Outer hair cells (OHC) from the organ of Corti are capable of fast voltage-induced length changes (Santos-Sacchi and Dilger, 1988), suggesting that an associated voltage sensor should reside in the OHC plasma membrane. Voltage-dependent mechanical responses and nonlinear charge movement of isolated OHCs from the guinea pig were analyzed using the whole-cell voltage-clamp technique. Ionic currents in the cells were blocked. Nonlinear voltage-dependent charge movement or, correspondingly, voltage-dependent capacitance was measured with step or AC analysis. OHC movements were measured either from video or using a differential photodiode technique. Maximum charge movements up to 2.5 pC were measured in OHCs from the low-frequency region of the cochlea. Both AC and step analyses indicated a peak nonlinear capacitance of 16-17 pF. The voltage dependence was fit to a Boltzmann relation with the step analysis indicating a maximum nonlinear capacitance at -23 mV step potential from a holding potential of about -120 mV, and AC analysis indicating a maximum at a holding potential near -40 mV. AC analysis probably provides a more accurate evaluation of voltage dependence. Measures of OHC motility magnitude versus voltage follow the nonlinear capacitance-voltage function obtained from AC measures. Treatment of the cells with gadolinium ions (0.5-1 mM) blocked OHC motility. This treatment also produced a shift of the nonlinear capacitance function along the voltage axis in the depolarizing direction, which can be explained by membrane surface charge screening. However, maximum capacitance was reduced as well and may correspond to the reduction or abolition of OHC motility in response to gadolinium treatment. Gadolinium effects were reversible. Nonlinear capacitance is not a function of membrane deformation due to length changes, since removal of OHC cytosol via the patch pipette abolished longitudinal movement but did not reduce nonlinear charge movement. It is interesting to note that the nonlinear capacitance will dynamically influence the time constant of the OHC during acoustically evoked receptor potential generation.

Myosin Vlla is a newly identified member of the myosin superfamily of actin-based motors. Recently, the

The unique electromotility of the outer hair cell (OHC) is believed to promote sharpening of the passive mechanical vibration of the mammalian basilar membrane. The cell also presents a voltage-dependent capacitance, or equivalently, a nonlinear gating current, which correlates well with its mechanical activity, suggesting that membrane-bound voltage sensor-motor elements control OHC length. We report that the voltage dependence of the gating charge and motility are directly related to membrane stress induced by intracellular pressure. A tracking procedure was devised to continuously monitor the voltage at peak capacitance (VpkCm) after obtaining whole cell voltage clamp configuration. In addition, nonlinear capacitance was more fully evaluated with a stair step voltage protocol. Upon whole cell configuration, VpkCm was typically near -20 mV. Negative patch pipette pressure caused a negative shift in VpkCm, which obtained a limiting value near the normal resting potential of the OHC (approximately -70 mV) at the point of cell collapse. Positive pressure in the pipette caused a positive shift that could reach values greater than 0 mV. Measures of the mechanical activity of the OHC mirrored those of charge movement. Similar membrane-tension dependent peak shifts were observed after the cortical cytoskeletal network was disrupted by intracellular dialysis of trypsin from the patch pipette. We conclude that unlike stretch receptors, which may sense tension through elastic cytoskeletal elements, the OHC motor senses tension directly. Furthermore, since the voltage dependence of the OHC nonlinear capacitance and motility is directly regulated by intracellular turgor pressure, we speculate that modification of intracellular pressure in vivo provides a mechanism for controlling the gain of the mammalian "cochlear amplifier".

No abstract

Salicylate, one of the most widely used drugs, is known to induce reversible tinnitus and hearing loss. Salicylate interferes with outer hair cells (OHCs), which are believed to underlie normal auditory frequency selectivity and sensitivity. In the present experiments, the effects of salicylate and lanthanides on OHC motility and nonlinear capacitance were investigated by using isolated guinea-pig OHCs while attempting to avoid inadvertent intracellular pressure change, which itself can affect OHC motility and capacitance. Either extracellularly or intracellularly applied salicylate reduced nonlinear peak capacitance (Cm pk ) and shifted the voltage at peak capacitance to depolarized levels. Concentration-response curves for reduction in Cm pk by salicylate and GdCl 3 revealed a half-maximal concentration and Hill coefficient of 1.6 mM and 1.0, and 0.6 mM and 1.2, respectively. In comparable groups of OHCs, the normal Cm pk values of which were near 40 pF, average Cm pk decreased to 28 and 36 pF for intracellularly and extracellularly applied salicylate, respectively. Salicylate reduced, but did not completely block, the voltage-induced length change. Extracellularly, but not intracellularly, applied lanthanide blocked voltage-induced movement and capacitance almost completely. After intracellular trypsin treatment, salicylate reduced voltage-dependent capacitance reversibly, suggesting that salicylate directly acts on the sensor/motor and not via effects on intracellular structures, such as the subsurface cisternae. The results are consistent with the hypothesis that the dissociated, charged form of salicylate directly interacts with the sensor/ motor on the inner aspect of the OHC plasma, whereas lanthanides interact on the outer aspect.

Outer hair cells underlie high frequency cochlear amplification in mammals. Fast somatic motility can be driven by voltage-dependent conformational changes in the motor protein, prestin, which resides exclusively within lateral plasma membrane of the cell. Yet, how a voltage-driven motor could contribute to high frequency amplification, despite the low-pass membrane filter of the cell, remains an enigma. The recent identification of prestin's Cl- sensitivity revealed an alternative mechanism in which intracellular Cl- fluctuations near prestin could influence the motor. We report the existence of a stretch-sensitive conductance within the lateral membrane that passes anions and cations and is gated at acoustic rates. The resultant intracellular Cl- oscillations near prestin may drive motor protein transitions, as evidenced by pronounced shifts in prestin's state-probability function along the voltage axis. The sensitivity of prestin's state probability to intracellular Cl- levels betokens a more complicated role for Cl- than a simple extrinsic voltage sensor. Instead, we suggest an allosteric modulation of prestin by Cl- and other anions. Finally, we hypothesize that prestin sensitivity to anion flux through the mechanically activated lateral membrane can provide a driving force that circumvents the membrane's low-pass filter, thus permitting amplification at high acoustic frequencies.

SUMMARY 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 hyper-methylation causes ROS-dependent activation of AMP kinase and the pro-apoptotic 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 transgenic-mtTFB1 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.

The outer hair cell lateral membrane motor, prestin, drives the cell's mechanical response that underpins mammalian cochlear amplification. Little is known about the protein's structure-function relations. Here we provide evidence that prestin is a 10-transmembrane domain protein whose membrane topology differs from that of previous models. We also present evidence that both intracellular termini of prestin are required for normal voltage sensing, with short truncations of either terminal resulting in absent or modified activity despite quantitative findings of normal membrane targeting. Finally, we show with fluorescence resonance energy transfer that prestin-prestin interactions are dependent on an intact N-terminus, suggesting that this terminus is important for homo-oligomerization of prestin. These domains, which we have perturbed, likely contribute to allosteric modulation of prestin via interactions among prestin molecules or possibly between prestin and other proteins, as well.