Mammals have evolved a highly sophisticated sense of hearing that is characterized by an extraordinary ability to process high-frequency sounds, such as echolocation. This represents the pinnacle of mechanosensory evolution made possible by a mechanism of sound amplification, which involves the specialized “electromotility” of the mammalian outer hair cells. The voltage-dependent motor protein prestin (also known as SLC26A5) is responsible for the electromotive behaviour of outer hair cells and underlies the cochlear amplifier. Disruption or loss of prestin function can lead to disabling hearing loss. Recent 3D structures of prestin have begun to unravel the mechanism through which it converts voltage changes into membrane areal expansion. However, the specific structural elements conferring voltage and mechanosensitivity to prestin remain elusive. To address this, we introduced mutations in dolphin and human prestin that shift its midpoint of activation across various membrane potentials (in increments of +100 mV). We solved the high-resolution structures of these mutants using single particle cryo-electron microscopy (EM) in detergents and various lipid compositions. Our investigation revealed the significance of conserved glycine residues located on one of the peripheral transmembrane helices, TM6. These glycine residues serve as hinges at the interface between the protein and the lipid bilayer, influencing prestin’s voltage sensitivity and, consequently, its mechanotransduction. Patch-clamp electrophysiology and molecular dynamics simulations further corroborate these structural data. Together, these findings provide valuable insights into the modulation of lipids and the functional mechanisms of this motor protein in the mammalian cochlea.

Prestin responds to transmembrane voltage fluctuations by changing its cross-sectional area, a process underlying the electromotility of outer hair cells and cochlear amplification. Prestin belongs to the SLC26 family of anion transporters yet is the only member capable of displaying electromotility. Prestin’s voltage-dependent conformational changes are driven by the putative displacement of residue R399 and a set of sparse charged residues within the transmembrane domain, following the binding of a Cl⁻ anion at a conserved binding site formed by the amino termini of the TM3 and TM10 helices. However, a major conundrum arises as to how an anion that binds in proximity to a positive charge (R399), can promote the voltage sensitivity of prestin. Using hydrogen–deuterium exchange mass spectrometry, we find that prestin displays an unstable anion-binding site, where folding of the amino termini of TM3 and TM10 is coupled to Cl⁻ binding.