The development, distribution and density of the plasma membrane calcium ATPase 2 calcium pump in rat cochlear hair cells

Abstract

Calcium is tightly regulated in cochlear outer hair cells (OHCs). It enters mainly via mechanotransducer (MT) channels and is extruded by the plasma membrane calcium ATPase (PMCA)2 isoform of the PMCA, mutations in which cause hearing loss. To assess how pump expression matches the demands of Ca(2+) homeostasis, the distribution of PMCA2 at different cochlear locations during development was quantified using immunofluorescence and post-embedding immunogold labeling. The PMCA2 isoform was confined to stereociliary bundles, first appearing at the base of the cochlea around post-natal day (P)0 followed by the middle and then the apex by P3, and was unchanged after P8. The developmental appearance matched the maturation of the MT channels in rat OHCs. High-resolution immunogold labeling in adult rats showed that PMCA2 was distributed along the membranes of all three rows of OHC stereocilia at similar densities and at about a quarter of the density in inner hair cell stereocilia. The difference between OHCs and inner hair cells was similar to the ratio of their MT channel resting open probabilities. Gold particle counts revealed no difference in PMCA2 density between low- and high-frequency OHC bundles despite larger MT currents in high-frequency OHCs. The PMCA2 density in OHC stereocilia was determined in low- and high-frequency regions from calibration of immunogold particle counts as 2200/μm(2) from which an extrusion rate of ∼200 ions/s per pump was inferred. The limited ability of PMCA2 to extrude the Ca(2+) load through MT channels may constitute a major cause of OHC vulnerability and high-frequency hearing loss.

Figure 1

Developmental appearance of PMCA2 in the rat cochlea. Whole mounts of apical (low-CF), middle (mid-CF) and basal (high-CF) cochlear regions at: A. postnatal day 0 (P0); B1, B2, P6; C, P21. Whole mounts were labeled with the NR2 antibody and with phalloidin to mark the actin and focused on the hair bundle (HB; A, B1, C) and at the cell body (CB; B2). Three rows of outer hair cell bundles (ohc) and one row of inner hair cell bundles (ihc) indicated by arrows in B1 and the corresponding cell bodies in B2. D. Plots of PMCA2 fluorescence intensity in the OHC stereociliary bundle as a function of postnatal age for the three cochlear regions. Each point is the mean ± SEM of 15 OHCs in each of three preparations. E. Plots of the fluorescence intensity in the OHC bundle at P21 as a function of confocal photo-multiplier gain showing no difference between apex, middle and base. F. Schematic diagram of the organ of Corti showing approximate positions of the confocal planes through the hair bundles (HB) and cell bodies (CB) of OHC and IHC.

Figure 2

Developmental appearance of PMCA in the rat cochlea. Whole mounts of apical low-CF cochlear region at P2 (A) P6 (B) P9 (C) and P14 (D). Whole mounts were labeled with the 5F10 monoclonal antibody to all PMCA isoforms and with phalloidin to mark actin and focused on the hair bundle (HB) and cell body (CB); the two confocal planes are shown schematically in Fig. 1F. The 50 μm scale bar applies to B, C and D. Changes in the fluorescence intensities in the OHC bundle (E) and IHC bundle (F) are shown as a function of postnatal age for the apical, middle and basal cochlear regions. Changes in the fluorescence intensities in the OHC (G) and the IHC (H) basolateral membranes are shown as a function of postnatal age for the three cochlear regions. Each point in Fig. 2E and Fig. 2G is the mean ± SEM of 15 OHCs in each of three preparations and in Fig. 2D and Fig. 2H is the mean ± SEM of 5 IHCs in three preparations.

Figure 3

Post-embedding immunogold labeling for PMCA2 in rat OHCs. A. Transmission electron micrograph of an OHC bundle showing tall and middle row stereocilia labeled with NR2 antibody. Note that labeling is concentrated along the stereociliary membrane of each row with a lower level of labeling found within the actin core. B. Transmission electron micrograph showing labeling in all three stereociliary rows of another OHC; sc, stereocilium; cp, cuticular plate. Scale bar applies to both (A) and (B). C. Histograms showing PMCA2 gold particle labeling in each stereociliary row (short, middle and tall) as a function of the distance from the top of the cuticular plate along the OHC stereociliary membranes. In each histogram, the black line indicates the range of stereociliary heights measurable for each row, the mean height being indicated by the arrow. All OHCs are from the middle region of the cochlea.

Figure 4

PMCA2 labeling in rat OHCs and IHCs. A. Radial sections through stereociliary bundles of two OHCs and one IHC from the middle-CF region of the cochlea labeled with NR2 antibody against PMCA2 at the same (1:50) dilution and in the same experiment. Note the heavier labeling of the OHCs than the IHC. The scale bar applies to all images. B. PMCA2 labeling in apical and basal OHCs. Radial sections through stereociliary bundles of OHCs from the apical low- CF (left) and basal high-CF (right) regions of the same rat cochlea labeled with the NR2 antibody (1:50 dilution) in the same experiment. Gold particles are present at roughly the same density in the hair bundles of apical and basal cells. Since the scale bar applies to both micrographs, note the stereocilia at the apex have a larger diameter than those at the base.

Figure 5

Calibration of the immunogold labeling. A. Stereociliary bundle of an apical low-CF OHC labeled with the NR2 anti-PMCA2 antibody. B. Stereociliary bundle of a basal high-CF OHC labeled with the same PMCA2 antibody as in (A). C. Labeling with PMCA2 antibody of a section of a gel containing 2 mg/ml of the antigenic peptide against which the antibody was raised (see Methods). Gel and cochlear sections were labeled at the same time with the same antibody dilution. Scale bar applies to all three micrographs. D. Gold particle density in gel, from both areal and linear measurements in gel for four peptide concentrations.

Figure 6

Freeze fracture of an outer hair cell stereocilium (sc). The fracture plane has gone through the middle of the plasma membrane (between the outer and the inner leaflets of the plasma membrane) to show the inner leaflet over most of the shaft of the stereocilium, with particles clearly visible. The outer leaflet of the membrane is obscuring the particles lower down. Scale bar = 200 nm.