Gof SLC26Dg (49) supports Gorbunov et al.’s structural interpretations of prestin. Given the deduced structure, Gorbunov et al. identified possible chloride-binding residues within prestin’s central permeation pathway. When those residues were mutated, the protein became anion-insensitive, yet maintained NLC. For example, in the rPres mutation, R399S, which maintains NLC, salicylate inhibition of NLC was abolished. These data are in line with our Decumbin cancer observations that truncated Cl?movements are not responsible for voltage sensing, namely, the generation of NLC. We suggest, instead, that various anions differentially modulate the transition rates of prestin, likely as a consequence of their different binding affinities, thereby influencing Vh (i.e., the distribution of compact and expanded states of prestin) and the apparent Qmax obtained via discrete frequency admittance measures. This concept may explain the wide variability in prestin’s Boltzmann parameters that we and others have found with various anions (13,47). Some of prestin’s voltage-dependent characteristics are inadequately assessed using admittance analysis The prevailing concept of prestin activity has been that of an ultrafast two-state Boltzmann process. This is not surprising, since the OHC can change its length at acoustic rates up to 80 kHz (8?1). Consequently, this reasoning has guided our assessment method of prestin activity, where AC NLC measures have usurped the more tedious gatingcurrent methods that were utilized early on (32,35,51). Nevertheless, even gating-charge evaluations have suffered from problems associated with inadequate integration times and the shallow voltage dependence of prestin, which makes adequate linear leakage subtraction nearly impossible. Our data now show that AC capacitance measures do not correspond to full motor capability (18). The inaccuracies that fast single or dual AC measurements provide needn’t preclude their use, however, now that we have uncovered their limitations. Thus, by measuring Cm with a range of frequencies, with proper calibration for stray capacitance effects and including long interrogation times, valid measures of Qmax can be obtained. We previously noted that Boltzmann fits to the Q-V function of prestin cannot reliably predict unitary motor charge, Qm, since even Langevin fits are reasonable, which would place Qm values 3 times higher relative to two-state fits (26). This has been clearly emphasized by the Gummer group (52). One outcome of our study indicates that regardless of unitary charge magnitude (which remains stable across frequency, as indicated by invariant z values), the larger Qmax estimates from long interrogation times point to a higher density of prestin within the OHC lateral membrane. Consequently, the changes in prestin charge density observed in previous studies by narrow-band admittancetechniques may have been due to the effects of altered kinetics rather than prestin membrane content. Frequency dependence of OHC charge movement The frequency dependence of OHC charge movement has been investigated previously (12,31). Using the methodology of Fernandez et al. (53), we showed that AZD3759 chemical information capacitive reactance of the OHC with 140 mM intracellular solution in the whole-cell voltage-clamp configuration was voltage- and frequency-dependent, rising as zero frequency was approached (31). Subsequently, Gale and Ashmore (12) measured NLC in OHC membrane patches, where clamp time constants were better suited to high.Gof SLC26Dg (49) supports Gorbunov et al.’s structural interpretations of prestin. Given the deduced structure, Gorbunov et al. identified possible chloride-binding residues within prestin’s central permeation pathway. When those residues were mutated, the protein became anion-insensitive, yet maintained NLC. For example, in the rPres mutation, R399S, which maintains NLC, salicylate inhibition of NLC was abolished. These data are in line with our observations that truncated Cl?movements are not responsible for voltage sensing, namely, the generation of NLC. We suggest, instead, that various anions differentially modulate the transition rates of prestin, likely as a consequence of their different binding affinities, thereby influencing Vh (i.e., the distribution of compact and expanded states of prestin) and the apparent Qmax obtained via discrete frequency admittance measures. This concept may explain the wide variability in prestin’s Boltzmann parameters that we and others have found with various anions (13,47). Some of prestin’s voltage-dependent characteristics are inadequately assessed using admittance analysis The prevailing concept of prestin activity has been that of an ultrafast two-state Boltzmann process. This is not surprising, since the OHC can change its length at acoustic rates up to 80 kHz (8?1). Consequently, this reasoning has guided our assessment method of prestin activity, where AC NLC measures have usurped the more tedious gatingcurrent methods that were utilized early on (32,35,51). Nevertheless, even gating-charge evaluations have suffered from problems associated with inadequate integration times and the shallow voltage dependence of prestin, which makes adequate linear leakage subtraction nearly impossible. Our data now show that AC capacitance measures do not correspond to full motor capability (18). The inaccuracies that fast single or dual AC measurements provide needn’t preclude their use, however, now that we have uncovered their limitations. Thus, by measuring Cm with a range of frequencies, with proper calibration for stray capacitance effects and including long interrogation times, valid measures of Qmax can be obtained. We previously noted that Boltzmann fits to the Q-V function of prestin cannot reliably predict unitary motor charge, Qm, since even Langevin fits are reasonable, which would place Qm values 3 times higher relative to two-state fits (26). This has been clearly emphasized by the Gummer group (52). One outcome of our study indicates that regardless of unitary charge magnitude (which remains stable across frequency, as indicated by invariant z values), the larger Qmax estimates from long interrogation times point to a higher density of prestin within the OHC lateral membrane. Consequently, the changes in prestin charge density observed in previous studies by narrow-band admittancetechniques may have been due to the effects of altered kinetics rather than prestin membrane content. Frequency dependence of OHC charge movement The frequency dependence of OHC charge movement has been investigated previously (12,31). Using the methodology of Fernandez et al. (53), we showed that capacitive reactance of the OHC with 140 mM intracellular solution in the whole-cell voltage-clamp configuration was voltage- and frequency-dependent, rising as zero frequency was approached (31). Subsequently, Gale and Ashmore (12) measured NLC in OHC membrane patches, where clamp time constants were better suited to high.