Plasticity in membrane cholesterol contributes toward electrical maturation of hearing

Abstract

Advances in refining the "fluid mosaic" model of the plasma membrane have revealed that it is wrought with an ordered lipid composition that undergoes remarkable plasticity during cell development. Despite the evidence that specific signaling proteins and ion channels gravitate toward these lipid microdomains, identification of their functional impact remains a formidable challenge. We report that in contrast to matured auditory hair cells, depletion of membrane cholesterol in developing hair cells produced marked potentiation of voltage-gated K(+) currents (I(Kv)). The enhanced magnitude of I(Kv) in developing hair cells was in keeping with the reduced cholesterol-rich microdomains in matured hair cells. Remarkably, potentiation of the cholesterol-sensitive current was sufficient to abolish spontaneous activity, a functional blueprint of developing and regenerating hair cells. Collectively, these findings provide evidence that developmental plasticity of lipid microdomains and the ensuing changes in K(+) currents are important determinants of one of the hallmarks in the maturation of hearing.

FIGURE 1.

MβCD potentiates I_Kv only in developing chicken hair cells. Currents were elicited by 250-ms depolarizing voltage steps in 10-mV increments from a holding potential of −80 mV. A, example of current traces recorded at E12 from the midsection of the basilar papilla. I_Kv currents were recorded before and after the application of 1 mm MβCD. The difference current reflects the MβCD-sensitive component. B, current traces recorded at P2 from the midsection of the basilar papilla before and after the application of 1 mm MβCD. The difference current seen in A was abolished in P2 hair cells. C, the mean ± S.D. steady-state I-V plots are also shown (n = 16). Note the increase in current amplitude with application of MβCD. D, in contrast, the I-V plots for the current were unchanged after application of MβCD (n = 12). E, the Boltzmann fits were derived from the tail currents and plotted with solid lines from data obtained from hair cells at the midsection of E12 basilar papilla. Half-activation voltages were −31.3 ± 3.6 mV and −35.1 ± 5.8 mV (p = 0.3, n = 9) for control currents and after application of MβCD, respectively. The maximum slope factors for the activation curves were 18.3 ± 1.8 mV and 16.8 ± 3.9 mV (p = 0.1, n = 9) for control currents and after application of MβCD, respectively. F, summary data of the mean current density measured at 0-mV step potential from data collected from hair cells at the midsection of the basilar papilla (pA/picofarads (pF)) at E12, E16, E18, and P2. The sensitivity to MβCD decreased as hair cells became more mature. E12, n = 16; E16, n = 14; E18, n = 13; and P2, n = 12. *, p < 0.05.

FIGURE 2.

I_Kv currents in developing hair cells are resistant to the cholesterol-MβCD complex. Current traces were obtained from E12 hair cells from the midsection of the basilar papilla. Currents were elicited as described in Fig. 1. Shown are the current traces and amplitude for control conditions (A) and after exposure to the cholesterol-MβCD 8:2 saturated complex (B). C, the difference current traces reflect the loss of MβCD effects seen in Fig. 1. D, summary data of the I-V relationship obtained from 12 hair cells at E12 from the midsection of the basilar papilla. The data suggest that MβCD exerts its effects on I_Kv by depletion of membrane cholesterol.

FIGURE 3.

MβCD modulates TEA-sensitive I_Kv in developing chicken hair cells. A, exemplar current traces recorded at E12 from the apical aspects of the basilar papilla. I_Kv currents were recorded before (panel a) and after (panel b) the application of 1 mm MβCD, followed by application of 5 mm TEA plus 1 mm MβCD (panel c). The difference current (panel b-a) reflects the MβCD-sensitive component, and panel b-c reflects the TEA-sensitive component (panelsb and c). B, summary data of steady-state I-V plots are also shown (n = 11). Note the increase in the current amplitude with application of MβCD and subsequent current sensitivity to 5 mm TEA. C, example of current traces recorded at E12 from the midsection of the basilar papilla. I_Kv currents were recorded before (panel a) and after (panel b) the application of 5 mm TEA, followed by the application of 1 mm MβCD plus 5 mm TEA (panel c). The difference current (panel a-b) reflects the TEA-sensitive component, panel c-b clearly demonstrates that the MβCD-sensitive component was abolished. D, summary data showing the steady-state I-V plots (n = 9). Note that in the presence of TEA, there is a loss of MβCD-sensitive current.

FIGURE 4.

Detection of plasma membrane cholesterol accumulation by filipin staining in developing and mature hair cells. Whole-mount preparations of E14 (upper panel) and P2 (upper middle panel) basilar papillae fixed with paraformaldehyde were stained with filipin (green) and phalloidin (blue) and examined by confocal microscopy. Filipin stains membrane cholesterol-rich domain(s). To cross-check on the developmental specificity of filipin staining, we pretreated the preparations with MβCD. Pretreatment of the E14 basilar papilla with MβCD rendered the plasma membrane negative to filipin staining (lower middle panel). Also shown is filipin staining of the P2 basilar papilla (lower panel). Scale bar = 5 μm.

FIGURE 5.

MβCD produces marked changes in the membrane properties of developing hair cells. Shown is an example of ∼100 s of spontaneous electrical activity observed in E12 hair cells before (A) and after (B) application of 1 mm MβCD. The effects of MβCD on spike activity were concentration-dependent. C, summary data of a histogram describing the effects of cholesterol depletion on the resting membrane potential (rmp) (C), spike frequency (F) (D), and half-spike width (E) of developing (E12) hair cells. The role of membrane cholesterol in supporting the spontaneous activity of developing hair cells is apparent. *, p < 0.05; **, p < 0.01.