Mammalian prestin (SLC26A5), the motor protein of cochlear outer hair cells, converts transmembrane voltage changes into a fast change in membrane-plane area, producing electromotility and its electrical signature, nonlinear capacitance. How this area-motor activity works at the level of identified molecular events remains unresolved: it is not settled whether the voltage sensor is extrinsic (a bound chloride ion whose charge moves in the membrane field), intrinsic (charged protein residues), or a defined combination, nor how voltage, membrane mechanics, and several evolutionarily remodeled structural elements — the placental IVS-loop with its negatively charged patch, the extracellular loop, TM5b and the TM5–TM5b loop, and STAS-domain substitutions — contribute to charge movement and area change. A 2025/2026 ancestral-sequence-reconstruction and structural-modeling study (Fuentes-Ugarte et al.) sharpened these questions by mapping the transporter-to-motor substitutions.

Background

Prestin (SLC26A5) is a membrane protein in the lateral wall of cochlear outer hair cells that acts as a piezoelectric-like molecular motor: changes in transmembrane voltage drive a fast (microsecond-scale) change in the protein's cross-sectional area, elongating or shortening the cell. This "electromotility" (EM), whose electrical signature is a voltage-dependent nonlinear capacitance (NLC — the charge movement that gates the conformational change, measured as a bell-shaped capacitance-versus-voltage curve), powers the cochlear amplifier that sensitizes and sharpens mammalian hearing. Prestin belongs to the SLC26 family of anion transporters. Non-mammalian orthologues (zebrafish, chicken, lizard) are bona fide electrogenic anion exchangers, so mammalian prestin is a transporter that was repurposed into a voltage-driven motor on a conserved scaffold, a transition completed along the lineage to placental mammals. Prestin functions as a homodimer. Each subunit has a transmembrane domain (TMD) built from a "core" (transport) domain and a "gate" (scaffold) domain — an inverted-repeat transporter architecture in which the core moves in an elevator-like fashion relative to the gate — plus cytoplasmic N- and C-terminal regions; the C-terminal STAS domain (sulfate transporter and anti-sigma factor antagonist domain, a structural hallmark of the SLC26 family) participates in dimerization and contacts the TMD. Within the STAS domain, the intervening sequence (IVS) is a variable insertion containing a flexible IVS-loop (residues roughly 580–614 in human prestin) that is disordered and unresolved in every prestin structure solved to date.

How prestin senses voltage is a long-standing controversy. Oliver et al. (2001) proposed that intracellular anions, chiefly chloride, constitute an extrinsic voltage sensor: bound Cl⁻ is displaced within the membrane field, and the anion's redistributed charge produces the measured gating current. Rybalchenko and Santos-Sacchi (2008) and Song and Santos-Sacchi (2013) demonstrated anion control of voltage sensing and chloride-driven state transitions. Cryo-electron microscopy (cryo-EM) studies — Gorbunov et al. (2014) on anion interaction architecture, Ge et al. (2021) on human prestin, Bavi et al. (2021) on the dolphin orthologue's conformational cycle, and Kuwabara et al. (2023) on elevator-like core-domain movements — located the conserved anion-binding site at the N-termini of transmembrane helices TM3 and TM10 and mapped the compact ("contracted") and expanded conformational states that underlie the area change. In the alternative, intrinsic view, prestin's own charged residues (notably R399 near the binding site) carry the sensing charge. Lin et al. (2023) showed that Cl⁻ binding folds the N-terminal segments of TM3 and TM10, implicating both the anion and intrinsic residues in one coupled process, and that this folding behavior is absent in the non-electromotile relative SLC26A9. Prestin is also strongly modulated by membrane mechanics — tension, thickness, and cholesterol — and structures in membranes of graded thickness (Bavi et al. 2025) show that membrane thinning alone drives the same compact-to-expanded transition that voltage drives.

Fuentes-Ugarte et al. (2026) (preprint 2025) approached the transporter-to-motor transition by ancestral sequence reconstruction (ASR — statistical inference of the amino-acid sequences of a protein's ancestors along a phylogeny), combined with AlphaFold2-multimer structure prediction (an AI-based method for modeling multi-chain protein structures) and molecular dynamics (MD) simulations. They identified more than 200 substitutions along the lineage to placental mammals: early substitutions concentrate in the TMD — notably lipid-facing residues, TM6/TM7 (which recent cryo-EM shows bend as a mechanosensitive "elbow" under membrane thinning), TM13/TM14, and TM5b — while late substitutions cluster in the STAS-domain IVS. In placental mammals the IVS-loop acquires a helix-breaking glycine at G583 and a unique negatively charged patch (E609/E610/D611 in human numbering) positioned near the intracellular entrance of the adjacent monomer's chloride-binding site, with a bimodal "close"/"far" conformational distribution and transient salt bridges (electrostatic arginine/lysine–glutamate/aspartate contacts) to conserved basic residues lining the chloride access pathway. The extracellular loop between TM3 and TM4 (EC-loop) also carries clustered early substitutions that decouple it from the TM5–TM5b loop in mammals; strikingly, grafting the gerbil prestin EC-loop into the related transporter pendrin (SLC26A4) is sufficient to confer NLC and EM on pendrin (Tang et al. 2011), yet no mechanistic explanation of this is available. The authors flag as open: the functional consequences of the IVS-loop conformations and its charged patch (mutations of these charged residues were previously found to shift voltage operating parameters only modestly; Bai et al. 2006), a mechanistic explanation of the EC-loop's role, the unexplored TM5b and TM5–TM5b loop, the functional role of the STAS-domain substitutions — a region that has received limited attention in studies of the function and regulation of prestin and other members of the SLC26 family — how voltage triggers the conformational change, the influence of membrane tension and lipid composition on the conformational equilibrium, and whether the IVS-loop modulates chloride dynamics by restricting ion exit or trapping ions near the binding site. The identity of the voltage sensor (extrinsic versus intrinsic), likewise unresolved, is the field-level controversy documented above.

Problem Statement

Determine the molecular mechanism by which mammalian prestin converts membrane voltage into a change in membrane-plane area — the area-motor activity underlying outer hair cell electromotility — at the level of identified molecular events. This comprises, as one linked objective: (i) the identity of the voltage sensor, i.e., whether the gating charge is carried by a bound anion (an extrinsic sensor, as proposed for intracellular chloride), by intrinsic charged protein residues, or by a defined combination, and quantitatively how voltage triggers the compact-to-expanded conformational change, including how this electrical driving is coupled to membrane tension, thickness, and lipid composition; and (ii) the functional roles, in nonlinear capacitance and electromotility, of the structural elements implicated by the evolutionary and structural analysis: the placental-mammalian IVS-loop of the STAS domain and its negatively charged patch (E609/E610/D611 in human numbering) — in particular whether it modulates chloride dynamics by restricting ion exit or trapping ions near the binding site, thereby influencing voltage sensing or allosteric coupling; the extracellular loop between TM3 and TM4, whose graft into the transporter pendrin suffices to confer NLC/EM; TM5b and the TM5–TM5b loop, which carry several early substitutions but remain structurally and functionally unexplored; and the lineage-specific STAS-domain substitutions as a whole (a set that includes the IVS-loop charged patch just described), whose functional role in prestin and other SLC26 family members remains open, existing mutational data being limited to modest shifts in voltage operating parameters. The mechanism must be consistent with the available experimental evidence (for example, mutagenesis, electrophysiology, and structures), attributing the measured charge movement and area change to specified molecular events and specifying the functional role of each of the enumerated structural elements.

References

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