Functional expression and microdomain localization of prestin in cultured cells.
BACKGROUND
Prestin is an essential component of the molecular motor of cochlear outer hair cells that contribute to frequency selectivity and sensitivity of mammalian hearing. A model system to study prestin employs its transfection into cultured HEK 293 cells. Our goal was to characterize prestin's trafficking pathway and localization in the plasma membrane.


METHODS
We used immuno-colocalization of prestin with intracellular and plasma membrane markers and sucrose density fractionation to analyze prestin in membrane compartments. Voltage clamping was used to measure nonlinear capacitance (NLC), prestin's electrical signature.


CONCLUSIONS
Prestin targets to the membrane by 24 hours post-transfection when NLC is measurable. Prestin then concentrates into membrane foci that colocalize and fractionate with membrane microdomains. Depleting membrane cholesterol content altered prestin localization and NLC.


CONCLUSIONS
Prestin activity in HEK 293 cells results from expression in the plasma membrane and altering membrane lipid content affects prestin localization and activity.
Sturm
Angela K
AK
Bobby R. Alford Department of Otolaryngology-Head and Neck Surgery, Baylor College of Medicine, Houston, TX 77030, USA.
Rajagopalan
Lavanya
L
Yoo
Donald
D
Brownell
William E
WE
Pereira
Fred A
FA
D027321:Anion Transport Proteins / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D015415:Biomarkers / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism* / Q000502:physiology; D002499:Centrifugation, Density Gradient; D002784:Cholesterol / Q000378:metabolism / Q000502:physiology; D025802:Electric Capacitance; D004721:Endoplasmic Reticulum / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D005456:Fluorescent Dyes; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism / Q000502:physiology; D008563:Membrane Lipids / Q000378:metabolism / Q000502:physiology; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D018408:Patch-Clamp Techniques; D021381:Protein Transport / Q000502:physiology; D000076988:Sulfate Transporters; D013997:Time Factors; D014162:Transfection; D047392:beta-Cyclodextrins / Q000494:pharmacology
D027321:Anion Transport Proteins; D015415:Biomarkers; D005456:Fluorescent Dyes; D008563:Membrane Lipids; C498719:SLC26A5 protein, human; D000076988:Sulfate Transporters; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D002784:Cholesterol