⚛️ biophysics

Conformational changes underlying electromechanical transduction in prestin resemble a transport transition in pendrin

This study combines multi-microsecond molecular dynamics simulations and cryo-electron microscopy to reveal that prestin's electromechanical transduction involves conformational changes and anion-binding mechanisms that closely parallel the transport cycle of the related SLC26 family member pendrin, despite differences in ion accessibility and transition speeds.

Original authors: Zhang, C., Mariadasse, R., Yang, J., Bai, J.-P., Santos-Sacchi, J., Navaratnam, D. S., Beckstein, O.

Published 2026-08-05

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CC BY 4.0

Original authors: Zhang, C., Mariadasse, R., Yang, J., Bai, J.-P., Santos-Sacchi, J., Navaratnam, D. S., Beckstein, O.

Original paper licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). ⚕️ This is an AI-generated explanation of a preprint that has not been peer-reviewed. It is not medical advice. Do not make health decisions based on this content. Read full disclaimer

Imagine your ear is a tiny, high-tech concert hall. Inside this hall, there are special cells called outer hair cells that act like the sound system's amplifiers. Without them, the music would be too quiet to hear clearly. These cells have a superpower: they can change their shape almost instantly in response to electrical signals, making the sound waves bounce around more effectively. This shape-shifting is driven by a molecular machine called prestin. Think of prestin as a microscopic piston or a spring-loaded door that snaps open and shut billions of times a second.

To understand how this machine works, scientists often look at its "cousins"—other proteins in the same family that act as transporters. These cousins, like pendrin, are like delivery trucks that shuttle ions (tiny charged particles) across the cell membrane. They work by changing shape to open a door on one side, drop off a package, close up, and then open on the other side. This process is called the "alternating access" mechanism. The big question scientists have been asking is: Does prestin work like a delivery truck that shuttles ions back and forth, or is it something entirely different, like a pure motor that just changes shape to do mechanical work? Understanding this helps us figure out the fundamental physics of how we hear.

Now, let's dive into what this new study discovered. The researchers used powerful computer simulations to watch prestin move in slow motion, essentially creating a digital movie of the protein's life. They started with a "contracted" (shrunken) version of prestin and watched what happened. Surprisingly, the protein didn't just sit there; it spontaneously and rapidly expanded into a larger shape. This expansion wasn't random; it looked exactly like the "inward-facing" shape of its cousin, pendrin, when that cousin is ready to pick up a package from inside the cell.

The study suggests that prestin and pendrin are more similar than we thought. They share a core structural trick: when they switch from a shrunken state to an expanded state, they get wider specifically on the inner side of the cell membrane. It's like a person wearing a puffy jacket that only inflates on the inside, pushing against the inner wall of the room. This specific expansion is crucial because it matches what happens when the cell's electrical voltage changes, which is exactly how prestin is supposed to work.

However, there's a twist. While the shapes look similar, the job might be different. In pendrin, the shape change opens a tunnel that lets ions travel all the way through the protein, from the outside of the cell to the inside. But in prestin, the simulations showed that even when it expands, there is no continuous tunnel connecting the outside to the inside. It's as if prestin has the same body shape as the delivery truck, but it's missing the cargo bay. Instead of transporting ions, it seems to use that shape change to generate force, acting like a motor rather than a transporter.

The team also found a new "parking spot" for ions on the outside of the protein, which they confirmed using a high-resolution 3D image (cryo-electron microscopy). In the cousin protein, pendrin, this spot helps guide ions into the main tunnel. In prestin, this spot exists, but it doesn't connect to the inside, suggesting it might just be a place where ions hang out without ever crossing the membrane.

One of the most exciting findings is just how fast this happens. The computer simulations showed prestin snapping from its shrunken state to its expanded state in less than a millionth of a second (sub-microsecond). This speed is fast enough to keep up with the ultrasonic frequencies that mammals can hear. While pendrin stays relatively still in its shapes during the simulations, prestin is constantly jittering and switching, which might be the secret to its incredible speed.

In short, the paper suggests that prestin and pendrin are structural twins that use the same basic "elevator" movement to change shape. But while pendrin uses this movement to ferry ions across the membrane, prestin seems to have evolved to use that same movement to push and pull on the cell membrane itself, acting as a lightning-fast motor that powers our hearing. The study confirms that prestin's expansion is localized to the inner membrane layer and happens spontaneously, supporting the idea that it is a molecular motor that doesn't need to transport ions to do its job, even though it shares the same blueprint as the ion-transporting cousins.

Technical Summary: Conformational Changes Underlying Electromechanical Transduction in Prestin

Problem Statement
Prestin (SLC26A5) is the motor protein responsible for electromotility in mammalian cochlear outer hair cells, a process essential for hearing sensitivity. Unlike other members of the SLC26 anion transporter family, prestin functions as a voltage-dependent molecular motor rather than a secondary active transporter. While it is established that prestin transitions between contracted and expanded conformations in a voltage-dependent manner, the structural basis of this transition and its relationship to the canonical alternating-access transport cycle of SLC26 family members (exemplified by pendrin, SLC26A4) remain unclear. Specifically, it is unknown how prestin's expansion-contraction cycle maps onto the inward-facing (IF) and outward-facing (OF) states of transporters, and whether prestin possesses the anion binding sites and conformational connectivity required for transport or if it operates via a distinct mechanism.

Methodology
The study employs a multi-pronged approach combining multi-microsecond all-atom molecular dynamics (MD) simulations, cryo-electron microscopy (cryo-EM), and electrophysiology:

Molecular Dynamics Simulations: The authors performed equilibrium MD simulations starting from a compact gerbil prestin structure (PDB:7SUN) and a human prestin structure (PDB:7LGU). Simulations were conducted on the Anton 2 supercomputer and using GROMACS, totaling several microseconds of sampling time. Order parameters ( $\zeta$ for translational elevator movement and $\phi$ for rotational movement) were defined relative to pendrin IF and OF reference structures to quantify conformational states. Cross-sectional area profiles were calculated using a stratified 2D Voronoi tessellation method.
Cryo-EM Structure Determination: To validate simulation predictions, the authors determined a new cryo-EM structure of gerbil prestin in the presence of thiocyanate (SCN⁻) anions at 3.27 Å resolution.
Electrophysiology: Non-linear capacitance (NLC) measurements were performed on pendrin expressed in CHO cells to confirm its voltage-dependent conformational switching, comparing its behavior in chloride versus sulfate solutions.
Comparative Analysis: Structural and dynamic data from prestin were directly compared with available experimental structures and MD simulations of pendrin (IF and OF states).

Key Contributions and Results

Spontaneous Conformational Transitions: MD simulations initiated from the compact prestin state revealed rapid, spontaneous transitions to an expanded state on the sub-microsecond to microsecond timescale. This expanded state structurally resembles the inward-facing (IF) conformation of pendrin.
Identification of a New Contracted State: The simulations identified a previously uncharacterized contracted conformation, termed "compact-2," which structurally resembles the outward-facing (OF) conformation of pendrin.
Localization of Area Expansion: The transition from contracted to expanded states involves a significant increase in the protein's cross-sectional area (~150 Å² per protomer, ~8.8% total). This expansion is localized primarily to the inner leaflet of the membrane, driven by the movement of specific transmembrane helices (TM1, 2, 4, 9, 11). This finding aligns with the "membrane bending model" of prestin function.
Extracellular Anion Binding Site: Simulations predicted an extracellular (EC) anion binding site in prestin near residues Lys227 and Arg236. This prediction was confirmed by the new cryo-EM structure (PDB:9YAY) in the presence of SCN⁻, which showed density for an anion at this location. This site is structurally conserved with the EC site in pendrin.
Divergent Ion Accessibility: A critical distinction was found between prestin and pendrin regarding ion pathways. In pendrin, the EC and intracellular (IC) anion binding sites are connected by a solvent-accessible pathway in the OF state, facilitating the alternating access transport cycle. In contrast, in all prestin conformations (expanded, compact, and compact-2), the EC and IC sites remain disconnected; no solvent pathway exists between them. This suggests prestin does not facilitate ion transport across the membrane in the same manner as pendrin.
Pendrin Exhibits NLC: Electrophysiological recordings confirmed that pendrin exhibits non-linear capacitance (NLC), indicating voltage-dependent conformational switching similar to prestin, though with a more hyperpolarized voltage dependence ( $V_h \approx -240$ mV).

Significance
The paper concludes that prestin and pendrin share core structural and functional properties, specifically the elevator-like movement of the core domain relative to the gate domain and the existence of conserved anion binding sites. The study suggests that prestin's expansion-contraction cycle parallels a partial transition of the pendrin transporter cycle (specifically the transition between OF-like and IF-like states).

However, the authors emphasize key differences that define prestin's unique role as a motor rather than a transporter:

Lack of Transport Pathway: Unlike pendrin, prestin lacks a continuous solvent pathway connecting the extracellular and intracellular anion sites, preventing the ion leak cycles associated with transport.
Intrinsic Speed: Prestin exhibits rapid, spontaneous conformational fluctuations on the sub-microsecond timescale, a property not observed in the simulated pendrin trajectories. This intrinsic structural lability may underpin prestin's ability to operate at ultrasonic frequencies required for mammalian hearing.
Mechanism of Force Generation: The localization of area expansion to the inner membrane leaflet supports the hypothesis that prestin generates force through asymmetric membrane distortion, coupling membrane potential changes directly to mechanical work without requiring net ion transport.

The findings provide a structural framework linking prestin's electromechanical function to the broader SLC26 family while delineating the specific adaptations that allow prestin to function as a high-speed molecular motor.

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