3.E.1.7.2
Channelrhodopsin-2 (chlamyrhodopsin-4 of 737 aas and 7 N-terminal TMSs; ChR2; CR2; Cop4; CSOB) (light-gated cation-selective ion channel (both monovalent and divalent cations (H⁺, Na⁺, K⁺, and Ca²⁺⁾ are transported)) (Nagel et al., 2003). Berndt et al. (2010) showed that ChR2 has two open states with differing ion selectivities. The channel is fairly nonspecific at the beginning of a light pulse, and becomes more specific for protons during longer periods of light exposure. Residues involved in channel closure have been identified (Bamann et al. 2010).  ChR2 is 712 aas long; the MR domain is N-terminal (Lee et al. 2015). The free energy profiles computed for proton transfer to the counterion, either via a direct jump or mediated by a water molecule, demonstrate that, when retinal is all-trans, water and protein electrostatic interactions largely favour the protonated retinal Schiff base state (Adam and Bondar 2018).    Blue light illumination of ChR2 activates an intrinsic leak channel conductive for cations. Sequence comparison of ChR2 with the related ChR1 protein revealed a cluster of charged amino acids within the predicted transmembrane domain 2 (TM2), which includes glutamates E90, E97 and E101. Charge inversion substitutions altered ChR2 function, replacement of E90 by lysine or alanine resulted in differential effects on H+- and Na+-mediated currents. These results are consistent with this glutamate side chain within TMS2 contributing to ion flux through and the cation selectivity of ChR2 (Ruffert et al., 2011). Glutamate residue-97 lies in the outer pore where it interacts with a cation to facilitate dehydration. This residue is also the primary binding target of Gd3+(Tanimoto et al., 2012).  Channelrhodopsin has been converted into a light-gated chloride channel (Wietek et al. 2014).  TMSs 2, 6 and 7 reorient or rearrange during the photocycle with no major differences near TMSs 3 and 4 at the dimer interface. TMS2 plays a key role in light-induced channel opening and closing in ChR2 (Müller et al. 2015).  Negative charges at the extracellular side of transmembrane domain 7 funnel cations into the pore (Richards and Dempski 2015).  CrChR2, is the most widely used optogenetic tool in neuroscience.  Water efflux and the cessation of the ion conductance are synchronized (Lórenz-Fonfría et al. 2015).  light and pH induce changes in the structure and accessibility of TMSB (Volz et al. 2016). Residues V86, K93 and N258 form a putative barrier to ion translocation. These residues contribute to cation selectivity (V86 and N258), the transition between the two open states (V86), open channel stability, and the hydrogen-bonding network (K93I and K93N) (Richards and Dempski 2017). The x-ray structure is available and reveals much about the mechanism of channel regulation (Gerwert 2017; Volkov et al. 2017). The mechanism of formation of the ion channel of ChR2 has been studed by molecular dynamics simulation and steering (Yang et al. 2019). The effects on ion channel activities of different protonation states of E90 in channelrhodopsin-2 have been described (Cheng et al. 2021). Yang et al. 2023 have designed a TRP-like biohybrid sensor by integrating upconversion nanoparticles (UCNP) and optogenetically engineered cells on a graphene transistor for infrared sensing and imaging. They used UCNP and ChR2 within the sensor in place of TRPs. (Yang et al. 2023). Light activation of ChR2 augments an influx of Na⁺ with a consequent inhibition of cell growth. In a K⁺ uptake deficient yeast strain, growth can be rescued in selective medium by the blue light induced K⁺ conductance of ChR (Höler et al. 2023).