Developmental expression of the outer hair cell motor prestin in the mouse

Abstract

The development of motor protein activity in the lateral membrane of the mouse outer hair cell (OHC) from postnatal day 5 (P5) to P18 was investigated under whole-cell voltage clamp. Voltage-dependent, nonlinear capacitance (C (v)), which represents the conformational fluctuations of the motor molecule, progressively increased during development. At P12, the onset of hearing in the mouse, C (v) was about 70% of the mature level. C (v) saturated at P18 when hearing shows full maturation. On the other hand, C (lin), which represents the membrane area of the OHC, showed a relatively small increase with development, reaching steady state at P10. This early maturation of linear capacitance is further supported by morphological estimates of surface area during development. These results, in light of recent prestin knockout experiments and our results with quantitative polymerase chain reaction, suggest that, rather than the incorporation of new motors into the lateral membrane after P10, molecular motors mature to augment nonlinear capacitance. Thus, current estimates of motor protein density based on charge movement may be exaggerated. A corresponding indicator of motor maturation, the motor's operating voltage midpoint, V (pkcm), tended to shift to depolarized potentials during postnatal development, although it was unstable prior to P10. However, after P14, V (pkcm) reached a steady-state level near -67 mV, suggesting that intrinsic membrane tension or intracellular chloride, each of which can modulate V (pkcm), may mature at P14. These developmental data significantly alter our understanding of the cellular mechanisms that control cochlear amplification and provide a foundation for future analysis of genetic modifications of mouse auditory development.

Fig. 1

(A) Examples of C_m functions from three postnatal days. In addition to an increase in peak capacitance, note the shift in V_pkcm to the right during maturation. Fitted parameters were Q_max = 0.091 pC, z = 0.89 at P7; Q_max = 0.263 pC, z = 0.95 at P9; Q_max = 0.542 pC, z = 0.89 at P14. (B) Images of patch-clamped OHCs from P7 to P18. Scale bar (7.5 µm) in P14 figure applies to all images

Fig. 2

Cell dimensions as a function of postnatal day. (A) Linear capacitance (with standard error bars) and surface area as a function of postnatal day. Note saturation of each measure at P10. (B) Inverse relationship between measured length and width of OHCs. Solid gray line denotes constant surface area constraint on width vs. length for the cylindrical model (see Materials and Methods). Surface area was constrained at 740 µm² (P10). (C) Relationship between linear capacitance and calculated surface area. Solid gray line denotes a specific membrane capacitance of 0.008 pF/µm²

Fig. 3

(A) NLC (C_v, colored symbols) and unitary charge density (open circles) as a function of postnatal day. Each function saturates at P18. Charge density was computed using C_lin to calculate surface area with the specific membrane capacitance of 0.008 pF/µm². Logistic fits were made with C_v or density = max/[1+(day/mid) × b]. Charge density fit (max, b, mid): 10,956, –5.65, 10.22. C_v: 7.66, – 5.33, 10.66. The number of cells was (from P5 to P18) 2, 5, 5, 9, 5, 6, 10, 9, 5, 10, 6, 9, 5 and 5. Standard error is plotted. (B) Relationship between C_v and C_lin. Note the continued increase in C_v after C_lin (or surface area) saturates

Fig. 4

Voltage at peak capacitance (V^pkcm) changes during development. Note variability during early stages and eventual shift toward depolarized voltages, stabilizing near −67 mV

Fig. 5

Expression of prestin changes with development and peaks at P10. QPCR amplification of cDNA from cochlea RNA obtained on P5 (black), P8 (red), P10 (green) and P15 (yellow) are shown. Inset shows the C_t values of prestin and the relative amplification of prestin corrected for GAPDH expression at P5, P8, P10 and P15. The lower C_t value (triangles) at P10 confirms the highest expression at this time point. The relative expression (circles) of prestin is compared to expression at P10