The outer hair cell (OHC) from the organ of Corti is believed to be responsible for the mammal's exquisite sense of hearing. A membrane-based motile response of this cell underlies the initial processing of acoustic energy. The voltage-dependent capacitance of the OHC, possibly reflecting charge movement of the motility voltage sensor, was measured in cells during intracellular dialysis of trypsin under whole cell voltage clamp. Within 10 min after dialysis, light and electron microscopic examination revealed that the subplasmalemmal structures, including the cytoskeletal framework and subsurface cisternae, were disrupted and/or detached from adjacent plasma membrane. Dialysis of heat-inactivated trypsin produced no changes in cell structure. 2) in order to block ionic conductances. Pipette solutions were composed of (mM) CsCl 140, EGTA 10, tetraethylammonium chloride 5, MgCl2 2, and Hepes 5 (300 mosM, pH 7.2). Gigohm seals were obtained at the middle portion of the lateral wall. In order to introduce trypsin into the cell, relatively large patch pipette tips were used (1.5-to 2-1km inner diameter; series resistance, 2-3 MW). For trypsin treatments, 300 ptg of trypsin (source: bovine pancreas, Mr 23,281; Calbiochem) was dissolved in 1 ml of pipette solution and the osmolarity and pH were readjusted to 300 mosM and pH 7.2. As a control, heat-inactivated trypsin (enzyme pipette solution heated for 30 min in 560C water bath) was also used. Electrode capacitance was compensated after gigohm seal formation, and series resistance compensation was used in whole cell configuration. A modified version of Clampex (Axon Instruments, Burlingame, CA) was used to apply voltage stimuli and collect data that were saved on disk for off-line analysis. Current was filtered at 5 kHz with an eight-pole Bessel filter. All experiments were videotaped.Three methods were used to evaluate the effects of trypsin treatment on cell capacitance. The first method simply measured cell capacitance near the cell's normal in vivo resting potential. The cell was nominally held at -80 mV and a -10 mV step command voltage (4 ins) was applied repeatedly over time. At this potential both linear and a substantial amount of nonlinear capacitance contributes to the generation of a transient capacitive current (12). From averaged (x20) current records, an on-line analysis of membrane capacitance (Cm), membrane resistance (Rm), and series resistance (Rs) was performed and saved to disk. The transient analysis calculations have been published elsewhere (16).The second method evaluated the voltage dependence of nonlinear capacitance using a voltage stair protocol. The technique has been fully described elsewhere (14). Briefly, the cell was stair-stepped from a holding potential of -170 mV to voltages between -160 mV and +50 mV, in increments of 10 mV. From each step response, Cm, Rm, and Rs were calculated as a function of membrane voltage. The Abbreviation: OHC, outer hair cell. *To whom reprint requests should be addressed.
12268The publication cos...
