The lateral plasma membrane of the outer hair cells of the organ of Corti contain "force-generation units" composed of small domains of a semicrystalline array of motor proteins. Pillars, connecting these motor proteins to an actin-spectrin meshwork inside the membrane, convey the forces generated in the plane of the membrane to the cell's interior. We propose that the pillars are composed of the anion-exchanger protein and 4.1-band proteins, but it's unclear whether these proteins are just force conveyors or are themselves the motor proteins.
Scientists studying cell motility have traditionally focused on conspicuous systems, such as muscle cells or cilia. These investigators have succeeded in identifying motors -- the mechanisms whereby chemical changes are converted into movement. But in the past decade, otologists studying the cells responsible for human hearing have discovered a much less conspicuous type of cell motion: a membrane-based contraction of the outer auditory hair cells.
Our laboratory has discovered and is now investigating the unique molecular mechanism underlying the operation of this most recently described cellular motor. This mechanism could be important in a variety of movements controlled by the cellular membrane, and is likely to prove vital to our understanding of the cellular basis of hearing disorders.
Historically, several lines of evidence have suggested that the hair cells of the mammalian hearing organ -- "the organ of Corti" -- are not simply passive transducers but also serve as amplifiers of the mechanical input they receive. As early as 1948, Gold (1) predicted that some active mechanical process must take place to overcome the damping effects that occur inside the fluid-filled cochlea, thus permitting a high degree of subjective frequency discrimination. He postulated that sound might emanate from the ear as a byproduct of the amplification process. In 1978, Kemp (2) recorded acoustic emissions from the human ear that were evoked by an acoustic-click stimulus. This discovery gave experimental support to the theory of an active cochlea. Recently, several reports have provided data about the way auditory hair cells can amplify sound within the cochlea. Propelling this renewed excitement in auditory research is our discovery of a novel force-generation mechanism located in the lateral plasma membrane of the outer hair cells of the organ of Corti in the cochlea. During sound stimulation, this membrane-based, voltage-dependent motor is capable of providing mechanical feedback that appears to modulate the sensory transduction response of inner hair cells, which are responsible for the bulk of sound detection and discrimination.
Sensory Transduction in Hair Cells
Hair cells are the receptors that convert mechanical movements into electrical signals in the auditory and vestibular sensory organs of the inner ear. These sensory cells were named "hair cells" because of the bundle of sensory stereo cilia -- resembling hairs -- that projects vertically from the apical cell surface. Each "hair" bundle is composed of about 100 stereo cilia (1 to 5 m long) and the hairs of each bundle are grouped in a staircase-like arrangement. The tips of the shorter hairs are connected to the sides of their longer neighbors by thin cables called tip-links (3). During an incoming mechanical stimulus, the hair bundle tilts toward the taller cilia, stretching the tip-links. Each stretched tip-link pulls open a mechanically gated ion channel, allowing an influx of cations that depolarize the cell by tens of millivolts (3). This depolarization is transmitted to the central nervous system by excited afferent synaptic connections.
Outer Hair Cells of the Mammalian Cochlea
In the mammalian cochlea, pure tone stimulation evokes mechanical waves that travel through the organ of Corti, from the base of the basilar membrane toward its apex, peaking in amplitude at a specific point along the organ's approximately 3-cm-long path. Inner and outer hair cells of the mammalian cochlea distributed in parallel rows along the length of the basilar membrane cooperate to analyze this wave. The row of inner hair cells is wired with afferent innervation and delivers the bulk of auditory sensory information to the brain. The three or more rows of outer hair cells in mammals receive only about 5% of the afferent innervation (4) but possess extensive efferent innervation. Outer hair cells were a complete enigma until the mid-1980s, when Brownell and others discovered that the cells boast an unusual talent: the ability to change length when subjected to changes in membrane potential. Both inner and outer hair cells respond to vibrations by producing such a change in membrane potential (5,8). However, only outer hair cells are also equipped to do the opposite, that is, to vibrate in response to a change in membrane potential, lengthening and shortening with small changes in membrane potential.
Another important difference between inner and outer hair cells is their location in the organ of Corti. Inner hair cells are located near the attachment site of the basilar membrane, an area where the membrane vibrates very little. Outer hair cells have a long, cylindrical shape and are positioned above the section of the basilar membrane that vibrates the most in response to sound. Researchers now believe that the outer hair cells can increase basilar-membrane vibrations by making contact with this sensitive section of the membrane as it vibrates, an action analogous to pushing a child on a swing. The outer hair cells could thus act like miniature amplifiers, sustaining the vibrations of the basilar membrane. This mechanism can indeed generate forces large enough to influence the basilar membrane (9) and can operate at frequencies that span the range of those produced by the human voice.
Fast Cellular Motor of Outer Hair Cells
The lengthening and shortening of outer hair cells depends on a unique motor that senses changes in membrane potential. The molecular mechanism behind this motor differs radically from those found in muscle-cell contraction and the motion of cilia, which require energy in the form of ATP and are based on filament structures such as actin and mictrotubules in the cytoskeleton. In contrast, the mechanism driving contractions of outer hair cells uses no ATP and is membrane based (6). The immediate questions that this novel cell-motility mechanism raise for us are: How do the cells detect voltage changes across their membranes, how do they convert voltage changes into mechanical forces, and where are the molecular motors that generate movement?
By combining patch-clamping and microscopy techniques (10), we found that the movement associated with outer hair cells can be observed directly in isolated membrane patches from the lateral surface of cells. In this way, we observed that the curvature of membrane patches inside a patch pipette increases (i.e., the membrane bows out) when the membrane is hyperpolarized -- regardless of the hydrostatic pressure on the patch. This observation demonstrates that the electric field across the membrane is providing the energy for the motion, leading us to conclude that the motor and voltage sensor may be one and the same molecule within the membrane.
The distribution of motor activity along the lateral plasma membrane of the outer hair cell coincides with the distribution of arrays of closely packed transmembrane proteins (10). The high density of such molecules should allow lateral interaction among them, so changes in the subunits or reordering of the particle array could generate lateral expansion of the membrane. For example, simple calculation shows that an alteration in the subunit packing of a tetrameric aggregate observed in the hair cells could produce a change in area of at least the required amount predicted by the data. Researchers in several labs have observed that the lateral plasma membrane is visibly connected by pillar-like protein structures to an anisotropic actin-spectrin meshwork on the interior of the cell (11). The actin meshwork extends the length of the cell and could convert membrane-area changes into axial cellular forces.
Ashmore (7) and others recorded charge movements in the lateral plasma membrane during outer-hair-cell electromotility. This intramembrane change in charge distribution is analogous to the gating current of voltage-dependent ion channels and confirms, in neurons, for example, the presence of an extremely high density of voltage-sensing proteins that can undergo conformational change.
Using immunocytochemistry, we have shown that the anion-exchanger protein (AE) and band 4.1 proteins are associated with the plasma membrane at the site of force generation (12) in contractions of outer hair cells. We have also proposed that these proteins form pillars that connect the plasma membrane to the cortical actin-spectrin lattice. Members of the AE-protein family are known to be involved in regulation of cellular volume and pH, as well as in the mechanical coupling of the plasma membrane to the cortical cytoskeleton. This coupling is essential for funneling forces generated in the plane of the plasma membrane into the longitudinal axis of the cell. Indeed, sulfhydryl agents, which selectively bind to the hydro-phobic pockets of AE proteins, inhibit electromotility of outer hair cells.
Using a different approach, we screen-ed an organ of Corti cDNA library for isoforms from the AE-protein family, and we identified a transcript that encodes an isoform with an unusual membrane-spanning domain. We have determined with specific anti-peptide antibodies that this transcript is indeed expressed in outer hair cells along the lateral plasma membrane. The characterization of this novel AE is currently the focus of our effort to understand the role that these proteins might play as membrane-cytoskeletal linkers in hair cells and their potential involvement in the force generation in outer-hair-cell electromotility.
This novel molecular mechanism for force generation, so conceptually different from other mechanisms of cell motility, may well prove important beyond the realm of auditory cell biology. Some form of this mechanism may be expressed in other cells that undergo membrane-potential changes. Understanding the mechanism by which voltage is converted into membrane protein-conformation change in this system may add to our knowledge of other analogous systems, such as the gating of voltage-sensitive channels.
A common disorder of hearing is "ringing of the ears," or tinnitus, which can range in effects from being a minor annoyance to being a cause of suicide. The biological source of tinnitus remains widely debated, but many scientists agree with the idea that spontaneous vibrations of outer hair cells may account for at least some types.
Although only 5% of the afferent fibers of the cochlear nerve terminate on outer hair cells, their essential contribution to hearing is apparent from studies with aminoglycoside antibiotics, such as gentomycin, that can selectively destroy outer hair cells. Clinical and experimental studies indicate that without outer hair cells, our hearing would be 100 times less sensitive. What sound we could hear would be significantly distorted and fuzzy. A better understanding of the motor function of the outer hair cell, so critical to hearing, could eventually help many individuals suffering from hearing impairment.
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