The mammalian hearing organ shows high sensitivity and frequency selectivity. One of the key processes responsible for these features is the mechanical amplification of sound carried out by the outer hair cells (OHCs) of the inner ear through electromotility (EM). Electromotility is driven by prestin, a membrane protein member of the SLC26 family of transporters that acts as a voltage-dependent area motor. Strikingly, prestin only plays this role in mammals whereas in non-mammals, it acts as an anion transporter without significant piezoelectric activity. Studies aimed at explaining the origin of these differences found at least three regions of interest located in the transmembrane domain (TMD). However, to date, the effect of these differences on prestin function has not been placed in the context of its structure and dynamics. Furthermore, a region of high variability among members of the SCL26 family has been practically overlooked: The the intervening sequence (IVS) on the Sulphate Transporter and AntiSigma factor antagonist (STAS) domain. In this study, we combined phylogenetic analysis, reconstruction of ancestral prestin sequences, and structural modeling to offer a comparative analysis of prestin evolution from a structural point of view and to shed light on how its unique sequence features may play a role in the generation and tuning of EM. Our results suggest that sequence/structural differences in the TMD are related to changes in the interactions within the protein domains and between the protein and lipids leading to a gradual reduction of its ion transport capability while enhancing its voltage sensitivity essential for electromotility. Regarding the IVS, our results suggest that, in placental mammals, a unique patch of negative residues may directly interact with residues that form the entrance of the intracellular cavity of the adjacent monomer and modulate the response to voltage or frequency changes.

The mammalian hearing organ shows high sensitivity and frequency selectivity. One of the key processes responsible for these features is the mechanical amplification of sound carried out by the outer hair cells (OHCs) of the inner ear through electromotility (EM). Electromotility is driven by prestin, a membrane protein member of the SLC26 family of transporters that acts as a voltage-dependent area motor. Strikingly, prestin only plays this role in mammals whereas in non-mammals, it acts as an anion transporter without significant piezoelectric activity. Studies aimed at explaining the origin of these differences found at least three regions of interest located in the transmembrane domain (TMD). However, to date, the effect of these differences on prestin function has not been placed in the context of its structure and dynamics. Furthermore, a region of high variability among members of the SCL26 family has been practically overlooked: The the intervening sequence (IVS) on the Sulphate Transporter and AntiSigma factor antagonist (STAS) domain. In this study, we combined phylogenetic analysis, reconstruction of ancestral prestin sequences, and structural modeling to offer a comparative analysis of prestin evolution from a structural point of view and to shed light on how its unique sequence features may play a role in the generation and tuning of EM. Our results suggest that sequence/structural differences in the TMD are related to changes in the interactions within the protein domains and between the protein and lipids leading to a gradual reduction of its ion transport capability while enhancing its voltage sensitivity essential for electromotility. Regarding the IVS, our results suggest that, in placental mammals, a unique patch of negative residues may directly interact with residues that form the entrance of the intracellular cavity of the adjacent monomer and modulate the response to voltage or frequency changes.