Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

The conformational cycle of prestin underlies outer-hair cell electromotility

Abstract

The voltage-dependent motor protein prestin (also known as SLC26A5) is responsible for the electromotive behaviour of outer-hair cells and underlies the cochlear amplifier1. Knockout or impairment of prestin causes severe hearing loss2,3,4,5. Despite the key role of prestin in hearing, the mechanism by which mammalian prestin senses voltage and transduces it into cellular-scale movements (electromotility) is poorly understood. Here we determined the structure of dolphin prestin in six distinct states using single-particle cryo-electron microscopy. Our structural and functional data suggest that prestin adopts a unique and complex set of states, tunable by the identity of bound anions (Cl or SO42−). Salicylate, a drug that can cause reversible hearing loss, competes for the anion-binding site of prestin, and inhibits its function by immobilizing prestin in a new conformation. Our data suggest that the bound anion together with its coordinating charged residues and helical dipole act as a dynamic voltage sensor. An analysis of all of the anion-dependent conformations reveals how structural rearrangements in the voltage sensor are coupled to conformational transitions at the protein–membrane interface, suggesting a previously undescribed mechanism of area expansion. Visualization of the electromotility cycle of prestin distinguishes the protein from the closely related SLC26 anion transporters, highlighting the basis for evolutionary specialization of the mammalian cochlear amplifier at a high resolution.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Fig. 1: The structure and function of dolphin prestin homodimer in Cl.
Fig. 2: SO42− drives prestin towards the down and intermediate states at zero membrane potential.
Fig. 3: The structural basis of prestin inhibition by salicylate, and the evolutionary origins of electromotility.
Fig. 4: The structural basis of prestin’s voltage sensitivity and somatic electromotility.

Data availability

The atomic structure coordinates have been deposited at the RCSB PDB under accession numbers 7S8X, 7S9A, 7S9B, 7S9C, 7S9D and 7S9E; and the EM maps have been deposited in the Electron Microscopy Data Bank under accession numbers EMD-24928, EMD-24930, EMD-24931, EMD-24932, EMD-24933 and EMD-24934. All materials generated during the current study are available from the corresponding author under a materials transfer agreement with The University of Chicago.

References

  1. Zheng, J. et al. Prestin is the motor protein of cochlear outer hair cells. Nature 405, 149–155 (2000).

    ADS  CAS  PubMed  Google Scholar 

  2. Liu, X. Z. et al. Prestin, a cochlear motor protein, is defective in non-syndromic hearing loss. Hum. Mol. Genet. 12, 1155–1162 (2003).

    CAS  PubMed  Google Scholar 

  3. Liberman, M. C. et al. Prestin is required for electromotility of the outer hair cell and for the cochlear amplifier. Nature 419, 300–304 (2002).

    ADS  CAS  PubMed  Google Scholar 

  4. Dallos, P. et al. Prestin-based outer hair cell motility is necessary for mammalian cochlear amplification. Neuron 58, 333–339 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Cheatham, M., Huynh, K., Gao, J., Zuo, J. & Dallos, P. Cochlear function in Prestin knockout mice. J. Physiol. 560, 821–830 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Masterton, B., Heffner, H. & Ravizza, R. The evolution of human hearing. J. Acoust. Soc. Am. 45, 966–985 (1969).

    ADS  CAS  PubMed  Google Scholar 

  7. Heffner, H. & Masterton, B. Hearing in glires: domestic rabbit, cotton rat, feral house mouse, and kangaroo rat. J. Acoust. Soc. Am. 68, 1584–1599 (1980).

    ADS  Google Scholar 

  8. Fettiplace, R. Diverse mechanisms of sound frequency discrimination in the vertebrate cochlea. Trends Neurosci. 43, 88–102 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Ashmore, J. A fast motile response in guinea‐pig outer hair cells: the cellular basis of the cochlear amplifier. J. Physiol. 388, 323–347 (1987).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Brownell, W. E., Bader, C. R., Bertrand, D. & De Ribaupierre, Y. Evoked mechanical responses of isolated cochlear outer hair cells. Science 227, 194–196 (1985).

    ADS  CAS  PubMed  Google Scholar 

  11. Kachar, B., Brownell, W. E., Altschuler, R. & Fex, J. Electrokinetic shape changes of cochlear outer hair cells. Nature 322, 365–368 (1986).

    ADS  CAS  PubMed  Google Scholar 

  12. He, D. Z. et al. Changes in plasma membrane structure and electromotile properties in prestin deficient outer hair cells. Cytoskeleton 67, 43–55 (2010).

    CAS  PubMed  Google Scholar 

  13. Gorbunov, D. et al. Molecular architecture and the structural basis for anion interaction in prestin and SLC26 transporters. Nat. Commun. 5, 3622 (2014).

    ADS  PubMed  Google Scholar 

  14. Hallworth, R. & Nichols, M. G. Prestin in HEK cells is an obligate tetramer. J. Neurophysiol. 107, 5–11 (2012).

    CAS  PubMed  Google Scholar 

  15. Zheng, J. et al. Analysis of the oligomeric structure of the motor protein prestin. J. Biol. Chem. 281, 19916–19924 (2006).

    CAS  PubMed  Google Scholar 

  16. Navaratnam, D., Bai, J.-P., Samaranayake, H. & Santos-Sacchi, J. N-terminal-mediated homomultimerization of prestin, the outer hair cell motor protein. Biophys. J. 89, 3345–3352 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. He, D. Z., Lovas, S., Ai, Y., Li, Y. & Beisel, K. W. Prestin at year 14: progress and prospect. Hear. Res. 311, 25–35 (2014).

    CAS  PubMed  Google Scholar 

  18. Liu, Z., Qi, F.-Y., Xu, D.-M., Zhou, X. & Shi, P. Genomic and functional evidence reveals molecular insights into the origin of echolocation in whales. Sci. Adv.4, eaat8821 (2018).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  19. Walter, J. D., Sawicka, M. & Dutzler, R. Cryo-EM structures and functional characterization of murine Slc26a9 reveal mechanism of uncoupled chloride transport. eLife 8, e46986 (2019).

    PubMed  PubMed Central  Google Scholar 

  20. Chi, X. et al. Structural insights into the gating mechanism of human SLC26A9 mediated by its C-terminal sequence. Cell Discov. 6, 55 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Rybalchenko, V. & Santos-Sacchi, J. Anion control of voltage sensing by the motor protein prestin in outer hair cells. Biophys. J. 95, 4439–4447 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Tan, X. et al. From zebrafish to mammal: functional evolution of prestin, the motor protein of cochlear outer hair cells. J. Neurophysiol. 105, 36–44 (2011).

    PubMed  Google Scholar 

  23. Santos-Sacchi, J., Song, L., Zheng, J. & Nuttall, A. L. Control of mammalian cochlear amplification by chloride anions. J. Neurosci. 26, 3992–3998 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Rybalchenko, V. & Santos‐Sacchi, J. Cl flux through a non‐selective, stretch‐sensitive conductance influences the outer hair cell motor of the guinea‐pig. J. Physiol. 547, 873–891 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Homma, K., Duan, C., Zheng, J., Cheatham, M. A. & Dallos, P. The V499G/Y501H mutation impairs fast motor kinetics of prestin and has significance for defining functional independence of individual prestin subunits. J. Biol. Chem. 288, 2452–2463 (2013).

    CAS  PubMed  Google Scholar 

  26. Homma, K. & Dallos, P. Evidence that prestin has at least two voltage-dependent steps. J. Biol. Chem. 286, 2297–2307 (2011).

    CAS  PubMed  Google Scholar 

  27. Iwasa, K. A two-state piezoelectric model for outer hair cell motility. Biophys. J. 81, 2495–2506 (2001).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  28. Ludwig, J. et al. Reciprocal electromechanical properties of rat prestin: the motor molecule from rat outer hair cells. Proc. Natl Acad. Sci. USA 98, 4178–4183 (2001).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  29. Cazals, Y. Auditory sensori-neural alterations induced by salicylate. Prog. Neurobiol. 62, 583–631 (2000).

    CAS  PubMed  Google Scholar 

  30. Chen, G.-D. et al. Salicylate-induced cochlear impairments, cortical hyperactivity and re-tuning, and tinnitus. Hear. Res. 295, 100–113 (2013).

    CAS  PubMed  Google Scholar 

  31. Oliver, D. et al. Intracellular anions as the voltage sensor of prestin, the outer hair cell motor protein. Science 292, 2340–2343 (2001).

    CAS  PubMed  Google Scholar 

  32. Kakehata, S. & Santos-Sacchi, J. Effects of salicylate and lanthanides on outer hair cell motility and associated gating charge. J. Neurosci. 16, 4881–4889 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Schaechinger, T. J. & Oliver, D. Nonmammalian orthologs of prestin (SLC26A5) are electrogenic divalent/chloride anion exchangers. Proc. Natl Acad. Sci. USA 104, 7693–7698 (2007).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  34. Song, L. & Santos-Sacchi, J. Conformational state-dependent anion binding in prestin: evidence for allosteric modulation. Biophys. J. 98, 371–376 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Bai, J.-P. et al. Current carried by the Slc26 family member prestin does not flow through the transporter pathway. Sci. Rep. 7, 46619 (2017).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  36. Bai, J.-P. et al. Prestin’s anion transport and voltage-sensing capabilities are independent. Biophys. J. 96, 3179–3186 (2009).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  37. Jogini, V. & Roux, B. Dynamics of the Kv1.2 voltage-gated K+ channel in a membrane environment. Biophys. J. 93, 3070–3082 (2007).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  38. Starace, D. M. & Bezanilla, F. A proton pore in a potassium channel voltage sensor reveals a focused electric field. Nature 427, 548–553 (2004).

    ADS  CAS  PubMed  Google Scholar 

  39. Bezanilla, F. How membrane proteins sense voltage. Nat. Rev. Mol. Cell Biol. 9, 323–332 (2008).

    CAS  PubMed  Google Scholar 

  40. Dong, X.-X., Ehrenstein, D. & Iwasa, K. Fluctuation of motor charge in the lateral membrane of the cochlear outer hair cell. Biophys. J. 79, 1876–1882 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Dong, X.-X. & Iwasa, K. Tension sensitivity of prestin: comparison with the membrane motor in outer hair cells. Biophys. J. 86, 1201–1208 (2004).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  42. Beurg, M., Tan, X. & Fettiplace, R. A prestin motor in chicken auditory hair cells: active force generation in a nonmammalian species. Neuron 79, 69–81 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Dallos, P. & He, D. Z. Two models of outer hair cell stiffness and motility. J. Assoc. Res. Otolaryngol. 1, 283–291 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Izumi, C., Bird, J. E. & Iwasa, K. H. Membrane thickness sensitivity of prestin orthologs: the evolution of a piezoelectric protein. Biophys. J. 100, 2614–2622 (2011).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  45. Fang, J., Izumi, C. & Iwasa, K. H. Sensitivity of prestin-based membrane motor to membrane thickness. Biophys. J. 98, 2831–2838 (2010).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  46. Santos‐Sacchi, J., Shen, W., Zheng, J. & Dallos, P. Effects of membrane potential and tension on prestin, the outer hair cell lateral membrane motor protein. J. Physiol. 531, 661–666 (2001).

    PubMed  PubMed Central  Google Scholar 

  47. Kakehata, S. & Santos-Sacchi, J. Membrane tension directly shifts voltage dependence of outer hair cell motility and associated gating charge. Biophys. J. 68, 2190–2197 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Ashmore, J. Cochlear outer hair cell motility. Physiol. Rev. 88, 173–210 (2008).

    CAS  PubMed  Google Scholar 

  49. He, D. Z., Evans, B. N. & Dallos, P. First appearance and development of electromotility in neonatal gerbil outer hair cells. Hear. Res. 78, 77–90 (1994).

    CAS  PubMed  Google Scholar 

  50. Dallos, P. The active cochlea. J. Neurosci. 12, 4575–4585 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Santos-Sacchi, J. Asymmetry in voltage-dependent movements of isolated outer hair cells from the organ of Corti. J. Neurosci. 9, 2954–2962 (1989).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Santos-Sacchi, J. & Dilger, J. Whole cell currents and mechanical responses of isolated outer hair cells. Hear. Res. 35, 143–150 (1988).

    CAS  PubMed  Google Scholar 

  53. Ge, J. et al. Molecular mechanism of prestin electromotive signal amplification. Cell 184, 4669–4679 (2021).

    CAS  PubMed  Google Scholar 

  54. Butan, C. et al. Single particle cryo-EM structure of the outer hair cell motor protein prestin. Preprint at bioRxiv https://doi.org/10.1101/2021.08.03.454998 (2021).

  55. Santos-Sacchi, J. On the frequency limit and phase of outer hair cell motility: effects of the membrane filter. J. Neurosci. 12, 1906–1916 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Holley, M. & Ashmore, J. F. On the mechanism of a high-frequency force generator in outer hair cells isolated from the guinea pig cochlea. Proc. R. Soc. Lond. B Biol. Sci. 232, 413–429 (1988).

    ADS  CAS  PubMed  Google Scholar 

  57. Kirchhofer, A. et al. Modulation of protein properties in living cells using nanobodies. Nat. Struct. Mol. Biol. 17, 133–138 (2010).

    CAS  PubMed  Google Scholar 

  58. Clark, M. D., Contreras, G. F., Shen, R. & Perozo, E. Electromechanical coupling in the hyperpolarization-activated K+ channel KAT1. Nature 583, 145–149 (2020).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  59. Scheres, S. H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).

    PubMed  PubMed Central  Google Scholar 

  62. Wagner, T. et al. SPHIRE-crYOLO is a fast and accurate fully automated particle picker for cryo-EM. Commun. Biol. 2, 218 (2019).

    PubMed  PubMed Central  Google Scholar 

  63. Scheres, S. H. & Chen, S. Prevention of overfitting in cryo-EM structure determination. Nat. Methods 9, 853–854 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. Rosenthal, P. B. & Henderson, R. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721–745 (2003).

    CAS  PubMed  Google Scholar 

  65. Kucukelbir, A., Sigworth, F. J. & Tagare, H. D. Quantifying the local resolution of cryo-EM density maps. Nat. Methods 11, 63–65 (2014).

    CAS  PubMed  Google Scholar 

  66. Biasini, M. et al. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res. 42, W252–W258 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. Stein, N. CHAINSAW: a program for mutating pdb files used as templates in molecular replacement. J. Appl. Crystallogr. 41, 641–643 (2008).

    CAS  Google Scholar 

  68. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).

    PubMed  Google Scholar 

  69. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  70. Brown, A. et al. Tools for macromolecular model building and refinement into electron cryo-microscopy reconstructions. Acta Crystallogr. D 71, 136–153 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  71. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  72. Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. D 74, 531–544 (2018).

    CAS  Google Scholar 

  73. Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    CAS  PubMed  Google Scholar 

  74. Goddard, T. D. et al. UCSF ChimeraX: meeting modern challenges in visualization and analysis. Protein Sci. 27, 14–25 (2018).

    CAS  PubMed  Google Scholar 

  75. Pettersen, E. F. et al. UCSF ChimeraX: Structure visualization for researchers, educators, and developers. Protein Sci. 30, 70–82 (2021).

    CAS  PubMed  Google Scholar 

  76. Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J. Mol. Graph. 14, 33–38 (1996).

    CAS  PubMed  Google Scholar 

  77. Lindau, M. & Neher, E. Patch-clamp techniques for time-resolved capacitance measurements in single cells. Pflügers Arch. 411, 137–146 (1988).

    CAS  PubMed  Google Scholar 

  78. Camacho, C. et al. BLAST+: architecture and applications. BMC Bioinform. 10, 421 (2009).

    Google Scholar 

  79. Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  80. Stamatakis, A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30, 1312–1313 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  81. Letunic, I. & Bork, P. Interactive Tree Of Life (iTOL) v4: recent updates and new developments. Nucleic Acids Res. 47, W256–W259 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  82. Sievers, F. et al. Fast, scalable generation of high‐quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7, 539 (2011).

    PubMed  PubMed Central  Google Scholar 

  83. Lomize, M. A., Pogozheva, I. D., Joo, H., Mosberg, H. I. & Lomize, A. L. OPM database and PPM web server: resources for positioning of proteins in membranes. Nucleic Acids Res. 40, D370–D376 (2012).

    CAS  PubMed  Google Scholar 

  84. Morozenko, A. & Stuchebrukhov, A. Dowser++, a new method of hydrating protein structures. Proteins Struct. Funct. Bioinform. 84, 1347–1357 (2016).

    CAS  Google Scholar 

  85. Shaw, D. E. et al. Anton, a special-purpose machine for molecular dynamics simulation. Commun. ACM 51, 91–97 (2008).

    Google Scholar 

  86. Phillips, J. C. et al. Scalable molecular dynamics with NAMD. J. Comput. Chem. 26, 1781–1802 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  87. Jo, S., Kim, T., Iyer, V. G. & Im, W. CHARMM‐GUI: a web‐based graphical user interface for CHARMM. J. Comput. Chem. 29, 1859–1865 (2008).

    CAS  PubMed  Google Scholar 

  88. MacKerell, Jr A. D., Feig, M. & Brooks, C. L. Improved treatment of the protein backbone in empirical force fields. JACS 126, 698–699 (2004).

    CAS  Google Scholar 

  89. MacKerell, Jr A. D. et al. All-atom empirical potential for molecular modeling and dynamics studies of proteins. J. Phys. Chem. B 102, 3586–3616 (1998).

    CAS  PubMed  Google Scholar 

  90. Jorgensen, W. L., Chandrasekhar, J., Madura, J. D., Impey, R. W. & Klein, M. L. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 79, 926–935 (1983).

    ADS  CAS  Google Scholar 

  91. Huang, L. & Roux, B. Automated force field parameterization for nonpolarizable and polarizable atomic models based on ab initio target data. J. Chem. Theory Comput. 9, 3543–3556 (2013).

    CAS  Google Scholar 

  92. Feller, S. E., Zhang, Y., Pastor, R. W. & Brooks, B. R. Constant pressure molecular dynamics simulation: the Langevin piston method. J. Chem. Phys. 103, 4613–4621 (1995).

    ADS  CAS  Google Scholar 

  93. Martyna, G. J., Tobias, D. J. & Klein, M. L. Constant pressure molecular dynamics algorithms. J. Chem. Phys. 101, 4177–4189 (1994).

    ADS  CAS  Google Scholar 

  94. Essmann, U. et al. A smooth particle mesh Ewald method. J. Chem. Phys. 103, 8577–8593 (1995).

    ADS  CAS  Google Scholar 

  95. Martyna, G. J., Klein, M. L. & Tuckerman, M. Nosé–Hoover chains: the canonical ensemble via continuous dynamics. J. Chem. Phys. 97, 2635–2643 (1992).

    ADS  Google Scholar 

  96. Shan, Y., Klepeis, J. L., Eastwood, M. P., Dror, R. O. & Shaw, D. E. Gaussian split Ewald: a fast Ewald mesh method for molecular simulation. J. Chem. Phys. 122, 054101 (2005).

    ADS  Google Scholar 

  97. Aksimentiev, A. & Schulten, K. Imaging α-hemolysin with molecular dynamics: ionic conductance, osmotic permeability, and the electrostatic potential map. Biophys. J. 88, 3745–3761 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  98. Roux, B. The membrane potential and its representation by a constant electric field in computer simulations. Biophys. J. 95, 4205–4216 (2008).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  99. Castillo, J. P. et al. Mechanism of potassium ion uptake by the Na+/K+-ATPase. Nat. Commun. 6, 7622 (2015).

    ADS  CAS  PubMed  Google Scholar 

  100. Khalili-Araghi, F. et al. Calculation of the gating charge for the Kv1. 2 voltage-activated potassium channel. Biophys. J. 98, 2189–2198 (2010).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  101. Morin, A. et al. Cutting edge: Collaboration gets the most out of software. eLife 2, e01456 (2013).

    PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank M. Zhao for discussions and assistance with imaging; K. Poole, C. Cox, T. Ngo, O. Bavi, R. Hulse, C. Bassetto, Y. Nikolaev, Y. Krishnan, P. Bezanilla, H. Mchaourab, P. Gueorguieva, S. Zhong, M. Karami, P. Haller and F. Galan, and the members of the Perozo laboratory for exchanging ideas and comments on the manuscript; P. Shi for sharing the Tursiops prestin plasmid; J. Fuller, J. Austin II and T. Lavoie at the University of Chicago Advanced Electron Microscopy Facility for microscope maintenance and training; and U. Baxa and T. J. Edwards at NCEF for cryo-EM data collection. Anton 2 computer time was provided by the Pittsburgh Supercomputing Center (PSC) through grant R01GM116961 from the National Institutes of Health. The Anton 2 machine at PSC was made available by D.E. Shaw Research. N.B. acknowledges the Biology of Inner Ear course (BIE2019) and Gordon Conference (Auditory System Gordon Research Conference) for inspiring him to study hearing and prestin. This work was supported by NIDCD grant R01 DC019833 to E.P. N.B. was the recipient of a Chicago Fellowship. M.D.C. was supported by F30MH116647 and T32GM007281. This research was in part supported by the National Cancer Institute’s National Cryo-EM Facility at the Frederick National Laboratory for Cancer Research under contract HSSN261200800001E.

Author information

Authors and Affiliations

Authors

Contributions

N.B., M.D.C. and E.P. conceived the project. N.B. expressed and purified the protein. N.B. and M.D.C. prepared cryo-grids. N.B. and B.G.R. performed EM data collection. N.B., B.G.R. and M.D.C. processed the cryo-EM data and built and refined the atomic models. N.B. and G.F.C. performed and analysed the electrophysiological experiments. R.S. carried out MD simulations and electrostatic calculations. N.B. and W.M. carried out molecular cloning, mutagenesis and created all of the expression constructs. N.B. and W.M. managed cell culture. All of the authors analysed the data. N.B. and E.P. wrote the manuscript with input from all of the other authors.

Corresponding author

Correspondence to Eduardo Perozo.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature thanks Rachelle Gaudet and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Function, biochemistry and structural features of electromotile prestin.

a, Electromotility analysis of HEK293 cells transfected with wild-type dolphin prestin compared to GFP-only transfected cell (Mock-GFP). The cellular displacement was normalized based on the cell largest diameter, d0 (Fig. 1b). The normalized electromotility was 0.05±02 (n = 6) versus 0.008±0.002 (n = 5) for wild-type prestin and Mock-GFP, respectively. These values were measured at the depolarizing voltage step changing from +120 mV to −120 mV (mean ± SD, is the number of independent cells. One-sided Student t-test, unpaired, P=0.005). b, Size-exclusion chromatography (SEC) curves of the full-length dolphin prestin purified in GDN, run on a Superose 6 column, in high Cl (red) and SO42− (blue) based solution. The fractions indicated by black dotted lines in both represent purified proteins that were used for cryo-EM imaging. c, Purified dolphin prestin cryo-EM samples, run on a Stain-free SDS-PAGE gel, indicating size of ~80 kDa for the full-length prestin monomer (representation of n = 3).d, Topology of dolphin prestin. Different domains are indicated by color; the gate domain is colored in blue, the core domain in red and the C- and N-termini as well as the STAS domain in grey. The transmembrane helices are numbered from 1 to 14. The N- and C-termini as well as the STAS domain are oriented towards the cytoplasm.

Extended Data Fig. 2 Flow chart for the cryo-EM data processing and structure determination of the dolphin prestin in high Cl condition.

a, The final reconstruction has a nominal resolution of 3.3 Å (at FSC=0.143). The yellow scale bar on the micrograph represents 200 Å. All the images in this figure were created in UCSF ChimeraX.

Extended Data Fig. 3 Structure of prestin in high Cl and comparison with the Intermediate.

a, Comparison between prestin (high Cl) (blue and Red) and SLC26A9 Intermediate state (6RTF, grey). The structures are aligned based on residues 460 to 505 of one subunit (TM13-TM14, dotted box). ChimeraX was used for illustration. b, Electrostatic potential and surface charge distribution of SLC26A9 intermediate state19 compared with that of prestin in high Cl panel c. The electrostatic charge distribution ranges from −5 to 5 kT from negative to positive charge. ChimeraX was used for illustration.

Extended Data Fig. 4 Flow chart for the cryo-EM data processing and structure determination of the dolphin prestin in SO42−.

a, b Cryo-EM data processing and structure determination of the dolphin prestin in Down I (SO42−) and Down II (SO42−) states. A was obtained from Dataset I, which was combined with Class B from Dataset II. The final reconstruction yielded two structures, Down I (SO42−) and Down II (SO42−), which have nominal resolutions of 4.2 and 6.7 Å, respectively (at FSC=0.143). See Supplementary Figure 5 for the steps on how Class A and B were further processed. Evidence of both states was found in dataset II, however merging of datasets was required to improve resolution of states. c, Flow chart for the cryo-EM data processing and structure determination of the dolphin prestin in the Intermediate state (SO42−) (See Methods for details). The final reconstruction has a nominal resolution of 4.6 Å (at FSC=0.143). UCSF ChimeraX was for illustration of all the structures. The yellow scale bar on all the micrographs represents 200 Å.

Extended Data Fig. 5 Prestin’s cross-sectional area changes upon transition from Down to Up states.

a, Upon the transition from Down to Up state and the movement of the anion-binding site, the most obvious changes are seen in the peripheral helices TM5b, TM6-TM7, and TM8. b, MD simulation of prestin in Up state is compared with the Inhibited II state (Cl and Salicylate) equilibrated in POPC lipid bilayers. The cross-sectional area of outer and inner monolayers with mapped leaflet coordinate in the Z direction (across the membrane thickness) using all-atom molecular dynamics simulations (1µs). Δz shows movement of the phosphate group of the lipids in the Z (thickness) direction. The comparison was made between Up (Cl) and Inhibited II (SO42−) states. The largest difference was observed at the location of the TM6 helix. c, Cross-sectional area calculations of the transmembrane domain of SLC26A9(12) along the hydrophobic thickness using CHARMM-membrane builder. Cross-sectional area change of SLC26A9 from Inward-facing to Intermediate states (6RTC and 6RTF) per monomer19. Note that prior to area calculation, the spatial arrangements of all the structures with respect to the hydrocarbon core of the lipid bilayer were first adjusted using the PPM server(30). The structures were aligned based on residues 460 to 505 (TM13-TM14). d, Comparison of the change in the micelle morphology between two salicylate-inhibited structures Inhibited I (Cl) and Inhibited II (SO42− + Salicylate) states. The overlay of the two states shows drastic changes in the micelle thickness especially around TM6 region in addition to the overall changes in the micelle in-lane direction, both indicative of major structural rearrangements between the two states. ChimeraX was used for illustration.

Extended Data Fig. 6 Salicylate outcompetes SO42− in binding to anion-binding pocket.

a, The NLC measurements of HEK293T cells transfected with dolphin prestin in SO42− (0.15±0.06; n = 6). The NLC of these cells were completely abrogated (0.01±0.01) by 10 mM Na-Salicylate (mean ± s.e.m.; n, is the number of independent cells. One-sided student’s t-test, unpaired, P=0.01) b, Density of Salicylate (orange) in the anion-binding site (blue) was resolved in the Inhibited II (SO42−) state of dolphin prestin. c, Sequence alignment of prestin and close SLC transporters across different species. Residues forming the anion-binding site are largely conserved (e.g. Q97, F101, F137). Putative voltage-sensing residue R399 in dolphin prestin is replaced by a valine in murine SLC26A9. Clustal Omega was used for the sequence alignments. ChimeraX was used for illustration.

Extended Data Fig. 7 Flow chart for the cryo-EM data processing and structure determination of the dolphin prestin in the Salicylate-Inhibited states.

Flow chart of the dolphin prestin in the a, Inhibited I state (Cl + Salicylate) and b, (SO42− + Salicylate) The final reconstructions have a nominal resolution of 3.8 Å and 3.7 Å, respectively (at FSC=0.143). All the images in this figure were created in UCSF ChimeraX. The yellow scale bar on all the micrographs represents 200 Å.

Extended Data Fig. 8 Electrostatic calculations and charge transfer of prestin across the membrane.

a, Mutation of the key residues in the anion binding pocket either completely abolishes the NLC (R399Q) or right shifts the V1/2 by more than 80 mV (F101Y) to around +25 ± 5 mV (mean ± s.e.m.; n, is the number of independent cells. One-sided Student t-test, P=0.001); a similar effect has been observed in other prestin homologues using patch-clamp electrophysiology (51). b, Snapshots from the MD trajectories of the systems, and calculation of the electrostatic potential across the membrane at two states, the Down I state (with SO42− in the left cavity, and without SO42− in the right cavity) versus Up (with Cl in the left cavity and without any Cl in the right cavity). The x-z plane is crossing the two central anion-binding sites. In both models, the positive field is mainly focused around the transmembrane mid-plane and around the anion-binding site, creating an attractive (blue) field for the binding of the anion. However, in the Up state the field is more positive around the mid-plane compared to the corresponding region in the Intermediate state. In both cases, the presence of the anion only partially neutralizes (~35%) the positive field around the bilayer mid-plane. Note that the actual size of the simulation box is larger than what is illustrated here (see Methods). c, Averaged 1-D fraction of membrane potential in the z direction along the two central binding sites (shown as dashed blue lines in panel A with the central binding sites highlighted using the red cross symbols). The 1-D and 2-D maps were directly extracted from the ensemble averaged 3-D fraction of membrane potential map. The location of the phosphate atoms of the outer and inner lipid leaflets along the z axis was highlighted with dashed gray lines). d, Displacement of charge for prestin in the Up and Down I conformations at different transmembrane potentials. The gating charge between the two states is 0.38 +0.25 e calculated as the offset constant between the linear fits. (n = 3; data are mean ± SD; One-sided Student’s t-test; P=0.05). e, R399 in both monomers have been mutated to Q, S and E in different systems to see the contribution of R399 residue to the positive charge at the bilayer mid-plane using electrostatic calculations. R399 mutation to polar residues shows that R399 has almost ~40% contribution the positive charge of the field at the bilayer mid-plane. The remainder likely comes from the TM3-TM10 helical dipole and other positive charges in this area.

Extended Data Fig. 9 Whole cell patch-clamp electrophysiology of the mutations of different glycine residues along the TM6 helix.

All the individual data points, that has been averaged in Fig. 3f, has been presented here. Compared to wild-type prestin, mutation of evolutionary conserved glycine residues, a, G274 and G275 and b, G263, G265 and G270 largely affects the NLC.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics

Supplementary information

Supplementary Information

This file contains further supportive results for the findings in this study, including Supplementary Figs. 1–10 (which include the uncropped gel source data).

Reporting Summary

Peer Review File

Supplementary Video 1

Electromotility measurements of HEK293 cells transfected with dolphin prestin using whole-cell patch-clamp electrophysiology. To evoke prestin-mediated electromotility, the membrane potential was held at −70 mV; 10 mV increase-in-amplitude voltage steps were applied up to the final steps, which was from +150 mV to −140 mV (Fig. 1b). The magenta square indicates the area that was chosen in our custom-written code to track the cellular displacements.

Supplementary Video 2

Structural changes from the expanded (down I) to the compact (up) conformation as a linear interpolation. The side front and top views of the dimer have been shown in one single frame. The anion-binding site is highlighted in red and Arg399 is shown in stick representation and the backbone has been coloured yellow. The videos were made in UCSF ChimeraX.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Bavi, N., Clark, M.D., Contreras, G.F. et al. The conformational cycle of prestin underlies outer-hair cell electromotility. Nature 600, 553–558 (2021). https://doi.org/10.1038/s41586-021-04152-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41586-021-04152-4

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing