Expression density and functional characteristics of the outer hair cell motor protein are regulated during postnatal development in rat

Abstract

1. The non-linear capacitance (Cnon-lin) of postnatal outer hair cells (OHCs) of the rat was measured by a patch-clamp lock-in technique. Cnon-lin is thought to result from a membrane protein that provides the molecular basis for the unique electromotility of OHCs by undergoing conformational changes in response to changes in membrane potential (Vm). Protein conformation is coupled to Vm by a charged voltage sensor, which imposes Cnon-lin on the OHC. Cnon-lin was investigated in order to characterize the surface expression and voltage dependence of this motor protein during postnatal development. 2. On the day of birth (P0), Cnon-lin was not detected in OHCs of the basal turn of the cochlea, whilst it was 89 fF in apical OHCs. Cnon-lin increased gradually during postnatal development and reached 2.3 pF (basal turn, P9) and 7.5 pF (apical turn, P14) at the oldest developmental stages covered by our measurements. The density of the protein in the plasma membrane, deduced from non-linear charge movement per membrane area, increased steeply between P6 and P11 and reached steady state (4200 e- microm-2) at about P12. 3. Voltage at peak capacitance (V) shifted with development from hyperpolarized potentials shortly after birth (-88.3 mV, P2) to the depolarized potential characteristic of mature OHCs (-40.8 mV, P14). This developmental difference in V was also observed in outside-out patches immediately after patch excision. During subsequent wash-out V shifted towards the depolarized value found in the adult state, suggesting a direct modulation of the molecular motor. 4. Thus, the density of the motor protein in the plasma membrane and also its voltage dependence change concomitantly in the postnatal period and reach adult characteristics right at the onset of hearing.

Figure 1. C_non-lin from OHCs of postnatal rats measured with the phase-tracking technique

A, C_non-lin from apical OHCs of the developmental stages indicated. Residual linear capacitance was subtracted for display. Inset: C_non-lin (P0) on an enlarged scale. The line is the fit of eqn (4) to the data. Fit parameters were: V_½= -88.7 mV, α= 27.8 mV, Q_max= 7.2 fC at P0; V_½= -66.3 mV, α= 30.0 mV, Q_max= 116.3 fC at P6; and V_½= -51.8 mV, α= 30.4 mV, Q_max= 522.1 fC at P11. B, capacitance (upper trace), conductance (middle trace), and DC current (lower trace) as obtained with the phase-tracking technique during a voltage ramp (1.2 s) from -120 to +70 mV (P7, apical). Squares overlaid onto the upper trace are capacitance data from the same cell obtained with the stairstep method. Fits to the capacitance (not shown) yielded V_½= -60.1 mV, α= 27.6 mV, Q_max= 192.3 fC (phase-tracking) and V_½= -62.2 mV, α= 26.9 mV, Q_max= 195.4 fC (stairstep). Cs⁺-based pipette solution was used throughout the experiments shown in A and B.

Figure 2. Increase in C_non-lin during postnatal development

Peak C_non-lin (A), linear capacitance (B) and charge transfer density (C, see Methods), plotted as a function of time after birth for OHCs from the basal turn (○) and the apical turn (•) of the rat cochlea. The numbers of cells measured at the various ages are 6, 6, 7, 5, 6, 5, 5, 6, 1 and 6 for basal cells and 8, 4, 8, 5, 7, 6, 5, 8, 9, 7, 1, 6, 6 and 12 for apical OHCs. Cells were obtained from individuals of 4 litters. Error bars indicate standard deviation.

Figure 4. Release of cell turgor reversibly shifts V_½

A P13 OHC was perfused with hyposmotic solution. Upon switching to hyperosmotic solution, the cell quickly collapsed as viewed through the experimental microscope, but recovered after return to control solution. V_½ was monitored continuously by taking a phase-tracking measurement of C_non-lin every 3.15 s. Note that V_½ does not reach the hyperpolarized values of neonatal OHCs.

Figure 3. Voltage dependence of the motor protein changes during postnatal development

A, C_non-lin from P4 and P10 apical OHCs normalized to the respective C_peak. Note the developmental shift of V_½ at constant slope. Parameters yielded by fits (not shown) as in Fig. 1 were V_½= -79.1 mV, α= 31.1 mV for P4 and V_½= -36.2 mV, α= 30.5 mV for P10. B, V_½ of apical OHCs plotted versus time after birth (n = 3, 4, 8, 5, 7, 6, 8, 10, 10 and 12, respectively). Measurements were obtained within 30 s of establishment of the whole-cell configuration. Error bars indicate standard deviation.

Figure 5. Changes in V_½ in outside-out patches during development and wash-out

A, normalized C_non-lin measured in outside-out patches from OHCs at P2 and P13. Lines represent fits to the data as in Fig. 1. Parameters were V_½= -70.3 mV, α= 24.2 mV, Q_max= 0.28 fC at P2, and V_½= -37.9 mV, α= 25.5 mV, Q_max= 5.41 fC at P13. B, V_½ shifts to depolarized membrane potentials following patch excision in both P2 and P13 patches. Measurements are from the same patches as in A. The pipette solution was K⁺ based.