An Electrostatic Switch for Gating the Electromechanical Activity of SLC26A5 (prestin)

Abstract

SLC26 comprises a family of multifunctional anion transporters that mediate passive or coupled transport of small anions. SLC26A5 (prestin) stands out by functioning as a voltage-driven mechanical actuator in sensory outer hair cells. The quasi-piezoelectric function of prestin depends on binding of intracellular anions, suggesting that the underlying dynamics may be derived from an ancestral anion transport mechanism. We previously identified the structural architecture of prestin as a 7 transmembrane domain inverted repeat architecture, a layout shared with SLC4 (e.g., AE1) and SLC23 transporters. To address the molecular mode of action of anions in electromechanical function, we conducted a glutamate scan of the putative anion binding site. While introduction of glutamate at most positions probed was disruptive for protein function, glutamate at a central position (S396E) was tolerated. Moreover, S396E rendered prestin anion-independent and insensitive to competitive anionic blockers. We conclude that S396E reveals location and coordination of substrate anions. Anion binding may enable the voltage-dependent conformational transition by perturbing a local hydrogen or electrostatic network. To gain insights into the conformational dynamics underlying electromechanical activity we performed cysteine accessibility scanning mutagenesis of transmembrane segments 3 and 10, which mainly form the binding site as defined by glutamate substitution. Accessibility was essentially restricted to intracellular reagents. However, a position within TM3 near the substrate binding site was accessible both from the inside and from the outside - contrary to the crystal structure of the bacterial homolog SLC26Dg predicting inaccessibility to extracellular solutes in the inside-open conformation. Two-sided accessibility thus indicates movement of the rigid TM bundle comprising TMs 3 and 10 towards the extracellular side, possibly in an elevator-like movement. We speculate that this partial movement constitutes the conformational transition underlying prestin's electromechanical activity.