Tuning of the Outer Hair Cell Motor by Membrane Cholesterol

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

Received for publication, June 20, 2007, and in revised form, September 28, 2007 Published, JBC Papers in Press, October 12, 2007, DOI 10.1074/jbc.M705078200

Lavanya Rajagopalan‡§1,2, Jennifer N. Greeson¶3, Anping Xia‡, Haiying Liu�, Angela Sturm‡, Robert M. Raphael¶4,Amy L. Davidson**2, John S. Oghalai‡¶5, Fred A. Pereira‡¶�, and William E. Brownell‡ ‡‡¶6

From the ‡Bobby R. Alford Department of Otolaryngology-Head and Neck Surgery, ‡‡Department of Neuroscience, and �HuffingtonCenter on Aging and Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, §W. M. Keck Center forInterdisciplinary Bioscience Training, Houston, Texas 77005, the ¶Department of Bioengineering, Rice University,Houston, Texas 77005, and the **Department of Chemistry, Purdue University, West Lafayette, Indiana 47907

Cholesterol affects diverse biological processes, inmany casesby modulating the function of integral membrane proteins. Weobserved that alterations of cochlear cholesterolmodulate hear-ing in mice. Mammalian hearing is powered by outer hair cell(OHC) electromotility, a membrane-based motor mechanismthat resides in the OHC lateral wall. We show that membranecholesterol decreases during maturation of OHCs. To studythe effects of cholesterol on hearing at the molecular level, wealtered cholesterol levels in the OHCwall, which contains themembrane protein prestin. We show a dynamic and reversi-ble relationship between membrane cholesterol levels andvoltage dependence of prestin-associated chargemovement inboth OHCs and prestin-transfected HEK 293 cells. Cholesterollevels also modulate the distribution of prestin within plasmamembrane microdomains and affect prestin self-association inHEK 293 cells. These findings indicate that alterations in mem-brane cholesterol affect prestin function and functionally tunethe outer hair cell.

Cholesterol is an important component of the plasma mem-branes of most animal cells. It modulates the mechanical prop-erties of the membrane and affects the function of membrane-associated proteins. Recent studies have shown modulation bymembrane cholesterol of such diverse membrane proteins asrhodopsin (1, 2), the serotonin receptor 1A (3) and serotonintransporter 5HT1 (4), the chloride channel ClC-2 (5), severalclasses of potassium channels (6, 7), the nicotinic acetylcholinereceptor (8), and several G-protein-coupled receptors (9, 10).

These studies indicate that cholesterol may act as follows: 1) bybinding directly to and influencing the conformation anddynamics ofmembrane proteins (11–14); 2) by altering the bio-physical and mechanical properties of membranes (15–19);and/or 3) by promoting the formation of cholesterol-richmicrodomains (9, 20, 21). These heterogeneous orderedmicrodomains contain distinctive lipid and protein compo-sitions in which cholesterol may contribute as much as 50%of the membrane lipids (22–24). Cholesterol-rich membranedomains might serve to compartmentalize cellular processes,promote protein-protein and lipid-protein interactions, andthereby regulate protein function (23, 25, 26).Cellular cholesterol levels are tightly regulated, and disrup-

tion of cholesterol homeostasis leads to a host of disease condi-tions. Several clinical and experimental studies carried outusing rabbits, chinchillas, guinea pigs, and human subjects havelinked sensorineural hearing loss and/or increase of hearingthresholds to hypercholesterolemia (27–31). Reduction of cho-lesterol by statins or apheresis has been shown to delay hearingloss in mice (32) and improve hearing recovery in humans (33).A study of hypercholesterolemic humans indicated that theeffects of cholesterol on hearingmight involve effect(s) on non-linear mechanical processes in the cochlea (34).Apiezoelectric-likemembrane-basedmotor in the outer hair

cell (OHC)7 contributes to the exquisite sensitivity and fre-quency selectivity of mammalian hearing. This motor mecha-nism is required to counteract viscous damping in the fluid-filled cochlea, which would otherwise impair mechanicaltuning. TheOHC lateral wall is specialized for electro-mechan-ical force transduction (35, 36). Here, the energy in the trans-membrane electric field is converted into mechanical energy.The organization of the OHC lateral wall is unique among haircells and among all adult mammalian cell types. It is an elegant,nanoscale (�100 nm thick), trilaminate structure. The outerand inner layers are the plasma membrane and subsurface cis-ternae, respectively, and sandwiched between them is a layer ofcytoskeletal proteins called the cortical lattice. The lipid com-position of the plasma membrane and the subsurface cisternaemembranes is unknown, but the constituent lipids are in the

* This work was supported in part by NIDCD Grant DC00354 from theNational Institutes of Health (to W. E. B. and F. A. P.), National Science Foun-dation Grant BES-0522862 (to F. A. P.), NIDCD Grant DC008134 from theNational Institutes of Health (to R. M. R. and F. A. P.), and the DeafnessResearch Foundation (to L. R.). The costs of publication of this article weredefrayed in part by the payment of page charges. This article must there-fore be hereby marked “advertisement” in accordance with 18 U.S.C. Sec-tion 1734 solely to indicate this fact.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental text and Figs. 1– 4.

1 Supported by a Keck Center for Interdisciplinary Bioscience training grant.2 Supported by a Welch Foundation award.3 Supported by National Institutes of Health NRSA Predoctoral Fellowship

DC07563-01.4 Supported by National Science Grants BES-0449379 and NSF BES-0321275.5 Supported by NIDCD Grant DC006671 from the National Institutes of

Health.6 To whom correspondence should be addressed: Baylor College of Medicine,

One Baylor Plaza, Houston, TX 77030. Tel.: 713-798-8540; Fax: 713-798-8553; E-mail: [email protected].

7 The abbreviations used are: OHC, outer hair cell; M�CD, methyl-�-cyclodex-trin; NLC, nonlinear capacitance; GFP, CFP, YFP, green, cyan, or yellow flu-orescent protein; HA, hemagglutinin; DPOAE, distortion product oto-acoustic 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.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 50, pp. 36659 –36670, December 14, 2007© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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fluid phase allowing for free diffusion (37–39). Labeling studiessuggest that lateral wall membranes contain less cholesterolthan the OHC apical and basal plasma membranes (40–43).The relatively low cholesterol level of the OHC lateral wallplasma membrane is unusual among animal cells, and mayserve to modulate the function of the membrane proteins thatreside there. These proteins include a modified anion exchangerAE2 (44), the Glut5 sugar transporter (45–47), stretch-activatedion channels (48–50), 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 mammalianhearing (53). Prestin greatly increases charge movement intoand out of, as opposed to through, the membrane (54, 55).Intracellular anions such as chloride and bicarbonate have beenshown to be the charge carrier (55) consistent with the mem-bership of prestin in the SLC26A family of anion transporters(56).When transfected into several mammalian cell lines, pres-tin confers a voltage-dependent nonlinear capacitance (NLC),the accepted electrical signature of electromotility (54, 55) (seesupplemental text for in-depth description).Motivated by the clinical effects of cholesterol on hearing

and the reduced cholesterol levels in the OHC lateral wall, wehave explored the effect of cholesterol on hearing at the organ,cellular, and molecular levels to clarify its biological basis ofaction. We observe that cholesterol affects otoacoustic emis-sions and functionally tunes nonlinear mechanical processes intheOHC,most likely through its effects on theOHCmembraneprotein prestin.

EXPERIMENTAL PROCEDURES

Materials

Methyl-�-cyclodextrin, water-soluble cholesterol (M�CDloaded with cholesterol), filipin, and bovine serum albuminwere obtained from Sigma. Primers were obtained from SigmaGenosys. Anti-flotillin-1 antibody (1:250 working dilution) waspurchased from BD Biosciences. Anti-HA (1:1000) was pur-chased from Cell Signaling Technology (Danvers, MA). Anti-GFP anti-mousemonoclonal antibodywas obtained fromSantaCruz Biotechnology (Santa Cruz, CA). AlexaFluor 594 phalloi-din (1:200), AlexaFluor 594 goat anti-mouse antibody (1:800),and concanavalinA-AlexaFluor 350 conjugate (working con-centration 50–200 �g/ml) were purchased from MolecularProbes (Carlsbad, CA). Peroxidase-labeled horse anti-mouseantibody was obtained from Vector Laboratories (Burlingame,CA). The ECL Western blotting detection kit was obtainedfrom Amersham Biosciences.

DPOAE Measurements

Mice used for DPOAE measurements were of a mixedgenetic background derived from two strains, 129SvEv andC57B6/J, and were 4–8 weeks old. Healthy mice were anesthe-tizedwith ketamine/xylazine and immobilized in a head holder.The pinna was resected and the middle ear bulla opened toexpose the round window. An earbar connected to two speak-ers and a probe tipmicrophone were inserted into the ear canalto within 2 mm of the tympanic membrane. The cubic distor-tion product amplitude was measured using an F2 frequency of

20 kHz with F1 � F2/1.2 (57). The intensities of the primarytones were equal. First, we ranged the primary tones from 20 to80 db in 10-db steps to verify that there was no notch in theDPOAE amplitude curve between 50 and 70 db. During theexperiment, we set the primary tones to 60-db sound pressurelevel, and the DPOAE amplitude was measured every 9 s. Aftera fewminutes, a borosilicatemicropipettewith a tip diameter of�50 �m containing the treatment solution (either 100 mMM�CD, 200 mM water-soluble cholesterol, �200 mM raffinose,or 10 mM water-soluble cholesterol) was carefully insertedthrough the round window membrane. The high concentra-tions of each treatment (in comparisonwith established in vitrostudies) were chosen to compensate for dilution of the solu-tions in the mouse perilymph. The treatment solutions wereallowed to diffuse passively into the perilymph. The middle earspace was monitored for fluid seepage, and any fluid was care-fully aspirated. DPOAE amplitudes were collected for up to 30min. In some cases, at the conclusion of the experiment, thebasilar membrane was perforated to eliminate DPOAEs,thereby verifying the measurements obtained. DPOAE ampli-tudes were then normalized so that the amplitude after themicropipette was inserted and all middle ear fluid was clearedwas 0 db. This time window is indicated as a gray box in eachpanel of Fig. 1.

Outer Hair Cell Isolation

Albino guinea pigs of either sex weighing 200–300 g andhaving a normal startle response to a hand clap were decapi-tated. The temporal bones were taken and themiddle ear bullaeopened. The otic capsule was removed, and the spiral ligamentwas peeled off to expose the organ of Corti. The modiolus withthe intact organ of Corti was removed from the temporal boneand subjected to mild trypsinization for �10 min at room tem-perature and trituration to detach OHCs. OHCs were platedonto the glass bottomof a coatedmicrowell Petri dish (MatTek,Ashland,MA). Isolated cells were selected for study on the basisof standard morphological criteria within 4 h of animal death.Under the light microscope, healthy cells display a character-istic birefringence, a uniformly cylindrical shape withoutregional swelling, a basally located nucleus, and no Brownianmotion 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-pres-tin) and into the pEGFP, pECFP, and pEYFP vectors (Clontech)as a GFP, CFP, or YFP fusion protein (prestin-EGFP), asdescribed previously (59–61). The prestin-ECFP and prestin-EYFP constructs were modified by site-directed mutagenesis(QuikChange mutagenesis kit, Stratagene, La Jolla, CA) toinclude a single amino acid substitution (A206K) on the CFP/YFP fusion protein, which renders CFP/YFP monomeric. Thesequences of the constructs were verified using five overlappingsequencing primers. NLC measurements confirmed that allconstructs used in this study are functional in HEK 293 cells.HEK 293 cell lines were transfected 24 h after passage withprestin-EGFP, prestin-ECFP, prestin-EYFP, or HA-prestin at a3:1 ratio of DNA with FuGENE 6 (Roche Applied Science).

Cholesterol Modulates OHC Function

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Cholesterol Manipulations

Outer Hair Cells—Because of the sensitivity of outer haircells to temperature and their deterioration with time after iso-lation, cholesterol manipulations were performed differently inOHCs than in HEK 293 cells. Cholesterol depletion was carriedout by pipetting M�CD into the external solution in the dishcontaining hair cells at a final concentration of �100 �M

(1/100th that used in HEK cells, see below) and incubating atroom temperature (see Fig. 3 for times of incubation). Higherconcentrations of M�CD produced drastic morphologicalchanges inOHCs and even cell death because of destabilizationof the cholesterol-rich apical and basalmembranes, causing thenucleus to be blown out of the cell. Cholesterol was loaded in asimilar manner at a final concentration of 1 mM of M�CD con-taining cholesterol (also referred to as water-soluble choles-terol). In both cases, treatment was carried out after forming awhole-cell patch on an OHC, and capacitance recordings weretaken throughout the incubation time.HEK 293 Cells—Steady-state electrophysiological measure-

ments were performed on prestin-transfected HEK 293 cells,48 h post-transfection, after treatment with 10 mM M�CD orwater-soluble cholesterol (at a 10 mM M�CD concentration)for 30 min at 37 °C. The effects of cholesterol manipulationswere followed kinetically in HEK 293 cells by pipetting M�CDor water-soluble cholesterol into the external solution at a finalconcentration of 10mMafter obtaining awhole-cell patch.HEK293 cells did not showmorphological changes as seen in OHCsupon cholesterol depletion. Filipin labeling of untreated, cho-lesterol-depleted, and cholesterol loaded HEK 293 cells (sup-plemental Fig. 1) shows changes in filipin fluorescence signalwith cholesterol manipulations, confirming that cholesterollevels are altered by our treatments.

Electrophysiological Measurements

Electrophysiological data were obtained from cells using thewhole-cell voltage clamp technique. Our recording techniquesare fully described earlier (60) and a brief description follows.Culture dishes containing transfected cells were placed on thestage of an inverted microscope (Carl Zeiss, Gottingen, Ger-many) under �100 magnification and extensively perfusedwith the extracellular solution containingCa2� andK� channelblockers prior to recording. All recordings were conducted atroom temperature (23 � 1 °C). Patch pipettes (quartz glass)with resistances ranging from 2 to 4 megohms were fabricatedusing a laser-based micropipette puller (P-2000, Sutter Instru-ment Company, Novato, CA) and filled with an intracellularsolution, also containing channel blockers. For cell membraneadmittance,Ywasmeasuredwith the patch-clamp technique inthe whole-cell mode using a DC voltage ramp with dual fre-quency stimulus (62) from �0.14 to 0.14 V with a holdingpotential of 0 V, and the cell parameters were calculated fromthe admittance as described earlier (63). The conductance, b,was also determined experimentally with a DC protocol, asdescribed 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 inEquation 1,

Cnorm � �C�V� � Cbaseline�/Cbaseline

Cfinal � Cnorm/Cnormpkc (Eq. 1)

where C(V) is the capacitance at voltage V; Cbaseline is capaci-tance at base-line voltage (defined above), andCnormpkc is equalto Cnorm at Vpkc.

Tissue Preparation and Filipin Labeling of Mouse OHCs

P6, P12, and adult ICR mice were sacrificed by cervical dis-location and decapitation. The temporal bone was removed,and the bony capsulewas stripped in fresh coldHanks’ balancedsalt solution (Invitrogen). The membranous labyrinth wasexposed in Dulbecco’s modified Eagle’s medium containing10% fetal bovine serum. The sensory epithelium was isolatedand affixed to round glass coverslips coated with Cell-TakTM(BD Biosciences). The tissue was washed twice with PBS, fixedwith 4% paraformaldehyde for 30 min, and stained with filipindye (4 mg/ml) and AlexaFluor 594 phalloidin for 30 min. Thesamples were then washed twice with PBS, mounted on glassslides with Fluoromount G antifade reagent, and sealed withnail polish. Images were captured on a Zeiss Axioplan micro-scope (Carl Zeiss Optics Company, Jena, Germany) with �63objective and analyzed with Applied Precision SoftWoRxdeconvolution software. Images of individual OHCs were ana-lyzed using NIH Image software, and pixel intensities along aline drawn through the middle of a single OHC were plotted asbar graphs in Fig. 2.

Immunofluorescence and Imaging

HA-prestin transfected cells on coverslips were eithertreated with or without 10 mMM�CD or water-soluble choles-terol for 30 min at 37 °C. Cells were then washed with PBS,stained with concanavalinA-AlexaFluor 350 conjugate (Molec-ular Probes, Carlsbad, CA) for 1 h on ice, washed with PBSagain, and then permeabilized with PBS/Triton X-100 beforefixing with 4% paraformaldehyde in PBS. The cells were thenstainedwith anti-HA antibody (1:1200; Cell Signaling Technol-ogy, 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 withFluoromount G antifade reagent (Electron Microscopy Sci-ences, Hatfield, PA) and fluorescent images captured on a ZeissLSM 510 deconvolution microscope (Carl Zeiss Optics Com-pany, Jena, Germany) with �63 objective and analyzed withApplied Precision SoftWoRx image restoration software.Images were also obtained using a Zeiss LSM 510 confocalmicroscope with �63 objective and analyzed using Zeiss AIMimaging software.

Membrane Fractionation

Cellmembraneswere fractionated as described byVetrivel etal. (64). Briefly, HEK 293 cells expressing HA-prestin, treatedwith or without M�CD or with water-soluble cholesterol (asdetailed above), were lysed in buffer (0.5% Lubrol WX, 25 mMTris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, and 1 mM phe-



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nylmethanesulfonyl fluoride). Membranes were fractionatedon a 5, 35, and 45% sucrose step gradient. Twelve 1-ml fractionswere collected, and excess lipids in each fraction were removedbymethanol/chloroformprecipitation before the proteinswereanalyzed by 7.5% SDS-PAGE. This experiment was repeated atleast six times with invariant results; a representative blot isshown in Fig. 6.

Cross-linking

Forty eight hours post-transfection with HA-prestin, HEK293 cells were either treated with methyl-�-cyclodextrin(M�CD) or with water-soluble cholesterol for 30 min at 37 °C,or left untreated, before gentle harvesting by scraping into 1 mlof PBS, pH 8.0. The cells were pelleted (2000� g for 5 min) andincubated with various concentrations of cross-linker bis(sul-fosuccinimidyl) suberate (0.078 to 5 mM of BS3) or withoutcross-linker for 30 min at room temperature. Reactions werequenched with 50 mM Tris, pH 7.5. The amount of protein ineach sample wasmeasured and normalized prior to gel loading.Samples were mixed with 8% 2� SDS sample buffer and incu-bated for 30min at room temperature before fractionation by a4–8% Tris-glycine PAGE and analysis by Western blotting.

Fluorescence Resonance Energy Transfer

Fluorescence resonance energy transfer (FRET), imple-mented on a Zeiss LSM 510 confocal microscope (Carl ZeissOptics Company, Jena, Germany), was used to measure thedegree of prestin self-association following cholesterol pertur-bations. HEK cells were cotransfected with prestin-CFP(donor) and prestin-YFP (acceptor). Both the CFP and YFPfusion proteins had an engineered mutation, A206K, that pre-vents CFP and YFP dimerization to allow easier interpretationof FRET results. Details of the acceptor photobleach techniqueutilized have been published previously (61). Briefly, a region ofinterest (ROI) on the cell membrane, exhibiting even, mem-brane localized fluorescence, was bleached to remove YFP sig-nal. CFP fluorescence intensity in the bleached ROI was meas-ured pre- and post-bleach to arrive at the value of FRETefficiency (Ef) from CFP to YFP. CFP intensity in an adjacentunbleached ROI was measured pre- and post-bleach to derivecontrol (Cf) values for each FRET measurement. For detaileddescription of methods, refer to Greeson et al. (61).

Quantification of Puncta

Membrane segments used for FRET experimentation werealso utilized in puncta quantification. Confocal images of pres-tin-YFP in living HEK 293 cell membranes were cropped to�25 by 75-pixel (1 pixel � 0.14 �m) membrane-containingregions (slice thicknesses � 3.3 �m) and analyzed using theMatlab (The Mathworks, Natick, MA) edge detection filter.The filter generated an image the same size as the input imagecomposed of ones where edges were detected and zeros every-where else (supplemental Fig. 3). Edge detection is based on oneof six specific methods, and the most advanced of the availablemethods, the Cannymethod, was used in this work. The Cannymethod identifies local maxima in the image gradients and usestwo different threshold values to define both strong and weakedges. A weak edge is only included in the output image if it is

connected to a strong edge. The use of two threshold valuesensures that this method is less likely to detect false positiveweak edges. Following application of the filter, the outputimage was analyzed for the presence of puncta. Puncta identi-fication was guided by the output image of the detection filterand supported by the membrane region image (supplementalFig. 3). The results of this analysis are shown in Fig. 5.

FIGURE 1. Effect of cochlear cholesterol loading/depletion on DPOAEamplitude. DPOAEs were measured continuously during the delivery of cho-lesterol, M�CD, or control solutions. The solid orange line indicates averagenoise, and the dotted orange line indicates the noise threshold, calculated asthree S.D. above average noise. The gray box indicates time that the micropi-pette was inserted through the round window membrane and any middle earfluid aspirated. During this time, the data demonstrate artifactual changesand changes in middle ear mechanics. After this time, the DPOAEs amplitudesare referenced to 0 db, and changes in the data represent changes in cochlearotoacoustic emissions. Top, injection of 200 mM raffinose (blue) or a dilute (10mM) M�CD/cholesterol solution (green) caused no changes in DPOAE ampli-tudes after the micropipette was inserted into the round window and allmiddle ear fluid aspirated. Middle, cholesterol depletion (using 100 mM

M�CD) caused progressive decreases in DPOAE amplitudes. Bottom, choles-terol loading (using 200 mM water-soluble cholesterol) caused initial slight(2–3 db) increases in DPOAE amplitudes, followed by a progressive decrease.The same traces, on a magnified y axis, are shown in the inset. DPOAE record-ings during depletion showed higher noise levels (2.4 db S.D. from the aver-age trend line) post-delivery, when compared with loading (0.34 db) andcontrol treatments (0.2 db), or to intrinsic noise in the recordings before deliv-ery (0.42, 0.89, and 0.31 for control, depletion, and loading treatments,respectively). Arrowheads indicate times at which the round window was per-forated to eliminate DPOAEs.



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Statistical Analysis

Vpkcs and capacitance gains inOHCs and prestin-transfectedHEK 293 cells subjected to different treatments (untreated,cholesterol loading, and cholesterol depletion) were comparedusing two-tailed t tests. Statistical significance (in comparisonto untreated) is indicated in Table 1. Two-way analysis of vari-ance was also used to evaluate statistical significance of FRETvalues in comparison to control FRET for each treatment, aswell as to FRET values of untreated cells. Statistical significanceis indicated by asterisks in Fig. 7D and Table 1.

RESULTS

Changes in Cholesterol Alter the Amplitude of DistortionProductOtoacoustic Emissions (DPOAEs)—To study the effectsof cholesterol on hearing at the organ level, we evaluated coch-lear function in vivo by measuring DPOAE amplitudes duringcholesterol alteration (Fig. 1). Cholesterol depletion resulted ina 20-db decrease in DPOAE amplitudes. Cholesterol loading,on the other hand, resulted in an initial 2–3-db increase inDPOAE 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, thelevel of cholesterol in the OHC lateral wall may be functionallyrelevant.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 labelingclearly indicates membrane cholesterol content, as shown byfilipin labeling of untreated, cholesterol-depleted and -loadedHEK 293 cells (supplemental Fig. 1). OHCs from P6 and P12mice showed distinct filipin labeling of the lateral wall, com-pared with cytoplasm. However, OHCs from adult miceshowed intracellular filipin staining surrounded by a region oflower staining corresponding to the membrane (Fig. 2, rightpanels). The filipin staining patterns are clearly visible in thepixel intensity graphs (Fig. 2, bottom row), which plot pixelintensities along the diameter of a single OHC in each case.These data indicate that the cholesterol in the OHC lateral wallis initially high and decreases during development. This timeframe parallels the onset andmaturation of OHC electromotil-ity (65), which includes a depolarizing shift in prestin-associ-ated charge movement.

FIGURE 2. OHC lateral wall cholesterol content decreases with maturation. Organ of Corti from P6 (left), P12 (middle), and adult (right) mice. Tissue wasstained with phalloidin (red; to visualize actin in stereocilia) and filipin (blue, to label cholesterol) and imaged using deconvolution microscopy. Sectionsthrough the cuticular plate (top row), lateral wall (middle row), and infra-nuclear region (bottom row) show marked filipin staining in the OHC lateral wall in P6and P12 versus adult mice. The single row of inner hair cells (IHC) and the three rows of outer hair cells (traces 1–3) are indicated in each set of panels. IndividualOHCs shown magnified in the insets are highlighted with asterisks. Pixel intensities along the indicated line in each of these insets are plotted as bar graphs atthe bottom. The gray bar in each of the graphs represents �8 �m, the average diameter of a single OHC. Shown are representative data from a singleexperiment; the experiment was repeated two times.



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Alterations in Cholesterol ContentModulate Nonlinear Capac-itance in Isolated OHCs—We directly evaluated the effect of cho-lesterol on prestin-associated charge movement at the cellularlevel by altering cholesterol content in isolated guinea pig OHCs.Weobservedcharacteristicbell-shapedvoltage-dependent capac-itance (NLC) with a peak, Vpkc, at about �0.050 V in untreatedOHCs (Fig. 3A), similar to that reported earlier (66). Depletionof cholesterol shiftedVpkc in the depolarizing direction to about�0.080 V, whereas loading excess cholesterol shifted Vpkctoward more hyperpolarizing voltages (less than �0.130 V, seeFig. 3A). Kinetic studies of these phenomena indicated that theshift in Vpkc occurred within minutes of adding water-solublecholesterol (loading) or M�CD (depletion) (Fig. 3B). Further-more, the effects of depletion and loading were reversible, andthe reversal of the Vpkc shift was equally rapid (Fig. 3C). Thesedata (statistics represented in Table 1) indicate a direct anddynamic correlation betweenOHCmembrane cholesterol con-tent and Vpkc.Changes in Membrane Cholesterol Reversibly Alter Vpkc in

Prestin-transfected HEK 293 Cells—We next investigated pres-tin-specific effects of cholesterol in HEK 293 cells. The Vpkc ofprestin-transfectedHEK 293 cells was approximately�0.070 V

(Fig. 4A). Upon cholesterol depletion, the Vpkc shifted towarddepolarized voltages, with an average peak at �0.004 V (Fig.4A). On the other hand, upon cholesterol loading, the Vpkcshifted toward hyperpolarized voltages, with an average valueof about �0.116 V (Fig. 4A). Importantly, these effects arereversible. Cholesterol depletion followed by loading, as well ascholesterol enrichment followed by depletion, both shifted thepeak voltage toward the control untreated average (Fig. 4B).The kinetics of these processes were similar to those measuredforOHCs, with changes in theVpkc occurringwithinminutes ofaddition of cholesterol orM�CD (Fig. 4C), and the process wasrapidly reversible (Fig. 4D). Comparisons of changes in Vpkc in

FIGURE 3. Cholesterol levels affect Vpkc of nonlinear capacitance in outer hair cells. A, peak (Vpkc) of NLC is at about �0.050 V in control untreated OHCs(black trace). Vpkc shifts to depolarized voltages upon cholesterol depletion (100 �M M�CD; red trace) and hyperpolarized voltages upon cholesterol loading (1mM water-soluble cholesterol; green trace). Traces have been normalized relative to peak capacitance. B, Vpkc begins to change within minutes after addition ofM�CD (red circle) or water-soluble cholesterol (green circle). Untreated cells (black circle) show no change over a comparable time course. Shown are Vpkcreadings from the same cell as a function of time post-treatment. C, reversibility of Vpkc shifts. Shown are changes in Vpkc of a single patched cell upon depletionof cholesterol (red circle), followed by loading (green circle). Arrows indicate time of treatment in B and C. Shown are representative data from single cells; samplesizes are indicated in Table 1.

TABLE 1Vpkc of nonlinear capacitance in OHCs and prestin-expressing HEK 293cellsMean values and S.D. are indicated. Sample sizes for each group are indicated inparentheses. Statistical significance of each group (in comparison to untreated cellsof the same type) is also indicated: ** � p 0.0001.

Vpkc (V)OHC HEK

Untreated �0.051 � 0.009 (n � 10) �0.070 � 0.0181 (n � 6)Depleted 0.092 � 0.017** (n � 5) 0.004 � 0.024** (n � 8)Loaded �0.130 � 0.002** (n � 3) �0.116 � 0.013** (n � 7)



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HEK 293 cells and OHCs are shown in Table 1. Our resultsdemonstrate that changes in cholesterol alter prestin-associ-ated charge movement in HEK 293 cells, as in the native envi-ronment of the OHC, indicating direct molecular level effectsof cholesterol on prestin function.Cholesterol Modulates the Distribution of Prestin in Mem-

brane Microdomains—We explored the possibility of choles-terol modulating the presence of prestin in membranemicrodomains by analyzingmembrane localization and the dis-tribution of prestin in HEK 293 cells (Fig. 5). Prestin colocalizeswith the plasma membrane (Fig. 5A), consistent with earlierobservations (59, 60), and is expressed in foci suggestive oflocalization to membrane microdomains (Fig. 5, A and D).Alterations in cholesterol content change the distribution ofthe foci; upon cholesterol depletion (Fig. 5, B and E), prestinshowed a less punctate (more uniform)membrane distribution,whereas upon cholesterol loading (Fig. 5, C and F), the numberof foci increased (Fig. 5G). Quantification of puncta (Fig. 5G)was based onmultiple images of membrane segments from dif-ferent batches of treatedHEK293 cells (supplemental Fig. 3). Inaddition, time-lapse images of a single transfected cell takenduring the course of depletion or loading show a clear decrease

and increase in puncta (supplemental Fig. 4). These resultsdemonstrate that cholesterol influences prestin distributionwithin the membrane.To assay prestin localization in membrane microdomains,

we characterized detergent-resistant membrane extracts iso-lated from prestin-transfected HEK 293 cells. As expected,prestin was detected in the dense endoplasmic reticulummem-brane fractions (Fig. 6A, lanes 8–10), which cofractionatedwiththe endoplasmic reticulum markers (59). Prestin was also seenin the less dense plasma membrane fractions and predomi-nantly localized in membrane microdomain fractions (Fig. 6A,lanes 4 and 5), where it cofractionated with flotillin-1, amicrodomain marker and structural component (67). Uponcholesterol depletion prestin, but not flotillin, redistributed outof the microdomain fractions (Fig. 6B). Upon cholesterol load-ing, prestin remained in microdomain fractions with higherintensities of all bands (Fig. 6C). These data indicate that prestincan localize to membrane microdomains and that cholesterolmodulates its distribution in these domains.Manipulation of Cholesterol Content Alters Prestin

Associations—Localization to microdomains raises the possi-bility that prestin may interact with itself or other proteins. To

FIGURE 4. Cholesterol affects membrane capacitance of HEK 293 cells expressing prestin. A, Vpkc is at about �0.070 V in untreated cells (black trace). TheVpkc shifts to depolarized voltages upon cholesterol depletion (10 mM M�CD; red trace, red arrow) and hyperpolarized voltages upon cholesterol loading (10 mM

water-soluble cholesterol; green trace, green arrow). Linear capacitance from an untransfected cell is shown for comparison (gray trace). B, effect of cholesterollevels on Vpkc shifts is reversible. Upon cholesterol depletion and reloading (red trace, red arrow) the Vpkc shifts back to a normal voltage. Similarly, uponcholesterol loading followed by depletion (green trace, green arrow), the Vpkc shifts back from hyperpolarized voltages to a normal value. Shown are repre-sentative average traces from single cells. Traces have been normalized to the capacitance at Vpkc. C, Vpkc changes after addition of M�CD (red circle) orwater-soluble cholesterol (green circle). Shown are Vpkc readings from the same cell as a function of time post-treatment. D, reversibility of Vpkc shifts. Shown arechanges in Vpkc of a single patched cell upon addition of cholesterol (red circle), followed by depletion (green circle). Arrows indicate time of treatment in C andD. Shown are representative data from single cells; sample sizes are indicated in Table 1.



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determine whether prestin-prestin interactions were altered bycholesterol, we analyzed prestin complex associations by cross-linking (Fig. 7). In the absence of cross-linker (lane 1), a weakprestin dimer band exists in untreated cell extracts (Fig. 7A).This band disappears upon cholesterol depletion (Fig. 7B) andis stronger upon cholesterol loading (Fig. 7C), indicating cho-lesterol favors prestin self-association in HEK 293 cells inde-pendent of cross-linking. Prestin dimers were present at rela-tively low BS3 cross-linker concentrations (Fig. 7A, lane 2), andthe dimer band intensifiedwith increasing cross-linker concen-trations (Fig. 7A, lanes 3–7). Cholesterol depletion caused adecrease in prestin cross-linking, with higher concentrations ofBS3 required to trap prestin as dimers (Fig. 7, compare lanes 2and 3 in B with lanes 2 and 3 in A). In contrast, loading choles-terol into the membrane caused the appearance of oligomericprotein bands even in the absence of cross-linker (Fig. 7C, lane

1) and bands intensified withincreasing cross-linker (Fig. 7C,lanes 2–8). In conjunction withPFO-polyacrylamide gels (supple-mental Fig. 2A) and coimmunopre-cipitation experiments (supplemen-tal Fig. 2B), and in accordance withearlier observations (61, 68, 69),these data provide strong evidenceof prestin self-association and indi-cate that prestin self-associationincreases with cholesterol content.Further evidence of cholesterol-

dependent prestin self-associationwas obtained using acceptor photo-bleach FRET. We found an averageFRET efficiency of 6.7% (Fig. 7D) inlive, untreated HEK 293 cellscotransfected with prestin-CFP andprestin-YFP. Cholesterol enrich-ment increased average FRET effi-ciency to 8.4%, whereas cholesteroldepletion resulted in a markeddecrease of FRET efficiency to 0.5%(Fig. 7D), indicating significant lossof prestin self-association. In thiscase, the measured FRET efficiencyis indistinguishable from corre-sponding control efficiency meas-urements (p 0.05). To determinewhether this elimination of prestinself-association is reversible, cellswere first depleted of membranecholesterol and then reloaded.Upon reloading, FRET efficiencyreturned to 6.8% (Fig. 7D). Theseresults confirm that modulation ofprestin self-association by mem-brane cholesterol is dynamic andreversible. Our organ, cellular, andmolecular level studies greatlyexpand our understanding of the

role of cholesterol in tuning the functionality of the outer haircell motor.

DISCUSSION

Cholesterol is crucial for membrane organization anddynamics and in the regulation of membrane protein sortingand function. Considering that electromotility is based in thehighly specialized and cholesterol-poor OHC lateral mem-brane, the established link between elevated cholesterol andauditory dysfunction (27, 28, 34, 70, 71) suggests direct effectsof cholesterol on the OHC membrane and proteins presenttherein. Models for OHC electromotility based solely on theelectromechanical transduction capabilities of the membranehave been proposed (72, 73). Following the discovery of themembrane protein prestin, numerous studies have demon-strated how alterations of membrane material properties affect

FIGURE 5. Prestin is expressed in punctate foci in the HEK 293 membrane. Immunofluorescence staining ofHA-prestin transfected HEK 293 cells was used to visualize membrane distribution and localization of prestin.Representative deconvolution images (A–C) show prestin fluorescence (red) coincides with that of concanava-lin-A (blue), a membrane marker. Enlarged images of the membrane are shown in insets. The bottom row (D–F)contains representative enlarged confocal images of the membrane of prestin-YFP transfected HEK 293 cells,showing puncta (arrowheads). A and D, HEK 293 cells transfected with prestin show punctate foci (arrowheads)of prestin fluorescence in the membrane. B and E, depletion of cholesterol by 10 mM M�CD causes a lesspunctate, more uniform prestin labeling. C and F, loading excess cholesterol (10 mM water-soluble cholesterol)causes an increase in number of punctate foci. G, quantification of number of foci. Puncta in membrane regionswere quantified as described using multiple images of membrane segments from different batches of treatedHEK 293 cells. The graph represents average number of puncta per 10.5 �m of membrane, calculated fromseveral live confocal images of membrane segments. Images used in puncta quantification are shown insupplemental Fig. 3; time-lapse images of a single transfected cell over the course of depletion and loadingtreatments are shown in supplemental Fig. 4.



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prestin function and/or OHC electromotility (67, 74–79).These studies point to a dynamic interplay between prestin andthe membrane in the generation of nonlinear capacitance andelectromotility; our study further characterizes this dynamicrelationship.Cholesterol Effects on Otoacoustic Emissions Correlate to

Effects on NLC—Cochlear cholesterol alterations influenceDPOAE amplitudes (Fig. 1). Manipulating cholesterol in iso-lated guinea pig OHCs revealed a dynamic and reversible rela-tionship between membrane cholesterol content and prestin-associated charge movement, with similar kinetics (Fig. 3).Cholesterol effects on DPOAEs may result from theobserved effects on prestin-associated charge movement asfollows. In normal untreated OHCs, the NLC peak is atapproximately �0.050 V; therefore, in the cell’s range ofreceptor potential (nominally between �0.060 and �0.080V) capacitance is sub-maximal. Shifting the NLC peak in thedepolarizing direction (as upon cholesterol depletion) wouldresult in a progressive lowering of capacitance and electro-motility in the range of the cell’s receptor potential. Thiswould reduce DPOAE amplitudes. On the other hand, shift-ing the NLC peak in the hyperpolarizing direction wouldresult in an initial increase of capacitance at the cell’s recep-tor potential followed by a decrease. Electromotility andDPOAE amplitudes would follow this pattern. This relation-ship is schematically presented in Fig. 8.

OHC Function May Be Modulated by Cholesterol ReductionduringMaturation—We show a lowering ofmembrane choles-terolwithmaturation (Fig. 2). During the post-natalmaturationof rodent OHCs, the distribution of prestin in the lateral wall isinitially inhomogeneous (45, 65). Concurrent with prestin dis-tribution becoming homogeneous, maturation of electromotil-ity and nonlinear capacitance is observed, which includes a shiftin Vpkc from an immature hyperpolarized value to the normaladult value (45, 65). Both effects may result from a decrease inOHC membrane cholesterol levels with maturation. Our datasuggest that the reduction in membrane cholesterol with mat-uration helps to tune the membrane-based motor to operate atmaximal gain in the OHC receptor potential range.HEK 293 Cells Provide a Model System for Studying Prestin

Function—Cholesterol manipulations in prestin-transfectedHEK 293 cells (Fig. 4) produced qualitatively similar results asin OHCs (Fig. 3; Table 1), validating the use of HEK 293 cells asa model system. The difference between Vpkc in cholesterol-depleted OHCs and HEK 293 cells may be due to structuraldifferences (membrane tension and turgor pressure) or differ-ences in cholesterol homeostasismechanisms, between the twocell types, which cause similar trends but different magnitudesin 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 ofmembranemicrodomains inHEK293 cells (Figs. 5 and 6). Similar foci havenot been observed in the adult OHC lateral wall membrane.Perturbing cholesterol content alters the distribution of pres-tin; prestin shifts out (cholesterol depletion, Fig. 6B) or remainsin (cholesterol enrichment, Fig. 6C) themicrodomain fractions,suggesting that prestin is capable of localizing to cholesterol-rich microdomains. In addition, a quantitative correlationexists between cholesterol content and the number of prestinpuncta in the membrane; cholesterol depletion results in areduction,whereas enrichment causes an increase in number offoci (Fig. 5G).Recent studies have suggested that prestinmay self-associate

and dimerize (61, 68, 69).We have obtained further evidence ofprestin self-association using a cross-linking reagent, whichrevealed the presence of prestin-prestin interactions that aredecreased upon cholesterol depletion and increased upon cho-lesterol addition (Fig. 7, A–C). Furthermore, FRET measure-ments provide direct evidence of the significant and reversibleeffect of cholesterol on prestin self-associations (Fig. 7D). Inlight of these data, the low cholesterol levels on the matureOHC lateral wall are consistent with the homogeneous distri-bution of prestin.Mechanism of Cholesterol Effects—The effects of cholesterol

onmembrane protein function have been the subject of numer-ous recent studies, and severalmechanisms have been put forthto explain the effects of cholesterol. In addition to the effects onmembrane material properties such as viscosity, elasticity,compressibility, and stiffness (80), cholesterol levels in themembrane influence the formation of ordered microdomains(81, 82) and partitioning of proteins into these domains byaltering the bending modulus of the membrane (16) andthereby influencing hydrophobicmismatch (15). The same fac-

FIGURE 6. Cholesterol affects membrane distribution of prestin.A, sucrose density gradient fractionation of membranes from HA-prestin-transfected HEK 293 cells. HA-prestin colocalizes with flotillin-1, a membranemicrodomain marker (lanes 4 and 5). B, depletion of cholesterol with 10 mM

M�CD causes a redistribution of HA-prestin into heavier membrane fractions.C, cholesterol enrichment (10 mM water-soluble cholesterol) enhances colo-calization into membrane microdomain fractions. The arrowhead and blackarrow point to unglycosylated and glycosylated monomeric prestin, respec-tively. The white arrow points to oligomeric species. Shown are representativedata from a single experiment; the experiment was repeated at least threetimes.



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tors may explain the effect of cholesterol content on prestin-associated charge movement. Because cholesterol is known tomodulate membrane material properties, which in turn affectthe dynamics of membrane proteins, cholesterol-dependentchanges in membrane stiffness or curvature could alter thedynamic fluctuations of prestin as would changes inmembranedipole potential and lipid packing density (19). Several studiesin the OHC have correlated changes in membrane tension,stiffness, and mechanics to changes in the Vpkc and electromo-tility (67, 75–77, 79). Molecular dynamics simulations suggestcholesterol affects lipid lateral pressure profiles (17, 18), andthis would impact the prestin conformational change that isassumed to accompany charge movement.Cholesterol-induced Vpkc shifts in both OHCs and HEK 293

cells are larger than those resulting from previous manipula-tions; these include exogenous chlorpromazine (77), fructose(46), and increasing intracellular pressure (76, 83), which shiftVpkc toward depolarizing potentials; and decreasing intracellu-lar pressure and exposure to the lipophilic ion, tetraphenylbo-rate (TPB�) (84), which move Vpkc in the hyperpolarizing

FIGURE 7. Effect of cholesterol on prestin self-association. A, HA-prestin is cross-linked as dimers and oligomers with increasing concentrations (0, 0.078,0.16, 0.32, 0.65, 1.25, 2.5, and 5 mM in lanes 1– 8, respectively) of the membrane-impermeable agent BS3. B, cholesterol depletion using 10 mM M�CD causes areduction in cross-linking; higher concentrations of BS3 are required for dimer formation. C, cholesterol loading using 10 mM water-soluble cholesterol causesan increase in cross-linking; oligomer bands appear even in the absence of cross-linker (lane 1). M, D, and T denote monomeric, dimeric, and trimeric prestinbands, respectively (based on molecular weight). D, acceptor photobleach FRET measurements to evaluate prestin self-association in live HEK 293 cells.Acceptor photobleach FRET efficiencies (f) and control (unbleached) FRET values (f) were measured from untreated (n � 22), cholesterol-depleted(n � 20), cholesterol-loaded (n � 16), and depleted and reloaded (n � 23) prestin-expressing HEK cells. Statistical significance (in comparison to controlFRET for each treatment) is represented; *, p 0.05. Shown are representative data from a single experiment; the experiment was repeated at leastthree times.

FIGURE 8. Schematic representation of correlation between NLC peakshifts and electromotility. The nonlinear capacitance of untreated OHCshas a peak at about �0.050 V, slightly depolarized from the resting poten-tial of the cell. The corresponding capacitance in the operating range(receptor potential) of the cell (indicated by gray box and sinusoidal wave)is therefore slightly sub-maximal. Upon depletion, the peak shifts furtheraway from this operating range, resulting in a progressive reduction incapacitance in this range. Upon loading, the peak initially shifts into theoperating range, resulting in small increase in capacitance, and then shiftsbeyond the operating range resulting in a decrease of capacitance in therange. Electromotility and otoacoustic emissions may be presumed tofollow the same pattern.



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direction. Because the magnitude of the Vpkc shifts is signifi-cantly greater than in previousmanipulations that are known tochange the material properties of the membrane, wemust con-sider the possibility that the Vpkc is also a function of self-asso-ciation. This contribution would reflect the relative amounts ofmonomers versus higher order oligomers, where the mono-meric form shifts Vpkc to depolarizing voltages, whereas higherorder oligomers shifts Vpkc to hyperpolarizing voltages. Theeffect of cholesterol on both prestin self-association and onprestin function is reversible, indicating a dynamic interactionof prestin with membrane components.Cholesterol also has a propensity to localize to membrane

microdomains. Prestin is present in microdomains in HEK 293cells, and its presence in these localized domains may facilitateits interaction with itself or with other proteins. It is likely thatprestin exists in a dynamic equilibrium between monomeric,dimeric, and perhaps higher order oligomeric forms. The effectof cholesterol might be to “cluster” prestin molecules, shiftingthe equilibrium toward dimeric or oligomeric species. Our dataindicate increased self-association in the presence of increasedcholesterol. The low cholesterol level observed in the matureOHC lateral wall suggests a preference for lowered prestin self-association, the functional consequences of which remain to bestudied.In summary, our study integrates systems-level, cellular and

molecular data to investigate the role of cholesterol in modu-lating the mechanical aspects of mammalian hearing. We havecharacterized interrelationships between prestin-prestin inter-actions andprestin-membrane interactions.Whether the effectof cholesterol is predominantly through formation of function-ally distinct microdomains, changes in membrane materialproperties, or both, the observable effects of changing the cho-lesterol content are a change in prestin self-association, areversible shift in Vpkc, and changes in otoacoustic emissions.This reinforces the concept of the molecular motor drivingelectromotility as an interdependent entity with protein andmembrane components working cooperatively to achieve non-linear charge movement and mechanical motion.

Acknowledgments—We thank Dominik Oliver, Huey Huang,Jonathan Sachs, Nathan Baker, and Henry Pownall for useful com-ments and Linda Lee for technical assistance.

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