Joseph Santos-Sacchi

Effects of Salicylate and Lanthanides on Outer Hair Cell Motility and Associated Gating Charge

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 (Cmpk) and shifted the voltage at peak capacitance to depolarized levels. Concentration–response curves for reduction in Cmpk by salicylate and GdCl3 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 Cmpk values of which were near 40 pF, average Cmpk 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.

Cl− flux through a non-selective, stretch-sensitive conductance influences the outer hair cell motor of the guinea-pig

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 prestins 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 prestins state‐probability function along the voltage axis. The sensitivity of prestins 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 membranes low‐pass filter, thus permitting amplification at high acoustic frequencies.

Mapping the distribution of the outer hair cell motility voltage sensor by electrical amputation

The outer hair cell (OHC) possesses a nonlinear charge movement whose characteristics indicate that it represents the voltage sensor responsible for OHC mechanical activity. OHC mechanical activity is known to exist along a restricted extent of the cells length. We have used a simultaneous partitioning microchamber and whole cell voltage clamp technique to electrically isolate sections of the OHC membrane and find that the nonlinear charge movement is also restricted along the cells length. Apical and basal portions of the OHC are devoid of voltage sensors, corresponding to regions of the cell where the subsurface cisternae and/or the mechanical responses are absent. We conclude that the physical domain of the motility voltage sensor corresponds to that of the mechanical effector and speculate that sensor and effector reside within one intra membranous molecular species, perhaps an evolved nonconducting or poorly conducting voltage-dependent ion channel.

Effects of membrane potential on the voltage dependence of motility-related charge in outer hair cells of the guinea-pig

1 Isolated outer hair cells (OHCs) from the guinea‐pig were whole‐cell voltage clamped to study the influence of initial voltage on the voltage dependence of motility‐related gating current or, equivalently, on the voltage dependence of membrane capacitance. 2 Prepulse delivery caused changes in the magnitude of motility‐related gating currents, which are due predominantly to shifts in the voltage at peak capacitance (VpkCm). Depolarization shifts VpkCm in the hyperpolarizing direction, and hyperpolarization does the opposite. The mean shift between ‐120 and +40 mV prepulse states with long‐term holding potentials (> 2 min) at −80 mV was 14.67 ± 0.95 mV (n= 10; mean ± s.e.m.). 3 The effect of initial membrane potential is sigmoidal, with a voltage dependence of 23 mV per e‐fold change in VpkCm, and maximum slope within the physiological range of OHC resting potentials. This indicates that the cell is poised to respond maximally to changes in resting potential. 4 The kinetics of prepulse effects are slow compared with motility‐related gating current kinetics. High‐resolution measurement of membrane capacitance (Cm) using two voltage sinusoids indicates that shifts in VpkCm induce Cm changes with time courses fitted by two exponentials (τ0, 0.070 ± 0.003 s; τ1, 1.28 ± 0.07 s; A0, 1.54 ± 0.13 pF; A1, 1.51 ± 0.13 pF; means ± s.e.m.; n= 22; step from +50 to −80 mV). Recovery of prepulse effects exhibits a similar time course. 5 Prepulse effects are resistant to intracellular enzymatic digestion, to fast intracellular calcium buffers, and to intracellular pressure. Through modelling, we indicate how the effect may be explained by an intrinsic voltage‐induced tension generated by the molecular motors residing in the lateral membrane.

Mitochondrial Stress Engages E2F1 Apoptotic Signaling to Cause Deafness

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 hypermethylation causes ROS-dependent activation of AMP kinase and the proapoptotic 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 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.

Harmonics of outer hair cell motility

The voltage-dependent mechanical activity of outer hair cells (OHC) from the organ of Corti is considered responsible for the peripheral auditory systems enhanced ability to detect and analyze sound. Nonlinear processes within the inner ear are presumed to be characteristic of this enhancement process. Harmonic distortion in the OHC mechanical response was analyzed under whole-cell voltage clamp. It is shown that the OHC produces DC, fundamental and second harmonic length changes in response to sinusoidal transmembrane voltage stimulation. Mechanical second harmonic distortion decreases with frequency, whereas the predicted transmembrane second harmonic voltage increases with frequency. Furthermore, the phase of the second harmonic distortion does not correspond to the phase of the predicted transmembrane voltage. In contradistinction, it has been previously shown (Santos-Sacchi, J. 1992. Neuroscience. 12:1906-1916) that fundamental voltage and evoked mechanical responses share magnitude and phase characteristics. OHC length changes are modeled as resulting from voltage-dependent cell surface area changes. The model suggests that the observed harmonic responses in the mechanical response are consistent with the nonlinearity of the voltage-to-length change (V-delta L) function. While these conclusions hold for the data obtained with the present voltage clamp protocol and help to understand the mechanism of OHC motility, modeling the electromechanical system of the OHC in the in vivo state indicates that the mechanical nonlinearity of the OHC contributes minimally to mechanical distortion. That is, in vivo, at moderate sound pressure levels and below, the dominant factor which contributes to nonlinearities of the OHC mechanical response resides within the nonlinear, voltage-generating, stereociliar transduction process.

Control of Mammalian Cochlear Amplification by Chloride Anions

Chloride ions have been hypothesized to interact with the membrane outer hair cell (OHC) motor protein, prestin on its intracellular domain to confer voltage sensitivity (Oliver et al., 2001). Thus, we hypothesized previously that transmembrane chloride movements via the lateral membrane conductance of the cell, GmetL, could serve to underlie cochlear amplification in the mammal. Here, we report on experimental manipulations of chloride-dependent OHC motor activity in vitro and in vivo. In vitro, we focused on the signature electrical characteristic of the motor, the nonlinear capacitance of the cell. Using the well known ototoxicant, salicylate, which competes with the putative anion binding or interaction site of prestin to assess level-dependent interactions of chloride with prestin, we determined that the resting level of chloride in OHCs is near or below 10 mm, whereas perilymphatic levels are known to be ∼140 mm. With this observation, we sought to determine the effects of perilymphatic chloride level manipulations of basilar membrane amplification in the living guinea pig. By either direct basolateral perfusion of the OHC with altered chloride content perilymphatic solutions or by the use of tributyltin, a chloride ionophore, we found alterations in OHC electromechanical activity and cochlear amplification, which are fully reversible. Because these anionic manipulations do not impact on the cation selective stereociliary process or the endolymphatic potential, our data lend additional support to the argument that prestin activity dominates the process of mammalian cochlear amplification.

Effects of membrane potential and tension on prestin, the outer hair cell lateral membrane motor protein

1 Under whole‐cell voltage clamp, the effects of initial voltage conditions and membrane tension on gating charge and voltage‐dependent capacitance were studied in human embryonic kidney cells (TSA201 cell line) transiently transfected with the gene encoding the gerbil protein prestin. Conformational changes in this membrane‐bound protein probably provide the molecular basis of the outer hair cell (OHC) voltage‐driven mechanical activity, which spans the audio spectrum. 2 Boltzmann characteristics of the charge movement in transfected cells were similar to those reported for OHCs (amax= 0.99 ± 0.16 pC, z = 0.88 ± 0.02; n= 5, means ±s.e.m.). Unlike that of the adult OHC, the voltage at peak capacitance (apkcm) was very negative (‐74.7 ± 3.8 mV). Linear capacitance in transfected cells was 43.7 ± 13.8 pF and membrane resistance was 458 ± 123 MΩ. 3 Voltage steps from the holding potential preceding the measurement of capacitance‐voltage functions caused a time‐ and voltage‐dependent shift in Vpkcm. For a prepulse to ‐150 mV, from a holding potential of 0 mV, Vpkcm shifted 6.4 mV, and was fitted by a single exponential time constant of 45 ms. A higher resolution analysis of this time course was made by measuring the change in capacitance during a fixed voltage step and indicated a double exponential shift (τ0= 51.6 ms, τ1= 8.5 s) similar to that of the native gerbil OHC. 4 Membrane tension, delivered by increasing pipette pressure, caused a positive shift in Vpkcm. A maximal shift of 7.5 mV was obtained with 2 kPa of pressure. The effect was reversible. 5 Our results show that the sensitivity of prestin to initial voltage and membrane tension, though present, is less than that observed in adult OHCs. It remains possible that some other interacting molecular species within the lateral plasma membrane of the native OHC amplifies the effect of tension and prior voltage on prestins activity.

Density of motility-related charge in the outer hair cell of the guinea pig is inversely related to best frequency

Whole cell voltage clamp and freeze fracture were used to study the electrophysiological and ultrastructural correlates of the outer hair cell (OHC) lateral membrane molecular motors. We find that specific voltage-dependent capacitance, which derives from motility-related charge movement, increases as cell length decreases. This increasing non-linear charge density predicts a corresponding increase in sensor-motor density. However, while OHC lateral membrane particle density increases, a quantitative correspondence is absent. Thus, the presumed equivalence of particle and motor is questionable. The data more importantly indicate that whereas the voltage driving OHC motility, i.e. the receptor potential, may decrease with frequency due to the OHCs low-pass membrane filter, the electrical energy (Q x V) supplied to the lateral membrane will tend to remain stable. This conservation of energy delivery is likely crucial for the function of the cochlear amplifier at high frequencies.

New tunes from Corti's organ: the outer hair cell boogie rules.

The amplification of acoustic stimuli is a feature of hair cells that evolved early on in vertebrates. Though standard stereocilia mechanisms to promote such amplification may persist in the mammal, an additional mechanism evolved to enhance high frequency sensation. Only in mammals, a special cell type, the outer hair cell, arose that possesses a remarkably fast somatic mechanical response, which probably endows the passive cochlea with a boost in sensitivity by a factor of 100 (40dB), at least. Experiments conducted over the past few years have shed light on many aspects of outer hair cell electromotility, including the molecular identification of the motor, the effects of a knockout, and underlying mechanisms of action. A review of this remarkable progress is attempted.