Folding of prestin’s anion-binding site and the mechanism of outer hair cell electromotility

Author:

Lin Xiaoxuan12ORCID,Haller Patrick R12,Bavi Navid12,Faruk Nabil2,Perozo Eduardo1234,Sosnick Tobin R1245ORCID

Affiliation:

1. Center for Mechanical Excitability, The University of Chicago

2. Department of Biochemistry and Molecular Biology, The University of Chicago

3. Institute for Neuroscience, The University of Chicago

4. Institute for Biophysical Dynamics, The University of Chicago

5. Prizker School for Molecular Engineering, The University of Chicago

Abstract

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. This event shortens the TM3–TM10 electrostatic gap, thereby connecting the two helices, resulting in reduced cross-sectional area. These folding events upon anion binding are absent in SLC26A9, a non-electromotile transporter closely related to prestin. Dynamics of prestin embedded in a lipid bilayer closely match that in detergent micelle, except for a destabilized lipid-facing helix TM6 that is critical to prestin’s mechanical expansion. We observe helix fraying at prestin’s anion-binding site but cooperative unfolding of multiple lipid-facing helices, features that may promote prestin’s fast electromechanical rearrangements. These results highlight a novel role of the folding equilibrium of the anion-binding site, and help define prestin’s unique voltage-sensing mechanism and electromotility.

Funder

National Science Foundation

National Institute of General Medical Sciences

National Institute on Deafness and Other Communication Disorders

Publisher

eLife Sciences Publications, Ltd

Subject

General Immunology and Microbiology,General Biochemistry, Genetics and Molecular Biology,General Medicine,General Neuroscience

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