Contribution of membrane cholesterol to outer hair cell lateral wall stiffness

The outer hair cell can be divided into three domains: the apex, the base, and the lateral wall. With the use of filipin, a polyene fluorescent Antibiotic that binds to Cholesterol, we found under fluorescence microscopy that the lateral wall membranes were less intensely stained than the apical and basal membranes. This difference in filipin fluorescence between the lateral walls and the ends diminished when cells were incubated in water-soluble Cholesterol before staining, suggesting that exogenous Cholesterol enters the lateral wall. Under confocal microscopy, we studied the incorporation pattern of a fluorescent Cholesterol analogue, NBD-cholesterol. NBD-cholesterol did not stain the apical membranes whereas it intensely labeled the lateral wall. The micropipette aspiration technique was used to assess the effect of Cholesterol on lateral wall stiffness. The lateral wall stiffness parameter of cells treated with water-soluble Cholesterol (n = 23) was significantly higher than that of controls (n = 27): 0.76+/-0.24 (mean +/- SD) versus 0.46+/-0.10 (Student's t-test, p < 0.001). In conclusion, Cholesterol has different distributions among outer hair cell membranes, and when water-soluble Cholesterol is incorporated into the cells, the outer hair cell lateral wall stiffness parameter increases.