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Channelrhodopsin (ChR) is a light-gated cation channel that responds to blue light. Since ChR can be readily expressed in specific neurons to precisely control their activities by light, it has become a powerful tool in neuroscience. Although the recently solved crystal structure of a chimeric ChR, C1C2, provided the structural basis for ChR, our understanding of the molecular mechanism of ChR still remains limited. Here we performed electrophysiological analyses and all-atom molecular dynamics (MD) simulations, to investigate the importance of the intracellular and central constrictions of the ion conducting pore observed in the crystal structure of C1C2. Our electrophysiological analysis revealed that two glutamate residues, Glu122 and Glu129, in the intracellular and central constrictions, respectively, should be deprotonated in the photocycle. The simulation results suggested that the deprotonation of Glu129 in the central constriction leads to ion leakage in the ground state, and implied that the protonation of Glu129 is important for preventing ion leakage in the ground state. Moreover, we modeled the 13-cis retinal bound; i.e., activated C1C2, and performed MD simulations to investigate the conformational changes in the early stage of the photocycle. Our simulations suggested that retinal photoisomerization induces the conformational change toward channel opening, including the movements of TM6, TM7 and TM2. These insights into the dynamics of the ground states and the early photocycle stages enhance our understanding of the channel function of ChR.

Krokinobacter eikastus rhodopsin 2 (KR2) is the first light-driven Na(+) pump discovered, and is viewed as a potential next-generation optogenetics tool. Since the positively charged Schiff base proton, located within the ion-conducting pathway of all light-driven ion pumps, was thought to prohibit the transport of a non-proton cation, the discovery of KR2 raised the question of how it achieves Na(+) transport. Here we present crystal structures of KR2 under neutral and acidic conditions, which represent the resting and M-like intermediate states, respectively. Structural and spectroscopic analyses revealed the gating mechanism, whereby the flipping of Asp116 sequesters the Schiff base proton from the conducting pathway to facilitate Na(+) transport. Together with the structure-based engineering of the first light-driven K(+) pumps, electrophysiological assays in mammalian neurons and behavioural assays in a nematode, our studies reveal the molecular basis for light-driven non-proton cation pumps and thus provide a framework that may advance the development of next-generation optogenetics.

Channelrhodopsins (ChRs) are light-gated cation channels derived from algae that have shown experimental utility in optogenetics; for example, neurons expressing ChRs can be optically controlled with high temporal precision within systems as complex as freely moving mammals. Although ChRs have been broadly applied to neuroscience research, little is known about the molecular mechanisms by which these unusual and powerful proteins operate. Here we present the crystal structure of a ChR (a C1C2 chimaera between ChR1 and ChR2 from Chlamydomonas reinhardtii) at 2.3 Å resolution. The structure reveals the essential molecular architecture of ChRs, including the retinal-binding pocket and cation conduction pathway. This integration of structural and electrophysiological analyses provides insight into the molecular basis for the remarkable function of ChRs, and paves the way for the precise and principled design of ChR variants with novel properties.

Channelrhodopsins(ChRs) are of great interest currently becauseof their important applications in optogenetics, the photostimulationof neurons. The absorption and circular dichroism (CD) spectra ofC1C2, a chimera of ChR1 and ChR2 of Chlamydomonas reinhardtii, have been studied experimentally and theoretically. The visibleabsorption spectrum of C1C2 shows vibronic fine structure in the 470nm band, consistent with the relatively nonpolar binding site. TheCD spectrum has a negative band at 492 nm (Δεmax= −6.17 M–1cm–1) anda positive band at 434 nm (Δεmax= +6.65 M–1cm–1), indicating exciton couplingwithin the C1C2 dimer. Time-dependent density functional theory (TDDFT)calculations are reported for three models of the C1C2 chromophore:(1) the isolated protonated retinal Schiff base (retPSB); (2) an ionpair, including the retPSB chromophore, two carboxylate side chains(Asp 292, Glu 162), modeled by acetate, and a water molecule; and(3) a hybrid quantum mechanical/molecular mechanical (QM/MM) modeldepicting the binding pocket, in which the QM part consists of thesame ion pair as that in (2) and the MM part consists of the proteinresidues surrounding the ion pair within 10 Å. For each of thesemodels, the CD of both the monomer and the dimer was calculated withTDDFT. For the dimer, DeVoe polarizability theory and exciton calculationswere also performed. The exciton calculations were supplemented bycalculations of the coupling of the retinal transition with aromaticand peptide group transitions. For the dimer, all three methods andthree models give a long-wavelength C2-axis-polarized band,negative in CD, and a short-wavelength band polarized perpendicularto the C2axis with positive CD, differing in wavelengthby 1–5 nm. Only the retPSB model gives an exciton couplet thatagrees qualitatively with experiment. The other two models give apredominantly or solely positive band. We further analyze an N-terminaltruncated mutant because it was assumed that the N-terminal domainhas a crucial role in the dimerization of ChRs. However, the CD spectrumof this mutant has an exciton couplet comparable to that of the wild-type,demonstrating that it is dimeric. Patch-clamp experiments suggestthat the N-terminal domain is involved in protein stabilization andchannel kinetics rather than dimerization or channel activity. [ABSTRACT FROM AUTHOR]