MUTANT NQ-RHODOPSIN KR 2

Abstract

The invention relates to mutant NQ-Rhodopsin having potassium pumping properties, 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 light-driven cation pump, wherein the light-driven cation pump comprises an amino acid sequence which has at least 70% sequence identity to the full length amino acid sequence of SEQ ID NO: 1 (Krokinobacter eikastus rhodopsin 2; KR2), and which comprises a substitution at a position corresponding to G263 in SEQ ID NO: 1, which substitution confers a potassium ion pumping ability.

17. The light-driven cation pump of claim 16, wherein the light-driven cation pump comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 1 (KR2), except for a substitution at position G263 in SEQ ID NO: 1, which substitution confers a potassium ion pumping ability.

18. The light-driven cation pump of claim 16, wherein the substitution is selected from G263F and G263Y, preferably wherein the substitution is G263F.

19. The light-driven cation pump of claim 16, wherein the light-driven cation pump does not comprise any substitution at a position in which the corresponding amino acid residue is identical among all NQ-rhodopsins in the alignment of FIG. 6.

20. The light-driven cation pump of claim 16, further comprising, in non-covalent linkage, a retinal or retinal derivative.

21. The light-driven cation pump of claim 20, wherein the retinal derivative is selected from the group consisting of 3,4-dehydroretinal, 13-ethylretinal, 9-dm-retinal, 3-hydroxyretinal, 4-hydroxyretinal, naphthylretinal; 3,7,11-trim ethyl -dodeca-2,4,6,8,10-pentaenal; 3,7-dimethyl-deca-2,4,6,8-tetraenal; 3,7-dimethyl-octa-2,4,6-trienal; and 6-7 rotation-blocked retinals, 8-9 rotation-blocked retinals, and 10-11 rotation-blocked retinals.

22. A nucleic acid construct, comprising a nucleotide sequence encoding the light-driven cation pump according to claim 16.

23. An expression vector, comprising a nucleotide sequence coding for the light-driven cation pump according to claim 16.

24. The expression vector of claim 23, wherein the vector is suitable for gene therapy.

25. The expression vector of claim 24, wherein the vector is suitable for virus-mediated gene transfer.

26. A recombinant host cell comprising the light-driven cation pump according to claim 16, the nucleic acid construct according to claim 22.

27. The recombinant host cell of claim 26, wherein the recombinant host cell is a mammalian cell which is (i) a neuron; or (ii) a muscle cell.

28. The recombinant host cell of claim 26, wherein the recombinant host cell is a mammalian cell which is (i) a bipolar neuron, a pseudounipolar neuron, a multipolar neuron, a an anaxonic neuron, a basket cell, Betz cell, Lugaro cell, medium spiny neuron, Purkinje cell, pyramidal neuron, Renshaw cell, unipolar brush cell, granule cell, anterior horn cell, spindle cell, an afferent neuron, efferent neuron, or an interneuron, photoreceptor cell, rods and cones; or (ii) a skeletal muscle cell, a smooth muscle cell, or a cardiac muscle cell.

29. A method of treatment, comprising the step of administering a light-driven cation pump according to claim 16.

30. A non-therapeutic method of actively transporting potassium ions across a membrane, comprising the step of introducing the light-driven cation pump according to claim 20 into said membrane.

31. The non-therapeutic method of claim 30, wherein said membrane is a biologic membrane.

32. The non-therapeutic method of claim 30, wherein the method is an in vitro method.

33. A method for silencing electrically excitable cells, comprising the step of introducing the light-driven cation pump according to claim 16 into an electrically excitable cell, and activating said light-driven cation pump, thereby silencing said electrically excitable cell.

34. The method of claim 33, wherein said method is an in vitro method.

35. An optogenetic research method, comprising the step of introducing the light-driven cation pump according to any claim 16 into a recombinant cell; or providing a recombinant host cell according to any claim 26, and activating said light-driven cation pump.

36. A method of high-throughput screening, providing the steps of providing a recombinant host cell according to claim 26, contacting the cell with a test substance, exciting the light-driven cation pump of the recombinant host cell, and identifying whether the test substance is an active agent which modulates the light-driven cation pump, a Ca.sup.++-inducible potassium channel, or a BK channel of the recombinant host cell.

Description

DESCRIPTION OF THE FIGURES

[0035] FIG. 1A, Overall architecture of KR2. KR2 fold and its orientation in the membrane. B. Tested mutations in the KR2 ion pathway. The mutants N61M, and G263F,L are described herein, the mutants R109A, N112A,D, D116A,E,N, Q123A,E and U2b1A,E,N have been tested by Inoue et al. (Nat Commun 4, 1678 (2013)).

[0036] FIG. 2 Comparison of KR2 structure with structures of other microbial rhodopsins. a, Comparison of the KR2 backbone structure in the type A crystals (yellow) with those of bacteriorhodopsin (BR, purple, PDB ID 1C3W) and halorhodopsin (HR, blue, PDB ID 1E12). Root mean square deviations of the KR2 transmembrane helix backbone N, C and C.sub.α atoms relative to BR and HR are 2.5 and 2.3 Å correspondingly. b Comparison of the cytoplasmic sides. c, Comparison of the retinal positions. In KR2, the retinal is closer to the helices C and D, similar to blue proteorhodopsins (grey, PDB ID 4JQ6, salmon, PDB ID 4KLY) and opposite to BR, HR and Exiguobacterium sibiricum rhodopsin (green, PDB ID 4HYJ). d, Function-defining residues in KR2, BR and HR. While BR's Asp-112 is present in all the three proteins, the residues Asp-85/Thr-89/Asp-96 are replaced with Asn-112/Asp-116/Gln-123 in KR2 and other sodium pumps, earning them the designation NQ- or NDQ-rhodopsins.

[0037] FIG. 3. Ion translocation pathway in KR2. a, Putative ion translocation pathway residues inside KR2. N-terminal helix is shown in blue, helix A is shown in transparent pink. b, Water molecules (red) and cavities (transparent red) inside KR2. The black arrows show the putative ion path. Helices F and G are not shown. The cavities were calculated using HOLLOW (Ho, B. & Gruswitz, F. BMC Structural Biology 8, 49 (2008)). In this illustration, structure of the monomeric blue KR2 was used.

[0038] FIG. 4. Ion uptake cavity in KR2. a, The ion uptake cavity (red). The protrusion is absent in other microbial rhodopsins due to bulky amino acids at KR2′s G263 position, such as Leu-224 in bacteriorhodopsin (Luecke, H., et al. Journal of Molecular Biology 291, 899-911 (1999)) (BR), Leu-250 in halorhodopsin (Kolbe, M., et al. Science 288, 1390-1396 (2000)) (HR) and Phe-233 in Exiguobacterium sibiricum rhodopsin (Gushchin, I. et al. PNAS 110, 12631-12636 (2013)). (ESR). b, Effects of the N61M, G263F and G263L mutations on the activity of the protein in E. coli cells. Addition of the protonophore CCCP allows the protons to enter the cell in exchange for the pumped ions, enhancing the alkalization of the media. c, Comparison of the wild type and G263F mutant photocurrents in the liposomes attached to BLMs in presence of the protonophore 1799. Illumination starts at 0.5 s and stops at 16.5 s (NaCl measurements) or 4.5 s (KCl measurements).

[0039] FIG. 5 Pump activities of KR2 mutants expressed in E. coli. The solutions contain 100 mM NaCl (blue), 100 mM NaCl and 30 μM CCCP (green), 100 mM KCl (red), 100 mM KCl and 30 μM CCCP (magenta). The cells were illuminated for 300 s (light area on the plots). From highest to lowest, the plots show: WT (green->blue->red); WT pH 5.6 (blue->green->red); N61M (green->blue->red); G263F (green->magenta->blue->red); G263L (green->blue/magenta/red).

[0040] FIG. 6 Sequence alignment using the alignment program COBALT (publicly available at http://www.st-va.ncbi.nlm.nih.gov/) and default settings. The aligned sequences (accession numbers in brackets) are: 1: Krokinobacter eikastus KR2 (Uniprot ID N0DKS8; SEQ ID NO: 1) as used in the examples as the parent enzyme; 2: Citromicrobium sp. JLT1363 (ZP_08702831; SEQ ID NO: 2); 3: Citromicrobium bathyomarinum JL354 (ZP_06860850; SEQ ID NO: 3); 4: Fulvimarina pelagi HTCC2506 (ZP_01440547; SEQ ID NO: 4); 5: Chaetoceros neogracile KOPRI AnM0002 (as derived from EL620625, SEQ ID to NO: 5); 6: Truepera radiovictrix DSM 17093 (YP_003705905, SEQ ID NO: 6); 7: T. radiovictrix DSM 17093 (YP_003706581; SEQ ID NO: 7); 8: Gillisia limnaea R-8282 (ZP_09669334, SEQ ID NO: 8); 9: Krokinobacter (Dokdonia) sp. 4H-3-7-5 (YP_004429763, SEQ ID NO: 9). The underlined residues in SEQ ID NO: 1 appear to be conserved among NQ-rhodopsines and are, therefore, in one embodiment preferably not subject to any substitution.

TABLE-US-00003 SEQUENCES Krokinobacter eikastus KR2 (Uniprot ID N0DKS8; SEQ ID NO: 1) MTQELGNANF ENFIGATEGF SEIAYQFTSH ILTLGYAVML AGLLYFILTI KNVDKKFQMS  60 NILSAVVMVS AFLLLYAQAQ NWTSSFTFNE EVGRYFLDPS GDLFNNGYRY LNWLIDVPML 120 LFQILFVVSL TTSKFSSVRN QFWFSGAMMI ITGYIGQFYE VSNLTAFLVW GAISSAFFFH 180 ILWVMKKVIN EGKEGISPAG QKILSNIWIL FLISWTLYPG AYLMPYLTGV DGFLYSEDGV 240 MARQLVYTIA DVSSKVIYGV LLGNLAITLS KNKELVEANS 280 Citromicrobium sp. JLT1363 (ZP_08702831; SEQ ID NO: 2) MVAPITDNTV VSPPIPEVNT ALANQLGNIE NVVTLTMQQY TLGSLILMVS YGACFAFILF  60 FLMSVQNLAP RYRLVPILSA VVMASAGLSL LQEFSLWKDS YAFVDGLYRP LAENETFTNA 120 YRYGNWTITV PILLTQLAIA MGLRQGEIQR RSLRMGVPAV LMIWTGLYGQ FGEVGDFSHL 180 NLWGVVSSIF FLWLILEVRQ TLIAGISSTP DILKPWPNNL WWFFLATWGL YPIAYALPQL 240 GATAEIVIAR QGIYSLADIA SKLIYGIILA RFVLRRSAYE GYMPAAEALA SAPEKPNTGG 300 PGLRRAP 307 Citromicrobium bathyomarinum JL354 (ZP_06860850; SEQ ID NO: 3) MAAPIADPTV AEPQIAQADA AISAIENLVT LTSQQYHLGS LILMVSYAAF FAFILFFLMS  60 VQNLAPRYRL VPILSAVVMA SAGLTLLQEF GLWKESYAYV DGLYRPLAQN DSFSNAYRYG 120 NWTITVPILL AQLAIAMGLR QAEVHRRALR MAVPGVLMIW TGLYGQFGAV GDFSHLNLWG 180 VISSIFFLWL ILEVRQTLIA GLATTPDILK SWPSNLWWFF LASWGLYPLA YALPQLGATG 240 DLVVARQAIY SFADIASKLI YGIILARFVL RRSAFEGYMP AAEALAPTPD KPNTGGPGLR 300 RAP 303 Fulvimarina pelagi HTCC2506 (ZP_01440547; SEQ ID NO: 4) MQDTELFADQ LGNIENLVSL TVGQYTMGSL ILMVSYGAHF AFVLYFLMTS LQLAPRYRIV  60 PIMSAIVMVS AGLSLLREFN AWEQSYEFVE GMYQPLAENS TFTNAYRYGN WTITVPILLT 120 QLPLAFGLLR PELHVRAARM CIPALLMIWT GLVGQFGETG NYLRLNVWGV ISTIFFVWLI 180 IEVRGVISRA IAMGPAELAA WPKNIWWFFL AFWGLYPIAY ALPQLGHTGD IVVIRQLLYS 240 IADVFSKLVY GIILSRYVLR RSALEGYKPA IEALSRTPVD LSMSKNGGAG YGDGDR 296 Chaetoceros neogracile KOPRI AnM0002 (as derived from EL620625, SEQ ID NO: 5) MSAVLNVEGV RAACDRETGA AIANMENCLQ YSPLQFELVG HALVIGYAAQ AAGFIYFAMT  60 MNMTKGRNYQ LCSIYGMIVM LSAFLLLYNQ WAAWEDSFVL NAAGLYESGG VKLFSNGYRY 120 LNWSIDVPLL LLQLVLVSGL EVGTGFNKNL QVTTSGLLMI YLGYIGQFYE NPDSMVPLIA 180 FGVVGTVAYA IMLAIVLQCL SHAKKNFKTE SAKFKMNLVF WIFLIFWTIY PISYFMPVFS 240 YTAEGVVVRQ FIYTIADVVS KVIYCIILTQ ICMTTVMLLR EQIA 284 Truepera radiovictrix DSM 17093 (YP_003705905, SEQ ID NO: 6) MLTMENLLTY SPVSYSIVSN TLTLGYAAMA AGLVYFVTTS KRAAPPYRLS STLSAVVMVS  60 AFLALYQLHQ TWLSAFTFNG EVWEGGATAF NNGYRYINWS IDVPILLTQL LIVMGFTGAR 120 FRRLWLQFVV AGLAMIYTGY AGQFYEATDS ARLYLWGAIS TAFFLWILVL VRRTIFDPPD 180 ALPERAAGLM RGVWWVLLGS WLLYPGAYLM PVFALSEGGV VARQISFTVA DVVSKVIYGV 240 MLSRVAELRS QADGYAHALE SETQPNPDRR GGAGVAAVQP QGGRAPRKGR VHRS 294 T. radiovictrix DSM 17093 (YP_003706581; SEQ ID NO: 7) MQPSPHRLGN ATFENYVGLE SGYTEMAYQM SAHVLTVGYA IMLAALIYFI LTIRQVRPRY  60 RMSSILSVVV MVSAFLLLLI QQLNWTSALQ FDPATARYRL APEGVEGIVT AGDLFNNGYR 120 YLNWLIDVPM LLFQILFVVT LSRSSFASVR NQFWFSGVAM IITGYVGQFY EVTRPGLFFL 180 WGSISTVFFI HILIVMRRVI KEGVENAPDS AKGMLGAIWP LFLISWMLYP GAYLMPYLYS 240 FSLEPALSES AVVARHLTYT VADVTSKVIY GVLLAAAATR MSKAEGYDWE VQTA 294 Gillisia limnaea R-8282 (ZP_09669334, SEQ ID NO: 8) MFGDFNRVDN PILSLSYFKI QLTGIGLCRL LKPCLNFSKN IKHIKKFIKI NGNKFEIDYF  60 DMTQELGNAN FENFIGATEG FSEIAYQFTS HILTLGYAVM LAGLLYFILT IKKVDKKYQM 120 SNILSAVVMV SAFLLLYAQA ENWTTSFTFD ISRGKYFLEP NGDLFNNGYR YLNWLIDVPM 180 LLFQILFVVS LTKSKFSSIR NQFWFSGAMM IITGYIGQFY EVSNLTAFFV WGAISSVFFF 240 HILWVMKKVI NEGKEGLSAD AQKILSNIWV LFLVSWFLYP GAYLMPYLTG LDGFFFSEDG 300 VMARQLTYTI ADVCSKVIYG VLLGNLALKL SNNKEMVELS N 341 Krokinobacter (Dokdonia) sp. 4H-3-7-5(YP_004429763, SEQ ID NO: 9) MTQELGNANF ENFIGATEGF SEIAYQFTSH ILTLGYAVML AGLLYFILTI KNVDKKFQMS  60 NILSAVVMVS AFLLLYAQAQ NWTSSFTFNE EVGRYFLDPS GDLFNNGYRY LNWLIDVPML 120 LFQILFVVSL TTSKFSSVRN QFWFSGAMMI ITGYIGQFYE VSNLTAFLVW GAISSAFFFH 180 ILWVMKKVIN EGKEGISPAG QKILSNIWIL FLISWFLYPG AYLMPYLTGV DGFLYSEDGV 240 MARQLVYTIA DVCSKVIYGV LLGNLAITLS KNKELVEANS 280 C-terminal tag (SEQ ID NO: 10) LEHHHHHH   8

EXAMPLES

Example 1

[0041] Expression Plasmid

[0042] Krokinobacter eikastus sodium pumping rhodopsin gene (Uniprot ID N0DKS8) coding DNA was synthesized de novo. The nucleotide sequence was optimized for E. coli expression using the GeneOptimizer™ software (Life Technologies, USA). The gene was introduced into the pSCodon1.2 expression vector (Staby™ Codon T7, Eurogentec, Belgium) via NdeI and XhoI restriction sites. Consequently, the expressed construct harbored an additional C-terminal tag with a sequence LEHHHHHH (SEQ ID NO: 10).

[0043] Protein Expression and Purification

[0044] E. coli cells of strain SE1 (Staby™ Codon T7, Eurogentec, Belgium) were transformed with the KR2 expression plasmid. Transformed cells were grown at 37° C. in shaking baffled flasks in an auto-inducing medium ZYP-5052 (Studier, F. W. Protein Expression and Purification 41, 207-234 (2005); incorporated herein by reference) containing 100 mg/L ampicillin. When glucose level in the growing bacterial culture dropped below 10 mg/L, 10 μM all-trans-retinal (Sigma-Aldrich, Germany) was added, the incubation temperature was reduced to 20° C. and incubation continued overnight. Collected cells were disrupted in M-110P Lab Homogenizer (Microfluidics, USA) at 25000 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 90000 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. Insoluble fraction was removed by ultracentrifugation at 90000 g for 1 h at 4° C. The supernatant was loaded on Ni-NTA column (Qiagen, Germany) and KR2 was eluted in a buffer containing 50 mM NaH.sub.2PO.sub.4/Na.sub.2HPO.sub.4 pH 7.5, 0.1 M NaCl, 0.5 M imidazole and 0.1% DDM. The eluate was subjected to size-exclusion chromatography on 125 ml Superdex 200 PG column (GE Healthcare Life Sciences, USA) in a buffer containing 50 mM NaH.sub.2PO.sub.4/Na.sub.2HPO.sub.4 pH 7.5, 0.1 M NaCl, 0.05% DDM. Protein-containing fractions with the minimal A280/A525 absorbance ratio were pooled and concentrated, e.g. to 40 mg/ml.

[0045] Measurements of the Pump Activity in E. coil Cells

[0046] E. coli cells of strain C41(DE3) (Lucigen, USA) were transformed with the KR2 expression plasmid. Transformed cells were grown at 37° C. in shaking baffled flasks in an auto-inducing medium ZYP-5052 (Studier, F. W. Protein Expression and Purification 41, 207-234 (2005); incorporated herein by reference) containing 100 mg/L ampicillin and induced at the optical density OD.sub.600 of 0.6-0.7 with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and 10 μM all-trans-retinal. 3 hours after the induction the cells were collected by centrifugation at 3000 g for 10 min and washed three times with unbuffered salt solution (100 mM NaCl or KCl, 10 mM MgCl.sub.2) 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 or KCl solution respectively and adjusted to OD.sub.600 of 8.5. The measurements were performed on three milliliters of stirred cell suspension kept at 1° C. The cells were illuminated for 5 minutes using the halogen lamp Intralux 5000-1 (VOLPI, Switzerland) and the light-induced pH changes were monitored with a pH meter LAB 850 (SCHOTT Instruments, Germany). Measurements were repeated under the same conditions after addition of 30 μM of CCCP and further addition of 20 mM of TPP.sup.+. For measurements at pH 5.6 the acidity was adjusted with 10 mM HCl and the cells were equilibrated for 30 minutes.

[0047] Upon dissolution of the crystal structure of KR2, an ion translocation pathway lined with polar and ionizable residues was hypothesized (FIG. 3a). There are three major cavities on this pathway (FIG. 3b). The first cavity protrudes from the protein surface towards Gln-123, situated at the position usually occupied by the proton donor residue in light-driven proton pumps (Lanyi, J. K. Annual Review of Physiology 66, 665-688 (2004); Bamann, C., et al. Biochimica et Biophysica Acta (BBA)-Bioenergetics 1837, 614-625 (2014)). Such wide opening of this cavity already in the ground state is unique among the microbial rhodopsins of known structure. The cavity is separated from the retinal by the hydrophobic residues Val-67 and Leu-120, conserved among bacterio- and proteorhodopsins. The second cavity precedes the Arg-109 side chain. The third cavity is situated in the region usually harboring the proton release group in light-driven proton pumps.

[0048] The cytoplasmic side of the protein is identical in all of the presented structures, and reveals a hydrophilic cavity protruding from the protein surface at the hydrophobic membrane core boundary towards the buried Gln-123, the residue replacing the proton donor residues of archaeal and bacterial proton pumps (FIG. 3b). Such protrusion is absent in other light-driven pumps, as there are much bulkier amino acids at the position of KR2's Gly-263 (FIG. 4a).

[0049] To determine the role of the cavity, we measured the activity of the mutants N61M, G263F and G263L (FIG. 4 and Fig.5). Mutation N61M, which makes the cavity less polar, results in lesser efficiency of sodium translocation and increased efficiency of proton translocation (FIG. 4b). Effects of the mutations of Gly-263 are more profound. While the sodium-pumping ability of the G263F mutant is severely impaired, the protein gained the potassium-pumping ability (FIG. 4b), as E. coli cells overexpressing KR2 alkalified the surrounding media under illumination in the KCI solution both in absence and presence of the protonophore carbonylcyanide-m-chlorophenylhydrazone (CCCP). This observation is supported by measurements of the photocurrents generated by KR2 reconstituted into liposomes.

[0050] Measurements of the Pump Activity in the Liposomes Attached to BLM

[0051] KR2 was reconstituted into liposomes (80% POPC, 10% POPG, 10% cholesterol (w/w), Avanti/Sigma Aldrich) at protein/lipid ratio of 7:100 (w/w). The detergent was removed by overnight stirring at 4° C. with the detergent-absorbing beads (Bio-Beads SM 2, BioRad). Optically BLMs were formed as described previously (Bamberg, E. et al. Biophys. Struct. Mechanism 5, 277-292 (1979); incorporated herein by reference). The electrolyte solution was 20 mM HEPES pH 7.4 without any Na.sup.+ or K.sup.+ ions. KR2-containing proteoliposomes were added to one of the compartments under gentle stirring. The system was illuminated with a mercury arc lamp (Osram HBO 100) at wavelengths >455 nm. Photosensivity of the samples developed over time and reached maximal current amplitudes after ˜30 minutes. 2 μM of the protonophore 1799 ((2,6-dihydroxy)-1,1,1,7,7,7-hexafluoro-2,6-bis(trifluoromethyl)-heptane-4-one) was added to both compartments, which effectively permeabilizes the compound membrane system for protons. Sodium and potassium titration was performed with NaCl and KCI solutions respectively by addition to both compartments of the cuvette. The system conductance remained constant during the titration. Subsequently, the membrane was made permeable for the cations, too, by the addition of the exchanger monensin. Photocurrents were measured under short-circuit conditions, so that no external driving force is generated. Further details of the system were as described (Bamberg, E. et al. Biophys. Struct. Mechanism 5, 277-292 (1979); incorporated herein by reference).

[0052] In the absence of Na.sup.+/K.sup.+ ions the wild type KR2 shows a stable stationary current that reflects the proton pumping activity of the protein. This pumping activity almost remains the same during potassium titration (FIG. 4c). However, the photocurrent amplitude rises significantly when the sodium concentration is raised (FIG. 4c). Declining curves at higher concentrations of sodium show that the protein is pumping sodium against the created chemical potential. This effect can be removed by the addition of the Na/H-exchanger monensin resulting in stable stationary currents.

[0053] The G263F mutant behaves differently. In the absence of Na.sup.+/K.sup.+ ions the stationary current is very low, showing that proton pumping is almost absent. Still the photocurrents preserve the dependence on sodium ions concentration (FIG. 4c), but the pumping became much less efficient. Addition of potassium ions results in drastic increase of the photocurrents (FIG. 4c), suggesting that G263F can pump potassium ions.

[0054] In contrast thereto, the G263L mutation resulted in the strongest inhibition of light-driven pumping. Based on the obtained data, we conclude that the ion uptake cavity plays a significant role in KR2 ion selectivity, and might be its selectivity filter.

Example 2

[0055] The KR2 G263F K.sup.+-pumping mutant is inserted into cultivated hippocampal cells together with the light-gated cation channel channelrhodopsin 2(ChR2). Expression of the KR2 G263F mutant is expected to allow silencing these electrically excitable cells by light. A similar effect was demonstrated by Zhang et.al (Nature. 446(7136), 633-639 (2007)). In this publication the hyperpolarizing pump halorhodopsin (NphR) was used to inhibit the light induced firing of neurons, which were activated by the light induced depolarization via ChR2. The outwardly directed KR2 G263F K.sup.+-pump, however, is supposed to act in a much less invasive manner as NphR, because under physiological conditions the pump can use after light activation the naturally occurring potassium gradient. Therefore, KR2 G263F mimicks the action of endogenous outwardly directed K.sup.+ channels, which are responsible for the repolarization(silencing) of electrically excitable cells. These properties make KR2 G263F to an optimal optogenetic tool for silencing of neurons and muscle cells.

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