Stant, k1, for Cl?binding (from 1e5 to 0.7e4). These results derive from the multiexponential kinetics of sensor charge PD173074 chemical information movement in the meno presto model, some slowly moving charge contributions being missed due to shorter interrogation times, and the fact that only an apparent Qmax was provided. Such behavior corresponds to our biophysical observations of OHCs and complements the biophysical data, which show that total sensor chargeFIGURE 4 Sensor charge movements estimated from two-sine admittance analysis, off-current integration, or eM show low-pass frequency characteristics. (A) The AC measured specific sensor charge (Qsp) corresponds to the integrated offcharge and shows that discrete measures of charge movement by AC admittance provide underestimates of the total prestin charge. (B) Qsp (circles) and eM (triangles), which is known to be driven by voltage, display magnitudes that correspond to the predictions of the meno presto model (gray lines). Interrogation time is the geometric average of periods of the dual-sine protocol, the integration time of sensor charge, or the eM fundamental frequency period (see Results). The biophysical data and model indicate that regardless of chloride concentration (but at above-zero concentrations), positive voltage will move prestin into the compact state, asymptoting at the maximum sensor charge dictated by prestin GS-4059 site Membrane content. Data are derived from averages of multi-dual-sine currents (circles) and eM (triangles) from n ?5? OHCs. To see this figure in color, go online.Biophysical Journal 110, 2551?561, June 7, 2016Santos-Sacchi and Songmovement is not directly linked to chloride concentration, but rather is misestimated due to prestin kinetics, in contradistinction to long-held concepts. Finally, to measure prestin’s frequency-dependent behavior in finer detail and expand on our data set, we measured NLC using chirp stimuli. Fig. 5 shows averaged results from another group of cells under each of the two chloride conditions (five to six cells per condition). NLC increases with a reduction of interrogating frequency, approaching that expected from zero-frequency or infiniteintegration estimates of sensor charge (Fig. 5, A and B). The meno presto model produces similar results (Fig. 5, Cand D), whereas a fast two-state Boltzmann model and a linear electrical resistor-capacitor (RC) model show no indication of frequency- or voltage/frequency-dependent capacitance, respectively (Fig. 5, E, G, and H). Appropriately setting the rate constants in a two-state model (forward/ backward rate constants of 0.5e3 s?) can produce a frequency-dependent roll-off within the measured bandwidth (Fig. 5 F); however, the resulting single-exponential transitions produce a different form of frequency dependence as compared to either the biophysical data or the meno presto model. These data confirm the validity of multi-dual-sine analysis of both linear electrical models and OHC NLC,FIGURE 5 Membrane capacitance versus frequency measured by high-resolution frequencydependent NLC of OHCs, the meno presto model, the fast two-state model, and the electrical model. (A) Averaged OHC NLC (n ?5) measured using the chirp protocol between 300 and 5000 Hz with 140 mM intracellular chloride. Note the rapid decline of peak capacitance. (B) Another group average of OHCs with 1 mM intracellular chloride (n ?6). The peak NLC decline is also evident in this condition. (C and D) Cm versus frequency as measured by the meno presto.Stant, k1, for Cl?binding (from 1e5 to 0.7e4). These results derive from the multiexponential kinetics of sensor charge movement in the meno presto model, some slowly moving charge contributions being missed due to shorter interrogation times, and the fact that only an apparent Qmax was provided. Such behavior corresponds to our biophysical observations of OHCs and complements the biophysical data, which show that total sensor chargeFIGURE 4 Sensor charge movements estimated from two-sine admittance analysis, off-current integration, or eM show low-pass frequency characteristics. (A) The AC measured specific sensor charge (Qsp) corresponds to the integrated offcharge and shows that discrete measures of charge movement by AC admittance provide underestimates of the total prestin charge. (B) Qsp (circles) and eM (triangles), which is known to be driven by voltage, display magnitudes that correspond to the predictions of the meno presto model (gray lines). Interrogation time is the geometric average of periods of the dual-sine protocol, the integration time of sensor charge, or the eM fundamental frequency period (see Results). The biophysical data and model indicate that regardless of chloride concentration (but at above-zero concentrations), positive voltage will move prestin into the compact state, asymptoting at the maximum sensor charge dictated by prestin membrane content. Data are derived from averages of multi-dual-sine currents (circles) and eM (triangles) from n ?5? OHCs. To see this figure in color, go online.Biophysical Journal 110, 2551?561, June 7, 2016Santos-Sacchi and Songmovement is not directly linked to chloride concentration, but rather is misestimated due to prestin kinetics, in contradistinction to long-held concepts. Finally, to measure prestin’s frequency-dependent behavior in finer detail and expand on our data set, we measured NLC using chirp stimuli. Fig. 5 shows averaged results from another group of cells under each of the two chloride conditions (five to six cells per condition). NLC increases with a reduction of interrogating frequency, approaching that expected from zero-frequency or infiniteintegration estimates of sensor charge (Fig. 5, A and B). The meno presto model produces similar results (Fig. 5, Cand D), whereas a fast two-state Boltzmann model and a linear electrical resistor-capacitor (RC) model show no indication of frequency- or voltage/frequency-dependent capacitance, respectively (Fig. 5, E, G, and H). Appropriately setting the rate constants in a two-state model (forward/ backward rate constants of 0.5e3 s?) can produce a frequency-dependent roll-off within the measured bandwidth (Fig. 5 F); however, the resulting single-exponential transitions produce a different form of frequency dependence as compared to either the biophysical data or the meno presto model. These data confirm the validity of multi-dual-sine analysis of both linear electrical models and OHC NLC,FIGURE 5 Membrane capacitance versus frequency measured by high-resolution frequencydependent NLC of OHCs, the meno presto model, the fast two-state model, and the electrical model. (A) Averaged OHC NLC (n ?5) measured using the chirp protocol between 300 and 5000 Hz with 140 mM intracellular chloride. Note the rapid decline of peak capacitance. (B) Another group average of OHCs with 1 mM intracellular chloride (n ?6). The peak NLC decline is also evident in this condition. (C and D) Cm versus frequency as measured by the meno presto.