Search

EP-4223768-B1 - NOVEL MUTANT BACTERIORHODOPSIN-LIKE-CHANNELRHODOPSIN ION CHANNEL

EP4223768B1EP 4223768 B1EP4223768 B1EP 4223768B1EP-4223768-B1

Inventors

  • MOSER, Tobias, Dr.
  • MAGER, Thomas, Dr.
  • ZERCHE, Maria, Dr.

Dates

Publication Date
20260506
Application Date
20220204

Claims (12)

  1. A mutant ion channel, wherein the mutant ion channel comprises: a 7-transmembrane-helix motif having at least 90% amino acid sequence identity to the full-length sequence of the 7-transmembrane-helix motif of the wild-type ion channel RI CCR1 set forth in SEQ ID NO: 9, and one or both of the following amino acid substitutions within said motif of the mutant ion channel: (i) a Leu at the position which corresponds to position T218 of RI CCR1 set forth in SEQ ID NO: 5, (ii) an Ala at the position which corresponds to position S220 of RI CCR1 set forth in SEQ ID NO: 5; and wherein the mutant ion channel is capable of being activated by light, and 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.
  2. The mutant ion channel of any one of the preceding claims, wherein the amino acid sequence of the 7-transmembrane-helix motif of the mutant ion channel has 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 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.
  3. The mutant ion channel of any one of the preceding claims, 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.
  4. The mutant ion channel of any one of the preceding claims, wherein the mutant ion channel comprises the following amino acid sequence motif: wherein: (a) Xaa1 is Leu, and Xaa2 is Ala; or (b) Xaa1 is Leu, and Xaa2 is Ser; or (c) Xaa1 is Thr, and Xaa2 is Ala.
  5. The mutant ion channel of any one of the preceding claims, 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 2 to determine the mean stationary current of the last 100ms 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 100ms of the 2s light pulse and the peak current of the 2s light pulse.
  6. The mutant ion channel of any one of the preceding claims, 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 2 to determine the mean stationary current of the last 100ms 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 100ms of the 2s light pulse and the peak current of the 2s light pulse.
  7. The mutant ion channel of any one of the preceding claims, 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 2 to determine the mean stationary current of the last 100ms of the 2s light pulse; and wherein the stationary photocurrent density is the quotient of the mean stationary current of the last 100ms of the 2s light pulse and the capacitance.
  8. The mutant ion channel of any one of the preceding claims, 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 2 to determine the mean stationary current of the last 100ms 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 100ms of the 2s light pulse and the capacitance of the NG108-15 cell.
  9. The mutant ion channel of any one of the preceding claims, 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.
  10. The mutant ion channel of any one of the preceding claims, 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.
  11. The mutant ion channel of claim 10, 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 RI CCR1 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 RI CCR1 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 2 to determine the mean stationary current of the last 100ms of the 2s light pulse; and wherein the stationary photocurrent density is the quotient of the mean stationary current of the last 100ms of the 2s light pulse and the capacitance.
  12. The mutant ion channel of claim 10 or claim 11, 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 RI CCR1 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 RI CCR1 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 2 to determine the mean stationary current of the last 100ms 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 100ms of the 2s light pulse and the capacitance of the NG108-15 cell.

Description

FIELD OF THE INVENTION The present invention relates to a novel mutant ion channel capable of being activated by light ('light-activated' ion channel) and having improved properties. BACKGROUND OF THE INVENTION Channelrhodopsins (ChRs) are light-activated ion channels comprising 7 transmembrane helices (7-transmembrane-helix motif, 7TM motif), which are used for the control of excitable cell activity with light (optogenetic control) [ref. 1-4]. Optogenetic approaches are of key importance for a deeper understanding of excitable cell networks and bear potential for the development of innovative medical treatments such as the recovery of vision and hearing [ref. 5 and 6]. ChRs enable both, light-controlled silencing and photostimulation of neurons. Neuronal silencing is carried out with anion selective ChRs [ref. 7] or potassium selective ChRs [ref. 8]. The optogenetic activation of neurons is accomplished using cation selective ChRs with low ion selectivity [ref. 2 and 9]. For different applications ChRs with suitable kinetics, ion selectivity and spectral properties were identified by the biophysical characterization of microbial type rhodopsins or generated by site directed mutagenesis of key residues for ChR function. Examples are the mutant L132C (CatCh; WO 2012/032103 and ref. 10) of the Chlamydomonas reinhardtii channelrhododopsin ChR2 (SEQ ID NO: 1, WO 03/084994 and ref. 2), the red light activated Chlamydomonas noctigama channelrhodopsin Chrimson (SEQ ID NO: 2, WO 2013/071231 and ref. 9), the Volvox channelrhodopsin (SEQ ID NO: 3, VChR1, ref. 11) and the chimera ReaChR (SEQ ID NO: 4, Red-absorbing ChannelRhodopsin; US 8,759,492 B2, and ref. 12). It was demonstrated that helix 6 modifications could accelerate channel closing in green algal ChRs (WO 2017/207761, WO 2017/207745, ref. 13). Examples are the mutations Y261F, S267M and Y268F and the combination of the corresponding mutations in Chrimson. Further examples are ChR2 F219Y, VChR1 F214Y and ReaChR F259Y. Optogenetic control of excitable cell activity faces limitations caused by the low single-channel conductance of green algal ChRs (ChR2, γ~40 fS, ref. 14). Further limitation is imposed by light dependent desensitization, which restricts photocurrent size. Robust membrane-targeted expression of the optogenetic activator and the application of high irradiance light pulses still allow for control of light induced spiking but can be challenging to implement in vivo and harbours the risk of photo- and cytotoxicity. Site directed mutagenesis yielded green algal ChR variants in which desensitization is significantly reduced and consequently allow for efficiency enhanced photostimulation [ref. 10 and 15] As light pulse induced phototoxicity decreases with increasing wavelength, this risk can be minimized by employing ChR variants which have action spectra that are red-shifted compared to the action spectrum of the most commonly used ChR variant ChR2 from Chlamydomonas reinhardtii (λmax ~460 nm, ref. 2). RI CCR1 ("ChRmine") (SEQ ID NO: 5) from the marine cryptophyte Rhodomonas lens is a so-called bacteriorhodopsin-like-channelrhodopsin [ref 16-18], also referred to as DTD Channelrhodopsin. RI CCR1 assembles as a trimer. Based on the high resolution cryo-electron microscopy structure and functional investigations the existence of a hydrophilic pore that extends through the center of the trimer in addition to three individual monomer pores was postulated. R/CCR1 has a red-shifted action spectrum (λmax = 520 nm) compared to ChR2 (λmax ~460 nm), and generates comparatively high photocurrents. However, its utility for chronic stimulation is impaired by a strong light-dependent desensitization of the photocurrent. Accordingly, there is need for variants of ChR R/CCR1 with reduced light-dependent desensitization which allow for sustainable neuronal photostimulation with enhanced efficiency. US 2020/087358 A1 describes engineered light-sensitive proteins, in particular channelrhodopsins and variants thereof, for expression in cells, tissues, organs and subjects and for treatment of neuronal and ocular disorders. SUMMARY OF THE INVENTION Helix 6 of the 7-transmembrane-helix motif is one of the moving helices upon light-activation in green algal ChRs [ref. 19 and 20], in which it plays a role in controlling light dependent protonation reactions, which govern open to closed state transitions [ref. 13, 21 and 22]. The inventors performed a study on the bacteriorhodopsin-like channelrhodopsin R/CCR1, in which the effect of helix 6 mutations on channel function was investigated. In this study, the present inventors surprisingly found, and experimentally verified in NG108-15 cells (see examples 1 and 2 herein), that mutation of positions 218 and 220 in helix 6 of R/CCR1 and the combination of the aforementioned mutations significantly reduced light-dependent desensitization of R/CCR1. The reduced light-dependent desensitization in R/CCR1 is a particular advantage of t