David Z. Z. He

Prestin is the motor protein of cochlear outer hair cells.

The outer and inner hair cells of the mammalian cochlea perform different functions. In response to changes in membrane potential, the cylindrical outer hair cell rapidly alters its length and stiffness. These mechanical changes, driven by putative molecular motors, are assumed to produce amplification of vibrations in the cochlea that are transduced by inner hair cells. Here we have identified an abundant complementary DNA from a gene, designated Prestin, which is specifically expressed in outer hair cells. Regions of the encoded protein show moderate sequence similarity to pendrin and related sulphate/anion transport proteins. Voltage-induced shape changes can be elicited in cultured human kidney cells that express prestin. The mechanical response of outer hair cells to voltage change is accompanied by a ‘gating current’, which is manifested as nonlinear capacitance. We also demonstrate this nonlinear capacitance in transfected kidney cells. We conclude that prestin is the motor protein of the cochlear outer hair cell.

Prestin is required for electromotility of the outer hair cell and for the cochlear amplifier.

Hearing sensitivity in mammals is enhanced by more than 40 dB (that is, 100-fold) by mechanical amplification thought to be generated by one class of cochlear sensory cells, the outer hair cells. In addition to the mechano-electrical transduction required for auditory sensation, mammalian outer hair cells also perform electromechanical transduction, whereby transmembrane voltage drives cellular length changes at audio frequencies in vitro. This electromotility is thought to arise through voltage-gated conformational changes in a membrane protein, and prestin has been proposed as this molecular motor. Here we show that targeted deletion of prestin in mice results in loss of outer hair cell electromotility in vitro and a 40–60 dB loss of cochlear sensitivity in vivo, without disruption of mechano-electrical transduction in outer hair cells. In heterozygotes, electromotility is halved and there is a twofold (about 6 dB) increase in cochlear thresholds. These results suggest that prestin is indeed the motor protein, that there is a simple and direct coupling between electromotility and cochlear amplification, and that there is no need to invoke additional active processes to explain cochlear sensitivity in the mammalian ear.

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.

First appearance and development of electromotility in neonatal gerbil outer hair cells

With the purpose of pinpointing the time of onset of electromotility, outer hair cells (OHCs) from apical and basal turns of the cochleae of postnatal gerbils, ranging in age from 6 to 19 days, were isolated and drawn into a glass microchamber. Length changes evoked by transcellular electrical stimulation were detected and measured with a photodiode detector. Motile responses first appeared in 3 out of 14 basal turn OHCs at 7 days after birth (DAB). At 8 DAB, 3 out of 13 apical turn cells also responded to the electrical stimulation. By 12 DAB, all the OHCs from both turns showed motile responses. Input-output functions relating applied stimulus and change in cell length revealed that the motile response threshold improved from 7 DAB to 12 DAB and the response amplitude kept increasing from 7 DAB until 13-14 DAB, when mature amplitudes were reached. Measurements of OHC length revealed only minor changes in basal turn hair cell length while apical hair cells continued to elongate until approximately 16 DAB. Since the onset of auditory function in gerbils occurs around 12 DAB and fine tuning develops between 14 and 17 DAB, our results suggest that the onset of OHC motility occurs earlier than that of auditory function and the maturation of the motility amplitude occurred earlier than the development of fine tuning. The maturation of OHC motility and the development of otoacoustic emissions are also compared and discussed.

Mechanoelectrical transduction of adult outer hair cells studied in a gerbil hemicochlea

Sensory receptor cells of the mammalian cochlea are morphologically and functionally dichotomized. Inner hair cells transmit auditory information to the brain, whereas outer hair cells (OHC) amplify the mechanical signal, which is then transduced by inner hair cells. Amplification by OHCs is probably mediated by their somatic motility in a mechanical feedback process. OHC motility in vivo is thought to be driven by the cells receptor potential. The first steps towards the generation of the receptor potential are the deflection of the stereociliary bundle, and the subsequent flow of transducer current through the mechanosensitive transducer channels located at their tips. Quantitative relations between transducer currents and basilar membrane displacements are lacking, as well as their variation along the cochlear length. To address this, we simultaneously recorded OHC transducer currents (or receptor potentials) and basilar membrane motion in an excised and bisected cochlea, the hemicochlea. This preparation permits recordings from adult OHCs at various cochlear locations while the basilar membrane is mechanically stimulated. Furthermore, the stereocilia are deflected by the same means of stimulation as in vivo. Here we show that asymmetrical transducer currents and receptor potentials are significantly larger than previously thought, they possess a highly restricted dynamic range and strongly depend on cochlear location.

Prestin and the Dynamic Stiffness of Cochlear Outer Hair Cells

The outer hair cell (OHC) lateral wall is a unique trilaminate structure consisting of the plasma membrane, the cortical lattice, and subsurface cisternae. OHCs are capable of altering their length in response to transmembrane voltage change. This so-called electromotile response is presumed to result from conformational changes of membrane-bound protein molecules, named prestin. OHC motility is accompanied by axial stiffness changes when the membrane potential of the cell is altered. During length changes, intracellular anions (mainly Cl-) act as extrinsic voltage sensors. In this study, we inquired whether the motor proteins are responsible for the voltage-dependent axial stiffness of OHCs, and whether ACh, the neurotransmitter of efferent neurons, modulates the stiffness of the cortical lattice and/or the stiffness of the motor protein. The experiments were done on isolated guinea pig OHCs in the whole-cell voltage-clamp mode. Axial stiffness was determined by loading a fiber of known stiffness onto the apical surface of the cells. Voltage-dependent stiffness and cell motility disappeared, and the axial stiffness of the cells significantly decreased after removal of intracellular Cl-. The result suggests that the stiffness of the motor protein is a major contributor to the global axial stiffness of OHCs. ACh was found to affect both the motor protein and other lateral wall stiffness components.

Motility-associated hair-bundle motion in mammalian outer hair cells.

Mammalian hearing owes its remarkable sensitivity and frequency selectivity to a local mechanical feedback process within the cochlea. Cochlear outer hair cells (OHCs) function as the key elements in the feedback loop in which the fast somatic motility of OHCs is thought to be the source of cochlear amplification. An alternative view is that amplification arises from active hair-bundle movement, similar to that seen in nonmammalian hair cells. We measured voltage-evoked hair-bundle motions in the gerbil cochlea to determine if such movements were also present in mammalian OHCs. The OHCs showed bundle movement with peak responses of up to 830 nm. The movement was insensitive to manipulations that would normally block mechanotransduction in the stereocilia, and it was absent in neonatal OHCs and prestin-knockout OHCs. These findings suggest that the bundle movement originated in somatic motility and that somatic motility has a central role in cochlear amplification in mammals.

Characterization of transcriptomes of cochlear inner and outer hair cells.

Inner hair cells (IHCs) and outer hair cells (OHCs) are the two types of sensory receptor cells that are critical for hearing in the mammalian cochlea. IHCs and OHCs have different morphology and function. The genetic mechanisms that define their morphological and functional specializations are essentially unknown. The transcriptome reflects the genes that are being actively expressed in a cell and holds the key to understanding the molecular mechanisms of the biological properties of the cell. Using DNA microarray, we examined the transcriptome of 2000 individually collected IHCs and OHCs from adult mouse cochleae. We show that 16,647 and 17,711 transcripts are expressed in IHCs and OHCs, respectively. Of those genes, ∼73% are known genes, 22% are uncharacterized sequences, and 5.0% are noncoding RNAs in both populations. A total of 16,117 transcripts are expressed in both populations. Uniquely and differentially expressed genes account for <15% of all genes in either cell type. The top 10 differentially expressed genes include Slc17a8, Dnajc5b, Slc1a3, Atp2a3, Osbpl6, Slc7a14, Bcl2, Bin1, Prkd1, and Map4k4 in IHCs and Slc26a5, C1ql1, Strc, Dnm3, Plbd1, Lbh, Olfm1, Plce1, Tectb, and Ankrd22 in OHCs. We analyzed commonly and differentially expressed genes with the focus on genes related to hair cell specializations in the apical, basolateral, and synaptic membranes. Eighty-three percent of the known deafness-related genes are expressed in hair cells. We also analyzed genes involved in cell-cycle regulation. Our dataset holds an extraordinary trove of information about the molecular mechanisms underlying hair cell morphology, function, pathology, and cell-cycle control.

Effect of acetylcholine and GABA on the transfer function of electromotility in isolated outer hair cells

Outer hair cells (OHC) from high- and low-frequency regions were separately isolated from guinea pig cochleas. The cells were inserted with their ciliary pole first into a partitioning microchamber so that only 20-50% of the cell length was excluded. Somatic length changes due to transcellular electrical stimulation were measured at the cuticular plate in the inserted portion of the cells. Transfer curves of electromotility of the OHCs were obtained by both a series of brief (2.5 ms) and longer (30 ms) square pulses with opposite polarity and linearly increasing size from 40 to 280 mV in both negative and positive directions. Alterations in the transient and steady-state electromotility transfer curves were examined by application of acetylcholine (ACh) and gamma-aminobutyric acid (GABA) to the synaptic pole. ACh, in the concentration range of 10-30 microM, evoked a significant magnitude and gain increase of electromotility in both transient and steady-state responses without a measurable shift in the operating point of the displacement-voltage transfer curve. A tonotopic response magnitude difference is found for ACh challenge. Basal turn OHCs responded with greater magnitude increase (+90% increase from control) than apical turn OHCs (+40%). GABA exerted an opposite effect, again in a location-dependent manner. Magnitude response decreased about 30% for long cells and 14% for short ones. Atropin, a muscarinic receptor antagonist, completely blocked the increase in electromotility response due to ACh. However, D-tubocurarine, a nicorinic receptor antagonist, while not blocking the ACh effect, altered the cells apparent operating point. Bicuculline methiodide, a GABAA-receptor antagonist, completely arrested GABA influences on the electromotility response. These results suggest that both ACh and GABA can change the electromotile activity of OHCs, in a tonotopically biased manner. ACh challenge evokes greater magnitude responses in basal turn OHCs, whereas GABA induces greater motility response decrease in apical turn OHCs. The control of the gain and magnitude of electromotility by the transmitter substances appear to involve at least two mechanisms. One is probably related to conformational changes of the voltage-to-movement converter molecules and a change in their number in an effective operational pool, the other operates via changing the electrical resistance of the basolateral cell membrane.

Mechanoelectric Transduction of Adult Inner Hair Cells

Inner hair cells (IHCs) are the true sensory receptors in the cochlea; they transmit auditory information to the brain. IHCs respond to basilar membrane (BM) vibration by producing a transducer current through mechanotransducer (MET) channels located at the tip of their stereocilia when these are deflected. The IHC MET current has not been measured from adult animals. We simultaneously recorded IHC transducer currents and BM motion in a gerbil hemicochlea to examine relationships between these two variables and their variation along the cochlear length. Results show that although maximum transducer currents of IHCs are uniform along the cochlea, their operating range is graded and is narrower in the base. The MET current displays adaptation, which along with response magnitude depends on extracellular calcium concentration. The rate of adaptation is invariant along the cochlear length. We introduce a new method of measuring adaptation using sinusoidal stimuli. There is a phase lead of IHC transducer currents relative to sinusoidal BM displacement, reflecting viscoelastic coupling of their cilia and their adaptation process.