Tonotopic Relationships Reveal the Charge Density Varies along the Lateral Wall of Outer Hair Cells

Proc. Natl. Acad. Sci. U.S.A.

2013

Outer hair cells (OHCs) drive cochlear amplification that enhances our ability to detect and discriminate sounds. The motor protein, prestin, which evolved from the SLC26 anion transporter family, underlies the OHC's voltage-dependent mechanical activity (eM). Here we report on simultaneous measures of prestin's voltagesensor charge movement (nonlinear capacitance, NLC) and eM that evidence disparities in their voltage dependence and magnitude as a function of intracellular chloride, challenging decades' old dogma that NLC reports on eM steady-state behavior. A very simple kinetic model, possessing fast anion-binding transitions and fast voltage-dependent transitions, coupled together by a much slower transition recapitulates these disparities and other biophysical observations on the OHC. The intermediary slow transition probably relates to the transporter legacy of prestin, and this intermediary gateway, which shuttles anion-bound molecules into the voltage-enabled pool of motors, provides molecular delays that present as phase lags between membrane voltage and eM. Such phase lags may help to effectively inject energy at the appropriate moment to enhance basilar membrane motion.hearing | molecular motor O uter hair cells (OHC) foster mechanical feedback within the mammalian cochlear partition that enhances perception of auditory stimuli by 2-3 orders of magnitude; this is known as cochlear amplification (1, 2). Following the molecular identification of prestin (3), an SLC26 family member (a5) that recapitulates the electromechanical properties of the native OHC's voltage sensor/motor (4, 5), a preponderance of evidence pointed to prestin-based electromotility (eM) as the basis of this boost (6, 7). During the last couple of decades, measures of nonlinear charge movement or capacitance (NLC), an electrical correlate of voltage-dependent conformational changes within the motor protein prestin, have served as surrogate, indeed as signature, of voltage-evoked eM of the OHC (8-12). Intracellular chloride plays an important role in prestin function (13), and recent evidence indicates that anions serve a modulatory, allosteric-like role (14-17). For the most part, anion effects, as well as many other influences on prestin, have been limited to the study of NLC, on the assumption that NLC reports on steady-state characteristics of OHC eM. We now show that this assumption is wrong.Using simultaneous measures of charge movement and eM, we show disparity between deduced characteristics of prestin motor protein conformation. That is, a dissociation in magnitude and voltage dependence of NLC and eM is revealed by lowering intracellular chloride to physiological levels (≤ 10 mM; ref. 6), all previous comparisons having been made with abnormally high intracellular chloride levels. Furthermore, this chloride effect, which presents as a function of rate and polarity of voltage stimulation, uncovers a mechanism predicted to be frequency dependent in vivo, and likely plays an important role in frequency selectivity and efficienc...

Section: Models Of Prestinmentioning

confidence: 97%

Disparities in voltage-sensor charge and electromotility imply slow chloride-driven state transitions in the solute carrier SLC26a5

Proc. Natl. Acad. Sci. U.S.A.

2013

“…Several mechanisms have been postulated to understand why the RC filter should not interfere with prestin activity at high frequencies. Previously, we envisioned three mechanisms that might help: 1), because turgor pressure within the OHC shifts the voltage dependence of NLC and electromotility (22,29,30), we suggested that alterations in turgor pressure could align V h and resting potential (22); 2), because prestin charge density increases in high-frequency OHCs (31,32), we reasoned that the rising electrical energy delivered to the lateral membrane could counter the reduced voltage drive; and 3), we reasoned that the flux of chloride through the stretch-activated chloride conductance, G metL , in the lateral membrane could gate prestin's chloride-dependent activity independent of the membrane RC (6). Though the first two possibilities remain tenable, the latter is unlikely, because we have recently discovered that the effects of chloride on prestin are low-pass filtered by a slow intermediary state transition (33).…”

Section: Discussionmentioning

confidence: 99%

An Electrical Inspection of the Subsurface Cisternae of the Outer Hair Cell

2015

Biophysical Journal

The cylindrical outer hair cell (OHC) of Corti's organ drives cochlear amplification by a voltage-dependent activation of the molecular motor, prestin (SLC26a5), in the cell's lateral membrane. The voltage-dependent nature of this process leads to the troublesome observation that the membrane resistor-capacitor filter could limit high-frequency acoustic activation of the motor. Based on cable theory, the unique 30 nm width compartment (the extracisternal space, ECS) formed between the cell's lateral membrane and adjacent subsurface cisternae (SSC) could further limit the influence of receptor currents on lateral membrane voltage. Here, we use dual perforated/whole-cell and loose patch clamp on isolated OHCs to sequentially record currents resulting from excitation at apical, middle, and basal loose patch sites before and after perforated patch rupture. We find that timing of currents is fast and uniform before whole-cell pipette washout, suggesting little voltage attenuation along the length of the lateral membrane. Prior treatment with salicylate, a disrupter of the SSC, confirms the influence of the SSC on current spread. Finally, a cable model of the OHC, which can match our data, indicates that the SSC poses a minimal barrier to current flow across it, thereby facilitating rapid delivery of voltage excitation to the prestin-embedded lateral membrane.

“…We have previously estimated that mechanical responses at high acoustic frequencies would be markedly smaller than basilar motion, based on the cell's RC time constant. This problem has been addressed by many investigators, and many ostensible resolutions to the RC time-constant problem have been proposed (15,25,(59)(60)(61)(62). However, we must now consider the slow kinetics of prestin at physiological chloride levels that we have uncovered.…”

Section: Frequency Dependence Of Ohc Charge Movementmentioning

confidence: 99%

Chloride Anions Regulate Kinetics but Not Voltage-Sensor Q max of the Solute Carrier SLC26a5

2016

Biophysical Journal

In general, SLC26 solute carriers serve to transport a variety of anions across biological membranes. However, prestin (SLC26a5) has evolved, now serving as a motor protein in outer hair cells (OHCs) of the mammalian inner ear and is required for cochlear amplification, a mechanical feedback mechanism to boost auditory performance. The mechanical activity of the OHC imparted by prestin is driven by voltage and controlled by anions, chiefly intracellular chloride. Current opinion is that chloride anions control the Boltzmann characteristics of the voltage sensor responsible for prestin activity, including Qmax, the total sensor charge moved within the membrane, and Vh, a measure of prestin's operating voltage range. Here, we show that standard narrow-band, high-frequency admittance measures of nonlinear capacitance (NLC), an alternate representation of the sensor's charge-voltage (Q-V) relationship, is inadequate for assessment of Qmax, an estimate of the sum of unitary charges contributed by all voltage sensors within the membrane. Prestin's slow transition rates and chloride-binding kinetics adversely influence these estimates, contributing to the prevalent concept that intracellular chloride level controls the quantity of sensor charge moved. By monitoring charge movement across frequency, using measures of multifrequency admittance, expanded displacement current integration, and OHC electromotility, we find that chloride influences prestin kinetics, thereby controlling charge magnitude at any particular frequency of interrogation. Importantly, however, this chloride dependence vanishes as frequency decreases, with Qmax asymptoting at a level irrespective of the chloride level. These data indicate that prestin activity is significantly low-pass in the frequency domain, with important implications for cochlear amplification. We also note that the occurrence of voltage-dependent charge movements in other SLC26 family members may be hidden by inadequate interrogation timescales, and that revelation of such activity could highlight an evolutionary means for kinetic modifications within the family to address hearing requirements in mammals.