Electrophysiological experiments for NLC measurements

We used the HEKA EPC 10 USB amplifier (HEKA Instruments Inc.) controlled by Patchmaster software (HEKA Instruments Inc.) to measure the NLC of prestin by whole-cell patch-clamp recordings at room temperature (22° to 26°C). Recording pipettes made of borosilicate glass were pulled with resistances of 2.5 to 4 megohms and filled with an internal solution containing 140 mM CsCl, 2 mM MgCl2, 10 mM EGTA, and 10 mM Hepes. During the recordings, cells were bathed in an external solution containing 120 mM NaCl, 20 mM TEA-Cl, 2 mM CoCl, 2 mM MgCl2, 10 mM Hepes, and 5 mM glucose. Solutions were adjusted to pH 7.2. The osmolarities of the internal and external solutions were adjusted with glucose to 300 and 320 mOsm/liter, respectively. We measured whole-cell membrane capacitance (Cm) using sine + DC software, a lock-in function of Patchmaster. Voltage-dependent NLC was assessed by recording Cm during voltage ramps, as previously described (48, 49). NLC curves were quantified by fitting with the derivative of a two-state Boltzmann functionEmbedded Imagewhere Qmax is the maximum charge transfer, V1/2 is the voltage at which the maximum charge is equally distributed across the membrane, Clin is the linear capacitance, and α is the slope factor of the voltage dependence of the charge transfer. Clin is proportional to the surface area of the membrane (cell size). To compare the magnitude of NLC obtained from different cells with different levels of prestin expression as a function of cell size, we normalized the NLC by the linear capacitance of the cells. Because differences in Qmax could have been caused by cell size, the charge movement was normalized to Clin. This quantity, designated as the charge density (Qmax/Clin), had units of femtocoulomb per picofarad. We used IgoPro software (IgoPro, WaveMetrics) for data processing and fitting.