Changes in plasma membrane structure and electromotile properties in prestin deficient outer hair cells

Abstract

Cochlear outer hair cells (OHCs) rapidly change their length and stiffness when their membrane potential is altered. Prestin, the motor protein for this electromotility, is present along the OHC lateral plasma membrane where there is a high density of intra-membrane protein particles (IMPs). However, it is not known to what extent prestin contributes to this unusual dense population of proteins and overall organization of the membrane to generate the unique electromechanical response of OHCs. We investigated the relationship of prestin with the IMPs, the underlying cortical cytoskeletal lattice, and electromotility in prestin-deficient mice. Using freeze-fracture, we observed a reduction in density and size of the IMPs that correlates with the reduction and absence of prestin in the heterozygous and homozygous mice, respectively. We also observed a reduction or absence of electromotility-related charge density, axial stiffness, and piezoelectric properties of the OHC. A comparison of the charge density with the number of IMPs suggests that prestin forms tetramers in the wild type but is likely to form lower number oligomers in the prestin-deficient OHCs from the heterozygous mice. Interestingly, the characteristic actin-based cortical cytoskeletal lattice that underlies the membrane is absent in the prestin-null OHCs, suggesting that prestin is also required for recruiting or maintaining the cortical cytoskeletal lattice. These results suggest that the majority of the IMPs are indeed prestin and that electrically evoked length and stiffness changes are interrelated and dependent on both prestin and on the cortical actin cytoskeletal lattice of the OHC lateral membrane.

Figure 1

Nonlinear capacitance (NLC) and ‘gating’ charge density measured from OHCs isolated from WT (+/+), heterozygous (+/−), and prestin-null (−/−) mice. (a) Images of isolated OHCs from the three genotypes. (b) Examples of the NLC measured from the three populations of OHCs. The NLC (lighter color lines) was fit with a two-state Boltzmann function (heavy lines). The NLC is color-coded; WT in black, heterozygous in blue, and prestin-null in red. The 6 parameters obtained from these cells are: Q_max 978.9±182.6 fC, α 31.1±4.9 mV⁻¹, z 0.79±0.12, V_1/2 −65.2±29.4 mV, C_non-lin 7.9±1.6 pF, C_lin 5.69±0.83 pF (for +/+, n=23); Q_max 720.1±159.6 fC, α 30.5±4.9 mV⁻¹, z 0.78±0.12, V_1/2 −68.9±33.9 mV, C_non-lin 5.9±0.86 pF, C_lin 5.39±0.55 pF (for +/−, n=22). The linear capacitance (C_lin) for homozygous OHCs is 4.8±0.34 pF (n=5). (b, inset) Schematic drawing of an OHC illustrates how the total plasma membrane area containing prestin is calculated. Magnification bar in a equals 10 µm.

Figure 2

Correlation between prestin expression levels and the presence of the high density of IMPs. (a–c) Prestin immunogold labeling of OHC lateral plasma membrane from +/+, +/−, and −/− mice. Prestin null OHCs show no immunogold labeling (c). Close up views of the protoplasmic freeze-fracture views of OHC lateral plasma membrane (d–e) from +/+, +/−, and −/− mice. In the WT, the protoplasmic fracture face characteristically shows a continuous high density of IMPs, where only very few and small areas devoid of IMPs can be found (delineated by the overlay trace in yellow). The protoplasmic fracture face of the lateral plasma membrane of the heterozygous OHC shows areas of high density and areas with normal density distribution of IMPs delineated by the overlay trace in yellow. The protoplasmic fracture face of the lateral plasma membrane of the prestin null OHCs lack the high-density distribution of IMPs (f). Magnification bars: 100 nm in (a–c) and 200 nm in (d, f)

Figure 3

Comparison of IMP size between wild type and prestin-deficient OHCs. High magnification images of the protoplasmic freeze-fracture face of the OHC lateral plasma membrane (a–c) from +/+,,+/−,and −/− mice as well as plasma membrane of Deiters’cell (d). Magnification bar equals 100 nm for a–d. (e) IMP diameter distribution for +/+, +/−, and −/− mice as well as plasma membrane of Deiters’cell.

Figure 4

Comparison of IMP size and shape between prestin and connexin oligomers. (a) Histogram of the diameters of IMPs in the OHC lateral membrane from the +/+ and −/− mice. (b) Estimated number of prestin molecules per IMP for the +/+ and +/− mice based on the ratio of non-linear capacitance and density of IMPs. (c) High magnification image of the freeze-fracture replica of a typical gap junction from an organ of Corti supporting cell. (d) High magnification image of the freeze-fracture replica of a typical region in the lateral plasma membrane from a WT OHC. In panel (c) both the exoplasmic (E-face) and protoplasmic (P-face) fracture faces can be visualized showing connexin IMPs in the P-face and complementary dimples in the E-face. (e) Histogram showing the distribution of IMP diameters for gap junction IMPs and for the IMPs from the prestin containing region of the lateral plasma membrane. (f) Exoplasmic fracture face of prestin containing region of the OHC lateral plasma membrane from WT mice shows IMPs dispersed on a scalloped surface likely resulting from the empty holes left when the IMPs were pulled out and portioned to the protoplasmic half of the membrane during freeze-fracture. Magnification bars equal: 50 nm in c and d and 100 nm in f.

Figure 5

Disruption of the cortical lattice in prestin-null OHCs. At the electron microscopic level the prestin-null OHC shows the characteristic organization of a typical OHC (a, b) with stereocilia (ST), cuticular plate (CP), tight-adherens junction (TAJ), subsurface cisternae underlying the lateral plasma membrane, attachment to the Deiters’ cell (DC), and efferent synapses (EF). (c) Schematic diagram of a portion of the lateral wall of the OHC (equivalent to the region delineated by the rectangle in a) showing the cortical actin-spectrin cytoskeletal lattice between the lateral plasma membrane and the underlying subsurface cisterna. The parallel actin filaments (arrows) and the periodic puncta (arrowheads) characteristic of the cortical actin-spectrin lattice are clearly visualized in thin section grazing or tangent to the curved surface of the OHC lateral wall (d, e) from the WT and hetereozygote but are absent in the prestin-null OHC (f). Magnification bars equal: 1 µm in a; 0.5 µm in b; and 200 nm in d–f.

Figure 6

Changes in axial stiffness and piezoelectric properties in prestin-deficient OHCs. (a) Measurement of passive axial stiffness. Examples of free-fiber (in light-color lines) and loaded-fiber motions (heavy lines) measured from the OHCs isolated from the cochleae of WT, heterozygous, and homozygous mice. The axial stiffness for the cells is 3.1 (+/+), 2.6 (+/−), and 0.9 (−/−) mN/m. (b) Examples of compression-evoked charge movement. Compression of the cell by a loaded fiber evoked an outward current in both +/+ and +/− OHCs. The current was absent in prestin-null OHCs. Bottom panel shows the waveform of the fiber motion. The black line indicates the direction of fiber motion (compression) and the outward current.

Figure 7

Examples of somatic motility and voltage-dependent stiffness changes obtained from WT and prestin-null OHCs. Downward deflections represent depolarization (cell contraction). On the left panels, somatic motility and voltage-dependent stiffness change was observed. The motility showed larger contraction than elongation at the holding potential of −70 mV. The stiffness change is reflected by changes of the amplitude of the loaded-fiber motion during contraction or elongation of the cell. On the right panels, no motility was observed. Note that the amplitude of the loaded-fiber motion was not altered when motility was absent.