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First published online June 23, 2005
doi: 10.1242/10.1242/jcs.02431

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The C-terminus of prestin influences nonlinear capacitance and plasma membrane targeting

¹ Auditory Physiology Laboratory, Department of Communication Sciences and Disorders, Northwestern University, Evanston, IL 60208 USA
² Feinberg School of Medicine, Northwestern University, Division of Molecular Medicine, Metabolism, and Diabetes, Chicago, IL 60611, USA
³ Department of Neurobiology and Physiology, The Neuroscience Institute, Northwestern University, Evanston, IL 60208, USA

View larger version (42K): [in a new window]   Fig. 1. A secondary structure and topology model of prestin's C-terminus based on a topological model previously described (Zheng et al., 2000). The properties of the amino acids side chains are indicated by different colors for polar (clear), non-polar (blue), acidic (red), basic (green) and cysteine residues (yellow) moieties. The location of the last transmembrane domain is based on the program TMPRED (transmembrane predictions). Regions of the C-terminus with a predicted secondary structure using the program PSIPRED (confidence 6-9) are indicated by colored backgrounds; -helix (blue), coil (red), and strand (green). The STAS domain (gray), and charge clusters (speckled) are indicated by background shading. The locations of the mutations created in prestin mutant proteins examined in this study are indicated with arrows in the cartoon. These include deletion mutants, chimera junction points and double point mutations.

View larger version (35K): [in a new window]   Fig. 2. (a) Voltage-dependent membrane capacitance of TSA-201 cells transiently transfected with wild type (positive control), Del719, Del709, Y520A/Y526A, as well as untransfected cells (negative control). All capacitance plots are normalized to the maximum of the corresponding (same transfection, same experiment) wild-type NLC function. (b). Membrane capacitance for constructs Del709, Y520A/Y526A, and a negative control plotted relative to their minimum capacitance value. The capacitance waveforms for these constructs are relatively flat and similar to the negative control. The voltage at the peak of the capacitance function represents most efficient charge transfer, while the peak itself relates to the amount of charge translocated. The double point mutation Y520A/Y526A and Del709 mutants exhibited no measurable NLC, whereas the Del719 mutation has normal capacitance relative to wild-type control cells from the same transfection batch. NLC measurements are analyzed by extracting parameters that describe the cell's Qmax, , V and Clin. The parameters are obtained from measurements where the voltage range was between –150 mV and +120 mV. The individual plots shown are representative of the average parameters (n>10).

View larger version (120K): [in a new window]   Fig. 3. Characterization of anti-N-mPres. (a) Immunofluorescence image of a gerbil cochlea stained with anti-N-mPres. The low-magnification image is taken with an epifluorescent microscope with 25x objective. The high-magnification images of OHCs are obtained by confocal microscopy, these are shown in b-e. Immunofluorescence and corresponding phase-contrast images of a normal gerbil (b and d) are compared with material obtained from a prestin knockout mouse (c and e). Both wild-type gerbil and prestin knockout cochlea samples are stained with anti-N-mPres. (f,g) Immunofluorescence and corresponding phase-contrast images of OK cells transiently transfected with wild type, then stained with anti-N-mPres. (h,i) Immunofluorescence and corresponding phase-contrast images of OK cells transiently transfected with the control vector pcDNA3.1-CAT. Bar, 20 µm.

View larger version (76K): [in a new window]   Fig. 4. Representative patterns of wild-type prestin and associated deletion mutants transfected into OK cells. (a) Immunofluorescence and corresponding phase-contrast images of OK cells transiently transfected with cytoplasmic protein EGFP, deletion mutant examples showing types I, and II morphology, and wild type (wt), representing type III morphology. All samples were stained with FITC-labeled anti-N-mPres, except the EGFP preparation. Cell boundaries are marked by arrows. `N' stands for nucleus. Bar, 20 µm. (b) Distribution of the frequency of occurrence of the three cell types for different mutations 48 hours after transfection. The prestin-expressing cells in different samples were classified as types I, II and III, according to the location of prestin (see text); white bars indicate type I, gray bars type II, and black bars type III cells. We note here that some cells classified as type III, found in the Del709 mutant (*), were actually found not to be so after testing for co-localization with the PM marker as described in Fig. 5 (also see text).

View larger version (144K): [in a new window]   Fig. 5. Subcellular localization of prestin and mutant proteins in type I, II and III cells. Immunofluorescence images of OK cells transiently transfected with wild type (wt-gPres) and three deletion mutants: Del516, Del709 and Del719. Three morphological phenotypes: types I, II and III are indicated for each mutant-expressing cell on the left side of the images. Red: anti-Golgi or anti-Na+/K+ ATPase markers. Na+/K+ ATPase is a plasma-membrane protein. Green: FITC-labeled anti-N-mPres, which indicates the location of prestin. Yellow images are superimposed from green and red images, indicating the co-localization of prestin and Na+/K+ ATPase or Golgi marker in the cell's plasma membrane or intracellular membranes. For better examination of the co-localization of prestin with the red markers, the images of the far right column are given at higher magnification. These segments correspond to the locations marked with white arrows. Note that in type II cells (example: Del709) the two labels are very close, but do not superimpose. Similarly, in some cells classified as type III, the two markers form parallel, adjacent, but not overlapping layers. In other cells, classified as type III (wild type and Del719), there is clear overlap, indicated by yellow color. Images were taken 48 hours after transfection. Bar, 20 µm.

View larger version (15K): [in a new window]   Fig. 6. Distribution of type I, II and III cells for the two chimeric and two deletion mutants. Deletion mutants close to the fusion sites are shown for comparison. White indicates type I cells, gray type II cells, and black type III cells.

View larger version (62K): [in a new window]   Fig. 7. Immunofluorescence images of OK cells transiently transfected with double point mutants: V499G/Y501H and Y520A/Y526A. Red, anti-Na+/K+ ATPase; green, prestin with EGFP attached to its C-terminus. Yellow images are superimposed from green and red images, indicating the co-localization of prestin and Na+/K+ ATPase in the cell's plasma membrane indicated by the arrow. There is no co-localization of prestin with the PM marker for Y520A/Y526A. Images were taken 24 hours after transfection. Bar, 20 µm.