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Prestin (SLC26a5) function evolved to enhance auditory sensitivity and frequency selectivity by providing mechanical feedback via outer hair cells (OHC) into the organ of Corti. Its effectiveness is governed by the voltage-dependent kinetics of the protein's charge movements, namely, nonlinear capacitance (NLC). We study the frequency response of NLC in the mouse OHC, a species with ultrasonic hearing. We find that the characteristic frequency cut-off (F is ) for the mouse in near 27 kHz. Single point mutations within the chloride binding pocket of prestin (e.g., S396E, S398E) lack the protein's usual anion susceptibility. In agreement, we now show absence of anion binding in these mutants through molecular dynamics (MD) simulations. NLC F is in the S396E knock-in mouse is unaltered, indicating that high frequency activity is not governed by chloride, but more likely by viscoelastic loads within the membrane. We also show that the allosteric action of chloride does not underlie piezoelectric-like behavior in prestin, since tension sensitivity of S396E NLC is comparable to that of WT. Because prestin structures of all species studied to-date are essentially indistinguishable, with analogous chloride binding pockets, auditory requirements of individual species for cochlear amplification likely evolved to enhance prestin performance by modifying, not its protein-anion interaction, but instead external mechanical loads on the protein., Significance: Prestin is believed to provide cochlear amplification in mammals that possess a wide range of frequency sensitivities. Previously, chloride anions have been shown to control prestin kinetics at frequencies below 10 kHz. However, now we find that chloride binding is not influential for prestin kinetics in the very high range of the mouse. We suggest that such high frequency prestin performance is governed by impinging mechanical loads within the membrane, and not interactions with anions.

Voltage-gated K + (K V ) channels govern K +  ion flux across cell membranes in response to changes in membrane potential. They are formed by the assembly of four subunits, typically from the same family. Electrically silent K V channels (K V S), however, are unable to conduct currents on their own. It has been assumed that these K V S must obligatorily assemble with subunits from the K V 2 family into heterotetrameric channels, thereby giving rise to currents distinct from those of homomeric K V 2 channels. Herein, we show that K V S subunits indeed also modulate the activity, biophysical properties and surface expression of recombinant K V 7 isoforms in a subunit-specific manner. Employing co-immunoprecipitation, and proximity labelling, we unveil the spatial coexistence of K V S and K V 7 within a single protein complex. Electrophysiological experiments further indicate functional interaction and probably heterotetramer formation. Finally, single-cell transcriptomic analyses identify native cell types in which this K V S and K V 7 interaction may occur. Our findings demonstrate that K V cross-family interaction is much more versatile than previously thought-possibly serving nature to shape potassium conductance to the needs of individual cell types., (© 2024. The Author(s).)