Biomedical subjects

William E Brownell

Publications and source records attributed to William E Brownell.

17 recordsLinked to original sources

PubMed2006

Essential helix interactions in the anion transporter domain of prestin revealed by evolutionary trace analysis.

Prestin, a member of the SLC26A family of anion transporters, is a polytopic membrane protein found in outer hair cells (OHCs) of the mammalian cochlea. Prestin is an essential component of the membrane-based motor that enhances electromotility of OHCs and contributes to frequency sensitivity and selectivity in mammalian hearing. Mammalian cells expressing prestin display a nonlinear capacitance (NLC), widely accepted as the electrical signature of electromotility. The associated charge movement requires intracellular anions reflecting the membership of prestin in the SLC26A family. We used the computational approach of evolutionary trace analysis to identify candidate functional (trace) residues in prestin for mutational studies. We created a panel of mutations at each trace residue and determined membrane expression and nonlinear capacitance associated with each mutant. We observe that several residue substitutions near the conserved sulfate transporter domain of prestin either greatly reduce or eliminate NLC, and the effect is dependent on the size of the substituted residue. These data suggest that packing of helices and interactions between residues surrounding the "sulfate transporter motif" is essential for normal prestin activity.

Amino Acid Sequence↗

PubMed2006

Voltage-dependent capacitance of human embryonic kidney cells.

We determine membrane capacitance, C as a function of dc voltage for the human embryonic kidney (HEK) cell. C was calculated from the admittance, Y, obtained during a voltage ramp when the HEK cell was held in whole-cell patch-clamp configuration. Y was determined at frequencies of 390.625 and from the measured current, i obtained with a dual-sinusoidal stimulus. We find that the fractional increase in the capacitance, C is small ( < 1%) and grows with the square of the voltage, Psi. C can be described by: C=C(0)(1+alpha(Psi+psi(s))2)[where C(0): Capacitance at 0 volts, psi(s): Difference in surface potential between cytoplasmic and extracellular leaflets and alpha: Proportionality constant]. We find that alpha and psi(s) are 0.120 (+/- 0.01) V(-2) and -0.073 (+/-0.017 V in solutions that contain ion channel blockers and 0.108 (+/- 0.29) V(-2) and -0.023 (+/- 0.009) V when 10 mM sodium salicylate was added to the extracellular solution. This suggests that salicylate does not affect the rate at which C grows with Psi, but reduces the charge asymmetry of the membrane. We also observe an additional linear differential capacitance of about (-46 fFV(-1)) in about 60% of the cells, this additional component acts simultaneously with the quadratic component and was not observed when salicylate was added to the solution. We suggest that the voltage dependent capacitance originates from electromechanical coupling either by electrostriction and/or Maxwell stress effects and estimate that a small electromechanical force (approximately equal to 1 pN) acts at physiological potentials. These results are relevant to understand the electromechanical coupling in outer hair cells (OHCs) of the mammalian cochlea, where an asymmetric bell-shaped C versus Psi relationship is observed upon application of a similar field. Prestin, a membrane protein expressed in OHCs is required to observe this function. When we compare the total charge contributions from HEK cell membrane (7 x 10(4) electrons, 10 pF cell) with that determined for prestin transfected cells (up to 5 x 10(6) electrons) we conclude that the charge contributions from the collective motion of membrane proteins and lipids in the field is dwarfed relative to that when prestin is present. We suggest that the capacitance-voltage relationships should be similar to that observed for HEK cells for OHCs that do not express prestin in their membranes.

Cell Line↗

PubMed2006

Photometric recording of transmembrane potential in outer hair cells.

Cochlear outer hair cells (OHCs) are polarized epithelial cells that have mechanoelectrical transduction channels within their apical stereocilia and produce electromotile force along their lateral wall. Phase shifts, or time delays, in the transmembrane voltage occurring at different axial locations along the cell may contribute to our understanding of how these cells operate at auditory frequencies. We developed a method to optically measure the phase of the OHC transmembrane potential using the voltage-sensitive dye (VSD) di-8-ANEPPS. The exit aperture of a fibre-optic light source was driven in two dimensions so that a 24 microm spot of excitation light could be positioned along the length of the OHC. We used the whole-cell patch-clamp technique in the current-clamp mode to stimulate the OHC at the base. The photometric response and the voltage response were monitored with a photodetector and patch-clamp amplifier, respectively. The photometric response was used to measure the regional changes in the membrane potential in response to maintained (dc) and sinusoidal (ac) current stimuli applied at the base of the cell. We used a neutral density filter to lower the excitation light intensity and reduce phototoxicity. A sensitive detector and lock-in amplifier were used to measure the small ac VSD signal. This permitted measurements of the ac photometric response below the noise floor of the static fluorescence. The amplitude and phase components of the photometric response were recorded for stimuli up to 800 Hz. VSD data at 400-800 Hz show the presence of a small phase delay between the stimulus voltage at the base of the cell and the local membrane potential measured along the lateral wall. Results are consistent with the hypothesis that OHCs exhibit inhomogeneous membrane potentials that vary with position in analogy with the voltage in nerve axons.

Animals↗

PubMed2006

Studies of plasma membrane mechanics and plasma membrane-cytoskeleton interactions using optical tweezers and fluorescence imaging.

We use optical tweezers in conjunction with an optical position-sensing system, which spectrally filters signals generated by a trapped fluorescent microsphere to study plasma membrane (PM) mechanics and its interactions with cytoskeleton. We dynamically measure the PM tethering force on human embryonic kidney cells that are a standard cultured cell line. Recorded tethering force vs. PM displacement profiles, revealed the tether formation process, initiated with linear deformation of the PM, followed by a nonlinear regime and terminated with the local separation of PM. Tethering force vs. displacement profiles were used to estimate tether formation force and stiffness parameter of the PM. Integration of the force-displacement profiles yielded the work of tether formation, including linear and nonlinear components. Our results demonstrate that spectral filtering of the optically trapped fluorescent microsphere image formed on the position-sensing system overcomes the artifacts introduced by the transillumination imaging and allows accurate measures of PM mechanics before and during the initial stages of tether formation.

Biomechanical Phenomena↗

PubMed2006

Dyslipidemia and auditory function.

The relationship between dyslipidemia and hearing is unclear. This study was conducted to investigate whether elevated serum lipid levels impact auditory function in humans and in guinea pigs. In the human study, a cross-sectional study of 40 volunteers with dyslipidemia was conducted. Pure tone thresholds, distortion product otoacoustic emissions, and lipid profiles were analyzed. When controlled for patient age and sex, we found that elevated triglycerides were associated with reduced hearing. In the guinea pig study, a prospective study of animals fed a high-fat diet for 14 weeks was conducted. Although the high-fat diet led to a dramatic elevation in the average weight and total cholesterol in all animals (from 61 to 589 mg/dl), there were no meaningful changes in distortion product otoacoustic emission magnitudes. These results suggest that whereas chronic dyslipidemia associated with elevated triglycerides may reduce auditory function, short-term dietary changes may not.

Adult↗

PubMed2005

Effects of chlorpromazine on mechanical properties of the outer hair cell plasma membrane.

An optical tweezers system was used to characterize the effects of chlorpromazine (CPZ) on the mechanical properties of the mammalian outer hair cell (OHC) through the formation of plasma membrane tethers. Such tethers exhibited force relaxation when held at a constant length for several minutes. We used a second-order generalized Kelvin body to model tether-force behavior from which several mechanical parameters were then calculated including stiffness, viscosity-associated measures, and force relaxation time constants. The results of the analysis portray a two-part relaxation process characterized by significantly different rates of force decay, which we propose is due to the local reorganization of lipids within the tether and the flow of external lipid into the tether. We found that CPZ's effect was limited to the latter phenomenon since only the second phase of relaxation was significantly affected by the drug. This finding coupled with an observed large reduction in overall tether forces implies a common basis for the drug's effects, the plasma membrane-cytoskeleton interaction. The CPZ-induced changes in tether viscoelastic behavior suggest that alterations in the mechanical properties of the OHC lateral wall could play a role in the modulation of OHC electromotility by CPZ.

Animals↗

PubMed2005

Effects of salicylate on plasma membrane mechanics.

High concentrations of the amphipathic drug salicylate (Sal) block outer hair cell (OHC) electromotility resulting in reversible hearing loss. We used optical tweezers to study the effects of Sal on the mechanics of the cell plasma membrane. Membrane tethers were formed from guinea pig OHCs and cultured human embryonic kidney (HEK) cells as controls. HEK cells are commonly used in functional expression studies of electromotility. Effective tether viscosity (eta(eff)), steady-state tethering force extrapolated to zero pulling rate F(ss0), and time constant for tether growth (tau(tg)) were estimated from the measurements of the instantaneous tethering force at different tether pulling rates. Average values of eta(eff), F(ss0), and tau(tg) for the OHC lateral wall plasma membrane and control cell plasma membrane remained the same after Sal perfusion, which is consistent with the hypothesis that Sal-induced reversible hearing loss appears to be more the result of its competition with essential anions and less the result of a change in plasma membrane mechanics.

Animals↗

PubMed2005

High-frequency force generation in the constrained cochlear outer hair cell: a model study.

Cochlear outer hair cell (OHC) electromotility is believed to be responsible for the sensitivity and frequency selectivity of the mammalian hearing process. Its contribution to hearing is better understood by examining the force generated by the OHC as a feedback to vibration of the basilar membrane (BM). In this study, we examine the effects of the constraints imposed on the OHC and of the surrounding fluids on the cell's high-frequency active force generated under in vitro and in vivo conditions. The OHC is modeled as a viscoelastic and piezoelectric cylindrical shell coupled with viscous intracellular and extracellular fluids, and the constraint is represented by a spring with adjustable stiffness. The solution is obtained in the form of a Fourier series. The model results are consistent with previously reported experiments under both low- and high-frequency conditions. We find that constrained OHCs achieve a much higher corner frequency than free OHCs, depending on the stiffness of the constraint. We analyze cases in which the stiffness of the constraint is similar to that of the BM, reticular lamina, and tectorial membrane, and find that the force per unit transmembrane potential generated by the OHC can be constant up to several tens of kHz. This model, describing the OHC as a local amplifier, can be incorporated into a global cochlear model that considers cochlear hydrodynamics and frequency modulation of the receptor potential, as well as the graded BM stiffness and OHC length.

Acoustics↗

PubMed2005

Mechanosensitive channels in the lateral wall can enhance the cochlear outer hair cell frequency response.

We present the results of a modeling study on the impact of mechanosensitive channels in the lateral wall of the outer hair cell on the cell frequency response. The model includes the electrical properties of the cell membrane, piezoelectricity associated with a membrane motor mechanism, and mechanosensitive channels in the cell lateral wall. The outer hair cell is loaded by the vibrating basilar and tectorial membranes, and this loading generates strain in the lateral wall. Our analysis reveals a property, the strain rate sensitivity, that, in concert with the piezoelectric effect, can enhance the cell frequency response. We discuss possible viscoelastic-type mechanisms of the channel's strain rate sensitivity that is consistent with the organization of the composite cell lateral wall. The parameters of our model are chosen on the basis of the previously estimated electrical and piezoelectric properties as well as typical conductance and density of the mechanosensitive channels in cells. We found that the strain rate sensitivity of the channels can result in receptor potentials greater than those predicted by the RC (resistance and capacitance) analysis. The effect of the channels is especially significant in an intermediate range of sound frequencies, and the channel-related gain is up to 3-4 times between 3 and 15 kHz.

Acoustics↗

PubMed2005

Modeling high-frequency electromotility of cochlear outer hair cell in microchamber experiment.

Cochlear outer hair cells (OHC) are critically important for the amplification and sharp frequency selectivity of the mammalian ear. The microchamber experiment has been an effective tool to analyze the OHC high-frequency performance. In this study, the OHC electrical stimulation in the microchamber is simulated. The model takes into account the inertial and viscous properties of fluids inside and outside the cell as well as the viscoelastic and piezoelectric properties of the cell composite membrane (wall). The closed ends of the cylindrical cell were considered as oscillatory rigid plates. The final solution was obtained in terms of Fourier series, and it was checked against the available results of the microchamber experiment. The conditions of the interaction between the cell and pipette was analyzed, and it was found that the amount of slip along the contact surface has a significant effect on the cell electromotile response. The cell's length changes were computed as a function of frequency, and their dependence on the viscosities of both fluids and the cell wall was analyzed. The distribution of the viscous losses inside the fluids was also estimated. The proposed approach can help in a better understanding of the high-frequency OHC electromotility under experimental and physiological conditions.

Cell Movement↗

PubMed2005

Effect of voltage-dependent membrane properties on active force generation in cochlear outer hair cell.

A computational model is proposed to analyze the active force production in an individual outer hair cell (OHC) under high-frequency conditions. The model takes into account important biophysical properties of the cell as well as constraints imposed by the surrounding environment. The biophysical properties include the elastic, piezoelectric, and viscous characteristics of the cell wall. The effect of the environment is associated with the stiffness of the constraint and the drag forces acting on the cell due to the interaction with the external and internal viscous fluids. The study concentrated on a combined effect of the transmembrane potential, frequency, and stiffness of the constraints. The effect of the voltage-dependent stiffness of the cell was particularly investigated and it was found to be twofold. First, it results in higher sensitivity and nonlinearity of the OHC active force production in the physiological range. Second, it determines smaller active forces in the hyperpolarization range. The resonant properties of the active force as functions of voltage and the constraint stiffness were also analyzed. The obtained results can be important for a better understanding of the OHC active force production and the contribution of cell electromotility to the cochlear amplification, sensitivity, and nonlinearity.

Acoustics↗

PubMed2003

Effect of outer hair cell piezoelectricity on high-frequency receptor potentials.

The low-pass voltage response of outer hair cells predicted by conventional equivalent circuit analysis would preclude the active force production at high frequencies. We have found that the band pass characteristics can be improved by introducing the piezoelectric properties of the cell wall. In contrast to the conventional analysis, the receptor potential does not tend to zero and at any frequency is greater than a limiting value. In addition, the phase shift between the transduction current and receptor potential tends to zero. The piezoelectric properties cause an additional, strain-dependent, displacement current in the cell wall. The wall strain is estimated on the basis of a model of the cell deformation in the organ of Corti. The limiting value of the receptor potential depends on the ratio of a parameter determined by the piezoelectric coefficients and the strain to the membrane capacitance. In short cells, we have found that for the low-frequency value of about 2-3 mV and the strain level of 0.1% the receptor potential can reach 0.4 mV throughout the whole frequency range. In long cells, we have found that the effect of the piezoelectric properties is much weaker. These results are consistent with major features of the cochlear amplifier.

Cell Size↗

PubMed2003

Outer hair cell piezoelectricity: frequency response enhancement and resonance behavior.

Stretching or compressing an outer hair cell alters its membrane potential and, conversely, changing the electrical potential alters its length. This bi-directional energy conversion takes place in the cell's lateral wall and resembles the direct and converse piezoelectric effects both qualitatively and quantitatively. A piezoelectric model of the lateral wall has been developed that is based on the electrical and material parameters of the lateral wall. An equivalent circuit for the outer hair cell that includes piezoelectricity shows a greater admittance at high frequencies than one containing only membrane resistance and capacitance. The model also predicts resonance at ultrasonic frequencies that is inversely proportional to cell length. These features suggest all mammals use outer hair cell piezoelectricity to support the high-frequency receptor potentials that drive electromotility. It is also possible that members of some mammalian orders use outer hair cell piezoelectric resonance in detecting species-specific vocalizations.

Animals↗

PubMed2002

Membrane tether formation from outer hair cells with optical tweezers.

Optical tweezers were used to characterize the mechanical properties of the outer hair cell (OHC) plasma membrane by pulling tethers with 4.5-microm polystyrene beads. Tether formation force and tether force were measured in static and dynamic conditions. A greater force was required for tether formations from OHC lateral wall (499 +/- 152 pN) than from OHC basal end (142 +/- 49 pN). The difference in the force required to pull tethers is consistent with an extensive cytoskeletal framework associated with the lateral wall known as the cortical lattice. The apparent plasma membrane stiffness, estimated under the static conditions by measuring tether force at different tether length, was 3.71 pN/microm for OHC lateral wall and 4.57 pN/microm for OHC basal end. The effective membrane viscosity was measured by pulling tethers at different rates while continuously recording the tether force, and estimated in the range of 2.39 to 5.25 pN x s/microm. The viscous force most likely results from the viscous interactions between plasma membrane lipids and the OHC cortical lattice and/or integral membrane proteins. The information these studies provide on the mechanical properties of the OHC lateral wall is important for understanding the mechanism of OHC electromotility.

Animals↗

PubMed2002

Urocortin-deficient mice display normal stress-induced anxiety behavior and autonomic control but an impaired acoustic startle response.

Corticotropin-releasing hormone (Crh) plays an important role in modulating physiological and behavioral responses to stress. Its actions are mediated through two receptors, Crhr1 and Crhr2. Urocortin (Ucn), a Crh-related neuropeptide and the postulated endogenous ligand for Crhr2, is a potential mediator of stress responses. We generated Ucn-deficient mice using embryonic stem cell technology to determine its role in stress-induced behavioral and autonomic responses. Unlike Crhr1- or Crhr2-deficient mice, Ucn-deficient mice exhibit normal anxiety-like behavior as well as autonomic regulation in response to stress. However, the mutant mice display an impaired acoustic startle response that is not due to an obvious hearing defect. Thus, our results suggest that Ucn does not play an essential role in stress-induced behavioral and autonomic responses. Ucn may modulate the acoustic startle response through the Ucn-expressing neuron projections from the region of the Edinger-Westphal nucleus.

Alleles↗

PubMed2002

Excess plasma membrane and effects of ionic amphipaths on mechanics of outer hair cell lateral wall.

The interaction between the outer hair cell (OHC) lateral wall plasma membrane and the underlying cortical lattice was examined by a morphometric analysis of cell images during cell deformation. Vesiculation of the plasma membrane was produced by micropipette aspiration in control cells and cells exposed to ionic amphipaths that alter membrane mechanics. An increase of total cell and vesicle surface area suggests that the plasma membrane possesses a membrane reservoir. Chlorpromazine (CPZ) decreased the pressure required for vesiculation, whereas salicylate (Sal) had no effect. The time required for vesiculation was decreased by CPZ, indicating that CPZ decreases the energy barrier required for vesiculation. An increase in total volume is observed during micropipette aspiration. A deformation-induced increase in hydraulic conductivity is also seen in response to micropipette-applied fluid jet deformation of the lateral wall. Application of CPZ and/or Sal decreased this strain-induced hydraulic conductivity. The impact of ionic amphipaths on OHC plasma membrane and lateral wall mechanics may contribute to their effects on OHC electromotility and hearing.

Animals↗

PubMedDate not supplied

Standing wave total internal reflection fluorescence microscopy to measure the size of nanostructures in living cells.

We present the first application of standing wave fluorescence microscopy (SWFM) to determine the size of biological nanostructures in living cells. The improved lateral resolution of less than 100 nm enables superior quantification of the size of subcellular structures. We demonstrate the ability of SWFM by measuring the diameter of biological nanotubes (membrane tethers formed between cells). The combination of SWFM with total internal reflection (TIR), referred to as SW-TIRFM, allows additional improvement of axial resolution by selective excitation of fluorescence in a layer of about 100 nm.

Equipment Design↗