A scenario wherein kinetic modifications within the family underlie prestin’s change to a molecular motor would be compelling. Interestingly, zebra fish prestin shows a lower-pass frequency response than rat prestin (33).In 2001, Oliver et al. (13) identified the chloride anion as a key element in prestin activation by voltage. They speculated that extrinsic anions serve as prestin’s voltage sensor (17), moving only partially through the membrane. Our observations and those of others over the ensuing years have challenged this concept, and we have suggested that chloride works as an allosteric-like modulator of prestin. These observations are as follows. 1) Monovalent, divalent, and trivalent anions, which support NLC, show no expected changes in z or Qmax (47). 2) A variety of sulfonic anions shift Vh in widely varying magnitudes and directions along the voltage axis (47). 3) The apparent anion affinity changes depending on the state of prestin, with anions being released from prestin upon hyperpolarization, opposite to the extrinsic sensor hypothesis (48). 4) (Z)-4-Hydroxytamoxifen biological activity mutations of charged residues alter z, our best estimate of unitary sensor charge (41). 5) Prestin shows transport properties ((40,41,43); however, see (39,42)). Despite these challenges, the extrinsic voltage-sensor hypothesis is still entertained. For example, Geertsma et al. (49) used their recently determined crystal structure of SLC26Dg, a prokaryotic fumarate transporter, to speculate on how prestin’s extrinsic voltage sensor might work. They reasoned that a switch to an outward-facing state could move a bound anion a small distance within the membrane. Unfortunately, there are no data showing an outward-facing state, only an inward-facing one. Indeed, if prestin did bind chloride but was incapable of reaching the outward-facing state (a defunct transporter), no chloride movements would occur upon voltage perturbation. Furthermore, the fact that the anion-binding pocket is in the center of the protein would mean that if an outward-facing state were achieved with no release of chloride, the monovalent anion would move a very small distance through the electric field of the membrane. However, z, from Boltzmann fits, indicates that the anion moves three-quarters of the distance through the electric field. Unless the electric field is inordinately concentrated only at the binding site, it is difficult to envisage this scenario. The data presented here clearly indicate that no direct relation between chloride level and Qmax exists, further suggesting that chloride does not serve as an extrinsic voltage sensor for prestin. Nevertheless, our recent work and meno presto model indicate that chloride binding to prestin is fundamental to the activation of this unusual motor. The model and data indicate that a stretched exponential intermediate transition between the chloride binding and the voltage-enabled state imposes lags that are expressed in whole-cell mechanical responses (28). This intermediate transition also accounts for our frequency- and chloride-dependent effects on measures of total charge movement, Qmax. Indeed, based on site-directed mutations of charged residues, we favor intrinsic charges serving as prestin’s voltage sensors (41). Recently, Gorbunov et al. (50), used cysteine accessibility scanning and molecular modeling to suggest structural homology of prestin to UraA. Notably, the crystal structureBiophysical Journal 110, 2551?561, June 7, get SB 202190 2016Santos-Sacchi and Son.A scenario wherein kinetic modifications within the family underlie prestin’s change to a molecular motor would be compelling. Interestingly, zebra fish prestin shows a lower-pass frequency response than rat prestin (33).In 2001, Oliver et al. (13) identified the chloride anion as a key element in prestin activation by voltage. They speculated that extrinsic anions serve as prestin’s voltage sensor (17), moving only partially through the membrane. Our observations and those of others over the ensuing years have challenged this concept, and we have suggested that chloride works as an allosteric-like modulator of prestin. These observations are as follows. 1) Monovalent, divalent, and trivalent anions, which support NLC, show no expected changes in z or Qmax (47). 2) A variety of sulfonic anions shift Vh in widely varying magnitudes and directions along the voltage axis (47). 3) The apparent anion affinity changes depending on the state of prestin, with anions being released from prestin upon hyperpolarization, opposite to the extrinsic sensor hypothesis (48). 4) Mutations of charged residues alter z, our best estimate of unitary sensor charge (41). 5) Prestin shows transport properties ((40,41,43); however, see (39,42)). Despite these challenges, the extrinsic voltage-sensor hypothesis is still entertained. For example, Geertsma et al. (49) used their recently determined crystal structure of SLC26Dg, a prokaryotic fumarate transporter, to speculate on how prestin’s extrinsic voltage sensor might work. They reasoned that a switch to an outward-facing state could move a bound anion a small distance within the membrane. Unfortunately, there are no data showing an outward-facing state, only an inward-facing one. Indeed, if prestin did bind chloride but was incapable of reaching the outward-facing state (a defunct transporter), no chloride movements would occur upon voltage perturbation. Furthermore, the fact that the anion-binding pocket is in the center of the protein would mean that if an outward-facing state were achieved with no release of chloride, the monovalent anion would move a very small distance through the electric field of the membrane. However, z, from Boltzmann fits, indicates that the anion moves three-quarters of the distance through the electric field. Unless the electric field is inordinately concentrated only at the binding site, it is difficult to envisage this scenario. The data presented here clearly indicate that no direct relation between chloride level and Qmax exists, further suggesting that chloride does not serve as an extrinsic voltage sensor for prestin. Nevertheless, our recent work and meno presto model indicate that chloride binding to prestin is fundamental to the activation of this unusual motor. The model and data indicate that a stretched exponential intermediate transition between the chloride binding and the voltage-enabled state imposes lags that are expressed in whole-cell mechanical responses (28). This intermediate transition also accounts for our frequency- and chloride-dependent effects on measures of total charge movement, Qmax. Indeed, based on site-directed mutations of charged residues, we favor intrinsic charges serving as prestin’s voltage sensors (41). Recently, Gorbunov et al. (50), used cysteine accessibility scanning and molecular modeling to suggest structural homology of prestin to UraA. Notably, the crystal structureBiophysical Journal 110, 2551?561, June 7, 2016Santos-Sacchi and Son.