Tuning of the Outer Hair Cell Motor by Membrane Cholesterol
2007; Elsevier BV; Volume: 282; Issue: 50 Linguagem: Inglês
10.1074/jbc.m705078200
ISSN1083-351X
AutoresLavanya Rajagopalan, Jennifer N. Greeson, Anping Xia, Haiying Liu, Angela Sturm, Robert M. Raphael, Amy L. Davidson, John S. Oghalai, Fred A. Pereira, William E. Brownell,
Tópico(s)Ion Channels and Receptors
ResumoCholesterol 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 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 (1Boesze-Battaglia K. Albert A.D. J. Biol. Chem. 1990; 265: 20727-20730Abstract Full Text PDF PubMed Google Scholar, 2Albert A.D. Boesze-Battaglia K. Prog. Lipid Res. 2005; 44: 99-124Crossref PubMed Scopus (90) Google Scholar), the serotonin receptor 1A (3Chattopadhyay A. Jafurulla M. Kalipatnapu S. Pucadyil T.J. Harikumar K.G. Biochem. Biophys. Res. 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When transfected into several mammalian cell lines, prestin confers a voltage-dependent nonlinear capacitance (NLC), the accepted electrical signature of electromotility (54Ludwig J. Oliver D. Frank G. Klocker N. Gummer A.W. Fakler B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4178-4183Crossref PubMed Scopus (130) Google Scholar, 55Oliver D. He D.Z. Klocker N. Ludwig J. Schulte U. Waldegger S. Ruppersberg J.P. Dallos P. Fakler B. Science. 2001; 292: 2340-2343Crossref PubMed Scopus (364) Google Scholar) (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. 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. 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 (57Oghalai J.S. Hear Res. 2004; 198: 59-68Crossref PubMed Scopus (28) Google Scholar). 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, 200 mm water-soluble cholesterol, ∼200 mm 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. 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 with in 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 (58Shehata W.E. Brownell W.E. Dieler R. Acta Otolaryngol. 1991; 111: 707-718Crossref PubMed Scopus (162) Google Scholar). 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 (59Sturm A.K. Rajagopalan L. Yoo D. Brownell W.E. Pereira F.A. Otolaryngol. Head Neck Surg. 2006; 136: 434-439Crossref Scopus (31) Google Scholar, 60Rajagopalan L. Patel N. Madabushi S. Goddard J.A. Anjan V. Lin F. Shope C. Farrell B. Lichtarge O. Davidson A.L. Brownell W.E. Pereira F.A. J. Neurosci. 2006; 26: 12727-12734Crossref PubMed Scopus (48) Google Scholar, 61Greeson J.N. Organ L.E. Pereira F.A. Raphael R.M. Brain Res. 2006; 1091: 140-150Crossref PubMed Scopus (31) Google Scholar). 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). 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 1 mm 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. 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 10 mm 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 data were obtained from cells using the whole-cell voltage clamp technique. Our recording techniques are fully described earlier (60Rajagopalan L. Patel N. Madabushi S. Goddard J.A. Anjan V. Lin F. Shope C. Farrell B. Lichtarge O. Davidson A.L. Brownell W.E. Pereira F.A. J. Neurosci. 2006; 26: 12727-12734Crossref PubMed Scopus (48) Google Scholar) 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 (62Santos-Sacchi J. Kakehata S. Takahashi S. J. Physiol. (Lond.). 1998; 510: 225-235Crossref Scopus (115) Google Scholar) 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 (63Farrell B. Do Shope C. Brownell W.E. Phys. Rev. E Stat. Nonlin. Soft Matter Phys. 2006; 73: 041930-1-041930-17Google Scholar). The conductance, b, was also determined experimentally with a DC protocol, as described earlier (60Rajagopalan L. Patel N. Madabushi S. Goddard J.A. Anjan V. Lin F. Shope C. Farrell B. Lichtarge O. Davidson A.L. Brownell W.E. Pereira F.A. J. Neurosci. 2006; 26: 12727-12734Crossref PubMed Scopus (48) Google Scholar). 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. 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. HA-prestin transfected cells on coverslips were either treated with or without 10 mm 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. Cell membranes were fractionated as described by Vetrivel et al. (64Vetrivel K.S. Cheng H. Lin W. Sakurai T. Li T. Nukia N. Wong P.C. Xu H. Thinakaran G. J. Biol. Chem. 2004; 279: 44945-44954Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). 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. 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 (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 (61Greeson J.N. Organ L.E. Pereira F.A. Raphael R.M. Brain Res. 2006; 1091: 140-150Crossref PubMed Scopus (31) Google Scholar). 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. (61Greeson J.N. Organ L.E. Pereira F.A. Raphael R.M. Brain Res. 2006; 1091: 140-150Crossref PubMed Scopus (31) Google Scholar). 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 every-where else (supplemental Fig. 3). Edge detection is based on one of six specific methods, and the most advan
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