NOVEL MUTANT BACTERIORHODOPSIN-LIKE-CHANNELRHODOPSIN ION CHANNEL

Abstract

The present invention relates to a mutant ion channel capable of being activated by light (‘light-activated’ ion channel) and having improved properties, nucleic acids and expression vectors encoding the mutant ion channel, cells comprising such nucleic acid or expression vector, devices containing the mutant ion channel, nucleic acid or expression vector as well as respective uses and methods.

Claims

1. A mutant ion channel, wherein the mutant ion channel comprises: a 7-transmembrane-helix motif having at least 70% amino acid sequence identity to the full-length sequence of the 7-transmembrane-helix motif of the wild-type ion channel RICCR1 set forth in SEQ ID NO: 9, and an amino acid substitution at one or both of the positions within said motif of the mutant ion channel which correspond to positions T218 and S220 of RICCR1 set forth in SEQ ID NO: 5; and wherein the mutant ion channel is capable of being activated by light.

2. The mutant ion channel of claim 1, wherein the mutant ion channel further shows reduced light-dependent desensitization compared to a reference ion channel which has a Thr at the amino acid position corresponding to T218 in SEQ ID NO:5 and a Ser at the amino acid position corresponding to S220 in SEQ ID NO:5 and otherwise is identical to the mutant ion channel.

3. The mutant ion channel of claim 1, wherein the amino acid sequence of the 7-transmembrane-helix motif of the mutant ion channel has at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99% identity to the full-length sequence of SEQ ID NO: 9; or wherein the mutant ion channel comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99% identity to the full-length sequence of SEQ ID NO: 5.

4. The mutant ion channel of claim 1, wherein the amino acid at the position corresponding to position T218 of SEQ ID NO: 5 is selected from Leu, Ile, Val, Met, Cys, Phe, Ala, Gly, Pro and Trp, and preferably is Leu.

5. The mutant ion channel of claim 1, wherein the amino acid at the position corresponding to position S220 of SEQ ID NO: 5 is selected from Ala, Gly, Leu, Val, Ile, Met, Pro, Cys and Trp, and preferably is Ala.

6. The mutant ion channel of claim 1, wherein the mutant ion channel further comprises one or more of the following additional amino acid substitutions: a Phe at the amino acid position corresponding to Y260 in SEQ ID NO:5, a His at the amino acid position corresponding to R136 in SEQ ID NO:5, a Trp at the amino acid position corresponding to S138 in SEQ ID NO:5, a Phe at the amino acid position corresponding to Y156 in SEQ ID NO:5, a Val at the amino acid position corresponding to T119 in SEQ ID NO:5, a Phe at the amino acid position corresponding to Y116 in SEQ ID NO:5; preferably, wherein the amino acid sequence of the 7-transmembrane-helix motif of the mutant ion channel is identical with the full-length sequence of SEQ ID NO: 9, except for the amino acid substitutions at one or both of the amino acid positions corresponding to positions T218 and S220 of SEQ ID NO: 5, and optionally one or more of the following additional amino acid substitutions: a Phe at the amino acid position corresponding to Y260 in SEQ ID NO:5, a His at the amino acid position corresponding to R136 in SEQ ID NO:5, a Trp at the amino acid position corresponding to S138 in SEQ ID NO:5, a Phe at the amino acid position corresponding to Y156 in SEQ ID NO:5, a Val at the amino acid position corresponding to T119 in SEQ ID NO:5, a Phe at the amino acid position corresponding to Y116 in SEQ ID NO:5.

7. The mutant ion channel of claim 1, wherein the mutant ion channel comprises the following amino acid sequence motif: Ala-Glu-His-Ser-Leu-His-Val-Leu-Lys-Phe-Ala-Val-Phe-Xaa1-Phe-Xaa2-MetLeu-Trp-Ile-Leu-Phe-Pro-Leu-Val-Trp-Ala-Ile (SEQ ID NO: 13) wherein: (a) Xaa1 is selected from Leu, Ile, Val, Met, Cys, Phe, Ala, Gly, Pro and Trp, and preferably is Leu, and Xaa2 is selected from Ala, Gly, Leu, Val, Ile, Met, Pro, Cys and Trp, and preferably is Ala; or (b) Xaa1 is selected from Leu, Ile, Val, Met, Cys, Phe, Ala, Gly, Pro and Trp, and preferably is Leu, and Xaa2 is selected from Ser, Thr, Tyr, Gln and Asn, and preferably is Ser; or (c) Xaa1 is selected from Thr, Ser, Tyr, Gln and Asn, and preferably is Thr, and Xaa2 is selected from Ala, Gly, Leu, Val, Ile, Met, Pro, Cys and Trp, and preferably is Ala.

8. The mutant ion channel of claim 1, wherein the mutant ion channel provides an at least 1.5-times, at least 1.7-times, or at least 2.0-times, and, optionally, up to 3.5-times, up to 3.0-times, or up to 2.9-times, higher stationary-peak-ratio than a reference ion channel which has a Thr at the amino acid position corresponding to T218 in SEQ ID NO:5 and a Ser at the amino acid position corresponding to S220 in SEQ ID NO:5 and otherwise is identical to the mutant ion channel; wherein the stationary-peak-ratio is measurable by whole-cell patch-clamp measurement of photocurrents in an NG108-15 cell expressing the mutant ion channel or the reference ion channel, respectively, at a membrane potential of −60 mV, in said whole-cell patch-clamp measurement, the photocurrents are measured upon illumination of the NG108-15 cell with a 2s light pulse of a wavelength of 532 nm at saturating intensity of 23 mW/mm.sup.2 to determine the mean stationary current of the last 100 ms of the 2s light pulse and the peak current of the 2s light pulse; and wherein the stationary-peak-ratio is the quotient of the mean stationary current of the last 100 ms of the 2s light pulse and the peak current of the 2s light pulse.

9. The mutant ion channel of claim 1, wherein the mutant ion channel provides an at least 1.5-times, at least 1.7-times, or at least 2.0-times, and, optionally, up to 3.5-times, up to 3.0-times, or up to 2.9-times, higher mean stationary-peak-ratio than a reference ion channel which has a Thr at the amino acid position corresponding to T218 in SEQ ID NO:5 and a Ser at the amino acid position corresponding to S220 in SEQ ID NO:5 and otherwise is identical to the mutant ion channel; wherein the mean stationary-peak-ratio is the mean of the stationary-peak-ratios of at least 5, at least 10, at least 15, e.g., 5-100, 10-75 or 15-60 individual NG108-15 cells expressing the mutant ion channel or from the stationary photocurrent densities of the same number of individual NG108-15 cells expressing the reference ion channel, respectively; wherein the stationary-peak-ratio of an individual NG108-15 cell is measurable by whole-cell patch-clamp measurement of photocurrents in the NG108-15 cell at a membrane potential of −60 mV, in said whole-cell patch-clamp measurement, the photocurrents are measured upon illumination of the NG108-15 cell with a 2s light pulse of a wavelength of 532 nm at saturating intensity of 23 mW/mm.sup.2 to determine the mean stationary current of the last 100 ms of the 2s light pulse and the peak current of the 2s light pulse; and wherein the stationary-peak-ratio of the NG108-15 cell is the quotient of the mean stationary current of the last 100 ms of the 2s light pulse and the peak current of the 2s light pulse.

10. The mutant ion channel of claim 1, wherein the mutant ion channel provides an at least 1.5-times, at least 1.7-times, or at least 2.0-times, and, e.g., up to 5.5-times, up to 5.0-times, or up to 4.5-times, higher stationary photocurrent density than a reference ion channel which has a Thr at the amino acid position corresponding to T218 in SEQ ID NO:5 and a Ser at the amino acid position corresponding to S220 in SEQ ID NO:5 and otherwise is identical to the mutant ion channel; wherein the stationary photocurrent density is measurable by whole-cell patch-clamp measurements with an NG108-15 cell expressing the mutant ion channel or the reference ion channel, respectively; in said whole-cell patch-clamp measurements: transient capacitive currents in response to voltage steps are measured to determine the capacitance of the NG108-15 cell, and photocurrents at a membrane potential of −60 mV are measured upon illumination of the NG108-15 cell with a 2s light pulse of a wavelength of 532 nm at saturating intensity of 23 mW/mm.sup.2 to determine the mean stationary current of the last 100 ms of the 2s light pulse; and wherein the stationary photocurrent density is the quotient of the mean stationary current of the last 100 ms of the 2s light pulse and the capacitance.

11. The mutant ion channel of claim 1, wherein the mutant ion channel provides an at least 1.5-times, at least 1.7-times, or at least 2.0-times, and, e.g., up to 5.5-times, up to 5.0-times, or up to 4.5-times, higher mean stationary photocurrent density than a reference ion channel which has a Thr at the amino acid position corresponding to T218 in SEQ ID NO:5 and a Ser at the amino acid position corresponding to S220 in SEQ ID NO:5 and otherwise is identical to the mutant ion channel; wherein the mean stationary photocurrent density is the mean of the stationary photocurrent densities of at least 5, at least 10, at least 15, e.g., 5-100, 10-75 or 15-60 individual NG108-15 cells expressing the mutant ion channel or from the stationary photocurrent densities of the same number of individual NG108-15 cells expressing the reference ion channel, respectively; wherein the stationary photocurrent density of an individual NG108-15 cell is measurable by whole-cell patch-clamp measurements; in said whole-cell patch-clamp measurements: transient capacitive currents in response to voltage steps are measured to determine the capacitance of the NG108-15 cell, and photocurrents at a membrane potential of −60 mV are measured upon illumination of the NG108-15 cell with a 2s light pulse of a wavelength of 532 nm at saturating intensity of 23 mW/mm.sup.2 to determine the mean stationary current of the last 100 ms of the 2s light pulse; and wherein the stationary photocurrent density of the NG108-15 cell is the quotient of the mean stationary current of the last 100 ms of the 2s light pulse and the capacitance of the NG108-15 cell.

12. The mutant ion channel of claim 1, wherein the mutant ion channel has: an Asp at the amino acid position corresponding to D115 in SEQ ID NO:5, a Thr or Val, preferably a Thr, at the amino acid position corresponding to T119 in SEQ ID NO:5, and an Asp at the amino acid position corresponding to D126 in SEQ ID NO:5.

13. The mutant ion channel of claim 1, wherein said capability of being activated by light is the capability of the mutant ion channel to provide a photocurrent in a cell which comprises the mutant ion channel in its plasma membrane and is exposed to light, in particular light of a wavelength in the range of 400-600 nm, 450-570 nm or 500-540 nm.

14. The mutant ion channel of claim 13, wherein said photocurrent is characterized in that the mutant ion channel provides a stationary photocurrent density of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the stationary photocurrent density provided by the wild-type ion channel RICCR1 set forth in SEQ ID NO: 5; wherein the stationary photocurrent density is measurable by whole-cell patch-clamp measurements with an NG108-15 cell expressing the mutant ion channel or RICCR1 set forth in SEQ ID NO: 5, respectively; in said whole-cell patch-clamp measurements: transient capacitive currents in response to voltage steps are measured to determine the capacitance of the NG108-15 cell, and photocurrents at a membrane potential of −60 mV are measured upon illumination of the NG108-15 cell with a 2s light pulse of a wavelength of 532 nm at saturating intensity of 23 mW/mm.sup.2 to determine the mean stationary current of the last 100 ms of the 2s light pulse; and wherein the stationary photocurrent density is the quotient of the mean stationary current of the last 100 ms of the 2s light pulse and the capacitance.

15. The mutant ion channel of claim 13, wherein said photocurrent is characterized in that the mutant ion channel provides a mean stationary photocurrent density of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the mean stationary photocurrent density provided by the wild-type ion channel RICCR1 set forth in SEQ ID NO: 5; wherein the mean stationary photocurrent density is the mean of the stationary photocurrent densities of at least 5, at least 10, at least 15, e.g., 5-100, 10-75 or 15-60 individual NG108-15 cells expressing the mutant ion channel or from the stationary photocurrent densities of the same number of individual NG108-15 cells expressing RICCR1 set forth in SEQ ID NO: 5, respectively; wherein the stationary photocurrent density of an individual NG108-15 cell is measurable by whole-cell patch-clamp measurements; in said whole-cell patch-clamp measurements: transient capacitive currents in response to voltage steps are measured to determine the capacitance of the NG108-15 cell, and photocurrents at a membrane potential of −60 mV are measured upon illumination of the NG108-15 cell with a 2s light pulse of a wavelength of 532 nm at saturating intensity of 23 mW/mm.sup.2 to determine the mean stationary current of the last 100 ms of the 2s light pulse; and wherein the stationary photocurrent density of the NG108-15 cell is the quotient of the mean stationary current of the last 100 ms of the 2s light pulse and the capacitance of the NG108-15 cell.

16. A method of using a nucleic acid encoding a mutant ion channel of claim 1 for treating or ameliorating loss of vision, or for treating or ameliorating loss of hearing.

17. The method of claim 16, wherein the nucleic acid is used for treating or ameliorating loss of hearing, and is introduced into spiral ganglion neurons of a human or a non-human animal in need of such treatment or amelioration to render said neurons light-sensitive.

18. The method of claim 17, wherein the human or non-human animal is equipped with an optical cochlear implant to allow for at least partial restoration of hearing.

19. The method of claim 16, wherein the amino acid at the position corresponding to position T218 of SEQ ID NO: 5 is selected from Leu, Ile, Val, Met, Cys, Phe, Ala, Gly, Pro and Trp, and preferably is Leu, and/or wherein the amino acid at the position corresponding to position S220 of SEQ ID NO: 5 is selected from is Ala, Gly, Leu, Val, Ile, Met, Pro, Cys and Trp, and preferably is Ala.

Description

DESCRIPTION OF THE FIGURES

[0319] FIG. 1: R/CCR1 mutants with reduced desensitization (increased Stationary-Peak-Ratio) at saturating intensity. NG108-15 cells transiently transfected with R/CCR1-EYFP (WT) (square), R/CCR1-EYFP S220A (circle), R/CCR1-EYFP T218L (triangle) and R/CCR1-EYFP T218L/S220A (rhombus) were investigated by whole-cell patch-clamp recordings at a membrane potential of −60 mV. Number of cells measured as indicated by n numbers in the legend. Photocurrents were measured upon illumination with a 2 s light pulse of a wavelength of λ=532 nm at saturating intensity of 23 mW/mm.sup.2. The stationary-peak-ratio was calculated as the quotient of the mean stationary current of the last 100 ms of the 2s light pulse and the peak current. Bars indicate mean and SD. Statistical analysis was performed by one-way ANOVA followed by post-hoc Bonferroni-test. P-values<0.05 were considered significant.

[0320] FIG. 2: R/CCR1 mutants with increased photocurrent density at saturating intensity. NG108-15 cells transiently transfected with R/CCR1-EYFP (WT) (square) (n=57), R/CCR1-EYFP S220A (circle) (n=38), R/CCR1-EYFP T218L (triangle) (n=18) and R/CCR1-EYFP T218L/S220A (rhombus) (n=21) were investigated by whole-cell patch-clamp recordings at a membrane potential of −60 mV. Photocurrents were measured upon illumination with a 2 s light pulse of a wavelength of λ=532 nm at saturating intensity of 23 mW/mm.sup.2. Number of cells measured as indicated by n numbers in the legend. Photocurrent densities shown were calculated as the quotient of the mean stationary current of the last 100 ms of the 2s light pulse normalized to cell capacitance. Bars indicate mean and SD. Statistical analysis was performed by one-way ANOVA followed by post-hoc Bonferroni-test. P-values<0.05 were considered significant.

[0321] FIG. 3: Desensitization of R/CCR1 variants at different light intensities. NG108-15 cells transiently transfected with R/CCR1-EYFP (WT) (square), R/CCR1-EYFP S220A (circle), R/CCR1-EYFP T218L (triangle) and R/CCR1-EYFP T218L/S220A (rhombus) were investigated by whole-cell patch-clamp recordings at a membrane potential of −60 mV. Photocurrents were measured upon illumination with a 2 s light pulse of a wavelength of λ=532 nm at different light intensity ranging from 0,0024 mW/mm.sup.2 to 23 mW/mm.sup.2. The stationary-peak-ratio was calculated as the quotient of the mean stationary current of the last 100 ms of the 2s light pulse and the peak current. Bars indicate mean and SD. (a), (c) and (e) show the stationary-peak-ratio across the range of light intensities for the R/CCR1 variants (a) R/CCR1-EYFP S220A (n=4), (b) R/CCR1-EYFP T218L (n=4) and (c) R/CCR1-EYFP T218L/S220A (n=4) in comparison to R/CCR1-EYFP (WT) (n=5). (b), (d) and (f) show exemplary photocurrent traces at saturating light intensity (23 mW/mm.sup.2) of (b) R/CCR1-EYFP S220A, (d) R/CCR1-EYFP T218L and (f) R/CCR1-EYFP T218L/S220A in comparison to R/CCR1-EYFP (WT) with the wild type trace depicted in light grey which is (nearly entirely) above the trace for the respective R/CCR1 variant in dark grey.

[0322] FIG. 4: Light intensity dependence of stationary photocurrent of R/CCR1 variants. NG108-15 cells transiently transfected with R/CCR1-EYFP (WT) (square), R/CCR1-EYFP S220A (circle), R/CCR1-EYFP T218L (triangle) and R/CCR1-EYFP T218L/S220A (rhombus) were investigated by whole-cell patch-clamp recordings at a membrane potential of −60 mV. Number of cells measured as indicated by n numbers in the legend. Stationary photocurrents were measured upon illumination with a 2 s light pulse of a wavelength of λ=532 nm at different light intensity ranging from 0,0024 mW/mm.sup.2 to 23 mW/mm.sup.2. The stationary photocurrent (I.sub.stat) was measured as the mean stationary current of the last 100 ms of the 2s light pulse and is depicted normalized to the maximum stationary current measured for each cell. Bars indicate mean and SD.

[0323] FIG. 5. Characterizing the optogenetic activation of the mouse auditory pathway by means of oABRs. A) Schematic representation of the approach to optogenetic manipulation and acquisition of optical auditory brainstem recordings (oABR) in mice. Spiral ganglion neurons (SGNs), were rendered light-sensitive by injection of AAV-ChR construct through the round window of mice at postnatal day 6. Following 6 to 13 weeks, the cochlea of mice was exposed, and a laser fiber, coupled to a 594 nm laser, was inserted into the round window. (B) oABRs driven with varying radiant flux (1 ms pulses at 10 and 20 Hz, grey levels code the radiant flux in mW) for an exemplary mouse injected with the AAV-ChR construct AAV2/9-R/CCR1-EYFP (WT). D) Threshold of minimum light intensity eliciting a detectable oABR response for R/CCR1-EYFP (WT) (light grey, left-hand side) and R/CCR1-EYFP T218L/S220A (dark gray, right-hand side). Student's t-test (**P≤0.005). E) P1-N1 amplitude, and F) P1 latency (1 ms pulses at 10 and 20 Hz; radiant flux from 20-37 mW). G) P1-N1 amplitudes with varying radiant flux (1 ms pulses at 10 and 20 Hz). H) Latency of oABR P1 with varying pulse duration (radiant flux 20-37 mW at 10 and 20 Hz). I) Latency of oABR P1 as a function of stimulus rate (1 ms pulses with radiant flux of 20-37 mW). Data represents mean (+/−SD) of n=6 mice injected with AAV2/9-R/CCR1-EYFP (WT), and n=8 mice injected with AAV2/9-R/CCR1-EYFP T218L/S220A.

[0324]

TABLE-US-00005 DESCRIPTION OF THE SEQUENCES SEQ ID NO: 1 (ChR2 (chop2-315); accession number of Chop2-737: AF461397; 315 aa) MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGTNGAQTASNVLQWLAAGF SILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFFEFKNPSMLYLATGHRVQWLRYAEWL LTCPVILIHLSNLTGLSNDYSRRTMGLLVSDIGTIVWGATSAMATGYVKVIFFCLGLCYGANTFF HAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVSWGMFPILFILGPEGFGVLSVYGSTVGHTIID LMSKNCWGLLGHYLRVLIHEHILIHGDIRKTTKLNIGGTEIEVETLVEDEAEAGAVNKGTGK SEQ ID NO: 2 (Chrimson; accession number: KF992060; 350 aa) MAELISSATRSLFAAGGINPWPNPYHHEDMGCGGMTPTGECFSTEWWCDPSYGLSDAGYGY CFVEATGGYLVVGVEKKQAWLHSRGTPGEKIGAQVCQWIAFSIAIALLTFYGFSAWKATCGW EEVYVCCVEVLFVTLEIFKEFSSPATVYLSTGNHAYCLRYFEWLLSCPVILIKLSNLSGLKNDYSK RTMGLIVSCVGMIVFGMAAGLATDWLKWLLYIVSCIYGGYMYFQAAKCYVEANHSVPKGHCR MVVKLMAYAYFASWGSYPILWAVGPEGLLKLSPYANSIGHSICDIIAKEFWTFLAHHLRIKIHE HILIHGDIRKTTKMEIGGEEVEVEEFVEEEDEDTV SEQ ID NO: 3 (VChR1; accession number: EU622855; 300 aa) MDYPVARSLIVRYPTDLGNGTVCMPRGQCYCEGWLRSRGTSIEKTIAITLQWVVFALSVACLG WYAYQAWRATCGWEEVYVALIEMMKSIIEAFHEFDSPATLWLSSGNGVVWMRYGEWLLTCP VLLIHLSNLTGLKDDYSKRTMGLLVSDVGCIVWGATSAMCTGWTKILFFLISLSYGMYTYFHAA KVYIEAFHTVPKGICRELVRVMAWTFFVAWGMFPVLFLLGTEGFGHISPYGSAIGHSILDLIAK NMWGVLGNYLRVKIHEHILLYGDIRKKQKITIAGQEMEVETLVAEEED SEQ ID NO: 4 (ReaChR; accession number KF448069; 350 aa) MVSRRPWLLALALAVALAAGSAGASTGSDATVPVATQDGPDYVFHRAHERMLFQTSYTLENN GSVICIPNNGQCFCLAWLKSNGTNAEKLAANILQWVVFALSVACLGWYAYQAWRATCGWEE VYVALIEMMKSIIEAFHEFDSPATLWLSSGNGVVWMRYGEWLLTCPVILIHLSNLTGLKDDYSK RTMGLLVSDVGCIVWGATSAMCTGWTKILFFLISLSYGMYTYFHAAKVYIEAFHTVPKGLCRQ LVRAMAWLFFVSWGMFPVLFLLGPEGFGHISPYGSAIGHSILDLIAKNMWGVLGNYLRVKIHE HILLYGDIRKKQKITIAGQEMEVETLVAEEEDKYESS SEQ ID NO: 5 (RICCR1; ChRmine (RICCR1-309); accession number of RICCR1-332: MN585304; accession number of RICCR1-309 (ChRmine): MN194599; 309 aa; T218 and S220 highlighted in bold; D115 ,Y116, T119, D126, R136, S138, Y156 and Y260 underlined) MAHAPGTDQMFYVGTMDGWYLDTKLNSVAIGAHWSCFIVLTITTFYLGYESWTSRGPSKRTS FYAGYQEEQNLALFVNFFAMLSYFGKIVADTLGHNFGDVGPFIIGFGNYRYADYMLTCPMLVY DLLYQLRAPYRVSCSAIIFAILMSGVLAEFYAEGDPRLRNGAYAWYGFGCFWFIFAYSIVMSIVA KQYSRLAQLAQDTGAEHSLHVLKFAVFTFSMLWILFPLVWAICPRGFGWIDDNWTEVAHCVC DIVAKSCYGFALARFRKTYDEELFRLLEQLGHDEDEFQKLELDMRLSSNGERLRRLS SEQ ID NO: 6 (RICCR1 T218L; 309 aa, mutated residue highlighted in bold) MAHAPGTDQMFYVGTMDGWYLDTKLNSVAIGAHWSCFIVLTITTFYLGYESWTSRGPSKRTS FYAGYQEEQNLALFVNFFAMLSYFGKIVADTLGHNFGDVGPFIIGFGNYRYADYMLTCPMLVY DLLYQLRAPYRVSCSAIIFAILMSGVLAEFYAEGDPRLRNGAYAWYGFGCFWFIFAYSIVMSIVA KQYSRLAQLAQDTGAEHSLHVLKFAVFLFSMLWILFPLVWAICPRGFGWIDDNWTEVAHCVC DIVAKSCYGFALARFRKTYDEELFRLLEQLGHDEDEFQKLELDMRLSSNGERLRRLS SEQ ID NO: 7 (RICCR1 S220A; 309 aa, mutated residue highlighted in bold) MAHAPGTDQMFYVGTMDGWYLDTKLNSVAIGAHWSCFIVLTITTFYLGYESWTSRGPSKRTS FYAGYQEEQNLALFVNFFAMLSYFGKIVADTLGHNFGDVGPFIIGFGNYRYADYMLTCPMLVY DLLYQLRAPYRVSCSAIIFAILMSGVLAEFYAEGDPRLRNGAYAWYGFGCFWFIFAYSIVMSIVA KQYSRLAQLAQDTGAEHSLHVLKFAVFTFAMLWILFPLVWAICPRGFGWIDDNWTEVAHCVC DIVAKSCYGFALARFRKTYDEELFRLLEQLGHDEDEFQKLELDMRLSSNGERLRRLS SEQ ID NO: 8 (RICCR1 T218L/S220A; 309 aa, mutated residues highlighted in bold) MAHAPGTDQMFYVGTMDGWYLDTKLNSVAIGAHWSCFIVLTITTFYLGYESWTSRGPSKRTS FYAGYQEEQNLALFVNFFAMLSYFGKIVADTLGHNFGDVGPFIIGFGNYRYADYMLTCPMLVY DLLYQLRAPYRVSCSAIIFAILMSGVLAEFYAEGDPRLRNGAYAWYGFGCFWFIFAYSIVMSIVA KQYSRLAQLAQDTGAEHSLHVLKFAVFLFAMLWILFPLVWAICPRGFGWIDDNWTEVAHCVC DIVAKSCYGFALARFRKTYDEELFRLLEQLGHDEDEFQKLELDMRLSSNGERLRRLS SEQ ID NO: 9 (7-transmembrane-helix motif of RICCR1; 7TM motif of RICCR1; Ser27 to Lys269 of SEQ ID NO:5; 243 aa) SVAIGAHWSCFIVLTITTFYLGYESWTSRGPSKRTSFYAGYQEEQNLALFVNFFAMLSYFGKIV ADTLGHNFGDVGPFIIGFGNYRYADYMLTCPMLVYDLLYQLRAPYRVSCSAIIFAILMSGVLAEF YAEGDPRLRNGAYAWYGFGCFWFIFAYSIVMSIVAKQYSRLAQLAQDTGAEHSLHVLKFAVFT FSMLWILFPLVWAICPRGFGWIDDNWTEVAHCVCDIVAKSCYGFALARFRK SEQ ID NO: 10 (7TM motif of RICCR1 T218L; Ser27 to Lys269 of SEQ ID NO: 6; 243 aa; mutated residue highlighted in bold) SVAIGAHWSCFIVLTITTFYLGYESWTSRGPSKRTSFYAGYQEEQNLALFVNFFAMLSYFGKIV ADTLGHNFGDVGPFIIGFGNYRYADYMLTCPMLVYDLLYQLRAPYRVSCSAIIFAILMSGVLAEF YAEGDPRLRNGAYAWYGFGCFWFIFAYSIVMSIVAKQYSRLAQLAQDTGAEHSLHVLKFAVFL FSMLWILFPLVWAICPRGFGWIDDNWTEVAHCVCDIVAKSCYGFALARFRK SEQ ID NO: 11 (7TM motif of RICCR1 S220A; Ser27 to Lys269 of SEQ ID NO: 7; 243 aa; mutated residue highlighted in bold) SVAIGAHWSCFIVLTITTFYLGYESWTSRGPSKRTSFYAGYQEEQNLALFVNFFAMLSYFGKIV ADTLGHNFGDVGPFIIGFGNYRYADYMLTCPMLVYDLLYQLRAPYRVSCSAIIFAILMSGVLAEF YAEGDPRLRNGAYAWYGFGCFWFIFAYSIVMSIVAKQYSRLAQLAQDTGAEHSLHVLKFAVFT FAMLWILFPLVWAICPRGFGWIDDNWTEVAHCVCDIVAKSCYGFALARFRK SEQ ID NO: 12 (7TM motif of RICCR1 T218L/S220A; Ser27 to Lys269 of SEQ ID NO: 8; 243 aa; mutated residue highlighted in bold) SVAIGAHWSCFIVLTITTFYLGYESWTSRGPSKRTSFYAGYQEEQNLALFVNFFAMLSYFGKIV ADTLGHNFGDVGPFIIGFGNYRYADYMLTCPMLVYDLLYQLRAPYRVSCSAIIFAILMSGVLAEF YAEGDPRLRNGAYAWYGFGCFWFIFAYSIVMSIVAKQYSRLAQLAQDTGAEHSLHVLKFAVFL FAMLWILFPLVWAICPRGFGWIDDNWTEVAHCVCDIVAKSCYGFALARFRK SEQ ID NO: 13 (helix 6 motif of RICCR1 T218X.sup.1/S220X.sup.2; Ala205 to Ile232 of SEQ ID NO: 5 with amino acid mutations at one or both of T218 and S220 of SEQ ID NO: 5; 28 aa; variable residue highlighted in bold) AEHSLHVLKFAVFX.sup.1FX.sup.2MLWILFPLVWAI

[0325] wherein [0326] (a) X.sup.1 is selected from L, I, V, M, C, F, A, G, P and W, and preferably is L, and [0327] X.sup.2 is selected from A, G, L, V, I, M, P, C and W, and preferably is A; or [0328] (b) X.sup.1 is selected from L, I, V, M, C, F, A, G, P and W, and preferably is L, and [0329] X.sup.2 is selected from S, T, Y, Q and N, and preferably is S; or [0330] (c) X.sup.1 is selected from T, S, Y, Q and N, and preferably is T, and [0331] X.sup.2 is selected from A, G, L, V, I, M, P, C and W, and preferably is A.

[0332] The present invention is illustrated by the following examples which should not be construed as limiting the scope of the invention which is defined by the claims.

EXAMPLES

Example 1—Photocurrent Desensitization and Photocurrent Density of R/CCR1 Mutants in NG108-15 Cells

[0333] Molecular biology. The humanized DNA sequence, coding for R/CCR1-309 (ChRmine, SEQ ID NO: 5, accession number: MN194599), C-terminally fused to EYFP was cloned into the mammalian expression vector pcDNA3.1 (-) (Invitrogen, Carlsbad, USA). EYFP was thereby flanked by the well-described targeting sequences TS and ES (i.e., TS-EYFP-ES) for optimized plasma membrane expression of the shown constructs (see ref. 26, 27). The resulting constructs are termed ‘RICCR1-EYFP’ herein (designation followed by mutation(s) if any). The mutations T218L and S220A were introduced into R/CCR1 by site-directed mutagenesis using the primers shown in table 1.

TABLE-US-00006 TABLE 1 List of primers used for RICCR1 mutant generation.  RICCR1 T218L/S220A RICCR1 T218L RICCR1 S220A humanized RICCR1 template humanized RICCR1 humanized RICCR1 T218L sequence 5′-GTTCGCCGTG 5′-GTGTTTACCTT 5′-CGTGTTTCTG of forward TTTCTGTTCTCCA CGCCATGCTG TTCGCCATGCT primer TGCTGTG-3′ TGGATTC-3′ GTGGATTCTG-3′ (SEQ ID NO: 14) (SEQ ID NO: 16) (SEQ ID NO: 18) sequence 5′-CACAGCATGG 5′-GAATCCACAG 5′-CAGAATCCAC of reverse AGAACAGAAACAC CATGGCGAAG AGCATGGCGAA primer GGCGAAC-3′ GTAAACAC-3′ CAGAAACACG-3′ (SEQ ID NO: 15) (SEQ ID NO: 17) (SEQ ID NO: 19)

[0334] NG108-15 cell culture and transfection. NG108-15 cells (ATCC, HB-12377TM, Manassas, USA) were cultured at 37° C. and 5% CO.sub.2 in DMEM (Sigma, St. Louis, USA) supplemented with 10% fetal calf serum (Sigma, St. Louis, USA), and 5% penicillin/streptomycin (Sigma, St. Louis, USA). The cells were seeded on 24-well plates one day prior to transient transfections. Two to three days prior to the patch-clamp experiments the NG108-15 cells were transiently transfected with pcDNA3.1(-) carrying R/CCR1 or R/CCR1 mutants using Lipofectamine LtX (Invitrogen, Carlsbad, USA).

[0335] Electrophysiological recordings from NG108-15. For the electrophysiological characterization of R/CCR1 wt and the aforementioned R/CCR1 mutants whole cell patch-clamp measurement were performed under voltage clamp conditions using the Axopatch 200B amplifier (Axon Instruments, Union City, USA) and the DigiData 1322A interface (Axon Instruments, Union City, USA). Patch pipettes with resistances of 2-6 MΩ were fabricated from thin-walled borosilicate glass on a horizontal puller (Model P-1000, Sutter Instruments, Novato, USA). The series resistance was <15MΩ. The bath solution contained 140 mM NaCl, 2 mM CaCl.sub.2, 2 mM MgCl.sub.2, 10 mM HEPES, pH 7.4. and the pipette solution contained 110 mM NaCl, 2 mM MgCl.sub.2, 10 mM EGTA, 10 mM HEPES, pH 7.4. All recordings were performed at room temperature (297 K). For determination and comparison of the off-kinetics, current densities and desensitization, NG108-15 cells heterologously expressing R/CCR1 and the aforementioned R/CCR1 mutants were investigated at a membrane potential of −60 mV. Photocurrents were measured in response to 3 ms or 2 s light pulses with a saturating intensity of 23 mW/mm.sup.2 using diode-pumped solid-state lasers (λ=532) focused into a 400-μm optic fiber. Light pulses were applied by a fast computer-controlled shutter (Uniblitz LS6ZM2, Vincent Associates, Rochester, USA). The τoff value was determined by a fit of the decaying photocurrent, which was elicited in response to a 3 ms light pulse, to a monoexponential function. The stationary-peak-ratio was calculated as the quotient of the mean stationary current of the last 100 ms of the 2 s light pulse and the peak current. The current density (J-60 mV) was determined by dividing the stationary current in response to a 2 s light pulse with a saturating intensity of 23 mW/mm.sup.2 by the capacitance of the cell. In order to avoid an experimental bias, the NG108-15 cells for the electrophysiological recordings were chosen independent of the brightness of their EYFP fluorescence.

[0336] The results are shown in FIGS. 1 and 2 and in Tables 2, 3 and 4 below.

[0337] As demonstrated by the shown results, mutations at positions T218 and S220 are significantly reducing photocurrent desensitization in R/CCR1. The stationary-peak-ratios of R/CCR1 T218L, R/CCR1 S220A and R/CCR1 T218L/S220A are significantly increased compared to the stationary-peak-ratio of R/CCR1 wt (FIG. 1 and Table 2). Accordingly, the mean stationary photocurrent densities of R/CCR1 T218L, R/CCR1 S220A and R/CCR1 T218L/S220A are significantly increased compared to the mean stationary photocurrent density of R/CCR1 wt (FIG. 2 and Table 3). For R/CCR1 T218L and R/CCR1 T218L/S220A the closing kinetics is unchanged compared to R/CCR1 wt (Table 4). The closing kinetics of R/CCR1 S220A is slower compared to the closing kinetics of R/CCR1 wt (Table 4).

TABLE-US-00007 TABLE 2 R/CCR1 mutants show reduced desensitization (increased Stationary- Peak-Ratio) at saturating intensity. NG018-15 cells transfected with R/CCR1-EYFP (WT) (n = 57), R/CCR1-EYFP S220A (n = 38), R/CCR1-EYFP T218L (n = 18) and R/CCR1-EYFP T218L/S220A (n = 21) were investigated by whole-cell patch-clamp recordings at a membrane potential of −60 mV. Photocurrents were measured upon illumination with a 2 s light pulse of a wavelength of λ = 532 nm at saturating intensity of 23 mW/mm.sup.2. The stationary- peak-ratio was calculated as the quotient of the mean stationary current of the last 100 ms of the 2 s light pulse and the peak current. Shown are mean and standard deviation (SD). Stationary-Peak-Ratio at saturating intensity Construct Mean SD n R/CCR1-EYFP WT 0.22 0.12 57 R/CCR1-EYFP S220A 0.62 0.14 38 R/CCR1-EYFP T218L 0.44 0.13 18 R/CCR1-EYFP T218L/S220A 0.62 0.15 21

TABLE-US-00008 TABLE 3 Photocurrent density of R/CCR1 mutants at saturating intensity. NG108-15 cells transfected with R/CCR1-EYFP (WT) (n = 44), R/CCR1-EYFP S220A (n = 35), R/CCR1-EYFP T218L (n = 18) and R/CCR1-EYFP T218L/S220A (n = 20) were investigated by whole-cell patch-clamp recordings at a membrane potential of −60 mV. Photocurrents were measured upon illumination with a 2 s light pulse of a wavelength of λ = 532 nm at saturating intensity of 23 mW/mm.sup.2. Photocurrent densities shown were calculated as the quotient of the mean stationary current of the last 100 ms of the 2 s light pulse normalized to cell capacitance. Shown are mean and standard deviation (SD). Current density [pA/pF] at saturating intensity Construct Mean R/CCR1-EYFP (WT) 21.58 15.76 44 R/CCR1-EYFP S220A 54.26 27.19 35 R/CCR1-EYFP T218L 64.83 38.84 18 R/CCR1-EYFP T218L/S220A 95.85 58.52 20

TABLE-US-00009 TABLE 4 Off-kinetics of the R/CCR1 variants at −60 mV. NG018-15 cells transfected with R/CCR1-EYFP (WT) (n = 7), R/CCR1- EYFP S220A (n = 6), R/CCR1- EYFP T218L (n = 7) and R/CCR1-EYFP T218L/S220A (n = 7) were investigated by whole-cell patch-clamp recordings at a membrane potential of −60 mV. Displayed are Toff values measured upon illumination with a 3 ms light pulse of a wavelength of λ = 532 nm at saturating intensity of 23 mW/mm.sup.2. Shown are mean and standard deviation (SD). T.sub.off at −60 mV [ms] Construct Mean SD n R/CCR1-EYFP (WT) 63 15 7 R/CCR1-EYFP S220A 152 18 6 R/CCR1-EYFP T218L 59 20 7 R/CCR1-EYFP T218L/S220A 58 12 7

Example 2—Light Dependence of R/CCR1 Variants in NG108-15 Cells

[0338] Conditions and procedures, as described in Example 1, also apply for Example 2. The photocurrents were measured upon illumination with 2s light pulses of a wavelength of λ=532 nm at light intensities ranging from 0,0024 mW/mm.sup.2 to 23 mW/mm.sup.2.

[0339] The results are shown in FIGS. 3 and 4 and in Table 5 below.

[0340] As demonstrated by the shown results, mutations at positions T218 and S220 are reducing photocurrent desensitization in R/CCR1 upon illumination with light pulses of different light intensities. At the indicated light intensities the stationary-peak-ratios of R/CCR1 T218L, R/CCR1 S220A and R/CCR1 T218L/S220A are increased compared to the stationary-peak-ratio of R/CCR1 wt (FIG. 3). The light sensitivities of the investigated R/CCR1 variants are depicted in table 5. The stationary photocurrent of R/CCR1 wt shows a non-hyperbolic dependence on light intensity is (FIG. 4). Photocurrent reduction at high light intensities indicates a, to the knowledge of the present inventors, previously undescribed substrate (light) inhibition mechanism in R/CCR1. The R/CCR1 mutants, as disclosed herein, show a hyperbolic dependence on light intensity, which indicates suppression of this substrate inhibition mechanism.

TABLE-US-00010 TABLE 5 Light sensitivity of R/CCR1 variants. NG108-15 cells, transfected with R/CCR1-EYFP (WT), R/CCR1-EYFP S220A, R/CCR1-EYFP T218L and R/CCR1-EYFP T218L/S220A, were investigated by whole-cell patch-clamp recordings at a membrane potential of −60 mV. Stationary photocurrents were measured upon illumination with a 2 s light pulse of a wavelength of λ = 532 nm at indicated light intensities. Half maximal activation (EC.sub.50) was determined by hyperbolic fitting. Shown are mean and standard deviation (SD) of the resulting EC.sub.50 values. EC.sub.50 [mW/mm.sup.2] Construct Mean SD n R/CCR1-EYFP (WT) 0.014 0.005 6 R/CCR1-EYFP S220A 0.031 0.017 4 R/CCR1-EYFP T218L 0.029 0.014 5 R/CCR1-EYFP T218L/S220A 0.020 0.007 4

Example 3—Optoqenetic Stimulation of the Mouse Auditory Pathway by R/CCR1 Variants

[0341] Animals. Data were obtained from 19 adult C57Bl/6 wild-type mice of either sex. Animals were kept in a 12 h light/dark cycle, with access to food and water ad libitum. For all procedures, animals were placed on a heating pad and the body temperature was monitored by a rectal thermometer and maintained at approximately 37° C. by a custom-made heating pad. All experimental procedures were done in compliance with the German national animal care guidelines and approved by the local animal welfare committee of the University Medical Center Göttingen, as well as the responsible authorities of the state of Lower Saxony, Germany (LAVES).

[0342] Postnatal AAV injection into the cochlea. The AAV-ChR construct injections into scala tympani of the left ear via the round window was performed at postnatal day 6 in C57BL/6 wild-type mice. The right ear served as a non-injected control. The ChRs R/CCR1 wt or R/CCR1 T218L/S220A C-terminally tagged with EYFP, wherein the EYFP is flanked by targeting sequences TS and ES (see ref. 26, 27), which enhance plasma membrane expression (ChR-TS-EYFP-ES, herein designated “R/CCR1-EYFP”) were expressed under the control of the human synapsin promoter and were delivered to spiral ganglion neurons (SGNs) using the viral capsid AAV2/9. In brief, mouse pups were randomly selected for virus injections. Under general isoflurane anesthesia (1-2%) combined buprenorphine (0.1 mg/kg) and carprofen (5 mg/kg) as well as local xylocaine for analgesia, the round window of the left ear was accessed via a retroauricular incision. The round window membrane was identified and gently punctured using a borosilicate capillary pipette, which was kept in place to inject the virus of varying titers: 1.96×10.sup.12 to 1.48×10.sup.13 genome copies/ml. After virus injection, the tissue surrounding the injection site was repositioned and the wound was sutured. Recovery of the animals was accompanied with carprofen (5 mg/kg) the day after the surgery.

[0343] Optical stimulation in vivo. Six to thirteen weeks after virus injections, in vivo optical stimulation and recordings were performed under anesthesia using isoflurane (5% for anesthesia induction, 1-2% for maintenance with frequent monitoring of the hind-limb withdrawal reflex and anesthesia adjustments, accordingly) and analgesia by subdermal injection of buprenorphine (0.1 mg/kg body weight) and carprofen (5 mg/kg body weight). The left cochlea was exposed by performing a retroauricular incision behind the pinna followed by a bullostomy, where the round window was visualized and punctured. A 200 μm optical fiber coupled to either a 594 nm (OBIS LS OPSL, 100 mW, Coherent Inc., Santa Clara, Calif., United States) laser. Laser power was calibrated prior to each experiment using a laser power meter (LaserCheck, Coherent Inc., Santa Clara, Calif., United States).

[0344] Auditory brainstem responses (ABR). Stimulus generation and delivery, as well as data acquisition was performed using a custom-written software (MATLAB, MathWorks, Natick, Mass., United States) employing National Instrument data acquisition cards and a custom-build laser-controller. Recordings were conducted in a soundproof chamber (IAC Acoustics, IL, United States). Optically evoked ABRs (oABRs) were recorded by placing needle electrodes behind the pinna, on the vertex, and on the back of the anesthetized mice. The difference in potential between the vertex and mastoid subdermal needles was amplified using a custom-designed amplifier, sampled at a rate of 50 kHz for 20 ms, filtered (300-3000 Hz) and averaged across 1000 stimulus presentations. The oABRs threshold was defined and determined as the lowest light intensity for which one of the 3 waves was reliably visible. The latency of a given wave was defined as the time delay between the stimulus onset and the peak of the wave of interest. The amplitude was defined as the difference response strength between positive peak (P) and the negative (N), of a wave of interest.

[0345] The results are shown in FIGS. 5A, 5B and 5D-5I.

[0346] Application to the auditory system, for which optogenetic hearing restoration represents a future clinical application of optogenetics, exemplifies the benefit of the T218L/S220A mutant of R/CCR1. Studying the dependence of optogenetically evoked spiral ganglion neuron (SGN) activity on radiant flux (see FIG. 5G), pulse duration (see FIG. 5H) and repetition rate (see FIG. 5I) was investigated on a population level by oABR 1-st wave analysis. As demonstrated by the shown results the optogenetic stimulation of the auditory pathway by the T218L/S220A mutant of R/CCR1 enables stimulation of the auditory pathway at substantially lower light intensity than wild-type R/CCR1 (see FIG. 5D). This benefit can be critical for the clinical application, as the daily energy budget of optogenetic hearing restoration needs to comply with what one battery pack can supply in order to meet the expectations of the users.

LIST OF REFERENCES

[0347] WO 2012/032103 [0348] WO 03/084994 [0349] WO 2013/071231 [0350] U.S. Pat. No. 8,759,492 B2 [0351] WO 2017/207761 [0352] WO 2017/207745 [0353] WO 2020/150093 [0354] [1] Nagel, G., Ollig, D., Fuhrmann, M., Kateriya, S., Musti, A. M., Bamberg, E., and Hegemann, P. (2002) Channelrhodopsin-1: a light-gated proton channel in green algae, Science 296, 2395-2398. [0355] [2] Nagel, G., Szellas, T., Huhn, W., Kateriya, S., Adeishvili, N., Berthold, P., Ollig, D., Hegemann, P., and Bamberg, E. (2003) Channelrhodopsin-2, a directly light-gated cation-selective membrane channel, Proc Natl Acad Sci USA 100, 13940-13945. [0356] [3] Boyden, E. S., Zhang, F., Bamberg, E., Nagel, G., and Deisseroth, K. (2005) Millisecond-timescale, genetically targeted optical control of neural activity, Nat Neurosci 8, 1263-1268. [0357] [4] Volkov, O., Kovalev, K., Polovinkin, V., Borshchevskiy, V., Bamann, C., Astashkin, R., Marin, E., Popov, A., Balandin, T., Willbold, D., Buldt, G., Bamberg, E., and Gordeliy, V. (2017) Structural insights into ion conduction by channelrhodopsin 2, Science 358. [0358] [5] Sahel, J. A., Boulanger-Scemama, E., Pagot, C., Arleo, A., Galluppi, F., Martel, J. N., Esposti, S. D., Delaux, A., de Saint Aubert, J. B., de Montleau, C., Gutman, E., Audo, I., Duebel, J., Picaud, S., Dalkara, D., Blouin, L., Taiel, M., and Roska, B. (2021) Partial recovery of visual function in a blind patient after optogenetic therapy, Nat Med 27, 1223-1229. [0359] [6] Dieter, A., Keppeler, D., and Moser, T. (2020) Towards the optical cochlear implant: optogenetic approaches for hearing restoration, EMBO Mol Med12, e11618. [0360] [7] Govorunova, E. G., Sineshchekov, O. A., Janz, R., Liu, X., and Spudich, J. L. (2015) NEUROSCIENCE. Natural light-gated anion channels: A family of is microbial rhodopsins for advanced optogenetics, Science 349, 647-650. [0361] [8] Govorunova, E. G., Gou, Y., Sineshchekov, O. A., Li, H., Wang, Y., Brown, L. S., Xue, M., Spudich, J. L. (2021) Kalium rhodopsins: Natural light-gated potassium channels, In bioRxiv. [0362] [9] Klapoetke, N. C., Murata, Y., Kim, S. S., Pulver, S. R., Birdsey-Benson, A., Cho, Y. K., Morimoto, T. K., Chuong, A. S., Carpenter, E. J., Tian, Z., Wang, J., Xie, Y., Yan, Z., Zhang, Y., Chow, B. Y., Surek, B., Melkonian, M., Jayaraman, V., Constantine-Paton, M., Wong, G. K., and Boyden, E. S. (2014) Independent optical excitation of distinct neural populations, Nat Methods 11, 338-346. [0363] [10] Kleinlogel, S., Feldbauer, K., Dempski, R. E., Fotis, H., Wood, P. G., Bamann, C., and Bamberg, E. (2011) Ultra light-sensitive and fast neuronal activation with the Ca(2)+-permeable channelrhodopsin CatCh, Nat Neurosci 14, 513-518. [0364] [11] Zhang, F., Prigge, M., Beyriere, F., Tsunoda, S. P., Mattis, J., Yizhar, O., Hegemann, P., and Deisseroth, K. (2008) Red-shifted optogenetic excitation: a tool for fast neural control derived from Volvox carten, Nat Neurosci 11, 631-633. [0365] [12] Lin, J. Y., Knutsen, P. M., Muller, A., Kleinfeld, D., and Tsien, R. Y. (2013) ReaChR: a red-shifted variant of channelrhodopsin enables deep transcranial optogenetic excitation, Nat Neurosc 16, 1499-1508. [0366] [13] Mager, T., Lopez de la Morena, D., Senn, V., Schlotte, J., A, D. E., Feldbauer, K., Wrobel, C., Jung, S., Bodensiek, K., Rankovic, V., Browne, L., Huet, A., Juttner, J., Wood, P. G., Letzkus, J. J., Moser, T., and Bamberg, E. (2018) High frequency neural spiking and auditory signaling by ultrafast red-shifted optogenetics, Nat Commun 9, 1750. [0367] [14] Feldbauer, K., Zimmermann, D., Pintschovius, V., Spitz, J., Bamann, C., and Bamberg, E. (2009) Channelrhodopsin-2 is a leaky proton pump, Proc Natl Acad Sci USA 106, 12317-12322. [0368] [15] Berndt, A., Schoenenberger, P., Mattis, J., Tye, K. M., Deisseroth, K., Hegemann, P., and Oertner, T. G. (2011) High-efficiency channelrhodopsins for fast neuronal stimulation at low light levels, Proc Natl Acad Sci USA 108, 7595-7600. [0369] [16] Marshel, J. H., Kim, Y. S., Machado, T. A., Quirin, S., Benson, B., Kadmon, J., Raja, C., Chibukhchyan, A., Ramakrishnan, C., Inoue, M., Shane, J. C., McKnight, D. J., Yoshizawa, S., Kato, H. E., Ganguli, S., and Deisseroth, K. (2019) Cortical layer-specific critical dynamics triggering perception, Science 365. [0370] [17] Sineshchekov, 0. A., Govorunova, E. G., Li, H., Wang, Y., Melkonian, M., Wong, G. K., Brown, L. S., and Spudich, J. L. (2020) Conductance Mechanisms of Rapidly Desensitizing Cation Channelrhodopsins from Cryptophyte Algae, mBio 11. [0371] [18] Kishi, K. E., Kim, Y. S., Fukuda, M., Kusakizako T., Thadhani, E., Byrne, E. F. X., Paggi, J. M., Ramakrishnan, C., Matsui, T. E., Yamashita, K., Nagata, T., Konno, M., Wang, P. Y., Inoue, M., Benster, T., Uemura, T., Liu, K., Shibata, M., Nomura, N., Iwata, S., Nureki, O., Dror, R. O., Inoue, K., Deisseroth, K., Kato, H. E. (2021) Structural basis for channel conduction in the pump-likechannelrhodopsin ChRmine, bioRxiv. [0372] [19] Müller, M., Bamann, C., Bamberg, E., and Kuhlbrandt, W. (2015) Light-induced helix movements in channelrhodopsin-2, J Mol Biol 427, 341-349. [0373] [20] Sattig, T., Rickert, C., Bamberg, E., Steinhoff, H. J., and Bamann, C. (2013) Light-induced movement of the transmembrane helix B in channelrhodopsin-2, Angew Chem Int Ed Engl 52, 9705-9708. [0374] [21] Lorenz-Fonfria, V. A., Resler, T., Krause, N., Nack, M., Gossing, M., Fischer von Mollard, G., Bamann, C., Bamberg, E., Schlesinger, R., and Heberle, J. (2013) Transient protonation changes in channelrhodopsin-2 and their relevance to channel gating, Proc Natl Acad Sci USA 110, E1273-1281. [0375] [22] Lorenz-Fonfria, V. A., Bamann, C., Resler, T., Schlesinger, R., Bamberg, E., and Heberle, J. (2015) Temporal evolution of helix hydration in a light-gated ion channel correlates with ion conductance, Proc Natl Acad Sci USA 112, E5796-5804. [0376] [23] Zhang, H., and Cohen, A. E. (2017) Optogenetic Approaches to Drug Discovery in Neuroscience and Beyond, Trends Biotechnol 35, 625-639. [0377] [24] Agus, V., and Janovjak, H. (2017) Optogenetic methods in drug screening: technologies and applications, Curr Opin Biotechnol 48, 8-14. [0378] [25] Kleinlogel, S., Vogl, C., Jeschke, M., Neef, J., and Moser T. (2020) Emerging Approaches for restoration of hearing and vision, Physiol Rev 100, 1467-1525. [0379] [26] Gradinaru, V., Zhang, F., Ramakrishnan, C., Mattis, J., Prakash, R., Diester, I., Goshen, I., Thompson, K. R., and Deisseroth, K. (2010) Molecular and cellular approaches for diversifying and extending optogenetics. Cell 141, 154-165 [0380] [27] Keppeler, D., Merino, R. M., Lopez de la Morena, D., Bali, B., Huet, A. T., Gehrt, A., Wrobel, C., Subramanian, S., Dombrowski, T., Wolf, F., Rankovic, V., Neef, A., and Moser, T. (2018) Ultrafast optogenetic stimulation of the auditory pathway by targeting-optimized Chronos. EMBO J.