Figure 3. Voltage-dependent charge movement by zprestin.

A, transient currents measured from a CHO cell expressing zprestin–GFP (left) or rprestin–GFP (right) were elicited by a series of depolarizing voltage steps of 3 ms duration and 10 mV increments starting at −30 mV (zprestin) and −130 mV (rprestin), as are illustrated in the lower panels (dashed lines indicate that larger voltage steps are omitted for clarity). Any residual linear – capacitive or resistive – currents were eliminated using a P/−8 protocol. Each trace is the average of 10 consecutive recordings. Note the inverse transient current at return to the initial voltage. Non-transfected cells had no detectable transient currents (data not shown). B, charge measured from a zprestin-expressing cell at the end of a voltage step (OFF) is plotted against the charge at the onset of the step (ON). A line of unity slope well described the relation, indicating equality of ON- and OFF-charges. C, charge measured as in A is plotted against step voltage. Data from individual cells were fitted with a 2-state Boltzmann function and normalized to saturating charge. The plot shows mean normalized data (±s.d.) from 5 cells (zprestin) and 4 cells (rprestin). Continuous lines show the best fits to the averaged data with V_1/2=94.7 mV, α=53.2 mV for zprestin and V_1/2=−74.6 mV, α=34.0 mV for rprestin. D, NLC measured from the same CHO cell expressing zprestin–GFP with the different stimulus frequencies indicated. Continuous lines are the best fits to the first derivative of a 2-state Boltzmann function. For fit results, see Results. Capacitance recordings are presented with linear membrane capacitance subtracted. E, NLC measured from a rprestin–GFP-expressing cell as described in (C). Fits (continuous lines) yielded peak NLC of 0.75 ± 0.35 pF, Q_max of 111 ± 47 fC, V_1/2 of −69 ± 14 mV and slope values of 35.5 ± 1.2 mV (at 2 kHz; n=6). Inset shows the lack of detectable NLC in a representative control cell (recordings for all four frequencies are superimposed). F, frequency dependence of NLC in cells expressing zprestin: peak NLC obtained with the various stimulation frequencies was normalized to peak NLC at 0.5 kHz stimulation frequency for each cell (n=7). G, transient currents measured in response to voltage steps (from −140 to −20 mV, rprestin; from −80 to +120 mV, zprestin). Traces are averaged from 4 and 10 repetitive recordings, respectively, and normalized to peak current. Superimposed curves show monoexponential fits to the current relaxation yielding time constants (τ_ON) of 26 μs (rprestin) and 120 μs (zprestin). For recording conditions, see Methods. H, time constants of charge transfer for a range of step potentials were obtained as in G. Data are from n=3 and 6 cells expressing rprestin and zprestin, respectively.