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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Biol Chem. Author manuscript; available in PMC 2009 May 8.
Published in final edited form as:
PMCID: PMC2679373
NIHMSID: NIHMS101524

Tuning of the Outer Hair Cell Motor by Membrane Cholesterol*,S

Abstract

Cholesterol affects diverse biological processes, in many cases by modulating the function of integral membrane proteins. We observed that alterations of cochlear cholesterol modulate hearing in mice. Mammalian hearing is powered by outer hair cell (OHC) electromotility, a membrane-based motor mechanism that resides in the OHC lateral wall. We show that membrane cholesterol decreases during maturation of OHCs. To study the effects of cholesterol on hearing at the molecular level, we altered cholesterol levels in the OHC wall, which contains the membrane protein prestin. We show a dynamic and reversible relationship between membrane cholesterol levels and voltage dependence of prestin-associated charge movement in both OHCs and prestin-transfected HEK 293 cells. Cholesterol levels also modulate the distribution of prestin within plasma membrane microdomains and affect prestin self-association in HEK 293 cells. These findings indicate that alterations in membrane cholesterol affect prestin function and functionally tune the outer hair cell.

Cholesterol is an important component of the plasma membranes of most animal cells. It modulates the mechanical properties of the membrane and affects the function of membrane-associated proteins. Recent studies have shown modulation by membrane cholesterol of such diverse membrane proteins as rhodopsin (1, 2), the serotonin receptor 1A (3) and serotonin transporter 5HT1 (4), the chloride channel ClC-2 (5), several classes of potassium channels (6, 7), the nicotinic acetylcholine receptor (8), and several G-protein-coupled receptors (9, 10). These studies indicate that cholesterol may act as follows: 1) by binding directly to and influencing the conformation and dynamics of membrane proteins (1114); 2) by altering the biophysical and mechanical properties of membranes (1519); and/or 3) by promoting the formation of cholesterol-rich microdomains (9, 20, 21). These heterogeneous ordered microdomains contain distinctive lipid and protein compositions in which cholesterol may contribute as much as 50% of the membrane lipids (2224). Cholesterol-rich membrane domains might serve to compartmentalize cellular processes, promote protein-protein and lipid-protein interactions, and thereby regulate protein function (23, 25, 26).

Cellular cholesterol levels are tightly regulated, and disruption of cholesterol homeostasis leads to a host of disease conditions. Several clinical and experimental studies carried out using rabbits, chinchillas, guinea pigs, and human subjects have linked sensorineural hearing loss and/or increase of hearing thresholds to hypercholesterolemia (2731). Reduction of cholesterol by statins or apheresis has been shown to delay hearing loss in mice (32) and improve hearing recovery in humans (33). A study of hypercholesterolemic humans indicated that the effects of cholesterol on hearing might involve effect(s) on nonlinear mechanical processes in the cochlea (34).

A piezoelectric-like membrane-based motor in the outer hair cell (OHC)7 contributes to the exquisite sensitivity and frequency selectivity of mammalian hearing. This motor mechanism is required to counteract viscous damping in the fluid-filled cochlea, which would otherwise impair mechanical tuning. The OHC lateral wall is specialized for electro-mechanical force transduction (35, 36). Here, the energy in the transmembrane electric field is converted into mechanical energy. The organization of the OHC lateral wall is unique among hair cells and among all adult mammalian cell types. It is an elegant, nanoscale (~100 nm thick), trilaminate structure. The outer and inner layers are the plasma membrane and subsurface cisternae, respectively, and sandwiched between them is a layer of cytoskeletal proteins called the cortical lattice. The lipid composition of the plasma membrane and the subsurface cisternae membranes is unknown, but the constituent lipids are in the fluid phase allowing for free diffusion (3739). Labeling studies suggest that lateral wall membranes contain less cholesterol than the OHC apical and basal plasma membranes (4043). The relatively low cholesterol level of the OHC lateral wall plasma membrane is unusual among animal cells, and may serve to modulate the function of the membrane proteins that reside there. These proteins include a modified anion exchanger AE2 (44), the Glut5 sugar transporter (4547), stretch-activated ion channels (4850), and prestin (45).

Prestin (SLC26A5), a critical component of the OHC lateral wall motor, is a polytopic integral membrane protein (45, 51, 52) and is essential for OHC electromotility and mammalian hearing (53). Prestin greatly increases charge movement into and out of, as opposed to through, the membrane (54, 55). Intracellular anions such as chloride and bicarbonate have been shown to be the charge carrier (55) consistent with the membership of prestin in the SLC26A family of anion transporters (56). When transfected into several mammalian cell lines, prestin confers a voltage-dependent nonlinear capacitance (NLC), the accepted electrical signature of electromotility (54, 55) (see supplemental text for in-depth description).

Motivated by the clinical effects of cholesterol on hearing and the reduced cholesterol levels in the OHC lateral wall, we have explored the effect of cholesterol on hearing at the organ, cellular, and molecular levels to clarify its biological basis of action. We observe that cholesterol affects otoacoustic emissions and functionally tunes nonlinear mechanical processes in the OHC, most likely through its effects on the OHC membrane protein prestin.

EXPERIMENTAL PROCEDURES

Materials

Methyl-β-cyclodextrin, water-soluble cholesterol (MβCD loaded with cholesterol), filipin, and bovine serum albumin were obtained from Sigma. Primers were obtained from Sigma Genosys. Anti-flotillin-1 antibody (1:250 working dilution) was purchased from BD Biosciences. Anti-HA (1:1000) was purchased from Cell Signaling Technology (Danvers, MA). Anti- GFP anti-mouse monoclonal antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). AlexaFluor 594 phalloidin (1:200), AlexaFluor 594 goat anti-mouse antibody (1:800), and concanavalinA-AlexaFluor 350 conjugate (working concentration 50–200 μg/ml) were purchased from Molecular Probes (Carlsbad, CA). Peroxidase-labeled horse anti-mouse antibody was obtained from Vector Laboratories (Burlingame, CA). The ECL Western blotting detection kit was obtained from Amersham Biosciences.

DPOAE Measurements

Mice used for DPOAE measurements were of a mixed genetic background derived from two strains, 129SvEv and C57B6/J, and were 4–8 weeks old. Healthy mice were anesthetized with ketamine/xylazine and immobilized in a head holder. The pinna was resected and the middle ear bulla opened to expose the round window. An earbar connected to two speakers and a probe tip microphone were inserted into the ear canal to within 2 mm of the tympanic membrane. The cubic distortion product amplitude was measured using an F2 frequency of 20 kHz with F1 = F2/1.2 (57). The intensities of the primary tones were equal. First, we ranged the primary tones from 20 to 80 db in 10-db steps to verify that there was no notch in the DPOAE amplitude curve between 50 and 70 db. During the experiment, we set the primary tones to 60-db sound pressure level, and the DPOAE amplitude was measured every 9 s. After a few minutes, a borosilicate micropipette with a tip diameter of ~50 μm containing the treatment solution (either 100 mM MβCD, 200mM water-soluble cholesterol, ~200mM raffinose, or 10 mM water-soluble cholesterol) was carefully inserted through the round window membrane. The high concentrations of each treatment (in comparison with established in vitro studies) were chosen to compensate for dilution of the solutions in the mouse perilymph. The treatment solutions were allowed to diffuse passively into the perilymph. The middle ear space was monitored for fluid seepage, and any fluid was carefully aspirated. DPOAE amplitudes were collected for up to 30 min. In some cases, at the conclusion of the experiment, the basilar membrane was perforated to eliminate DPOAEs, thereby verifying the measurements obtained. DPOAE amplitudes were then normalized so that the amplitude after the micropipette was inserted and all middle ear fluid was cleared was 0 db. This time window is indicated as a gray box in each panel of Fig. 1.

FIGURE 1
Effect of cochlear cholesterol loading/depletion on DPOAE amplitude

Outer Hair Cell Isolation

Albino guinea pigs of either sex weighing 200–300 g and having a normal startle response to a hand clap were decapitated. The temporal bones were taken and the middle ear bullae opened. The otic capsule was removed, and the spiral ligament was peeled off to expose the organ of Corti. The modiolus with the intact organ of Corti was removed from the temporal bone and subjected to mild trypsinization for ~10 min at room temperature and trituration to detach OHCs. OHCs were plated onto the glass bottom of a coated microwell Petri dish (MatTek, Ashland, MA). Isolated cells were selected for study on the basis of standard morphological criteria within 4 h of animal death. Under the light microscope, healthy cells display a characteristic birefringence, a uniformly cylindrical shape without regional swelling, a basally located nucleus, and no Brownian motion of subcellular cytoplasmic particles (58).

Prestin Constructs and Transfection

Gerbil prestin was cloned into the pIRES-hrGFP vector (Stratagene, La Jolla, CA) as a HA tag fusion protein (HA-prestin) and into the pEGFP, pECFP, and pEYFP vectors (Clontech) as a GFP, CFP, or YFP fusion protein (prestin-EGFP), as described previously (5961). The prestin-ECFP and prestin-EYFP constructs were modified by site-directed mutagenesis (QuikChange mutagenesis kit, Stratagene, La Jolla, CA) to include a single amino acid substitution (A206K) on the CFP/YFP fusion protein, which renders CFP/YFP monomeric. The sequences of the constructs were verified using five overlapping sequencing primers. NLC measurements confirmed that all constructs used in this study are functional in HEK 293 cells. HEK 293 cell lines were transfected 24 h after passage with prestin-EGFP, prestin-ECFP, prestin-EYFP, or HA-prestin at a 3:1 ratio of DNA with FuGENE 6 (Roche Applied Science).

Cholesterol Manipulations

Outer Hair Cells

Because of the sensitivity of outer hair cells to temperature and their deterioration with time after isolation, cholesterol manipulations were performed differently in OHCs than in HEK 293 cells. Cholesterol depletion was carried out by pipetting MβCD into the external solution in the dish containing hair cells at a final concentration of ~100 μM (1/100th that used in HEK cells, see below) and incubating at room temperature (see Fig. 3 for times of incubation). Higher concentrations of MβCD produced drastic morphological changes in OHCs and even cell death because of destabilization of the cholesterol-rich apical and basal membranes, causing the nucleus to be blown out of the cell. Cholesterol was loaded in a similar manner at a final concentration of 1mM of MβCD containing cholesterol (also referred to as water-soluble cholesterol). In both cases, treatment was carried out after forming a whole-cell patch on an OHC, and capacitance recordings were taken throughout the incubation time.

FIGURE 3
Cholesterol levels affect Vpkc of nonlinear capacitance in outer hair cells

HEK 293 Cells

Steady-state electrophysiological measurements were performed on prestin-transfected HEK 293 cells, 48 h post-transfection, after treatment with 10 mM MβCD or water-soluble cholesterol (at a 10 mM MβCD concentration) for 30 min at 37 °C. The effects of cholesterol manipulations were followed kinetically in HEK 293 cells by pipetting MβCD or water-soluble cholesterol into the external solution at a final concentration of 10mM after obtaining a whole-cell patch. HEK 293 cells did not show morphological changes as seen in OHCs upon cholesterol depletion. Filipin labeling of untreated, cholesterol- depleted, and cholesterol loaded HEK 293 cells (supplemental Fig. 1) shows changes in filipin fluorescence signal with cholesterol manipulations, confirming that cholesterol levels are altered by our treatments.

Electrophysiological Measurements

Electrophysiological data were obtained from cells using the whole-cell voltage clamp technique. Our recording techniques are fully described earlier (60) and a brief description follows. Culture dishes containing transfected cells were placed on the stage of an inverted microscope (Carl Zeiss, Gottingen, Germany) under ×100 magnification and extensively perfused with the extracellular solution containing Ca2+ and K+ channel blockers prior to recording. All recordings were conducted at room temperature (23 ± 1 °C). Patch pipettes (quartz glass) with resistances ranging from 2 to 4 megohms were fabricated using a laser-based micropipette puller (P-2000, Sutter Instrument Company, Novato, CA) and filled with an intracellular solution, also containing channel blockers. For cell membrane admittance, Y was measured with the patch-clamp technique in the whole-cell mode using a DC voltage ramp with dual frequency stimulus (62) from −0.14 to 0.14 V with a holding potential of 0 V, and the cell parameters were calculated from the admittance as described earlier (63). The conductance, b, was also determined experimentally with a DC protocol, as described earlier (60).

In all representations, capacitances were normalized with respect to base-line capacitance (taken as the capacitance at 0.1 V), and peak capacitance (differs according to treatment), as in Equation 1,

Cnorm=(C(V)Cbaseline)/CbaselineCfinal=Cnorm/Cnormpkc
(Eq. 1)

where C(V) is the capacitance at voltage V; Cbaseline is capacitance at base-line voltage (defined above), and Cnormpkc is equal to Cnorm at Vpkc.

Tissue Preparation and Filipin Labeling of Mouse OHCs

P6, P12, and adult ICR mice were sacrificed by cervical dislocation and decapitation. The temporal bone was removed, and the bony capsule was stripped in fresh cold Hanks’ balanced salt solution (Invitrogen). The membranous labyrinth was exposed in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum. The sensory epithelium was isolated and affixed to round glass coverslips coated with Cell-Tak (BD Biosciences). The tissue was washed twice with PBS, fixed with 4% paraformaldehyde for 30 min, and stained with filipin dye (4 mg/ml) and AlexaFluor 594 phalloidin for 30 min. The samples were then washed twice with PBS, mounted on glass slides with Fluoromount G antifade reagent, and sealed with nail polish. Images were captured on a Zeiss Axioplan microscope (Carl Zeiss Optics Company, Jena, Germany) with ×63 objective and analyzed with Applied Precision SoftWoRx deconvolution software. Images of individual OHCs were analyzed using NIH Image software, and pixel intensities along a line drawn through the middle of a single OHC were plotted as bar graphs in Fig. 2.

FIGURE 2
OHC lateral wall cholesterol content decreases with maturation

Immunofluorescence and Imaging

HA-prestin transfected cells on coverslips were either treated with or without 10mM MβCD or water-soluble cholesterol for 30 min at 37 °C. Cells were then washed with PBS, stained with concanavalinA-AlexaFluor 350 conjugate (Molecular Probes, Carlsbad, CA) for 1 h on ice, washed with PBS again, and then permeabilized with PBS/Triton X-100 before fixing with 4% paraformaldehyde in PBS. The cells were then stained with anti-HA antibody (1:1200; Cell Signaling Technology, Inc., Danvers, MA), followed by AlexaFluor 594 goat anti-mouse secondary antibody (1:800; Molecular Probes, Carlsbad, CA). Coverslips were mounted inverted on glass slides with Fluoromount G antifade reagent (Electron Microscopy Sciences, Hatfield, PA) and fluorescent images captured on a Zeiss LSM 510 deconvolution microscope (Carl Zeiss Optics Company, Jena, Germany) with ×63 objective and analyzed with Applied Precision SoftWoRx image restoration software. Images were also obtained using a Zeiss LSM 510 confocal microscope with ×63 objective and analyzed using Zeiss AIM imaging software.

Membrane Fractionation

Cell membranes were fractionated as described by Vetrivel et al. (64). Briefly, HEK 293 cells expressing HA-prestin, treated with or without MβCD or with water-soluble cholesterol (as detailed above), were lysed in buffer (0.5% Lubrol WX, 25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, and 1 mM phenylmethanesulfonyl fluoride). Membranes were fractionated on a 5, 35, and 45% sucrose step gradient. Twelve 1-ml fractions were collected, and excess lipids in each fraction were removed by methanol/chloroform precipitation before the proteins were analyzed by 7.5% SDS-PAGE. This experiment was repeated at least six times with invariant results; a representative blot is shown in Fig. 6.

FIGURE 6
Cholesterol affects membrane distribution of prestin

Cross-linking

Forty eight hours post-transfection with HA-prestin, HEK 293 cells were either treated with methyl-β-cyclodextrin (MβCD) or with water-soluble cholesterol for 30 min at 37 °C, or left untreated, before gentle harvesting by scraping into 1 ml of PBS, pH 8.0. The cells were pelleted (2000 × g for 5 min) and incubated with various concentrations of cross-linker bis(sulfosuccinimidyl) suberate (0.078 to 5 mM of BS3) or without cross-linker for 30 min at room temperature. Reactions were quenched with 50 mM Tris, pH 7.5. The amount of protein in each sample was measured and normalized prior to gel loading. Samples were mixed with 8% 2× SDS sample buffer and incubated for 30 min at room temperature before fractionation by a 4–8% Tris-glycine PAGE and analysis by Western blotting.

Fluorescence Resonance Energy Transfer

Fluorescence resonance energy transfer (FRET), implemented on a Zeiss LSM 510 confocal microscope (Carl Zeiss Optics Company, Jena, Germany), was used to measure the degree of prestin self-association following cholesterol perturbations. HEK cells were cotransfected with prestin-CFP (donor) and prestin-YFP (acceptor). Both the CFP and YFP fusion proteins had an engineered mutation, A206K, that prevents CFP and YFP dimerization to allow easier interpretation of FRET results. Details of the acceptor photobleach technique utilized have been published previously (61). Briefly, a region of interest (ROI) on the cell membrane, exhibiting even, membrane localized fluorescence, was bleached to remove YFP signal. CFP fluorescence intensity in the bleached ROI was measured pre- and post-bleach to arrive at the value of FRET efficiency (Ef) from CFP to YFP. CFP intensity in an adjacent unbleached ROI was measured pre- and post-bleach to derive control (Cf) values for each FRET measurement. For detailed description of methods, refer to Greeson et al. (61).

Quantification of Puncta

Membrane segments used for FRET experimentation were also utilized in puncta quantification. Confocal images of prestin- YFP in living HEK 293 cell membranes were cropped to ~25 by 75-pixel (1 pixel = 0.14 μm) membrane-containing regions (slice thicknesses = 3.3 μm) and analyzed using the Matlab (The Mathworks, Natick, MA) edge detection filter. The filter generated an image the same size as the input image composed of ones where edges were detected and zeros everywhere else (supplemental Fig. 3). Edge detection is based on one of six specific methods, and the most advanced of the available methods, the Canny method, was used in this work. The Canny method identifies local maxima in the image gradients and uses two different threshold values to define both strong and weak edges. A weak edge is only included in the output image if it is connected to a strong edge. The use of two threshold values ensures that this method is less likely to detect false positive weak edges. Following application of the filter, the output image was analyzed for the presence of puncta. Puncta identification was guided by the output image of the detection filter and supported by the membrane region image (supplemental Fig. 3). The results of this analysis are shown in Fig. 5.

FIGURE 5
Prestin is expressed in punctate foci in the HEK 293 membrane

Statistical Analysis

Vpkcs and capacitance gains in OHCs and prestin-transfected HEK 293 cells subjected to different treatments (untreated, cholesterol loading, and cholesterol depletion) were compared using two-tailed t tests. Statistical significance (in comparison to untreated) is indicated in Table 1. Two-way analysis of variance was also used to evaluate statistical significance of FRET values in comparison to control FRET for each treatment, as well as to FRET values of untreated cells. Statistical significance is indicated by asterisks in Fig. 7D and Table 1.

FIGURE 7
Effect of cholesterol on prestin self-association
TABLE 1
Vpkc of nonlinear capacitance in OHCs and prestin-expressing HEK 293 cells

RESULTS

Changes in Cholesterol Alter the Amplitude of Distortion Product Otoacoustic Emissions (DPOAEs)

To study the effects of cholesterol on hearing at the organ level, we evaluated cochlear function in vivo by measuring DPOAE amplitudes during cholesterol alteration (Fig. 1). Cholesterol depletion resulted in a 20-db decrease in DPOAE amplitudes. Cholesterol loading, on the other hand, resulted in an initial 2–3-db increase in DPOAE amplitude, followed by a decrease of up to 20 db (Fig. 1). These results confirm that cholesterol modulates hearing. Because DPOAE amplitudes reflect OHC electromotility, the level of cholesterol in the OHC lateral wall may be functionally relevant.

OHC Lateral Wall Cholesterol Content Decreases with Maturation

To visualize cholesterol in the OHC lateral wall, we used filipin labeling of mouse OHCs (Fig. 2). Filipin labeling clearly indicates membrane cholesterol content, as shown by filipin labeling of untreated, cholesterol-depleted and -loaded HEK 293 cells (supplemental Fig. 1). OHCs from P6 and P12 mice showed distinct filipin labeling of the lateral wall, compared with cytoplasm. However, OHCs from adult mice showed intracellular filipin staining surrounded by a region of lower staining corresponding to the membrane (Fig. 2, right panels). The filipin staining patterns are clearly visible in the pixel intensity graphs (Fig. 2, bottom row), which plot pixel intensities along the diameter of a single OHC in each case. These data indicate that the cholesterol in the OHC lateral wall is initially high and decreases during development. This time frame parallels the onset and maturation of OHC electromotility (65), which includes a depolarizing shift in prestin-associated charge movement.

Alterations in Cholesterol Content Modulate Nonlinear Capacitance in Isolated OHCs

We directly evaluated the effect of cholesterol on prestin-associated charge movement at the cellular level by altering cholesterol content in isolated guinea pig OHCs. We observed characteristic bell-shaped voltage-dependent capacitance (NLC) with a peak, Vpkc, at about −0.050 V in untreated OHCs (Fig. 3A), similar to that reported earlier (66). Depletion of cholesterol shifted Vpkc in the depolarizing direction to about +0.080 V, whereas loading excess cholesterol shifted Vpkc toward more hyperpolarizing voltages (less than −0.130 V, see Fig. 3A). Kinetic studies of these phenomena indicated that the shift in Vpkc occurred within minutes of adding water-soluble cholesterol (loading) or MβCD (depletion) (Fig. 3B). Furthermore, the effects of depletion and loading were reversible, and the reversal of the Vpkc shift was equally rapid (Fig. 3C). These data (statistics represented in Table 1) indicate a direct and dynamic correlation between OHC membrane cholesterol content and Vpkc.

Changes in Membrane Cholesterol Reversibly Alter Vpkc in Prestin-transfected HEK 293 Cells

We next investigated prestin- specific effects of cholesterol in HEK 293 cells. The Vpkc of prestin-transfected HEK 293 cells was approximately −0.070 V (Fig. 4A). Upon cholesterol depletion, the Vpkc shifted toward depolarized voltages, with an average peak at +0.004 V (Fig. 4A). On the other hand, upon cholesterol loading, the Vpkc shifted toward hyperpolarized voltages, with an average value of about −0.116 V (Fig. 4A). Importantly, these effects are reversible. Cholesterol depletion followed by loading, as well as cholesterol enrichment followed by depletion, both shifted the peak voltage toward the control untreated average (Fig. 4B). The kinetics of these processes were similar to those measured for OHCs, with changes in the Vpkc occurring within minutes of addition of cholesterol or MβCD (Fig. 4C), and the process was rapidly reversible (Fig. 4D). Comparisons of changes in Vpkc in HEK 293 cells and OHCs are shown in Table 1. Our results demonstrate that changes in cholesterol alter prestin-associated charge movement in HEK 293 cells, as in the native environment of the OHC, indicating direct molecular level effects of cholesterol on prestin function.

FIGURE 4
Cholesterol affects membrane capacitance of HEK 293 cells expressing prestin

Cholesterol Modulates the Distribution of Prestin in Membrane Microdomains

We explored the possibility of cholesterol modulating the presence of prestin in membrane microdomains by analyzing membrane localization and the distribution of prestin in HEK 293 cells (Fig. 5). Prestin colocalizes with the plasma membrane (Fig. 5A), consistent with earlier observations (59, 60), and is expressed in foci suggestive of localization to membrane microdomains (Fig. 5, A and D). Alterations in cholesterol content change the distribution of the foci; upon cholesterol depletion (Fig. 5, B and E), prestin showed a less punctate (more uniform) membrane distribution, whereas upon cholesterol loading (Fig. 5, C and F), the number of foci increased (Fig. 5G). Quantification of puncta (Fig. 5G) was based on multiple images of membrane segments from different batches of treated HEK 293 cells (supplemental Fig. 3). In addition, time-lapse images of a single transfected cell taken during the course of depletion or loading show a clear decrease and increase in puncta (supplemental Fig. 4). These results demonstrate that cholesterol influences prestin distribution within the membrane.

To assay prestin localization in membrane microdomains, we characterized detergent-resistant membrane extracts isolated from prestin-transfected HEK 293 cells. As expected, prestin was detected in the dense endoplasmic reticulum membrane fractions (Fig. 6A, lanes 8–10), which cofractionated with the endoplasmic reticulum markers (59). Prestin was also seen in the less dense plasma membrane fractions and predominantly localized in membrane microdomain fractions (Fig. 6A, lanes 4 and 5), where it cofractionated with flotillin-1, a microdomain marker and structural component (67). Upon cholesterol depletion prestin, but not flotillin, redistributed out of the microdomain fractions (Fig. 6B). Upon cholesterol loading, prestin remained in microdomain fractions with higher intensities of all bands (Fig. 6C). These data indicate that prestin can localize to membrane microdomains and that cholesterol modulates its distribution in these domains.

Manipulation of Cholesterol Content Alters Prestin Associations

Localization to microdomains raises the possibility that prestin may interact with itself or other proteins. To determine whether prestin-prestin interactions were altered by cholesterol, we analyzed prestin complex associations by cross-linking (Fig. 7). In the absence of cross-linker (lane 1), a weak prestin dimer band exists in untreated cell extracts (Fig. 7A). This band disappears upon cholesterol depletion (Fig. 7B) and is stronger upon cholesterol loading (Fig. 7C), indicating cholesterol favors prestin self-association in HEK 293 cells independent of cross-linking. Prestin dimers were present at relatively low BS3 cross-linker concentrations (Fig. 7A, lane 2), and the dimer band intensified with increasing cross-linker concentrations (Fig. 7A, lanes 3–7). Cholesterol depletion caused a decrease in prestin cross-linking, with higher concentrations of BS3 required to trap prestin as dimers (Fig. 7, compare lanes 2 and 3 in B with lanes 2 and 3 in A). In contrast, loading cholesterol into the membrane caused the appearance of oligomeric protein bands even in the absence of cross-linker (Fig. 7C, lane 1) and bands intensified with increasing cross-linker (Fig. 7C, lanes 2–8). In conjunction with PFO-polyacrylamide gels (supplemental Fig. 2A) and coimmunoprecipitation experiments (supplemental Fig. 2B), and in accordance with earlier observations (61, 68, 69), these data provide strong evidence of prestin self-association and indicate that prestin self-association increases with cholesterol content.

Further evidence of cholesterol-dependent prestin self-association was obtained using acceptor photobleach FRET. We found an average FRET efficiency of 6.7% (Fig. 7D) in live, untreated HEK 293 cells cotransfected with prestin-CFP and prestin-YFP. Cholesterol enrichment increased average FRET efficiency to 8.4%, whereas cholesterol depletion resulted in a marked decrease of FRET efficiency to 0.5% (Fig. 7D), indicating significant loss of prestin self-association. In this case, the measured FRET efficiency is indistinguishable from corresponding control efficiency measurements (p > 0.05). To determine whether this elimination of prestin self-association is reversible, cells were first depleted of membrane cholesterol and then reloaded. Upon reloading, FRET efficiency returned to 6.8% (Fig. 7D). These results confirm that modulation of prestin self-association by membrane cholesterol is dynamic and reversible. Our organ, cellular, and molecular level studies greatly expand our understanding of the role of cholesterol in tuning the functionality of the outer hair cell motor.

DISCUSSION

Cholesterol is crucial for membrane organization and dynamics and in the regulation of membrane protein sorting and function. Considering that electromotility is based in the highly specialized and cholesterol-poor OHC lateral membrane, the established link between elevated cholesterol and auditory dysfunction (27, 28, 34, 70, 71) suggests direct effects of cholesterol on the OHC membrane and proteins present therein. Models for OHC electromotility based solely on the electromechanical transduction capabilities of the membrane have been proposed (72, 73). Following the discovery of the membrane protein prestin, numerous studies have demonstrated how alterations of membrane material properties affect prestin function and/or OHC electromotility (67, 7479). These studies point to a dynamic interplay between prestin and the membrane in the generation of nonlinear capacitance and electromotility; our study further characterizes this dynamic relationship.

Cholesterol Effects on Otoacoustic Emissions Correlate to Effects on NLC

Cochlear cholesterol alterations influence DPOAE amplitudes (Fig. 1). Manipulating cholesterol in isolated guinea pig OHCs revealed a dynamic and reversible relationship between membrane cholesterol content and prestin-associated charge movement, with similar kinetics (Fig. 3). Cholesterol effects on DPOAEs may result from the observed effects on prestin-associated charge movement as follows. In normal untreated OHCs, the NLC peak is at approximately −0.050 V; therefore, in the cell’s range of receptor potential (nominally between −0.060 and −0.080 V) capacitance is sub-maximal. Shifting the NLC peak in the depolarizing direction (as upon cholesterol depletion) would result in a progressive lowering of capacitance and electromotility in the range of the cell’s receptor potential. This would reduce DPOAE amplitudes. On the other hand, shifting the NLC peak in the hyperpolarizing direction would result in an initial increase of capacitance at the cell’s receptor potential followed by a decrease. Electromotility and DPOAE amplitudes would follow this pattern. This relationship is schematically presented in Fig. 8.

FIGURE 8
Schematic representation of correlation between NLC peak shifts and electromotility

OHC Function May Be Modulated by Cholesterol Reduction during Maturation

We show a lowering of membrane cholesterol with maturation (Fig. 2). During the post-natal maturation of rodent OHCs, the distribution of prestin in the lateral wall is initially inhomogeneous (45, 65). Concurrent with prestin distribution becoming homogeneous, maturation of electromotility and nonlinear capacitance is observed, which includes a shift in Vpkc from an immature hyperpolarized value to the normal adult value (45, 65). Both effects may result from a decrease in OHC membrane cholesterol levels with maturation. Our data suggest that the reduction in membrane cholesterol with maturation helps to tune the membrane-based motor to operate at maximal gain in the OHC receptor potential range.

HEK 293 Cells Provide a Model System for Studying Prestin Function

Cholesterol manipulations in prestin-transfected HEK 293 cells (Fig. 4) produced qualitatively similar results as in OHCs (Fig. 3; Table 1), validating the use of HEK 293 cells as a model system. The difference between Vpkc in cholesterol-depleted OHCs and HEK 293 cells may be due to structural differences (membrane tension and turgor pressure) or differences in cholesterol homeostasis mechanisms, between the two cell types, which cause similar trends but different magnitudes in the effects of depletion.

Prestin-transfected HEK 293 cells allowed for histological and biochemical analyses following alterations in cholesterol. Prestin appears to be present in foci characteristic of membrane microdomains in HEK 293 cells (Figs. 5 and and6).6). Similar foci have not been observed in the adult OHC lateral wall membrane. Perturbing cholesterol content alters the distribution of prestin; prestin shifts out (cholesterol depletion, Fig. 6B) or remains in (cholesterol enrichment, Fig. 6C) the microdomain fractions, suggesting that prestin is capable of localizing to cholesterol rich microdomains. In addition, a quantitative correlation exists between cholesterol content and the number of prestin puncta in the membrane; cholesterol depletion results in a reduction, whereas enrichment causes an increase in number of foci (Fig. 5G).

Recent studies have suggested that prestin may self-associate and dimerize (61, 68, 69). We have obtained further evidence of prestin self-association using a cross-linking reagent, which revealed the presence of prestin-prestin interactions that are decreased upon cholesterol depletion and increased upon cholesterol addition (Fig. 7, A–C). Furthermore, FRET measurements provide direct evidence of the significant and reversible effect of cholesterol on prestin self-associations (Fig. 7D). In light of these data, the low cholesterol levels on the mature OHC lateral wall are consistent with the homogeneous distribution of prestin.

Mechanism of Cholesterol Effects

The effects of cholesterol on membrane protein function have been the subject of numerous recent studies, and several mechanisms have been put forth to explain the effects of cholesterol. In addition to the effects on membrane material properties such as viscosity, elasticity, compressibility, and stiffness (80), cholesterol levels in the membrane influence the formation of ordered microdomains (81, 82) and partitioning of proteins into these domains by altering the bending modulus of the membrane (16) and thereby influencing hydrophobic mismatch (15). The same factors may explain the effect of cholesterol content on prestin-associated charge movement. Because cholesterol is known to modulate membrane material properties, which in turn affect the dynamics of membrane proteins, cholesterol-dependent changes in membrane stiffness or curvature could alter the dynamic fluctuations of prestin as would changes in membrane dipole potential and lipid packing density (19). Several studies in the OHC have correlated changes in membrane tension, stiffness, and mechanics to changes in the Vpkc and electromotility (67, 7577, 79). Molecular dynamics simulations suggest cholesterol affects lipid lateral pressure profiles (17, 18), and this would impact the prestin conformational change that is assumed to accompany charge movement.

Cholesterol-induced Vpkc shifts in both OHCs and HEK 293 cells are larger than those resulting from previous manipulations; these include exogenous chlorpromazine (77), fructose (46), and increasing intracellular pressure (76, 83), which shift Vpkc toward depolarizing potentials; and decreasing intracellular pressure and exposure to the lipophilic ion, tetraphenylborate (TPB) (84), which move Vpkc in the hyperpolarizing direction. Because the magnitude of the Vpkc shifts is significantly greater than in previous manipulations that are known to change the material properties of the membrane, we must consider the possibility that the Vpkc is also a function of self-association. This contribution would reflect the relative amounts of monomers versus higher order oligomers, where the monomeric form shifts Vpkc to depolarizing voltages, whereas higher order oligomers shifts Vpkc to hyperpolarizing voltages. The effect of cholesterol on both prestin self-association and on prestin function is reversible, indicating a dynamic interaction of prestin with membrane components.

Cholesterol also has a propensity to localize to membrane microdomains. Prestin is present in microdomains in HEK 293 cells, and its presence in these localized domains may facilitate its interaction with itself or with other proteins. It is likely that prestin exists in a dynamic equilibrium between monomeric, dimeric, and perhaps higher order oligomeric forms. The effect of cholesterol might be to “cluster” prestin molecules, shifting the equilibrium toward dimeric or oligomeric species. Our data indicate increased self-association in the presence of increased cholesterol. The low cholesterol level observed in the mature OHC lateral wall suggests a preference for lowered prestin selfassociation, the functional consequences of which remain to be studied.

In summary, our study integrates systems-level, cellular and molecular data to investigate the role of cholesterol in modulating the mechanical aspects of mammalian hearing. We have characterized interrelationships between prestin-prestin interactions and prestin-membrane interactions. Whether the effect of cholesterol is predominantly through formation of functionally distinct microdomains, changes in membrane material properties, or both, the observable effects of changing the cholesterol content are a change in prestin self-association, a reversible shift in Vpkc, and changes in otoacoustic emissions. This reinforces the concept of the molecular motor driving electromotility as an interdependent entity with protein and membrane components working cooperatively to achieve nonlinear charge movement and mechanical motion.

Supplementary Material

SuppFig1

SuppFig2

SuppFig3

SuppFig4

Acknowledgments

We thank Dominik Oliver, Huey Huang, Jonathan Sachs, Nathan Baker, and Henry Pownall for useful comments and Linda Lee for technical assistance.

Footnotes

*This work was supported in part by NIDCD Grant DC00354 from the National Institutes of Health (to W. E. B. and F. A. P.), National Science Foundation Grant BES-0522862 (to F. A. P.), NIDCD Grant DC008134 from the National Institutes of Health (to R. M. R. and F. A. P.), and the Deafness Research Foundation (to L. R.).

SThe on-line version of this article (available at http://www.jbc.org) contains supplemental text and Figs. 14.

7The abbreviations used are: OHC, outer hair cell;MβCD, methyl-β-cyclodextrin; NLC, nonlinear capacitance; GFP, CFP, YFP, green, cyan, or yellow fluorescent protein; HA, hemagglutinin; DPOAE, distortion product otoacoustic emissions; FRET, fluorescence resonance energy transfer; ROI, region of interest; BS3, bis (sulfosuccinimidyl) suberate; PFO, perfluoro-octanoic acid; PBS, phosphate-buffered saline; P, postnatal day; prestin-E, prestin-enhanced.

References

1. Boesze-Battaglia K, Albert AD. J Biol Chem. 1990;265:20727–20730. [PubMed]
2. Albert AD, Boesze-Battaglia K. Prog Lipid Res. 2005;44:99–124. [PubMed]
3. Chattopadhyay A, Jafurulla M, Kalipatnapu S, Pucadyil TJ, Harikumar KG. Biochem Biophys Res Commun. 2005;327:1036–1041. [PubMed]
4. Scanlon SM, Williams DC, Schloss P. Biochemistry. 2001;40:10507–10513. [PubMed]
5. Hinzpeter A, Fritsch J, Borot F, Trudel S, Vieu DL, Brouillard F, Baudoin-Legros M, Clain J, Edelman A, Ollero M. J Biol Chem. 2007;282:2423–2432. [PubMed]
6. Romanenko VG, Fang Y, Byfield F, Travis AJ, Vandenberg CA, Rothblat GH, Levitan I. Biophys J. 2004;87:3850–3861. [PubMed]
7. Hajdu P, Varga Z, Pieri C, Panyi G, Gaspar R., Jr Pfluegers Arch. 2003;445:674–682. [PubMed]
8. Rankin SE, Addona GH, Kloczewiak MA, Bugge B, Miller KW. Biophys J. 1997;73:2446–2455. [PubMed]
9. Pucadyil TJ, Chattopadhyay A. Prog Lipid Res. 2006;45:295–333. [PubMed]
10. Burger K, Gimpl G, Fahrenholz F. Cell Mol Life Sci. 2000;57:1577–1592. [PubMed]
11. Addona GH, Sandermann H, Jr, Kloczewiak MA, Miller KW. Biochim Biophys Acta. 2003;1609:177–182. [PubMed]
12. Addona GH, Sandermann H, Jr, Kloczewiak MA, Husain SS, Miller KW. Biochim Biophys Acta. 1998;1370:299–309. [PubMed]
13. Jones OT, McNamee MG. Biochemistry. 1988;27:2364–2374. [PubMed]
14. Radhakrishnan A, Sun LP, Kwon HJ, Brown MS, Goldstein JL. Mol Cell. 2004;15:259–268. [PubMed]
15. Allende D, Vidal A, McIntosh TJ. Trends Biochem Sci. 2004;29:325–330. [PubMed]
16. Song J, Waugh RE. Biophys J. 1993;64:1967–1970. [PubMed]
17. Niemela PS, Ollila S, Hyvonen MT, Karttunen M, Vattulainen I. Plos Comput Biol. 2007;3:e34. [PMC free article] [PubMed]
18. Samuli Ollila OH, Rog T, Karttunen M, Vattulainen I. J Struct Biol 2007 [PubMed]
19. Starke-Peterkovic T, Turner N, Vitha MF, Waller MP, Hibbs DE, Clark RJ. Biophys J. 2006;90:4060–4070. [PubMed]
20. Lee AG. Biochim Biophys Acta. 2004;1666:62–87. [PubMed]
21. Ohvo-Rekila H, Ramstedt B, Leppimaki P, Peter Slotte J. Prog Lipid Res. 2002;41:66–97. [PubMed]
22. Pike LJ. Biochem J. 2004;378:281–292. [PubMed]
23. Pike LJ. J Lipid Res. 2003;44:655–667. [PubMed]
24. Simons K, Ikonen E. Science. 2000;290:1721–1726. [PubMed]
25. Lucero HA, Robbins PW. Arch Biochem Biophys. 2004;426:208–224. [PubMed]
26. Simons K, Ikonen E. Nature. 1997;387:569–572. [PubMed]
27. Saito T, Sato K, Saito H. Arch Otorhinolaryngol. 1986;243:242–245. [PubMed]
28. Sikora MA, Morizono T, Ward WD, Paparella MM, Leslie K. Acta Otolaryngol. 1986;102:372–381. [PubMed]
29. Morizono T, Paparella MM. Ann Otol Rhinol Laryngol. 1978;87:804–814. [PubMed]
30. Morizono T, Sikora MA, Ward WD, Paparella MM, Jorgensen J. Acta Otolaryngol. 1985;99:516–524. [PubMed]
31. Marcucci R, Alessandrello Liotta A, Cellai AP, Rogolino A, Berloco P, Leprini E, Pagnini P, Abbate R, Prisco D. J Thromb Haemostasis. 2005;3:929–934. [PubMed]
32. Syka J, Ouda L, Nachtigal P, Solichova D, Semecky V. Neurosci Lett. 2007;411:112–116. [PubMed]
33. Suckfull M. Ther Apher Dial. 2001;5:377–383. [PubMed]
34. Preyer S, Baisch A, Bless D, Gummer AW. Hear Res. 2001;152:139–151. [PubMed]
35. Brownell WE. In: Vertebrate Hair Cells. Eatock RA, editor. Springer-Verlag Inc; New York: 2006. pp. 313–347.
36. Brownell WE, Spector AA, Raphael RM, Popel AS. Annu Rev Biomed Eng. 2001;3:169–194. [PubMed]
37. de Monvel JB, Brownell WE, Ulfendahl M. Biophys J. 2006;91:364–381. [PubMed]
38. Oghalai JS, Tran TD, Raphael RM, Nakagawa T, Brownell WE. Hear Res. 1999;135:19–28. [PubMed]
39. Oghalai JS, Zhao HB, Kutz JW, Brownell WE. Science. 2000;287:658–661. [PMC free article] [PubMed]
40. Santi PA, Mancini P, Barnes C. J Histochem Cytochem. 1994;42:705–716. [PubMed]
41. Nguyen TV, Brownell WE. Otolaryngol Head Neck Surg. 1998;119:14–20. [PubMed]
42. Oghalai JS, Patel AA, Nakagawa T, Brownell WE. J Neurosci. 1998;18:48–58. [PubMed]
43. Brownell WE, Oghalai JS. In: Cell and Molecular Biology of the Ear. Lim D, editor. Academic/Plenum Press; New York: 2000. pp. 69–83.
44. Kalinec F, Kalinec G, Negrini C, Kachar B. Hear Res. 1997;110:141–146. [PubMed]
45. Belyantseva IA, Adler HJ, Curi R, Frolenkov GI, Kachar B. J Neurosci. 2000;20:RC116. [PubMed]
46. Geleoc GS, Casalotti SO, Forge A, Ashmore JF. Nat Neurosci. 1999;2:713–719. [PubMed]
47. Nakazawa K, Spicer SS, Schulte BA. Hear Res. 1995;82:93–99. [PubMed]
48. Ding JP, Salvi RJ, Sachs F. Hear Res. 1991;56:19–28. [PubMed]
49. Iwasa KH, Li MX, Jia M, Kachar B. Neurosci Lett. 1991;133:171–174. [PubMed]
50. Rybalchenko V, Santos-Sacchi J. J Physiol (Lond) 2003;547:873–891. [PubMed]
51. Yu N, Zhu ML, Zhao HB. Brain Res. 2006;1095:51–58. [PMC free article] [PubMed]
52. Adler HJ, Belyantseva IA, Merritt RCJ, Frolenkov GI, Dougherty GW, Kachar B. Hear Res. 2003;184:27–40. [PubMed]
53. Liberman MC, Gao J, He DZ, Wu X, Jia S, Zuo J. Nature. 2002;419:300–304. [PubMed]
54. Ludwig J, Oliver D, Frank G, Klocker N, Gummer AW, Fakler B. Proc Natl Acad Sci U S A. 2001;98:4178–4183. [PubMed]
55. Oliver D, He DZ, Klocker N, Ludwig J, Schulte U, Waldegger S, Ruppersberg JP, Dallos P, Fakler B. Science. 2001;292:2340–2343. [PubMed]
56. Zheng J, Shen W, He DZ, Long KB, Madison LD, Dallos P. Nature. 2000;405:149–155. [PubMed]
57. Oghalai JS. Hear Res. 2004;198:59–68. [PubMed]
58. Shehata WE, Brownell WE, Dieler R. Acta Otolaryngol. 1991;111:707–718. [PubMed]
59. Sturm AK, Rajagopalan L, Yoo D, Brownell WE, Pereira FA. Otolaryngol Head Neck Surg. 2006;136:434–439. [PMC free article] [PubMed]
60. Rajagopalan L, Patel N, Madabushi S, Goddard JA, Anjan V, Lin F, Shope C, Farrell B, Lichtarge O, Davidson AL, Brownell WE, Pereira FA. J Neurosci. 2006;26:12727–12734. [PMC free article] [PubMed]
61. Greeson JN, Organ LE, Pereira FA, Raphael RM. Brain Res. 2006;1091:140–150. [PubMed]
62. Santos-Sacchi J, Kakehata S, Takahashi S. J Physiol (Lond) 1998;510:225–235. [PubMed]
63. Farrell B, Do Shope C, Brownell WE. Phys Rev E Stat Nonlin Soft Matter Phys. 2006;73:041930-1–041930-17. [PMC free article] [PubMed]
64. Vetrivel KS, Cheng H, Lin W, Sakurai T, Li T, Nukia N, Wong PC, Xu H, Thinakaran G. J Biol Chem. 2004;279:44945–44954. [PMC free article] [PubMed]
65. Oliver D, Fakler B. J Physiol (Lond) 1999;519:791–800. [PubMed]
66. Santos-Sacchi J. J Neurosci. 1991;11:3096–3110. [PubMed]
67. Rajendran L, Masilamani M, Solomon S, Tikkanen R, Stuermer CA, Plattner H, Illges H. Proc Natl Acad Sci U S A. 2003;100:8241–8246. [PubMed]
68. Navaratnam D, Bai JP, Samaranayake H, Santos-Sacchi J. Biophys J. 2005;89:3345–3352. [PubMed]
69. Zheng J, Du GG, Anderson CT, Keller JP, Orem A, Dallos P, Cheatham M. J Biol Chem. 2006;281:19916–19924. [PubMed]
70. Evans MB, Tonini R, Shope CD, Oghalai JS, Jerger JF, Insull W, Jr, Brownell WE. Otol Neurotol. 2006;27:609–614. [PMC free article] [PubMed]
71. Tami TA, Fankhauser CE, Mehlum DL. Otolaryngol Head Neck Surg. 1985;93:235–239. [PubMed]
72. Raphael RM, Popel AS, Brownell WE. Biophys J. 2000;78:2844–2862. [PubMed]
73. Zhang PC, Keleshian AM, Sachs F. Nature. 2001;413:428–432. [PubMed]
74. Santos-Sacchi J, Wu M. J Membr Biol. 2004;200:83–92. [PubMed]
75. Santos-Sacchi J, Shen W, Zheng J, Dallos P. J Physiol (Lond) 2001;531:661–666. [PubMed]
76. Kakehata S, Santos-Sacchi J. Biophys J. 1995;68:2190–2197. [PubMed]
77. Lue AJC, Zhao HB, Brownell WE. Otolaryngol Head Neck Surg. 2001;125:71–76. [PubMed]
78. Liao Z, Popel AS, Brownell WE, Spector AA. J Acoust Soc Am. 2005;118:3737–3746. [PubMed]
79. Murdock DR, Ermilov SA, Spector AA, Popel AS, Brownell WE, Anvari B. Biophys J. 2005;89:4090–4095. [PubMed]
80. Needham D, Nunn RS. Biophys J. 1990;58:997–1009. [PubMed]
81. Barenholz Y. Prog Lipid Res. 2002;41:1–5. [PubMed]
82. Barenholz Y. Subcell Biochem. 2004;37:167–215. [PubMed]
83. Iwasa KH. Biophys J. 1993;65:492–498. [PubMed]
84. Wu M, Santos-Sacchi J. J Membr Biol. 1998;166:111–118. [PubMed]