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. 2007 Mar;136(3):434-9.
doi: 10.1016/j.otohns.2006.10.030.

Functional expression and microdomain localization of prestin in cultured cells

Angela K Sturm  1 , Lavanya Rajagopalan, Donald Yoo, William E Brownell, Fred A Pereira
Affiliations

Functional expression and microdomain localization of prestin in cultured cells

Angela K Sturm et al. Otolaryngol Head Neck Surg. 2007 Mar.

Abstract

Introduction: 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.

Results & discussion: 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.

Conclusion: Prestin activity in HEK 293 cells results from expression in the plasma membrane and altering membrane lipid content affects prestin localization and activity.

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Figures

Figure 1
Figure 1
Temporal expression and membrane localization of prestin in HEK 293 cells. Prestin was expressed from two separate constructs, prestin-GFP and HA-prestin, in HEK 293 cells. Cellular localization of prestin was detected by immunofluorescence to the HA tag (red) or epifluorescence of prestin-EGFP (green) and images captured by deconvolution microscopy. Both prestin-EGFP and HA-prestin are expressed by 8 hours post-transfection (A), are seen in the plasma membrane by 24 hours (B), and show progressively less uniform and more punctate distribution by 24 to 96 hours (C–E). In addition, both prestin-EGFP and HA-prestin colocalize as evinced by overlay (yellow) of their fluorescence signals (F).
Figure 2
Figure 2
Subcellular localization of prestin. Prestin trafficks through the Golgi and endoplasmic reticulum (data not shown) as expected before extensively colocalizing to clathrin vesicles and to endosomes. Prestin is seen to colocalize to the plasma membrane with integrin2α/VLA-2α and lipid raft marker CTX-B, which binds GM1 glycosphingolipids. Colocalization is visualized as yellow fluorescence (arrowheads) due to overlap of red and green fluorescence. Images were captured using a Zeiss Axioplan II deconvolution microscope with 63× oil immersion objective.
Figure 3
Figure 3
Prestin localizes with microdomain markers. Prestin colocalizes in lanes 4/5 with flotillin-1, a raft localized protein, and membrane proteins Na+/K+ATPase and integrin2α/VLA-2α. All prestin-EGFP in lane 4/5 redistributes into higher-density fractions containing ER membranes (BiP/GRP 78 positive) after depletion of cholesterol with MβCD.
Figure 4
Figure 4
Cholesterol depletion causes a shift in peak capacitance in prestin NLC. Prestin-EGFP transfected cells were treated with MβCD for 30 minutes at 37°C to deplete cholesterol before being patched to measure nonlinear capacitance. The peak of the capacitance shows a significant shift towards depolarizing voltages (arrow). The average voltage at maximum capacitance was −71.25 mV ± 13.6 mV, n = 4 (untreated), and 10.25 mV ± 8.7 mV, n = 4 (cholesterol depleted). The electrophysiological characteristics of the cells (membrane resistance, reversal potential, etc.) remained unaltered.
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