NEW OPTOGENETIC TOOL

Abstract

The invention relates to newly characterized light-inducible inward proton pumps and their use in medicine, their utility as optogenetic tools, nucleic acid constructs encoding same, expression vectors carrying the nucleic acid construct, cells comprising said nucleic acid construct or expression vector, and their respective uses.

Claims

1-15. (canceled)

16. A method of medical treatment, comprising administering a light-driven inward directed proton pump having at least 59% sequence similarity over the full length of SEQ ID NO: 1 (NsXeR) to a patient.

17. The method of claim 16, wherein the light-driven inward directed proton pump has at least 90% sequence similarity to the full length of SEQ ID NO: 1 (NsXeR).

18. The method of claim 16, wherein the light-driven inward directed proton pump has at least 90% sequence identity to the full length of SEQ ID NO: 1 (NsXeR).

19. The method of claim 16, wherein light-driven inward directed proton pump is not mutated at position E4, H48, S55, W73, D76, S80, A87, P209, C212, K214, and D220 of SEQ ID NO: 1; or wherein light-driven inward directed proton pump is not truncated at the N-terminus; or wherein light-driven inward directed proton pump is not mutated at position E4, H48, S55, W73, D76, S80, A87, P209, C212, K214, and D220 of SEQ ID NO: 1, and is not truncated at the N-terminus.

20. The method of claim 16, wherein the light-driven inward directed proton pump comprises an amino acid sequence selected from SEQ ID NO: 1 (NsXeR), 2 (HrvXeR1), 9 (HrvXeR), 10 (AlkXeR), 11 (AlkXeR1), 12 (AlkXeR2), 13 (AlkXeR3), 14 (AlkXeR4), and 15 (AlkXeR5).

21. The method of claim 16, wherein the light-driven inward directed proton pump consists of an amino acid sequence selected from SEQ ID NO: 1 (NsXeR), 2 (HrvXeR1), 9 (HrvXeR), 10 (AlkXeR), 11 (AlkXeR1), 12 (AlkXeR2), 13 (AlkXeR3), 14 (AlkXeR4), and 15 (AlkXeR5).

22. The method of claim 16, wherein the light-driven inward directed proton pump is active between pH 6 and pH 8.

23. The method of claim 16, wherein the absorption maximum of the light-driven inward directed proton pump is between 560 nm and 580 nm.

24. The method of claim 16, wherein the photocycle of the light-driven inward directed proton pump is less than 50 ms, if measured in proteo-nanodiscs exhibiting a molar ratio of DMPC:MSP1E3:light-driven inward directed proton pump of 100:2:3 at 20 C. and pH 7.5, providing pulses of 5 ns duration at 532 nm wavelength and energy of 3 mJ/pulse.

25. The method of claim 16, wherein the light-driven inward directed proton pump has a turnover rate of more than 250 s.sup.1, if measured in rat hippocampal neurons by patch-clamp measurements in the whole cell configuration using patch pipettes with resistances of 3-8 M, filled with 129 mM potassium gluconate, 10 mM HEPES, 10 mM KCl, 4 mM MgATP and 0.3 mM Na.sub.3GTP, titrated to pH 7.3, and an extracellular solution contained 125 mM NaCl, 2 mM KCl, 2 mM CaCl.sub.2, 1 mM MgCl2, 1 mM MgCl.sub.2, 30 mM glucose and 25 mM HEPES, titrated to pH 7.3.

26. The method of claim 16, wherein the light-driven inward directed proton pump is capable of triggering action potentials in a frequency of more than 40 Hz, if measured in rat hippocampal neurons by patch-clamp measurements in the whole cell configuration using patch pipettes with resistances of 3-8 M, filled with 129 mM potassium gluconate, 10 mM HEPES, 10 mM KCl, 4 mM MgATP and 0.3 mM Na.sub.3GTP, titrated to pH 7.3, and an extracellular solution contained 125 mM NaCl, 2 mM KCl, 2 mM CaCl.sub.2, 1 mM MgCl.sub.2, 1 mM MgCl.sub.2, 30 mM glucose and 25 mM HEPES, titrated to pH 7.3.

27. The method of claim 16, wherein the light-driven inward directed proton pump is capable of being triggered with a pulse width of 3 ms of =532 nm and an intensity of 23 mW/mm.sup.2, if measured in rat hippocampal neurons by patch-clamp measurements in the whole cell configuration using patch pipettes with resistances of 3-8 M, filled with 129 mM potassium gluconate, 10 mM HEPES, 10 mM KCl, 4 mM MgATP and 0.3 mM Na.sub.3GTP, titrated to pH 7.3, and an extracellular solution contained 125 mM NaCl, 2 mM KCl, 2 mM CaCl.sub.2, 1 mM MgCl.sub.2, 1 mM MgCl.sub.2, 30 mM glucose and 25 mM HEPES, titrated to pH 7.3.

28. The method of claim 16, wherein the medical treatment is selected from restoring auditory activity, recovery of vision, treating or alleviating alkalosis, treating or alleviating neurological injury, treating or alleviating brain damage, treating or alleviating seizure, and treating or alleviating a degenerative neurological disorder.

29. The method of claim 28, wherein the neurological disorder is Parkinson's disease or Alzheimer's disease.

30. A nucleic acid construct, comprising a nucleotide sequence coding for a light-driven inward directed proton pump having at least 59% sequence similarity over the full length of SEQ ID NO: 1 (NsXeR), wherein the nucleotide sequence is codon-optimized for expression in human cells.

31. An expression vector, comprising a nucleotide sequence coding for a light-driven inward directed proton pump having at least 59% sequence similarity over the full length of SEQ ID NO: 1 (NsXeR), wherein the nucleotide sequence is optimized for expression in human cells.

32. The expression vector of claim 31, wherein the vector is a viral vector.

33. The expression vector of claim 31, wherein the coding sequence of the light-driven inward directed proton pump is under the control of a neuronal cell specific human promotor.

34. The expression vector of claim 33, wherein the neuronal cell specific human promotor is the human synapsin promotor.

35. A mammalian cell expressing a light-driven inward directed proton pump having at least 59% sequence similarity over the full length of SEQ ID NO: 1 (NsXeR).

36. A mammalian cell comprising a nucleic acid construct according to claim 30 an expression vector according to claim 31.

37. The mammalian cell of claim 36, wherein the cell is (a) a hippocampal cell, a photoreceptor cell, a retinal rod cell, a retinal cone cell, a retinal ganglion cell, a bipolar neuron, a ganglion cell, a pseudounipolar neuron, a multipolar neuron, a pyramidal neuron, a Purkinje cell, or a granule cell; or (b) a neuroblastoma cell; a HEK293 cell; a COS cell; a BHK cell; a CHO cell; a myeloma cell; or a MDCK cell.

38. A liposome, comprising a light-driven inward directed proton pump having at least 59% sequence similarity over the full length of SEQ ID NO: 1 (NsXeR).

39. A non-human mammal, comprising a cell according to claim 35.

40. The non-human animal of claim 39, wherein the cell is an endogenous cell.

Description

DESCRIPTION OF THE FIGURES

[0093] FIG. 1: Sequence alignment of microbial rhodopsins. The sequence alignment was performed with Clustal Omega. Helices regions are marked with + sign, N-terminal and transmembrane helices are subscribed. The motif amino acids and the H48-D220 proton acceptor pair are highlighted in bold.

[0094] Specification of UniProtIDs for the sequences. NsXeR (G0QG75), HrvXeR1 (H0AAK5), ASR (Q8YSC4), HsBR (P02945), PR (Q9F7P4), NpHR (P15647), DeKR2 (N0DKS8), NpSR2 (P42196), HrvXeR (Ghai et al., supra), AlkXeR (Vavourakis et al., supra), AlkXeRs1-5 (Vavourakis et al., supra).

[0095] FIG. 2: Electrogenic properties of XeR. a. pH changes upon illumination in E. coli cell suspensions expressing different XeRs. Graphs show the pH changes with and without the addition of CCCP. b. pH changes upon illumination in liposome suspension with reconstructed NsXeR (with and without CCCP). c. pH changes upon illumination in liposomes suspension measured under different pH values.

[0096] FIG. 3: Spectroscopic characterization of NsXeR. a. Absorption spectra of representatives of xenorhodopsin family solubilized in the detergent DDM. The corresponding positions of absorption maximum is indicated in the legend. b. Transient absorption changes of NsXeR (pH 7.5, T=20 C.) at three characteristic wavelengths 378, 408, and 564 nm. Black lines are experimental data and light gray and dark gray lines represent the result of global fit using five exponents. The photocycles were measured for the two preparations: NsXeR in nanodiscs (light gray) and in liposomes (dark gray). Note that the differences in amplitudes between the samples are due to the approximately two times higher concentration of NsXeR in liposomes than in nanodiscs (see FIG. 4). c. Proposed model of NsXeR photocycle in nanodiscs.

[0097] FIG. 4: Photocycles of the NsXeR in nanodiscs (ND, upper row) and liposomes (LIP, lower row) preparations (20 C., pH 7.5). Five kinetically distinct protein states (red lines) are obtained via global multi exponential analysis of the flash photolysis data exemplified in the FIG. 3b. An each panel contains for the reference the correspondent spectrum on unexcited protein (P.sub.0, black lines). The spectra of P.sub.i=1 . . . 5 states were calculated from correspondent spectra of exponents, which were further converted to the differential spectra of the states assuming the sequential irreversible model of the photocycle. The half-times of reactions are depicted between the panels. The fraction of cycled molecules was 12.5% in ND, and 15% in LIP.

[0098] FIG. 5: Photocurrents in HEK293 and NG108-15 cells. Photocurrents in cells expressing NsXeR at the membrane potentials changed in 20 mV steps from 100 mV and corresponding I-V curves. a. HEK293 with pipette solution 1 and bath solution 1. b. NG108-15 cells with pipette solution 2 and bath solution 2 (control measurements to confirm that protons are responsible for inwardly directed current).

[0099] FIG. 6: Spiking traces at different light-pulse frequencies. Rat hippocampal neurons heterologously expressing NsXeR were investigated by patch-clamp experiments in the whole cell configuration under current clamp conditions. Action potentials were triggered by 40 light-pulses at indicated frequencies. The light pulses had a pulse width of 3 ms, a wavelength of =532 nm and an intensity of 23 mW/mm.sup.2.

[0100] FIG. 7: Variability of spike latency. Exemplary spiking traces measured in different neuronal cells. The light pulses had a pulse width of A) 3 ms and B) 10 ms. Rat hippocampal neurons heterologously expressing NsXeR were investigated by patch-clamp experiments in the whole cell configuration under current clamp conditions. The spikes were triggered by light pulses with a wavelength of =532 nm and an intensity of 23 mW/mm.sup.2.

[0101] FIG. 8: On and Off kinetics of NsXeR measured in NG108 cells at indicated membrane potentials. Ultrashort nanosecond light pulses were generated by the Opolette 355 at the wavelength of =570 nm. Corresponding I-V curve is shown on the right, peak photocurrent is plotted against membrane potential.

DESCRIPTION OF THE SEQUENCES

[0102]

TABLE-US-00002 SEQIDNO:1(NsXeR;UniProtIDG0QG75,N-terminalhelixunderlined;motif aminoacidsandH48-D220protonacceptorpairinbold) MVYEAITAGGFGSQPFILAYIITAMISGLLFLYLPRKLDVPQKFGIIHFF IVVWSGLMYTNFLNQSFLSDYAWYMDWMVSTPLILLALGLTAFHGADTKR YDLLGALLGAEFTLVITGLLAQAQGSITPYYVGVLLLLGVVYLLAKPFRE IAEESSDGLARAYKILAGYIGIFFLSYPTVWYISGIDALPGSLNILDPTQ TSIALVVLPFFCKQVYGFLDMYLIHKAE SEQIDNO:2(HrvXeR1;UniProtIDH0AAK5,N-terminalhelixunderlined;motif aminoacidsandH48-D220protonacceptorpairinbold) MVYEAIAASGSTPYLMAYIATAFLSGLLYLFLRKVWWTNVPLKFPIIHFF IVTWSGIMYLNFLNGTALSDFGWYMDWMISTPLILLALGLTAMHGRETRW DLLGALMGLQFMLVITGIISQESGMTYAYWIGNALLLGVFYLVWGPLREM AKETSDVLARSYTTLSAYISVFFVLYPTVWYLSETIYPAGPGIFGAFETS VAFVILPFFCKQAYGFLDMYLIHEAEEQM SEQIDNO:3(ASR;UniProtIDQ8YSC4) MNLESLLHWIYVAGMTIGALHFWSLSRNPRGVPQYEYLVAMFIPIWSGLA YMAMAIDQGKVEAAGQIAHYARYIDWMVTTPLLLLSLSWTAMQFIKKDWT LIGFLMSTQIVVITSGLIADLSERDWVRYLWYICGVCAFLIILWGIWNPL RAKTRTQSSELANLYDKLVTYFTVLWIGYPIVWIIGPSGFGWINQTIDTF LFCLLPFFSKVGFSFLDLHGLRNLNDSRQTTGDRFAENTLQFVENITLFA NSRRQQSRRRV SEQIDNO:4(HsBR;UniPotIDP02945) MLELLPTAVEGVSQAQITGRPEWIWLALGTALMGLGTLYFLVKGMGVSDP DAKKFYAITTLVPAIAFTMYLSMLLGYGLTMVPFGGEQNPIYWARYADWL FTTPLLLLDLALLVDADQGTILALVGADGIMIGTGLVGALTKVYSYRFVW WAISTAAMLYILYVLFFGFTSKAESMRPEVASTFKVLRNVTVVLWSAYPV VWLIGSEGAGIVPLNIETLLFMVLDVSAKVGFGLILLRSRAIFGEAEAPE PSAGDGAAATSD SEQIDNO:5(PR;UniProtID:Q9F7P4) MKLLLILGSVIALPTFAAGGGDLDASDYTGVSFWLVTAALLASTVFFFVE RDRVSAKWKTSLTVSGLVTGIAFWHYMYMRGVWIETGDSPTVFRYIDWLL TVPLLICEFYLILAAATNVAGSLFKKLLVGSLVMLVFGYMGEAGIMAAWP AFIIGCLAWVYMIYELWAGEGKSACNTASPAVQSAYNTMMYIIIFGWAIY PVGYFTGYLMGDGGSALNLNLIYNLADFVNKILFGLIIWNVAVKESSNA SEQIDNO:6(NpHR;UniProtIDP15647) MTETLPPVTESAVALQAEVTQRELFEFVLNDPLLASSLYINIALAGLSIL LFVFMTRGLDDPRAKLIAVSTILVPVVSIASYTGLASGLTISVLEMPAGH FAEGSSVMLGGEEVDGVVTMWGRYLTWALSTPMILLALGLLAGSNATKLF TAITFDIAMCVTGLAAALTTSSHLMRWFWYAISCACFLVVLYILLVEWAQ DAKAAGTADMFNTLKLLTVVMWLGYPIVWALGVEGIAVLPVGVTSWGYSF LDIVAKYIFAFLLLNYLTSNESVVSGSILDVPSASGTPADD SEQIDNO:7(DeKR2;UniProtIDN0DKS8) MTQELGNANFENFIGATEGFSEIAYQFTSHILTLGYAVMLAGLLYFILTI KNVDKKFQMSNILSAVVMVSAFLLLYAQAQNWTSSFTFNEEVGRYFLDPS GDLFNNGYRYLNWLIDVPMLLFQILFVVSLTTSKFSSVRNQFWFSGAMMI ITGYIGQFYEVSNLTAFLVWGAISSAFFFHILWVMKKVINEGKEGISPAG QKILSNIWILFLISWTLYPGAYLMPYLTGVDGFLYSEDGVMARQLVYTIA DVSSKVIYGVLLGNLAITLSKNKELVEANS SEQIDNO:8(NpSR2;UniProtIDP42196) MVGLTTLFWLGAIGMLVGTLAFAWAGRDAGSGERRYYVTLVGISGIAAVA YVVMALGVGWVPVAERTVFAPRYIDWILTTPLIVYFLGLLAGLDSREFGI VITLNTVVMLAGFAGAMVPGIERYALFGMGAVAFLGLVYYLVGPMTESAS QRSSGIKSLYVRLRNLTVILWAIYPFIWLLGPPGVALLTPTVDVALIVYL DLVTKVGFGFIALDAAATLRAEHGESLAGVDTDAPAVAD SEQIDNO;9(HrvXeR,N-terminalhelixunderlined;motifaminoacidsandH48- D220protonacceptorpairinbold) MVFEAIAGSGTEMYIQAYIATAFLSGLLYLYLSRVWWDNVPLKFPIVHFFIVTWSGIMYLN FLNESLFSNFAWYMDWLISTPLIVLALGMTALHHADKKHYDLLGMLMGLQFMLVVTGIISQ STGATLAYWVGNALLLGVIYLLWFPFREIAEQGSERLAKSYKTLAAYISIFFVLYPAAWYL GTPGPMEVLSDFQTSLAFVVLPFFCKQVYGFLDLYMIHHAED SEQIDNO:10(AlkXeR,N-terminalhelixunderlined;motifaminoacidsandH48- D220protonacceptorpairinbold) MVLPELATLTSQTIAAYIAATALSAVAFLWMSKNWGDVPKKFYLIHFFIVSWSGLMYMNIL YDTSIAELAFYADWLVSTPLIVLALGLSAYIASDSTDWSMVGSLMGLQFMLIAAGLLAHVA ETAAATWAFYGISCLFMFGVIYMIWGPLMRVTESNDALNREYHKLGLFVILTWLSYPTIWA LGDVGGYGLGVLSDYQVTLGYVILPFLCKAGFGFLDIYLLDRISDDI SEQIDNO:11(AlkXeR1,N-terminalhelixunderlined;motifaminoacidsandH48- D220protonacceptorpairinbold) MVYEAIAGSGSSPYTWAYIVTAFLSGLAFLYLSRVWDNVPRRFPIVHFFIVTWSGLMYLNF VEGQTILSNYAWYVDWMVSTPLIVLALALTATYKSEKNHYDLIAALMGLQFMLIVTGIISQ EAAASTAYAFWIGCGLLAGVAYLLWVPFRKIAEETSEVLAKKYKLLAGYITVFFALYPLVW YLSGTVYPSGPGMLGAFETSLAFVILPFFCKQVYGFLDMYLIHKAGEDL SEQIDNO:12(AlkXeR2,N-terminalhelixunderlined;motifaminoacidsandH48- D220protonacceptorpairinbold) MVYEAIAASGSSPYIWAYIITAFLSGLAFLYLSRIWDNVPRRFPIVHFFIVTWSGLMYLNF VEGQTLISDYAWYVDWMISTPLIVLALAMTATYKSEKNHYDLIAALMGLQFMLIVTGIISQ EAAASTAYAFWIGCGLLAGVAYLLWVPFRKIAEETSDVLAKKYKLLAGYITVFFALYPAAW YLSEVVYPEGPAMLGAFETSLAFVILPFFCKQVYGFLDMYLIQKAGEEI SEQIDNO:13(AlkXeR3,N-terminalhelixunderlined;motifaminoacidsandH48- D220protonacceptorpairinbold) MIGVILIYEVTSRLFMVYEAIAASGSSPYIWAYIATALLSGLAYLFLYRVWDNVPRRFPII HFFIVSWSALMYLSFVEGQTLFSDYVWYMDWIISTPLIVLALVLTATYKSEGSHYDLIGAA MGLQFMLIVTGIVSQDTAMSADFVGIPVAFWLGCVWLAGLIYLLWGPFKEIAEQTSHHLAQ KYKILAGYISLFFALYPTAWYLSETVYPEGPAVLGAFETSLAFVILPFFCKQVYGFLDMYM IHQAGEEM SEQIDNO:14(AlkXeR4,N-terminalhelixunderlined;motifaminoacidsandH48- D220protonacceptorpairinbold) MVYEAIAASGSSPYIWAYIITAFLSGLAFLYLSRIWDNVPRRFPIVHFFIVTWSGLMYLNF VEGQTLISDYAWYVDWMISTPLIVLALAMTATYKSEKNHYDLIAALMGLQFMLIVTGIISQ EAAASTAYAFWIGCGLLAGVAYLLWVPFRKIAEETSDVLAKKYKLLAGYITVFFALYPAAW YLSEVVYPEGPAMLGAFETSLAFVILPFFCKQVYGFLDMYLIQKAGEEI SEQIDNO:15(AlkXeR5,N-terminalhelixunderlined;motifaminoacidsandH48- D220protonacceptorpairinbold) MVYEAIAASGSSPYIWAYIATAFLSGLAFLYLSKVWDNVPRRFPIVHFFIVTWSGLMYLNF VEGQTLISDYAWYVDWMVSTPLIVLALALTATYKSEKNHYDLIGALMGLQFMLVVTGIISQ EAAATTAYAFWIGCGLLVGVAYLLWVPFRKIAEETSEVLAKKYKILAGYITVFFALYPLVW YLSGTVYPEGPGMLGAFETSLAFVILPFECKQVYGELDMYLIQKAGKEL SEQIDNO:16(humancodon-optimizedNsXeR) atggtgtacgaggccatcacagccggcggattcggcagccagcctttcatcctggcctac 60 atcatcaccgccatgatcagcggcctgctgttcctgtacctgccccggaagctggacgtg 120 ccccagaagttcggcatcatccactttttcatcgtcgtgtggagcggcctgatgtatacc 180 aacttcctgaaccagagcttcctgagcgactacgcctggtacatggactggatggtgtcc 240 acccccctgatcctgctggccctgggactgacagctttccacggcgccgacaccaagaga 300 tacgacctgctgggagcactgctgggcgccgagtttaccctcgtgatcactggactgctg 360 gctcaggcccagggctccatcaccccttactatgtgggcgtgctcctgctgctgggggtg 420 gtgtatctgctggccaagcccttcagagagatcgccgaggaaagcagcgacggcctggcc 480 agagcctacaagatcctggccggctatatcggcatcttctttctgtcctaccccaccgtg 540 tggtacatcagcggcatcgacgccctgcccggcagcctgaatatcctggaccctacccag 600 acctctatcgccctggtggtgctgccattcttctgtaaacaagtgtacggcttcctggac 660 atgtacctgatccacaaggctgag 684

EXAMPLES

Example 1Characterization of Xenorhodopsins

[0103] pH Changes in E. coli Suspensions

[0104] NsXeR (Uniprot ID G0QG75), HrvXeR (Ghai, R. et al. Sci. Rep. 1, (2011)) and AlkXeR (Vavourakis, C. D. et al. Front. Microbiol. 7, (2016)), coding DNAs were synthesized commercially (Eurofins). The nucleotide sequences were optimized for E. coli expression using the GeneOptimizer software (Life Technologies, USA). The genes together with the 5 ribosome-binding sites and the 3 extensions coding additional LEHHHHHH* tags were introduced into the pET15b expression vector (Novagen) via XbaI and BamHI restriction sites.

[0105] The protein was expressed as described previously (Gushchin, I. et al. Crystal structure of a light-driven sodium pump. Nat. Struct. Mol. Biol. 22, 390-395 (2015); incorporated herein in its entirety by reference) with modifications. E. coli cells of strain C41(DE3) (Lucigen) were transformed with the expression plasmids. Transformed cells were grown at 37 C. in shaking baffled flasks in an autoinducing medium, ZYP-5052 (Studier, F. W. Protein production by auto-induction in high-density shaking cultures. Protein Expr. Purif. 41, 207-234 (2005); incorporated herein in its entirety by reference) containing 100 mg/L ampicillin, and were induced at optical density OD600 of 0.6-0.7 with 1 mM isopropyl -d-1-thiogalactopyranoside (IPTG) and supplemented with 10 M all-trans-retinal. Three hours after induction, the cells were collected by centrifugation at 3,000 g for 10 min and were washed three times with an unbuffered salt solution (100 mM NaCl, and 10 mM MgCl2) with 30-min intervals between the washes to allow exchange of the ions inside the cells with the bulk. After that, the cells were resuspended in 100 mM NaCl solution and adjusted to an OD600 of 8.5. The measurements were performed in 3 ml aliquots of stirred cell suspension kept at 1 C. The cells were illuminated for 5 min with a halogen lamp (Intralux 5000-1, VOLPI) and the light-induced pH changes were monitored with a pH meter (LAB 850, Schott Instruments).

[0106] The pH of the cell suspension increased upon illumination and decreased back when the light was turned off (FIG. 2a). The effect of the pH change was completely abolished when repeated under the same conditions after addition of 30 M of carbonylcyanide m-chlorophenylhydrazone (CCCP). Similar experiments previously done with other proton pumps such as bacteriorhodopsine (BR) gave the opposite behavior of pH upon illumination of the cells (data not shown). Two other members of the xenorhodopsin family, HrvXeR and AlkXeR, studied in the present work gave the same results as NsXeR (FIG. 2a). Thus, pH experiments provide evidence that Nanohaloarchaea rhodopsins are inwardly directed proton pumps.

[0107] pH Changes in Liposome Suspension

[0108] The protein was expressed as described above. However, three hours after induction, the cells were collected by centrifugation at 3,000 g for 30 min. The collected cells were disrupted in M-110P Lab Homogenizer (Microfluidics, USA) at 25,000 psi in a buffer containing 20 mM Tris-HCl pH 8.0, 5% glycerol, 0.5% Triton X-100 (Sigma-Aldrich, USA) and 50 mg/L DNase I (Sigma-Aldrich, USA). The membrane fraction of cell lysate was isolated by ultracentrifugation at 90,000 g for 1 h at 4 C. The pellet was resuspended in a buffer containing 50 mM NaH.sub.2PO.sub.4/Na.sub.2HPO.sub.4 pH 8.0, 0.1 M NaCl and 1% DDM (Anatrace, Affymetrix, USA) and stirred overnight for solubilization. The insoluble fraction was removed by ultracentrifugation at 90,000 g for 1 h at 4 C. The supernatant was loaded on Ni-NTA column (Qiagen, Germany) and xenorhodopsins were eluted in a buffer containing 50 mM NaH.sub.2PO.sub.4/Na.sub.2HPO.sub.4 pH 7.5, 0.1 M NaCl, 0.3 M imidazole and 0.2% DDM. The eluate was dialysed against 100 volumes of 50 mM NaH.sub.2PO.sub.4/Na.sub.2HPO.sub.4 pH 7.5, 0.1 M NaCl buffer twice for 2 hours to dispose imidazole.

[0109] Purified NsXeR were reconstituted in soybean liposomes as described previously (Huang, K. S., Bayley, H. & Khorana, H. G. Delipidation of bacteriorhodopsin and reconstitution with exogenous phospholipid. Proc. Natl. Acad. Sci. 77, 323-327 (1980); incorporated herein by reference). Briefly, phospholipids (asolectin from soybean, Sigma-Aldrich) were dissolved in CHCl.sub.3 (Chloroform ultrapure, Applichem Panreac) and dried under a stream of N.sub.2 in a glass vial. Residual solvent was removed with a vacuum pump overnight. The dried lipids were resuspended at a final concentration of 1% (w/v) in 0.15 M NaCl supplemented with 2% (w/v) sodium cholate. The mixture was clarified by sonication at 4 C. and xenorhodopsin was added at a protein/lipid ratio of 7:100 (w/w). The detergent was removed by overnight stirring with detergent-absorbing beads (Amberlite XAD 2, Supelco). The mixture was dialyzed against 0.15 M NaCl (adjusted to a desired pH) at 4 C. for 1 day (four 200 ml changes) to obtain certain pH.

[0110] The measurements were performed on 2 ml of stirred proteoliposome suspension at 0 C. Proteoliposomes were illuminated for 18 minutes with a halogen lamp (Intralux 5000-1, VOLPI) and then were kept in the dark for another 18 minutes. Changes in pH were monitored with a pH meter (LAB 850, Schott Instruments). Measurements were repeated for different starting pH and in the presence of 40 uM of CCCP under the same conditions.

[0111] The pH changes upon illumination showed acidification of the solution outside the membrane (FIG. 2b). These pH changes were abolished, when CCCP was added to the suspension. Since in similar experiments all the known outwardly directed proton pumps (like BR and PR) show the opposite pH behavior (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)), we conclude that NsXeR are real inwardly directed proton pumps. Interesting is that in a wide range of pH values (between pH 5 and 9) our experiments still show inward proton pumping (FIG. 2c).

[0112] Absorption Spectra and Photocycle

[0113] Here we report results of the analysis of 2 data sets: the XeR protein reconstituted in nanodiscs and liposomes. The proteo-nanodiscs were assembled using standard protocol (Ritchie, T. K. et al. in Methods in Enzymology (ed. Dzgnes, N.) 464, 211-231 (Academic Press, 2009); incorporated herein by reference). 1,2-dimyristoyl-sn-glycero-3-phosphocholine, DMPC (Avanti Polar Lipids, USA) was used as lipid. An elongated MSP1E3 version of apolipoprotein-1 was used. The molar ratio during assembly was DMPC:MSP1E3:NsXeR=100:2:3. Liposomes were prepared as described above.

[0114] The absorption spectra were recorded using the Shimadzu UV-2401PC spectrophotometer. The laser flash photolysis setup was similar to that described by Chizhov and co-workers (Chizhov, I. et al. Spectrally silent transitions in the bacteriorhodopsin photocycle. Biophys. J. 71, 2329-2345 (1996); incorporated herein by reference). The excitation/detection systems were composed as such: a Surelite II-10 Nd:YAG laser (Continuum Inc, USA) was used providing pulses of 5 ns duration at 532 nm wavelength and energy of 3 mJ/pulse. Samples (55 mm spectroscopic quartz cuvette (Hellma GmbH & Co, Germany)) were placed in a thermostated house between two collimated and mechanically coupled monochromators ( m model 77250, Oriel Corp., USA). The probing light (Xe-arc lamp, 75 W, Osram, Germany) passed the first monochromator, sample and arrived after a second monochromator at a PMT detector (R3896, Hamamatzu, Japan). The current-to-voltage converter of the PMT determines the time resolution of the measurement system of ca 50 ns (measured as an apparent pulse width of the 5 ns laser pulse). Two digital oscilloscopes (LeCroy 9361 and 9400A, 25 and 32 kilobytes of buffer memory per channel, respectively) were used to record the traces of transient transmission changes in two overlapping time windows. The maximal digitizing rate was 10 ns per data point. Transient absorption changes were recorded from 10 ns after the laser pulses until full completion of the photo-transformation. At each wavelength, 25 laser pulses were averaged to improve the signal-to-noise ratio. The quasi-logarithmic data compression reduced the initial number of data points per trace (50000) to 600 points evenly distributed in a log time scale giving 100 points per time decade. The wavelengths were varied from 300 to 730 nm in steps of 2 nm (altogether, 216 spectral points) using a computer-controlled step-motor. Absorption spectra of the samples were measured before and after each experiment on standard spectrophotometer (Beckman DU-800).

[0115] Each data set was independently analyzed using the global multi-exponential nonlinear least-squares fitting program MEXFIT (Gordeliy, V. I. et al. Molecular basis of transmembrane signalling by sensory rhodopsin II-transducer complex. Nature 419, 484-487 (2002); incorporated herein by reference). The number of exponential components was incremented until the standard deviation of weighted residuals did not further improve. After establishing the apparent rate constants and their assignment to the internal irreversible transitions of a single chain of relaxation processes, the amplitude spectra of exponents were transformed to the difference spectra of the corresponding intermediates in respect to the spectrum of final state. Subsequently, the absolute absorption spectra of states were determined by adding the difference spectra divided by the fraction of converted molecules to the spectra of the final states. Criteria for the determination of the fraction value were the absence of negative absorbencies and contributions from the initial state to the calculated spectra of final state. For further details of the methods see (Chizhov, I. et al. Biophys. J. 71, 2329-2345 (1996)).

[0116] The absorption maximum of NsXeR in solubilized form is 565 nm (FIG. 3a). Its position does not shift when the pH of the buffer is varied in the range from 4.5 to 9.0. NsXeR does not exhibit light and dark adaptation. The homologue AlkXeR is a red-shifted variant, its absorption maximum is 577 nm (FIG. 3a). Transient absorption changes of NsXeR (pH 7.5, T=20 C.) are shown at three characteristic wavelengths 378, 408, and 564 nm with NsXeR prepared in two different ways: in nanodiscs (light gray) and in single lipid vesicles (dark gray) (FIG. 3b). The results of global fit using five exponents are shown in FIG. 4. The photocycle of NsXeR in nanodiscs is faster (27 ms) than in lipid vesicles (50 ms). The photocycle of NsXeR in nanodiscs is shown in FIG. 3c.

[0117] The photocycle of NsXeR contains a microsecond part, which is usually assigned to the multistep reaction of a release of the energized ion (the H.sup.+ in our case) and a millisecond part of relaxation and re-uptake of the ion.

[0118] However, the NsXeR photocycle reveals some distinct features, which to our knowledge have never been reported in the previous studies of retinal proteins (FIG. 4). After the microsecond part of the photocycle (P.sub.1, P.sub.2, P.sub.3) that includes archetypical intermediates with the K and L-like spectral shifts (P.sub.1, .sub.max=570 nm, P.sub.2, .sub.max=530 nm), in the millisecond time domain we obtained two spectrally and kinetically different M intermediates (P.sub.4 and P.sub.5). The first M-form (P.sub.4) has a characteristic three-band absorption spectrum with the maximal at 360, 378 and 398 nm. This state with the half-time of 2 in nanodiscs (3 in lipid vesicles) milliseconds converts to the state P.sub.5 with a single maximum at 392 nm. Both intermediates should correspond to the de-protonated state of the retinal Schiff-base. It is interesting that the state P.sub.4 has the same spectral features as a previously reported spectrum of retro-retinal in bacteriorhodopsin (BR). This form of retinal is characterized by conversion of the backbone carbon C.sub.14 from the CH to the CH.sub.2 form with the corresponding change of the C.sub.14=C.sub.13 double bond to a single one and the alteration of the -electron conjugation along the retinal. It was reported that the retro-retinal form of BR was achieved by deep UV illumination of the sample and (or) addition to the solution of the HCl acid. This retro-BR does not exhibit a photo-activity. On the other hand an alteration of the -electron conjugation in the excited state of the retinal might cause similar spectral features in the de-protonated state without covalent bond changes on the C.sub.14. The solution mechanism of the charge separation along the retinal upon photoexcitation and the accompanying alteration of the -electron conjugation was proposed (Chernavskii, D. S. An alternative model of the bacteriorhodopsin action and unusual properties of the K-610-intermediate. Biofizika (1994)) and further theoretically corroborated (Buda, F., de Groot, H. J. M. & Bifone, A. Charge Localization and Dynamics in Rhodopsin. Phys. Rev. Lett. 77, 4474-4477 (1996)). Perhaps, we are observing the first experimental evidence of the proposed mechanism. It is interesting that contrary to other retinal proton pumps (BR, pSRII, PR) we don't see any additional intermediates (N or O-like) on the path of re-protonation. The MII (P.sub.5) state directly converts to the ground state of NsXeR with a half-time of 27 (50 in lipid vesicles) milliseconds.

Example 2Crystallization of NsXeR

[0119] The NsXeR protein was prepared and purified as described in Example 1. Finally proteins were concentrated to 70 mg/ml for crystallization. NsXeR crystals grew in meso approach (Landau, E. M. & Rosenbusch, J. P. Lipidic cubic phases: A novel concept for the crystallization of membrane proteins. Proc. Natl. Acad. Sci. 93, 14532-14535 (1996); and Caffrey, M. & Cherezov, V. Crystallizing membrane proteins using lipidic mesophases. Nat. Protoc. 4, 706-731 (2009); each incorporated herein by reference), similar to that used in previous works (Gordeliy, V. I. et al. Molecular basis of transmembrane signalling by sensory rhodopsin II-transducer complex. Nature 419, 484-487 (2002); incorporated by reference). The solubilized protein in the crystallization buffer was mixed with premelted at 47 C. monoundecenoin (Nu-Chek Prep) to form a lipidic mesophase. 100 nl aliquots of a protein-mesophase mixture were spotted on a 96-well LCP glass sandwich plate (Marienfeld) and overlaid with 600 nL of precipitant solution by means of the NT8 crystallization robot (Formulatrix). The best crystals were obtained with a protein concentration of 20 mg/ml and 2.0 M sodium malonate, pH 8.0 (Hampton Research). The crystals were grown at 22 C. and appeared in 1-4 weeks.

[0120] X-ray diffraction data (wavelengths 0.969 and 0.972 ) were collected at ID23-1 beamline of the ESRF at 100 K, with a PILATUS 6M detector. Diffraction images were processed with XDS (Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125-132 (2010); incorporated by reference). The reflection intensities were scaled with SCALA from the CCP4 suite (Winn, M. D. et al. Overview of the CCP 4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235-242 (2011); incorporated by reference). Crystallographic data collection and refinement statistics is shown in the following table.

TABLE-US-00003 Data collection Space group P2.sub.12.sub.12.sub.1 Cell dimensions a, b, c () 64.50, 94.71, 198.36 , , () 90, 90, 90 Resolution () 47-3.4 (3.67-3.4) R.sub.merge (%) 17.5 (150.2) I/I 6.0 (1.0) Completeness (%) 99.8 (99.8) Redundancy 4.5 (4.6) Refinement Resolution () 47.37-3.4 No. reflections 31932 R.sub.work/R.sub.free (%) 28.5/30.8 No. atoms Protein and retinal 4991 B factors (.sup.2) Protein and retinal 107.6 r.m.s. deviations Bond lengths () 0.011 Bond angles () 1.486

[0121] The structure was refined to the resolution of 3.4 . Reference model (archaerhodopsin-2, PDB 2EI4) for molecular replacement was chosen with the MoRDa pipeline (Vagin, A. & Lebedev, A. MoRDa, an automatic molecular replacement pipeline. Acta Crystallogr. Sect. Found. Adv. 71, s19-s19 (2015); incorporated by reference). Initial phases were successfully obtained in P2.sub.12.sub.12.sub.1 space group by an Automated Model Building and Rebuilding using Autobuild (Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213-221 (2010); incorporated by reference). The initial model was iteratively refined using REFMAC5 (Murshudov, G. N. et al. REFMAC 5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D Biol. Crystallogr. 67, 355-367 (2011); incorporated by reference), PHENIX and Coot (Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126-2132 (2004); incorporated by reference).

[0122] P2.sub.12.sub.12.sub.1 space group crystals contain one trimer of NsXeR in the asymmetric unit. Positions of the residues 95-97 in loop CD, 154-156 in loop EF are not resolved.

[0123] The light-driven inward proton pump XeR has seven transmembrane -helices (A-G) and a co-factor retinal covalently bound to 213 Lysine via the Schiff base. The helix A is preceded with a small N-terminal -helix, which is capping the protein on the extracellular side.

[0124] Comparison with BR structure (PDB 1C3W) indicates considerable differences in helices localization and form, A and G helices are significantly distorted, presumably due to the presence of prolines in those helices. Xenorhodopsins have a conservative residue Pro-209 (position in NsXeR), which is located at the position of Asp212 in BR. Our experiments showed that its replacement with Asp makes the protein unstable. If changed to glycine, the pumping activity dramatically decreases (see below table). Thus, Pro209 is crucial for proton pumping.

TABLE-US-00004 Proton translocation validation H48X Not folded, cells not colored D76E, D76N, Not folded, cells not colored D76S, D76T D220N No pumping Colored cells D220E Fully functional C212A, C212D Not folded, cells not colored C212S Lowered pumping Conservative aminoacids mutations W73R Not folded, cells not colored W73A Strongly lowered Colored cells pumping W73Y Lowered pumping S55A Not folded, cells not colored P209G Strongly lowered pumping P209D Not folded, cells not colored N-terminus 2-12 Lowered pumping Colored, not stable when solubilized E4Q Fully functional E4A, E4L Lowered pumping

[0125] Proton-Uptake Region and Active Center

[0126] Retinal is in 13-cis conformation. However, due to insufficient resolution we cannot distinguish whether it is in 15-syn or 15-anti conformation. NsXeR has a big proton-uptake cavity, which is separated from the bulk with an N-term very short helix on the extracellular part of the protein. We suggest that the cavity is filled with water molecules. The putative proton donor Asp76 might be available from that cavity. Mutations of Asp76 to Glu, Ser, Thr and Asn do not allow the protein to fold correctly (mutants were not colored, see above table). This is evidence of not only functional, but also significant structural role of these amino acids. Ser55 is located close to Asp76 and it may stabilize this residue. Substitution of Ser55 with Alanine (Ala53 in BR) also breaks protein folding.

[0127] Residues Tyr3 from N-terminal helix and Trp73, which is the analogue of highly conservative amino acid Arg82 (position in BR), separate the proton-uptake cavity from the bulk of the extracellular part of the protein, so that the proton may enter the protein through the space between the helices A and B and loop BC. Substitution of Trp73 with Arg was fatal for protein folding (W73R mutant is not colored). W73A mutant binds retinal and has the color of the wild-type protein, but demonstrates no pumping activity, which means this residue is critical for proton translocation.

[0128] Proton-Release Region

[0129] Another major difference of NsXeR from other known microbial retinal proton pumps is that it has no charged amino acid at the position equivalent to Asp96 in BR (in NsXeR it is Ala71). However, the residues His48 (10 from the Schiff base in the ground state) and Asp220 (12 ), which are connected via a hydrogen bond are located close to the expected proton acceptor position. Substitution of Asp220 with Asn demolishes proton pumping completely.

[0130] His48 is a unique residue, which is not present at a similar place in other known microbial rhodopsins. Our experiments showed that substitution of Histidine-48 with any other amino acid crushes protein structure (all mutants are not colored), which indicated its crucial role in protein architecture. We suggest that the pair His48-Asp220 is a proton acceptor, and the protonation processes from the Schiff base through the His48 residue, more precisely, through the pair His48-Asp220.

[0131] Remarkably, it is exactly the same proton acceptor pair as in proteorhodopsins. However, contrary to XeRs it is placed at the extracellular part of the protein close to the Schiff base and serves as a Schiff base proton acceptor, which is accessible from the bulk through a big proton-release cavity, so a further proton release may easily proceed directly to the bulk along the proton gradient. Thus, a unique and unusual set of key residues in NsXeR results in inwardly directed proton pumping

[0132] Putative Mechanism of Inwardly Directed Proton Transport

[0133] The structure and experiments with mutated amino acids provide insights into the mechanism of inwardly directed proton transport. Upon illumination retinal isomerizes and the Schiff base which is surrounded by a hydrophobic environment deprotonates and the proton is translocated to the deprotonated His48-Asp220 pair. It happens in MI and MII intermediate states since both intermediates correspond to the deprotonated state of the retinal Schiff base. Indeed, it is known that Asp-His interaction substantially lowers the pK.sub.a of Asp by stabilizing its deprotonated state. A key role of the Asp-His pair in proton translocation is supported by the mentioned above experiments with the mutated Asp and His. We suggest that after re-isomerization of the retinal the protonated Asp-His pair, connected to a hydrophilic cavity (proton release cavity, releases a proton directly to the cytoplasm. After isomerization of the retinal Asp76 protonates through the hydrophilic cavity. Re-isomerization of the retinal results also in re-protonation of the Schiff base from D76.

Example 3Optogenetic Implications

[0134] Experiments with Human Embryonic Kidney (HEK) and Neuroblastoma Glioma (NG) Cells

[0135] The human codon optimized NsXeR gene was synthesized commercially (Eurofins). The gene was cloned into the pcDNA3.1() vector bearing an additional membrane trafficking signal (Gradinaru, V. et al. Molecular and Cellular Approaches for Diversifying and Extending Optogenetics. Cell 141, 154-165 (2010), incorporated herein by reference), a P2A self-cleaving peptide (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. Bioorganic Chem. 39, 406-416 (2013); incorporated by reference) and a GFP variant at the C-terminus (Shcherbo, D. et al. Bright far-red fluorescent protein for whole-body imaging. Nat. Methods 4, 741-746 (2007); incorporated by reference). The gene was cloned under the CMV promoter. The sequence was verified by sequencing.

[0136] The HEK293 and NG108-15 cells at confluency of 80% were transfected with the plasmid and Lipofectamine LTX according to the manufacturer's protocol (ThermoFisher Scientific, USA). The cells were incubated under 5% CO.sub.2 at 37 C. for two days before measurements.

[0137] For the electrophysiological characterization of NsXeR whole cell patch-clamp recordings were performed (Axopatch 200B interface, Axon Instruments). Patch pipettes with resistances of 2-5 M were fabricated from thin-walled borosilicate glass (GB150F-8P) on a horizontal puller (Model P-1000, Sutter Instruments). For experiments in HEK293 cells the pipette solution contained 110 mM NaCl, 2 mM MgCl.sub.2, 10 mM EGTA, 10 mM HEPES, pH 7.4 (pipette solution 1) and the bath solution contained 140 mM NaCl, 2 mM MgCl.sub.2, 10 mM HEPES, pH 7.4 (bath solution 1). For the experiments in NG108-15 cells the pipette solution contained 110 mM Na.sub.2SO.sub.4, 4 mM MgSO.sub.4, 10 mM EGTA, 10 mM HEPES, pH 7.4 (with H.sub.2SO.sub.4) (pipette solution 2) and the bath solution contained 140 mM N-methyl-D-glucamine, 4 mM MgSO.sub.4, 10 mM HEPES, pH 7.4 (with H.sub.2SO.sub.4) (bath solution 2).

[0138] Photocurrents were measured in response to light pulses with a saturating intensity of 23 mW/mm.sup.2 using diode-pumped solid-state lasers (=532 nm) focused into a 400-m optic fiber. Light pulses were applied by a fast computer-controlled shutter (Uniblitz LS6ZM2, Vincent Associates). Ultrashort nanosecond light pulses were generated by the Opolette 355 tunable laser system (OPTOPRIM). For the measurement of the actionspectra the pulse energies at the different wavelengths were set to values which corresponded to equal photon counts of 10.sup.19 photons/m.sup.2. Moreover photocurrent-voltage relationships at membrane potentials ranging from 100 mV to +60 mV were measured (except for On/Off kinetics, where membrane potentials ranged from 80 mV to +80 mV).

[0139] FIG. 5a shows photocurrents generated by NsXeR in the HEK293 cell. Typical photocurrent values vary from 40 to 150 pA at 60 mV applied potential, whereas the currents normalized to the capacitance (meaning the size) of the cell are about 1-2 pA/pF. An additional control experiment in NG108-15 cells was performed. To exclude the transport of Cl.sup. ions (which may account for apparent inward current) chloride salts in buffers were replaced by sulfate. To exclude monovalent ion transport into the cell we replaced Na.sup.+ in the bath solution by large N-methyl-D-glucamine. The pH of the solutions was symmetric (pH 7.4). However, similar photocurrents were recorded in this experimental configuration (FIG. 5b), convincing us that the transport of protons is responsible for the effect. Thus, the experiments with HEK and NG cells also confirm that NsXeR is an inwardly directed pump and show that it is able to generate significant currents through plasma membranes upon illumination of the cells.

[0140] Light-Triggered Spiking in Rat Hippocampal Neurons

[0141] We heterologously expressed NsXeR in rat hippocampal neurons by means of adeno-associated virus mediated gene transfer. Hippocampi were isolated from postnatal P1 Sprague-Dawley rats and treated with papain (20 U ml.sup.1) for 20 min at 37 C. The hippocampi were washed with DMEM (Invitrogen/Gibco, high glucose) supplemented with 10% fetal bovine serum and titrated in a small volume of this solution. 96,000 cells were plated on poly-D-lysine/laminin coated glass cover slips in 24-well plates. After 3 hours the plating medium was replaced by culture medium (Neurobasal A containing 2% B-27 supplement, and 2 mM Glutamax-I).

[0142] rAAV2/1 virus was prepared using a pAAV2 vector with a human synapsin promoter containing the humanized DNA sequence of NsXeR, C-terminally fused to the Kir2.1 membrane trafficking signal, a P2A self-cleaving peptide and a GFP variant. Briefly 510.sup.9 genome copies/ml (GC/ml) of rAAV2/1 virus was added to each well 4-9 days after plating. The electrophysiological recordings were performed 19-23 days after transduction.

[0143] For the electrophysiological characterization we performed a whole cell patch clamp experiments under the current clamp conditions. Briefly, patch pipettes with resistances of 3-8 M were filled with 129 mM potassium gluconate, 10 mM HEPES, 10 mM KCl, 4 mM MgATP and 0.3 mM Na.sub.3GTP, titrated to pH 7.3. The extracellular solution contained 125 mM NaCl, 2 mM KCl, 2 mM CaCl.sub.2, 1 mM MgCl.sub.2, 30 mM glucose and 25 mM HEPES, titrated to pH 7.3. Electrophysiological signals were filtered at 10 kHz, digitized with an Axon Digidata 1322A (50 kHz) and acquired and analyzed using pClamp9 software (Axon Instruments).

[0144] The NsXeR-mediated, light-triggered inward transport of protons led to the depolarization of the membrane potential. Therefore, light-triggered spiking in rat hippocampal neurons was possible (FIG. 6). NsXeR enabled a fast, neural photostimulation with a firing success rate of 100% up to a frequency of 40 Hz. Spike failures at higher stimulation frequencies can be explained by intrinsic properties of the rat hippocampal neurons as a vast majority of rat hippocampal neurons have a maximal firing frequency of 40-60 Hz (Gunaydin, L. A. et al. Ultrafast optogenetic control. Nat. Neurosci. 13, 387-392 (2010)).

[0145] An important observation is that light-triggered spiking could be achieved with a pulse width of only 3 ms (FIG. 7a), which approximately corresponds to the turnover time of the pump (FIG. 8). Hence, the extent of depolarization due to the transport of a single proton by each NsXeR is sufficient to successfully trigger action potentials. However a variability of the spike latencies was observed (FIG. 7), which in some cases required longer pulse widths for the light-triggered spiking (FIG. 7b). Longer spike latencies could be explained by a comparatively lower expression of NsXeR in those neurons.

Example 4Optogenetic Stimulation of the Auditory Pathway

[0146] Hernandez et al. J Clin Invest. 124, 1114-1129 (2014) (incorporated herein by reference), demonstrates a strategy for optogenetic stimulation of the auditory pathway in rodents. In particular, the authors describe animal models to characterize optogenetic stimulation, which is the optical stimulation of neurons genetically engineered to express the light-gated ion channel channelrhodopsin-2 (ChR2). Optogenetic stimulation of spiral ganglion neurons (SGNs) activates the auditory pathway, as demonstrated by recordings of single neuron and neuronal population responses. Furthermore, optogenetic stimulation of SGNs restore auditory activity in deaf mice. Approximation of the spatial spread of cochlear excitation by recording local field potentials (LFPs) in the inferior colliculus in response to suprathreshold optical, acoustic, and electrical stimuli indicate that optogenetic stimulation achieves better frequency resolution than monopolar electrical stimulation.

[0147] Introducing the coding sequence for the light-inducible inward proton pump of the present disclosure, such as NsXeR, into the constructs as described, e.g., by Hernandez et al. represents routine practice.

Example 5Optogenetic Approach for the Recovery of Vision

[0148] Mac et al. Mol Ther. 23, 7-16 (2015) (incorporated herein by reference), describes optogenetic reactivation of retinal neurons mediated by adeno-associated virus (AAV) gene therapy. Most inherited retinal dystrophies display progressive photoreceptor cell degeneration leading to severe visual impairment. Optogenetic reactivation of retinal neurons mediated by adeno-associated virus (AAV) gene therapy has the potential to restore vision regardless of patient-specific mutations. The challenge for clinical translatability is to restore a vision as close to natural vision as possible, while using a surgically safe delivery route for the fragile degenerated retina. To preserve the visual processing of the inner retina, ON bipolar cells are targeted, which are still present at late stages of disease. For safe gene delivery, a recently engineered AAV variant is used that can transduce the bipolar cells after injection into the eye's easily accessible vitreous humor. It is shown that AAV encoding channelrhodopsin under the ON bipolar cell-specific promoter mediates long-term gene delivery restricted to ON-bipolar cells after intravitreal administration. Channelrhodopsin expression in ON bipolar cells leads to restoration of ON and OFF responses at the retinal and cortical levels. Moreover, light-induced locomotory behavior is restored in treated blind mice.

[0149] Introducing the coding sequence for the light-inducible inward proton pump of the present disclosure, such as NsXeR, into the constructs as described, e.g., by Mace et al. represents routine practice. The new light-inducible inward proton pumps of the present disclosure are inserted in the cassettes for the activation of ON bipolar cells as well as for the Ganglion cells in the retina.

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