prestin is a crucial part of the motor complex driving OHCs. Other cells transfected with prestin acquire electromechanical properties similar to those in the native cell. While the mechanism of prestin has yet to be fully understood, the charge transfer is its critical component. Here we investigate the effect of the mechanics of the surrounding membrane on electric charge transfer by prestin. We simulate changes in the membrane mechanics via the corresponding changes in the free energy of the prestin system. The free energy gradient enters a Fokker-Planck equation that describes charge transfer in our model. We analyze the effects of changes in the membrane tension and membrane elastic moduli. In the case of OHC, we simulate changes in the longitudinal and/or circumferential stiffness of the cell’s orthotropic composite membrane. In the case of cells transfected with prestin, we vary the membrane areal modulus. As a result, we show the effects of the membrane mechanics on the probabilistic characteristics of prestin-associated charge transfer for both stationary and high-frequency conditions. We compare our computational results with the available experimental data and find good agreement with the experiment.     

An anion antiporter model of prestin, the outer hair cell motor protein

Cochlear amplification in mammalian hearing relies on an active mechanical feedback process generated by outer hair cells, driven by a protein, prestin (SLC26A5), in the lateral membrane. We have used kinetic models to understand the mechanism by which prestin might function. We show that the two previous hypotheses of prestin, which assume prestin cannot operate as a transporter, are insufficient to explain previously published data. We propose an alternative model of prestin as an electrogenic anion exchanger, exchanging one Cl(-) ion for one divalent or two monovalent anions. This model can reproduce the key aspects of previous experimental observations. The experimentally observed charge movements are produced by the translocation of one Cl(-) ion combined with intrinsic positively charged residues, while the transport of the counteranion is electroneutral. We tested the model with measurements of the Cl(-) dependence of charge movement, using SO(4)(2-) to replace Cl(-). The data was compatible with the predictions of the model, suggesting that prestin does indeed function as a transporter.     

Lateral wall protein content mediates alterations in cochlear outer hair cell mechanics before and after hearing onset

Specialized outer hair cells (OHCs) housed within the mammalian cochlea exhibit active, nonlinear, mechanical responses to auditory stimulation termed electromotility. The extraordinary frequency resolution capacity of the cochlea requires an exquisitely equilibrated mechanical system of sensory and supporting cells. OHC electromotile length change, stiffness, and force generation are responsible for a 100-fold increase in hearing sensitivity by augmenting vibrational input to non-motile sensory inner hair cells. Characterization of OHC mechanics is crucial for understanding and ultimately preventing permanent functional deficits due to overstimulation or as a consequence of various cochlear pathologies. The OHCs' major structural assembly is a highly-specialized lateral wall. The lateral wall consists of three structures; a plasma membrane highly-enriched with the motor-protein prestin, an actin-spectrin cortical lattice, and one or more layers of subsurface cisternae. Technical difficulties in independently manipulating each lateral wall constituent have constrained previous attempts to analyze the determinants of OHCs' mechanical properties. Temporal separations in the accumulation of each lateral wall constituent during postnatal development permit associations between lateral wall structure and OHC mechanics. We compared developing and adult gerbil OHC axial stiffness using calibrated glass fibers. Alterations in each lateral wall component and OHC stiffness were correlated as a function of age. Reduced F-actin labeling was correlated with reduced OHC stiffness before hearing onset. Prestin incorporation into the PM was correlated with increased OHC stiffness at hearing onset. Our data indicate lateral wall F-actin and prestin are the primary determinants of OHC mechanical properties before and after hearing onset, respectively.