A novel theoretical framework reveals more than one voltage-sensing pathway in the lateral membrane of outer hair cells.

Outer hair cell (OHC) electromotility amplifies acoustic vibrations throughout the frequency range of hearing. Electromotility requires that the lateral membrane protein prestin undergo a conformational change upon changes in the membrane potential to produce an associated displacement charge. The magnitude of the charge displaced and the mid-reaction potential (when one half of the charge is displaced) reflects whether the cells will produce sufficient gain at the resting membrane potential to boost sound in vivo. Voltage clamp measurements performed under near-identical conditions ex vivo show the charge density and mid-reaction potential are not always the same, confounding interpretation of the results. We compare the displacement charge measurements in OHCs from rodents with a theory shown to exhibit good agreement with in silico simulations of voltage-sensing reactions in membranes. This model equates the charge density to the potential difference between two pseudo-equilibrium states of the sensors when they are in a stable conformation and not contributing to the displacement current. The model predicts this potential difference to be one half of its value midway into the reaction, when one equilibrium conformation transforms to the other pseudo-state. In agreement with the model, we find the measured mid-reaction potential to increase as the charge density decreases to exhibit a negative slope of ∼1/2. This relationship suggests that the prestin sensors exhibit more than one stable hyperpolarized state and that voltage sensing occurs by more than one pathway. We determine the electric parameters for prestin sensors and use the analytical expressions of the theory to estimate the energy barriers for the two voltage-dependent pathways. This analysis explains the experimental results, supports the theoretical approach, and suggests that voltage sensing occurs by more than one pathway to enable amplification throughout the frequency range of hearing.

Scientist and data architect collaborate to curate and archive an inner ear electrophysiology data collection.

In the past scientists reported summaries of their findings; they did not provide their original data collections. Many stakeholders (e.g., funding agencies) are now requesting that such data be made publicly available. This mandate is being adopted to facilitate further discovery, and to mitigate waste and deficits in the research process. At the same time, the necessary infrastructure for data curation (e.g., repositories) has been evolving. The current target is to make research products FAIR (Findable, Accessible, Interoperable, Reusable), resulting in data that are curated and archived to be both human and machine compatible. However, most scientists have little training in data curation. Specifically, they are ill-equipped to annotate their data collections at a level that facilitates discoverability, aggregation, and broad reuse in a context separate from their creation or sub-field. To circumvent these deficits data architects may collaborate with scientists to transform and curate data. This paper's example of a data collection describes the electrical properties of outer hair cells isolated from the mammalian cochlea. The data is expressed with a variant of The Ontology for Biomedical Investigations (OBI), mirrored to provide the metadata and nested data architecture used within the Hierarchical Data Format version 5 (HDF5) format. Each digital specimen is displayed in a tree configuration (like directories in a computer) and consists of six main branches based on the ontology classes. The data collections, scripts, and ontological OWL file (OBI based Inner Ear Electrophysiology (OBI_IEE)) are deposited in three repositories. We discuss the impediments to producing such data collections for public use, and the tools and processes required for effective implementation. This work illustrates the impact that small collaborations can have on the curation of our publicly-funded collections, and is particularly salient for fields where data is sparse, throughput is low, and sacrifice of animals is required for discovery.

Electromechanics and Volume Dynamics in Nonexcitable Tissue Cells.

Cell volume regulation is fundamentally important in phenomena such as cell growth, proliferation, tissue homeostasis, and embryogenesis. How the cell size is set, maintained, and changed over a cell's lifetime is not well understood. In this work we focus on how the volume of nonexcitable tissue cells is coupled to the cell membrane electrical potential and the concentrations of membrane-permeable ions in the cell environment. Specifically, we demonstrate that a sudden cell depolarization using the whole-cell patch clamp results in a 50% increase in cell volume, whereas hyperpolarization results in a slight volume decrease. We find that cell volume can be partially controlled by changing the chloride or the sodium/potassium concentrations in the extracellular environment while maintaining a constant external osmotic pressure. Depletion of external chloride leads to a volume decrease in suspended HN31 cells. Introducing cells to a high-potassium solution causes volume increase up to 50%. Cell volume is also influenced by cortical tension: actin depolymerization leads to cell volume increase. We present an electrophysiology model of water dynamics driven by changes in membrane potential and the concentrations of permeable ions in the cells surrounding. The model quantitatively predicts that the cell volume is directly proportional to the intracellular protein content.

Force Measurements for Cancer Cells.

During cytoskeleton remodeling, cancer cells generate force at the plasma membrane that originates from chemical motors (e.g., actin). This force (pN) and its time course reflect the on and off-rates of the motors. We describe the design and calibration of a force-measuring device (i.e., optical tweezers) that is used to monitor this force and its time course at the edge of a cell, with particular emphasis on the temporal resolution of the instrument.

Measuring forces at the leading edge: a force assay for cell motility.

Cancer cells become mobile by remodelling their cytoskeleton to form migratory structures. This transformation is dominated by actin assembly and disassembly (polymerisation and depolymerisation) in the cytoplasm. Synthesis of filamentous actin produces a force at the leading edge that pushes the plasma membrane forward. We describe an assay to measure the restoring force of the membrane in response to forces generated within the cytoplasm adjacent to the membrane. A laser trap is used to form a long membrane nanotube from a living cell and to measure the axial membrane force at the end of the tube. When the tube, resembling a filopodium, is formed and in a relaxed state the axial membrane force exhibits a positive stationary value. This value reflects the influence of the cytoskeleton that acts to pull the tube back to the cell. A dynamic sawtooth force that rides upon the stationary value is also observed. This force is sensitive to a toxin that affects actin assembly and disassembly, but not affected by agents that influence microtubules and myosin light chain kinase. We deduce from the magnitude and characteristics of dynamic force measurements that it originates from depolymerisation and polymerisation of F-actin. The on- and off-rates, the number of working filaments, and the force per filament (2.5 pN) are determined. We suggest the force-dependent transitions are thermodynamically uncoupled as both the on- and off-rates decrease exponentially with a compressive load. We propose kinetic schemes that require attachment of actin filaments to the membrane during depolymerisation. This demonstrates that actin kinetics can be monitored in a living cell by measuring force at the membrane, and used to probe the mobility of cells including cancer cells.

Effects of cholesterol on nano-mechanical properties of the living cell plasma membrane.

In this study, we investigated the effects of membrane cholesterol content on the mechanical properties of cell membranes by using optical tweezers. We pulled membrane tethers from human embryonic kidney cells using single and multi-speed protocols, and obtained time-resolved tether forces. We quantified various mechanical characteristics including the tether equilibrium force, bending modulus, effective membrane viscosity, and plasma membrane-cytoskeleton adhesion energy, and correlated them to the membrane cholesterol level. Decreases in cholesterol concentration were associated with increases in the tether equilibrium force, tether stiffness, and adhesion energy. Tether diameter and effective viscosity increased with increasing cholesterol levels. Disruption of cytoskeletal F-actin significantly changed the tether diameters in both non-cholesterol and cholesterol-manipulated cells, while the effective membrane viscosity was unaffected by F-actin disruption. The findings are relevant to inner ear function where cochlear amplification is altered by changes in membrane cholesterol content.

Tonotopic relationships reveal the charge density varies along the lateral wall of outer hair cells.

Outer hair cells amplify and improve the frequency selectivity of sound within the mammalian cochlea through a sound-evoked receptor potential that induces an electromechanical response in their lateral wall membrane. We experimentally show that the membrane area and linear membrane capacitance of outer hair cells increases exponentially with the electrically evoked voltage-dependent charge movement (Q(T)) and peak membrane capacitance (C(peak)). We determine the size of the different functional regions (e.g., lateral wall, synaptic basal pole) of the polarized cells from the tonotopic relationships. We then establish that Q(T) and C(peak) increase with the logarithm of the lateral wall area (A(LW)) and determine from the functions that the charge (σ(LW,) pC/µm(2)) and peak (ρ(LW,) pF/µm(2)) densities vary inversely with A(LW) (σ(LW) = 1.3/A(LW) and ρ(LW) = 9/A(LW)). This shows contrary to conventional wisdom that σ(LW) and ρ(LW) are not constant along the length of an individual outer hair cell.

Power efficiency of outer hair cell somatic electromotility.

Cochlear outer hair cells (OHCs) are fast biological motors that serve to enhance the vibration of the organ of Corti and increase the sensitivity of the inner ear to sound. Exactly how OHCs produce useful mechanical power at auditory frequencies, given their intrinsic biophysical properties, has been a subject of considerable debate. To address this we formulated a mathematical model of the OHC based on first principles and analyzed the power conversion efficiency in the frequency domain. The model includes a mixture-composite constitutive model of the active lateral wall and spatially distributed electro-mechanical fields. The analysis predicts that: 1) the peak power efficiency is likely to be tuned to a specific frequency, dependent upon OHC length, and this tuning may contribute to the place principle and frequency selectivity in the cochlea; 2) the OHC power output can be detuned and attenuated by increasing the basal conductance of the cell, a parameter likely controlled by the brain via the efferent system; and 3) power output efficiency is limited by mechanical properties of the load, thus suggesting that impedance of the organ of Corti may be matched regionally to the OHC. The high power efficiency, tuning, and efferent control of outer hair cells are the direct result of biophysical properties of the cells, thus providing the physical basis for the remarkable sensitivity and selectivity of hearing.

Voltage and frequency dependence of prestin-associated charge transfer.

Membrane protein prestin is a critical component of the motor complex that generates forces and dimensional changes in cells in response to changes in the cell membrane potential. In its native cochlear outer hair cell, prestin is crucial to the amplification and frequency selectivity of the mammalian ear up to frequencies of tens of kHz. Other cells transfected with prestin acquire voltage-dependent properties similar to those of the native cell. The protein performance is critically dependent on chloride ions, and intrinsic protein charges also play a role. We propose an electro-diffusion model to reveal the frequency and voltage dependence of electric charge transfer by prestin. The movement of the combined charge (i.e., anion and protein charges) across the membrane is described with a Fokker-Planck equation coupled to a kinetic equation that describes the binding of chloride ions to prestin. We found a voltage- and frequency-dependent phase shift between the transferred charge and the applied electric field that determines capacitive and resistive components of the transferred charge. The phase shift monotonically decreases from zero to -90 degrees as a function of frequency. The capacitive component as a function of voltage is bell-shaped, and decreases with frequency. The resistive component is bell-shaped for both voltage and frequency. The capacitive and resistive components are similar to experimental measurements of charge transfer at high frequencies. The revealed nature of the transferred charge can help reconcile the high-frequency electrical and mechanical observations associated with prestin, and it is important for further analysis of the structure and function of this protein.

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.

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.

Estimating the time course of pore expansion during the spike phase of exocytotic release in mast cells of the beige mouse.

Our objective is to determine the time course of exocytotic fusion pore opening (P) in mast cells of the beige mouse from the measured efflux of the spike phase of exocytotic release (J). We show that a pore whose meridian or radius grows linearly with time cannot reproduce the efflux. We also show that a pore that opens very quickly [relative to the diffusivity of 5-hydroxytryptamine (5-HT)] and completely (P = pi) also does not mimic the experimental efflux, and estimate maximum pore angles of 70 (+/- 20) degrees. We show that a larger class of opening functions reproduces the rising phase and part of the decay phase and calculate pore expansion rate, pore radius and pore angle, none of which can be readily measured. In the initial stages of the spike phase (50-200 ms) when the gel matrix has not expanded significantly, this model suggests that the pore radius increases exponentially with a time constant of 82(+/- 62) ms with pore expansion reaching its maximum velocity of 20 (+/- 7) nm ms-1. We conclude that the release process is dynamic and suggest that the velocity of pore opening (V) and the diffusivity of 5-HT (D), in addition to the size of the vesicle (R, radius), vary with time. We discuss assumptions and improvements to the model and propose that this methodology is applicable for determining P from measured J in other endocrine cells and neurons when D within the secretory vesicle is much less than D within the pore neck.