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.
Highlights
These studies indicate that cholesterol may act as follows: 1) by binding directly to and influencing the conformation and dynamics of membrane proteins [11,12,13,14]; 2) by altering the biophysical and mechanical properties of membranes [15,16,17,18,19]; and/or 3) by promoting the formation of cholesterol-rich microdomains [9, 20, 21]
Motivated by the clinical effects of cholesterol on hearing and the reduced cholesterol levels in the outer hair cell (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
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)
Summary
Methyl--cyclodextrin, water-soluble cholesterol (MCD loaded with cholesterol), filipin, and bovine serum albumin were obtained from Sigma. 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). AntiGFP 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 g/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
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