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.     

Sound-induced motility of isolated cochlear outer hair cells is frequency-specific

The inner ear is capable of highly selective frequency discrimination. This is achieved not only by the travelling wave of the basilar membrane in the cochlear partition, but also by the active participation of nonlinear and vulnerable elements that enhance frequency selectivity. It has been shown that isolated mammalian outer hair cells respond with a change in length when subjected to sound stimulation at a fixed frequency. Here we investigate the motile behaviour of isolated cells when the stimulus frequency is varied between 200 and 10,000 Hz. By varying the frequency and the intensity of the tone, it is possible to obtain 'tuning curves' for the motile response. We demonstrate that the cell body of solitary hair cells, free from contact with the basilar membrane, shows a sharply tuned motile behaviour. We suggest that frequency selectivity in the organ of Corti is amplified by the tuned motility of the cell body of outer hair cells.     

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.     

Force generation by mammalian hair bundles supports a role in cochlear amplification

It is generally accepted that the acute sensitivity and frequency discrimination of mammalian hearing requires active mechanical amplification of the sound stimulus within the cochlea. The prevailing hypothesis is that this amplification stems from somatic electromotility of the outer hair cells attributable to the motor protein prestin. Thus outer hair cells contract and elongate in synchrony with the sound-evoked receptor potential. But problems arise with this mechanism at high frequencies, where the periodic component of the receptor potential will be attenuated by the membrane time constant. On the basis of work in non-mammalian vertebrates, force generation by the hair bundles has been proposed as an alternative means of boosting the mechanical stimulus. Here we show that hair bundles of mammalian outer hair cells can also produce force on a submillisecond timescale linked to adaptation of the mechanotransducer channels. Because the bundle motor may ultimately be limited by the deactivation rate of the channels, it could theoretically operate at high frequencies. Our results show the existence of another force generator in outer hair cells that may participate in cochlear amplification.     

Voltage-induced membrane movement

Thermodynamics predicts that transmembrane voltage modulates membrane tension and that this will cause movement. The magnitude and polarity of movement is governed by cell stiffness and surface potentials. Here we confirm these predictions using the atomic force microscope to dynamically follow the movement of voltage-clamped HEK293 cells in different ionic-strength solutions. In normal saline, depolarization caused an outward movement, and at low ionic strength an inward movement. The amplitude was proportional to voltage (about 1 nm per 100 mV) and increased with indentation depth. A simple physical model of the membrane and tip provided an estimate of the external and internal surface charge densities (-5 x 10(-3) C x m(-2) and -18 x 10(-3) C x m(-2), respectively). Salicylate (a negative amphiphile) inhibited electromotility by increasing the external charge density by -15 x 10(-3) C x m(-2). As salicylate blocks electromotility in cochlear outer hair cells at the same concentration, the role of prestin as a motor protein may need to be reassessed.     

Ionic basis of membrane potential in outer hair cells of guinea pig cochlea

Mammalian hearing involves features not found in other species, for example, the separation of sound frequencies depends on an active control of the cochlear mechanics. The force-generating component in the cochlea is likely to be the outer hair cell (OHC), one of the two types of sensory cell through which current is gated by mechano-electrical transducer channels sited on the apical surface. Outer hair cells isolated in vitro have been shown to be motile and capable of generating forces at acoustic frequencies. The OHC membrane is not, however, electrically tuned, as found in lower vertebrates. Here we describe how the OHC resting potential is determined by a Ca2+-activated K+ conductance at the base of the cell. Two channel types with unitary sizes of 240 and 45 pS underlie this Ca2+-activated K+ conductance and we suggest that their activity is determined by a Ca2+ influx through the apical transducer channel, as demonstrated in other hair cells. This coupled system simultaneously explains the large OHC resting potentials observed in vivo and indicates how the current gated by the transducer may be maximized to generate the forces required in cochlear micromechanics.     

Structure of the C-terminal domain of FliG, a component of the rotor in the bacterial flagellar motor.

Many motile species of bacteria are propelled by flagella, which are rigid helical filaments turned by rotary motors in the cell membrane. The motors are powered by the transmembrane gradient of protons or sodium ions. Although bacterial flagella contain many proteins, only three-MotA, MotB and FliG-participate closely in torque generation. MotA and MotB are ion-conducting membrane proteins that form the stator of the motor. FliG is a component of the rotor, present in about 25 copies per flagellum. It is composed of an amino-terminal domain that functions in flagellar assembly and a carboxy-terminal domain (FliG-C) that functions specifically in motor rotation. Here we report the crystal structure of FliG-C from the hyperthermophilic eubacterium Thermotoga maritima. Charged residues that are important for function, and which interact with the stator protein MotA, cluster along a prominent ridge on FliG-C. On the basis of the disposition of these residues, we present a hypothesis for the orientation of FliG-C domains in the flagellar motor, and propose a structural model for the part of the rotor that interacts with the stator.     

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 cell's 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.     

Auditory collusion and a coupled couple of outer hair cells.

The discrepancies between measured frequency responses of the basilar membrane in the inner ear and the frequency tuning found in psychophysical experiments led to Bekesy's idea of lateral inhibition in the auditory nervous system. We now know that basilar membrane tuning can account for neural tuning, and that sharpening of the passive travelling wave depends on the mechanical activity of outer hair cells (OHCs)3, but the mechanism by which OHCs enhance tuning remains unclear. OHCs generate voltage-dependent length changes at acoustic rates, which deform the cochlear partition. Here we use an electrical correlate of OHC mechanical activity, the motility-related gating current, to investigate mechano-electrical interactions among adjacent OHCs. We show that the motility caused by voltage stimulation of one cell in a group evokes gating currents in adjacent OHCs. The resulting polarization in adjacent cells is opposite to that within the stimulated cell, which may be indicative of lateral inhibition. Also such interactions promote distortion and suppression in the electrical and, consequently, the mechanical activity of OHCs. Lateral interactions may provide a basis for enhanced frequency selectivity in the basilar membrane of mammals.     

Mechanism of shape determination in motile cells

The shape of motile cells is determined by many dynamic processes spanning several orders of magnitude in space and time, from local polymerization of actin monomers at subsecond timescales to global, cell-scale geometry that may persist for hours. Understanding the mechanism of shape determination in cells has proved to be extremely challenging due to the numerous components involved and the complexity of their interactions. Here we harness the natural phenotypic variability in a large population of motile epithelial keratocytes from fish (Hypsophrys nicaraguensis) to reveal mechanisms of shape determination. We find that the cells inhabit a low-dimensional, highly correlated spectrum of possible functional states. We further show that a model of actin network treadmilling in an inextensible membrane bag can quantitatively recapitulate this spectrum and predict both cell shape and speed. Our model provides a simple biochemical and biophysical basis for the observed morphology and behaviour of motile cells.     

Electrokinetic shape changes of cochlear outer hair cells

Rapid mechanical changes have been associated with electrical activity in a variety of non-muscle excitable cells. Recently, mechanical changes have been reported in cochlear hair cells. Here we describe electrically evoked mechanical changes in isolated cochlear outer hair cells (OHCs) with characteristics which suggest that direct electrokinetic phenomena are implicated in the response. OHCs make up one of two mechanosensitive hair cell populations in the mammalian cochlea; their role may be to modulate the micromechanical properties of the hearing organ through mechanical feedback mechanisms. In the experiments described here, we applied sinusoidally modulated electrical potentials across isolated OHCs; this produced oscillatory elongation and shortening of the cells and oscillatory displacements of intracellular organelles. The movements were a function of the direction and strength of the electrical field, were inversely related to the ionic concentration of the medium, and occurred in the presence of metabolic uncouplers. The cylindrical shape of the OHCs and the presence of a system of membranes within the cytoplasm--laminated cisternae--may provide the anatomical substrate for electrokinetic phenomena such as electro-osmosis.     

Crystal structure of bacterial multidrug efflux transporter AcrB

AcrB is a major multidrug exporter in Escherichia coli. It cooperates with a membrane fusion protein, AcrA, and an outer membrane channel, TolC. We have determined the crystal structure of AcrB at 3.5 A resolution. Three AcrB protomers are organized as a homotrimer in the shape of a jellyfish. Each protomer is composed of a transmembrane region 50 A thick and a 70 A protruding headpiece. The top of the headpiece opens like a funnel, where TolC might directly dock into AcrB. A pore formed by three alpha-helices connects the funnel with a central cavity located at the bottom of the headpiece. The cavity has three vestibules at the side of the headpiece which lead into the periplasm. In the transmembrane region, each protomer has twelve transmembrane alpha-helices. The structure implies that substrates translocated from the cell interior through the transmembrane region and from the periplasm through the vestibules are collected in the central cavity and then actively transported through the pore into the TolC tunnel.     

Graded and nonlinear mechanical properties of sensory hairs in the mammalian hearing organ

It is generally agreed that frequency selectivity of the mammalian hearing organ is mainly due to a graded elasticity of the basilar membrane. Recent measurements of basilar membrane motion hair cell receptor potentials and neural tuning curves show that frequency selectivity can be extremely sharp. It has been suggested that in non-mammalian species there are additional tuning mechanisms in the sensory hair cells themselves, either by virtue of their electrical membrane properties or through a gradation in length of their sensory hairs. Indeed, sensory hair mechanical tuning has been demonstrated in the lizard. We have investigated the mechanical properties of sensory hair bundles in the guinea pig organ of Corti, and report here that hair-bundle stiffness increases longitudinally towards the high-frequency end of the cochlea, decreases radially towards the outer rows of cells, and is greater for excitatory than for inhibitory deflection. On the basis of these findings, we suggest that sensory hairs confer frequency-specific, nonlinear mechanical properties on the hearing organ.     

Atomic scale movement of the voltage-sensing region in a potassium channel measured via spectroscopy

Voltage-gated ion channels are transmembrane proteins that are essential for nerve impulses and regulate ion flow across cell membranes in response to changes in membrane potential. They are made up of four homologous domains or subunits, each of which contains six transmembrane segments. Studies of potassium channels have shown that the second (S2) and fourth (S4) segments contain several charged residues, which sense changes in voltage and form part of the voltage sensor. Although these regions clearly undergo conformational changes in response to voltage, little is known about the nature of these changes because voltage-dependent distance changes have not been measured. Here we use lanthanide-based resonance energy transfer to measure distances between Shaker potassium channel subunits at specific residues. Voltage-dependent distance changes of up to 3.2 A were measured at several sites near the S4 segment. These movements directly correlated with electrical measurements of the voltage sensor, establishing the link between physical changes and electrical charge movement. Measured distance changes suggest that the region associated with the S4 segment undergoes a rotation and possible tilt, rather than a large transmembrane movement, in response to voltage. These results demonstrate the first in situ measurement of atomic scale movement in a trans-membrane protein.     

朝鲜鹌鹑耳蜗的组织学观察

本实验对成体朝鲜鹌鹑耳蜗的组织学结构进行了观察,朝鲜鹌鹑耳蜗由基乳突、听壶和淋巴管三部分组成。听壶由毛细胞、支持细胞和基膜组成;毛细胞呈柱状,支持细胞呈指状。基乳突由毛细胞、支持细胞和基膜组成;毛细胞分成高、中间和矮毛细胞三型。高毛细胞呈柱状,分布在除近端以外各处,在远端全为高毛细胞;中间毛细胞呈壶形和柱形的中间形态,分布在高、矮毛细胞交界处;矮毛细胞呈壶形,分布在除远端以外各处。基乳突的基膜主要由排列疏松的纤维构成。     

Structure of the membrane domain of respiratory complex I

Complex I is the first and largest enzyme of the respiratory chain, coupling electron transfer between NADH and ubiquinone to the translocation of four protons across the membrane. It has a central role in cellular energy production and has been implicated in many human neurodegenerative diseases. The L-shaped enzyme consists of hydrophilic and membrane domains. Previously, we determined the structure of the hydrophilic domain. Here we report the crystal structure of the Esherichia coli complex I membrane domain at 3.0?? resolution. It includes six subunits, NuoL, NuoM, NuoN, NuoA, NuoJ and NuoK, with 55 transmembrane helices. The fold of the homologous antiporter-like subunits L, M and N is novel, with two inverted structural repeats of five transmembrane helices arranged, unusually, face-to-back. Each repeat includes a discontinuous transmembrane helix and forms half of a channel across the membrane. A network of conserved polar residues connects the two half-channels, completing the proton translocation pathway. Unexpectedly, lysines rather than carboxylate residues act as the main elements of the proton pump in these subunits. The fourth probable proton-translocation channel is at the interface of subunits N, K, J and A. The structure indicates that proton translocation in complex I, uniquely, involves coordinated conformational changes in six symmetrical structural elements.     

Novel form of growth cone motility involving site-directed actin filament assembly.

Regulation of cytoskeletal structure and motility by extracellular signals is essential for all directed forms of cell movement and underlies the developmental process of axonal guidance in neuronal growth cones. Interaction with polycationic microbeads can trigger morphogenic changes in neurons and muscle cells normally associated with formation of pre- and postsynaptic specializations. Furthermore, when various types of microscopic particles are applied to the lamellar surface of a neuronal growth cone or motile cell they often exhibit retrograde movement at rates of 1-6 microns min-1 (refs 3-6). There is strong evidence that this form of particle movement results from translocation of membrane proteins associated with cortical F-actin networks, not from bulk retrograde lipid flow and may be a mechanism behind processes such as cell locomotion, growth cone migration and capping of cell-surface antigens. Here we report a new form of motility stimulated by polycationic bead interactions with the growth-cone membrane surface. Bead binding rapidly induces intracellular actin filament assembly, coincident with a production of force sufficient to drive bead movements. These extracellular bead movements resemble intracellular movements of bacterial parasites known to redirect host cell F-actin assembly for propulsion. Our results suggest that site-directed actin filament assembly may be a widespread cellular mechanism for generating force at membrane-cytoskeletal interfaces.     

Deafness and renal tubular acidosis in mice lacking the K-Cl co-transporter Kcc4

Hearing depends on a high K(+) concentration bathing the apical membranes of sensory hair cells. K(+) that has entered hair cells through apical mechanosensitive channels is transported to the stria vascularis for re-secretion into the scala media(). K(+) probably exits outer hair cells by KCNQ4 K(+) channels(), and is then transported by means of a gap junction system connecting supporting Deiters' cells and fibrocytes() back to the stria vascularis. We show here that mice lacking the K(+)/Cl(-) (K-Cl) co-transporter Kcc4 (coded for by Slc12a7) are deaf because their hair cells degenerate rapidly after the beginning of hearing. In the mature organ of Corti, Kcc4 is restricted to supporting cells of outer and inner hair cells. Our data suggest that Kcc4 is important for K(+) recycling() by siphoning K(+) ions after their exit from outer hair cells into supporting Deiters' cells, where K(+) enters the gap junction pathway. Similar to some human genetic syndromes(), deafness in Kcc4-deficient mice is associated with renal tubular acidosis. It probably results from an impairment of Cl(-) recycling across the basolateral membrane of acid-secreting alpha-intercalated cells of the distal nephron.     

Outer hair cells in the mammalian cochlea and noise-induced hearing loss

Hair cells in the mammalian cochlea transduce mechanical stimuli into electrical signals leading to excitation of auditory nerve fibres. Because of their important role in hearing, these cells are a possible site for the loss of cochlear sensitivity that follows acoustic overstimulation. We have recorded from inner and outer hair cells (IHC, OHC) in the guinea pig cochlea during and after exposure to intense tones. Our results show functional changes in the hair cells that may explain the origin of noise-induced hearing loss. Both populations of hair cells show a reduction in amplitude and an increase in the symmetry of their acoustically evoked receptor potentials. In addition, the OHCs also suffer a sustained depolarization of the membrane potential. Significantly, the membrane and receptor potentials of the OHCs recover in parallel with cochlear sensitivity as measured by the IHC receptor potential amplitude and the auditory nerve threshold. Current theories of acoustic transduction suggest that the mechanical input to IHCs may be regulated by the OHCs. Consequently, the modified function of OHCs after acoustic overstimulation may determine the extent of the hearing loss following loud sound.