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 voltage-sensor 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.
Chloride is the major anion in cells, with many diseases arising from disordered Cl- regulation. For the non-invasive investigation of Cl- flux, YFP-H148Q and its derivatives chameleon and Cl-Sensor ...previously were introduced as genetically encoded chloride indicators. Neither the Cl- sensitivity nor the pH-susceptibility of these modifications to YFP is optimal for precise measurements of Cl- under physiological conditions. Furthermore, the relatively poor photostability of YFP derivatives hinders their application for dynamic and quantitative Cl- measurements. Dynamic and accurate measurement of physiological concentrations of chloride would significantly affect our ability to study effects of chloride on cellular events.
In this study, we developed a series of YFP derivatives to remove pH interference, increase photostability and enhance chloride sensitivity. The final product, EYFP-F46L/Q69K/H148Q/I152L/V163S/S175G/S205V/A206K (monomeric Cl-YFP), has a chloride Kd of 14 mM and pKa of 5.9. The bleach time constant of 175 seconds is over 15-fold greater than wild-type EYFP. We have used the sensor fused to the transmembrane protein prestin (gerbil prestin, SLC26a5), and shown for the first time physiological (mM) chloride flux in HEK cells expressing this protein. This modified fluorescent protein will facilitate investigations of dynamics of chloride ions and their mediation of cell function.
Modifications to YFP (EYFP-F46L/Q69K/H148Q/I152L/V163S/S175G/S205V/A206K (monomeric Cl-YFP) results in a photostable fluorescent protein that allows measurement of physiological changes in chloride concentration while remaining minimally affected by changes in pH.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The voltage-dependent protein SLC26a5 (prestin) underlies outer hair cell electromotility (eM), which is responsible for cochlear amplification in mammals. The electrical signature of eM is a ...bell-shaped nonlinear capacitance (NLC), deriving from prestin sensor-charge (Q
) movements, which peaks at the membrane voltage, V
, where charge is distributed equally on either side of the membrane. Voltage dependencies of NLC and eM differ depending on interrogation frequency and intracellular chloride, revealing slow intermediate conformational transitions between anion binding and voltage-driven Q
movements. Consequently, NLC exhibits low-pass characteristics, substantially below prevailing estimates of eM frequency response. Here we study in guinea pig and mouse of either sex synchronous prestin electrical (NLC, Q
) and mechanical (eM) activity across frequencies under voltage clamp (whole cell and microchamber). We find that eM and Q
magnitude and phase correspond, indicating tight piezoelectric coupling. Electromechanical measures (both NLC and eM) show dual-Lorentzian, low-pass behavior, with a limiting (τ
) time constant at V
of 32.6 and 24.8 μs, respectively. As expected for voltage-dependent kinetics, voltage excitation away from V
has a faster, flatter frequency response, with our fastest measured τ
for eM of 18.2 μs. Previous observations of ultrafast eM (τ ≈ 2 μs) were obtained at offsets far removed from V
We hypothesize that trade-offs in eM gain-bandwith arising from voltage excitation at membrane potentials offset from V
influence the effectiveness of cochlear amplification across frequencies.
Of two types of hair cells within the organ of Corti, inner hair cells and outer hair cells, the latter evolved to boost sensitivity to sounds. Damage results in hearing loss of 40-60 dB, revealing amplification gains of 100-1000× that arise from voltage-dependent mechanical responses electromotility (eM). eM, driven by the membrane protein prestin, may work beyond 70 kHz. However, this speed exceeds, by over an order of magnitude, kinetics of typical voltage-dependent membrane proteins. We find eM is actually low pass in nature, indicating that prestin bears kinetics typical of other membrane proteins. These observations highlight potential difficulties in providing sufficient amplification beyond a cutoff frequency near 20 kHz. Nevertheless, observed trade-offs in eM gain-bandwith may sustain cochlear amplification across frequency.
The mammalian outer hair cell (OHC) protein prestin (Slc26a5) differs from other Slc26 family members due to its unique piezoelectric-like property that drives OHC electromotility, the putative ...mechanism for cochlear amplification. Here, we use cryo-electron microscopy to determine prestin's structure at 3.6 Å resolution. Prestin is structurally similar to the anion transporter Slc26a9. It is captured in an inward-open state which may reflect prestin's contracted state. Two well-separated transmembrane (TM) domains and two cytoplasmic sulfate transporter and anti-sigma factor antagonist (STAS) domains form a swapped dimer. The transmembrane domains consist of 14 transmembrane segments organized in two 7+7 inverted repeats, an architecture first observed in the bacterial symporter UraA. Mutation of prestin's chloride binding site removes salicylate competition with anions while retaining the prestin characteristic displacement currents (Nonlinear Capacitance), undermining the extrinsic voltage sensor hypothesis for prestin function.
Cochlear amplification denotes a boost to auditory sensitivity and selectivity that is dependent on outer hair cells from Corti's organ. Voltage-driven electromotility of the cell is believed to feed ...energy back into the cochlear partition via a cycle-by-cycle mechanism at very high acoustic frequencies. Here we show using wide-band macro-patch voltage-clamp to drive prestin, the molecular motor underlying electromotility, that its voltage-sensor charge movement is unusually low pass in nature, being incapable of following high-frequency voltage changes. Our data are incompatible with a cycle-by-cycle mechanism responsible for high-frequency tuning in mammals.
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•Outer hair cells (OHC) boost auditory sensation for very high acoustic frequencies•We studied the frequency response of OHC's electromechanical nonlinear capacitance•The response is incommensurate with cycle-by-cycle feedback at very high frequencies•OHCs likely use another mechanism to drive cochlear amplification at high frequencies
Auditory Evoked Response; Bioelectronics; Mechanobiology
Large‐conductance calcium‐activated potassium (BK) channels play a critical role in electrical resonance, a mechanism of frequency selectivity in chicken hair cells. We determine that BK currents are ...dependent on inward flow of Ca2+, and intracellular buffering of Ca2+. Entry of Ca2+ is further amplified locally by calcium‐induced Ca2+ release (CICR) in close proximity to plasma membrane BK channels. Ca2+ imaging reveals peripheral clusters of high concentrations of Ca2+ that are suprathreshold to that needed to activate BK channels. Protein kinase A (PKA) activation increases the size of BK currents likely by recruiting more BK channels due to spatial spread of high Ca2+ concentrations in turn from increasing CICR. STORM imaging confirms the presence of nanodomains with ryanodine and IP3 receptors in close proximity to the Slo subunit of BK channels. Together, these data require a rethinking of how electrical resonance is brought about and suggest effects of CICR in synaptic release. Both genders were included in this study.
In hair cells calcium entering through voltage gated calcium channels activate BK channels and also induce calcium induced calcium release. Calcium induced calcium release further extends the spatial range of BK channel activation in proximity to voltage gated calcium channels.
Abstract
The outer hair cell (OHC) membrane harbors a voltage-dependent protein, prestin (SLC26a5), in high density, whose charge movement is evidenced as a nonlinear capacitance (NLC). NLC is ...bell-shaped, with its peak occurring at a voltage, V
h
, where sensor charge is equally distributed across the plasma membrane. Thus, V
h
provides information on the conformational state of prestin. V
h
is sensitive to membrane tension, shifting to positive voltage as tension increases and is the basis for considering prestin piezoelectric (PZE). NLC can be deconstructed into real and imaginary components that report on charge movements in phase or 90 degrees out of phase with AC voltage. Here we show in membrane macro-patches of the OHC that there is a partial trade-off in the magnitude of real and imaginary components as interrogation frequency increases, as predicted by a recent PZE model (Rabbitt in Proc Natl Acad Sci USA 17:21880–21888, 2020). However, we find similar behavior in a simple 2-state voltage-dependent kinetic model of prestin that lacks piezoelectric coupling. At a particular frequency, F
is
, the complex component magnitudes intersect. Using this metric, F
is
, which depends on the frequency response of each complex component, we find that initial V
h
influences F
is
; thus, by categorizing patches into groups of different V
h
, (above and below − 30 mV) we find that F
is
is lower for the negative V
h
group. We also find that the effect of membrane tension on complex NLC is dependent, but differentially so, on initial V
h
. Whereas the negative group exhibits shifts to higher frequencies for increasing tension, the opposite occurs for the positive group. Despite complex component trade-offs, the low-pass roll-off in absolute magnitude of NLC, which varies little with our perturbations and is indicative of diminishing total charge movement, poses a challenge for a role of voltage-driven prestin in cochlear amplification at very high frequencies.
In the developing auditory system, spontaneous activity generated in the cochleae propagates into the central nervous system to promote circuit formation. The effects of peripheral firing patterns on ...spontaneous activity in the central auditory system are not well understood. Here, we describe wide-spread bilateral coupling of spontaneous activity that coincides with the period of transient efferent modulation of inner hair cells from the brainstem medial olivocochlear system. Knocking out α9/α10 nicotinic acetylcholine receptors, a requisite part of the efferent pathway, profoundly reduces bilateral correlations. Pharmacological and chemogenetic experiments confirm that the efferent system is necessary for normal bilateral coupling. Moreover, auditory sensitivity at hearing onset is reduced in the absence of pre-hearing efferent modulation. Together, these results demonstrate how afferent and efferent pathways collectively shape spontaneous activity patterns and reveal the important role of efferents in coordinating bilateral spontaneous activity and the emergence of functional responses during the prehearing period.
The outer hair cell from Corti's organ possesses voltage-dependent intramembranous molecular motors evolved from the SLC26 anion transporter family. The motor, identified as prestin (SLC26a5), is ...responsible for electromotility of outer hair cells and mammalian cochlear amplification, a process that heightens our auditory responsiveness. Here, we describe experiments designed to evaluate the effects of anions on the motor's voltage-sensor charge movement, focusing on prestin's voltage-dependent Boltzmann characteristics. We find that the nature of the anion, including species, valence, and structure, regulates characteristics of the charge movement, signifying that anions play a more complicated role than simple voltage sensing in cochlear amplification.
Outer hair cell (OHC) nonlinear capacitance (NLC) represents voltage sensor charge movements of prestin (SLC26a5), the protein responsible for OHC electromotility. Previous measures of NLC frequency ...response have employed methods which did not assess the influence of dielectric loss (sensor charge movements out of phase with voltage) that may occur, and such loss conceivably may influence prestin's frequency dependent activity. Here we evaluate prestin's complex capacitance out to 30 kHz and find that prestin's frequency response determined using this approach coincides with all previous estimates. We also show that membrane tension has no effect on prestin's frequency response, despite substantial shifts in its voltage operating range, indicating that prestin transition rate alterations do not account for the shifts. The magnitude roll-off of prestin activity across frequency surpasses the reductions of NLC caused by salicylate treatments that are known to abolish cochlear amplification. Such roll-off likely limits the effectiveness of prestin in contributing to cochlear amplification at the very high acoustic frequencies processed by some mammals.
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