Generation of an electrochemical proton gradient is the first step of cell bioenergetics. In prokaryotes, the gradient is created by outward membrane protein proton pumps. Inward plasma membrane native proton pumps are yet unknown. We describe comprehensive functional studies of the representatives of the yet noncharacterized xenorhodopsins from Nanohaloarchaea family of microbial rhodopsins. They are inward proton pumps as we demonstrate in model membrane systems, Escherichia coli cells, human embryonic kidney cells, neuroblastoma cells, and rat hippocampal neuronal cells. We also solved the structure of a xenorhodopsin from the nanohalosarchaeon Nanosalina (NsXeR) and suggest a mechanism of inward proton pumping. We demonstrate that the NsXeR is a powerful pump, which is able to elicit action potentials in rat hippocampal neuronal cells up to their maximal intrinsic firing frequency. Hence, inwardly directed proton pumps are suitable for light-induced remote control of neurons, and they are an alternative to the well-known cation-selective channelrhodopsins.

Division for Structural Biochemistry Hannover Medical School Hannover Germany

European Synchrotron Radiation Facility 38027 Grenoble France

Evolutionary Genomics Group Departamento de Producción Vegetal y Microbiología Universidad Miguel Hernández San Juan de Alicante Alicante Spain

Friedrich Miescher Institute for Biomedical Research Basel Switzerland

Institut de Biologie Structurale Jean Pierre Ebel Université Grenoble Alpes Commissariat à l'Energie Atomique et aux Energies Alternatives CNRS Grenoble France

Institute for Biodiversity and Ecosystem Dynamics University of Amsterdam Amsterdam Netherlands

Institute for Biophysical Chemistry Hannover Medical School Hannover Germany

Institute of Complex Systems ICS 6 Structural Biochemistry Research Centre Jülich Jülich Germany

Institute of Crystallography RWTH Aachen University Aachen Germany

Institute of Hydrobiology Department of Aquatic Microbial Ecology Biology Centre of the Czech Academy of Sciences České Budějovice Czech Republic

Joint Institute for Nuclear Research Dubna Russia

Max Planck Institute of Biophysics Frankfurt am Main Germany

Moscow Institute of Physics and Technology Dolgoprudny Russia

See more in PubMed

Ernst O. P., Lodowski D. T., Elstner M., Hegemann P., Brown L. S., Kandori H., Microbial and animal rhodopsins: Structures, functions, and molecular mechanisms. Chem. Rev. 114, 126–163 (2014). PubMed PMC

Mitchell P., Chemiosmotic coupling in oxidative and photosynthetic phosphorylation. Biol. Rev. 41, 445–501 (1966). PubMed

Oesterhelt D., Stoeckenius W., Rhodopsin-like protein from the purple membrane of Halobacterium halobium. Nat. New Biol. 233, 149–152 (1971). PubMed

Inoue K., Ito S., Kato Y., Nomura Y., Shibata M., Uchihashi T., Tsunoda S. P., Kandori H., A natural light-driven inward proton pump. Nat. Commun. 7, 13415 (2016). PubMed PMC

Ugalde J. A., Podell S., Narasingarao P., Allen E. E., Xenorhodopsins, an enigmatic new class of microbial rhodopsins horizontally transferred between archaea and bacteria. Biol. Direct 6, 52 (2011). PubMed PMC

Vogeley L., Sineshchekov O. A., Trivedi V. D., Sasaki J., Spudich J. L., Luecke H., Anabaena sensory rhodopsin: A photochromic color sensor at 2.0 Å. Science 306, 1390–1393 (2004). PubMed PMC

Ghai R., Pašić L., Fernández A. B., Martin-Cuadrado A.-B., Mizuno C. M., McMahon K. D., Papke R. T., Stepanauskas R., Rodriguez-Brito B., Rohwer F., Sánchez-Porro C., Ventosa A., Rodríguez-Valera F., New abundant microbial groups in aquatic hypersaline environments. Sci. Rep. 1, 135 (2011). PubMed PMC

Vavourakis C. D., Ghai R., Rodriguez-Valera F., Sorokin D. Y., Tringe S. G., Hugenholtz P., Muyzer G., Metagenomic insights into the uncultured diversity and physiology of microbes in four hypersaline soda lake brines. Front. Microbiol. 7, 211 (2016). PubMed PMC

Nagel G., Szellas T., Huhn W., Kateriya S., Adeishvili N., Berthold P., Ollig D., Hegemann P., Bamberg E., Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. Proc. Natl. Acad. Sci. U.S.A. 100, 13940–13945 (2003). PubMed PMC

Huang K. S., Bayley H., Khorana H. G., Delipidation of bacteriorhodopsin and reconstitution with exogenous phospholipid. Proc. Natl. Acad. Sci. U.S.A. 77, 323–327 (1980). PubMed PMC

Racker E., Stoeckenius W., Reconstitution of purple membrane vesicles catalyzing light-driven proton uptake and adenosine triphosphate formation. J. Biol. Chem. 249, 662–663 (1974). PubMed

Moise A. R., Kuksa V., Imanishi Y., Palczewski K., Identification of all-trans-retinol:All-trans-13,14-dihydroretinol saturase. J. Biol. Chem. 279, 50230–50242 (2004). PubMed PMC

V. I. Gordeliy, R. Schlesinger, R. Efremov, G. Büldt, J. Heberle, in Membrane Protein Protocols, B. S. Selinsky, Ed. (Humana Press, 2003), vol. 228, pp. 305–316. PubMed

Luecke H., Schobert B., Richter H.-T., Cartailler J.-P., Lanyi J. K., Structure of bacteriorhodopsin at 1.55 Å resolution. J. Mol. Biol. 291, 899–911 (1999). PubMed

Gushchin I., Chervakov P., Kuzmichev P., Popov A. N., Round E., Borshchevskiy V., Ishchenko A., Petrovskaya L., Chupin V., Dolgikh D. A., Arseniev A. S., Kirpichnikov M., Gordeliy V., Structural insights into the proton pumping by unusual proteorhodopsin from nonmarine bacteria. Proc. Natl. Acad. Sci. U.S.A. 110, 12631–12636 (2013). PubMed PMC

Balashov S. P., Petrovskaya L. E., Lukashev E. P., Imasheva E. S., Dioumaev A. K., Wang J. M., Sychev S. V., Dolgikh D. A., Rubin A. B., Kirpichnikov M. P., Lanyi J. K., Aspartate–histidine interaction in the retinal schiff base counterion of the light-driven proton pump of Exiguobacterium sibiricum. Biochemistry 51, 5748–5762 (2012). PubMed PMC

Gradinaru V., Zhang F., Ramakrishnan C., Mattis J., Prakash R., Diester I., Goshen I., Thompson K. R., Deisseroth K., Molecular and cellular approaches for diversifying and extending optogenetics. Cell 141, 154–165 (2010). PubMed PMC

Kuzmich A. I., Vvedenskii A. V., Kopantzev E. P., Vinogradova T. V., Quantitative comparison of gene co-expression in a bicistronic vector harboring IRES or coding sequence of porcine teschovirus 2A peptide. Russ. J. Bioorg. Chem. 39, 406–416 (2013). PubMed

Shcherbo D., Merzlyak E. M., Chepurnykh T. V., Fradkov A. F., Ermakova G. V., Solovieva E. A., Lukyanov K. A., Bogdanova E. A., Zaraisky A. G., Lukyanov S., Chudakov D. M., Bright far-red fluorescent protein for whole-body imaging. Nat. Methods 4, 741–746 (2007). PubMed

Gunaydin L. A., Yizhar O., Berndt A., Sohal V. S., Deisseroth K., Hegemann P., Ultrafast optogenetic control. Nat. Neurosci. 13, 387–392 (2010). PubMed

Kleinlogel S., Terpitz U., Legrum B., Gökbuget D., Boyden E. S., Bamann C., Wood P. G., Bamberg E., A gene-fusion strategy for stoichiometric and co-localized expression of light-gated membrane proteins. Nat. Methods 8, 1083–1088 (2011). PubMed

Gushchin I., Shevchenko V., Polovinkin V., Kovalev K., Alekseev A., Round E., Borshchevskiy V., Balandin T., Popov A., Gensch T., Fahlke C., Bamann C., Willbold D., Büldt G., Bamberg E., Gordeliy V., Crystal structure of a light-driven sodium pump. Nat. Struct. Mol. Biol. 22, 390–395 (2015). PubMed

Studier F. W., Protein production by auto-induction in high-density shaking cultures. Protein Expr. Purif. 41, 207–234 (2005). PubMed

Ritchie T. K., Grinkova Y. V., Bayburt T. H., Denisov I. G., Zolnerciks J. K., Atkins W. M., Sligar S. G., Chapter 11–Reconstitution of membrane proteins in phospholipid bilayer nanodiscs. Methods Enzymol. 464, 211–231 (2009). PubMed PMC

Chizhov I., Chernavskii D. S., Engelhard M., Mueller K.-H., Zubov B. V., Hess B., Spectrally silent transitions in the bacteriorhodopsin photocycle. Biophys. J. 71, 2329–2345 (1996). PubMed PMC

Gordeliy V. I., Labahn J., Moukhametzianov R., Efremov R., Granzin J., Schlesinger R., Büldt G., Savopol T., Scheidig A. J., Klare J. P., Engelhard M., Molecular basis of transmembrane signalling by sensory rhodopsin II–transducer complex. Nature 419, 484–487 (2002). PubMed

Kabsch W., XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010). PubMed PMC

Winn M. D., Ballard C. C., Cowtan K. D., Dodson E. J., Emsley P., Evans P. R., Keegan R. M., Krissinel E. B., Leslie A. G. W., McCoy A., McNicholas S. J., Murshudov G. N., Pannu N. S., Potterton E. A., Powell H. R., Read R. J., Vagin A., Wilson K. S., Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011). PubMed PMC

Vagin A., Lebedev A., MoRDa, an automatic molecular replacement pipeline. Acta Crystallogr. A Found. Adv. 71, s19 (2015).

Adams P. D., Afonine P. V., Bunkóczi G., Chen V. B., Davis I. W., Echols N., Headd J. J., Hung L.-W., Kapral G. J., Grosse-Kunstleve R. W., McCoy A. J., Moriarty N. W., Oeffner R., Read R. J., Richardson D. C., Richardson J. S., Terwilliger T. C., Zwart P. H., PHENIX : A comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010). PubMed PMC

Murshudov G. N., Skubák P., Lebedev A. A., Pannu N. S., Steiner R. A., Nicholls R. A., Winn M. D., Long F., Vagin A. A., REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D Biol. Crystallogr. 67, 355–367 (2011). PubMed PMC

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

Proteorhodopsin insights into the molecular mechanism of vectorial proton transport

The Evolutionary Kaleidoscope of Rhodopsins

Neurophotonic tools for microscopic measurements and manipulation: status report

Structure-based insights into evolution of rhodopsins

Schizorhodopsins: A family of rhodopsins from Asgard archaea that function as light-driven inward H+ pumps