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Molecular Dynamics of Channelrhodopsin at the Early Stages of Channel Opening.

Takemoto M, Kato HE, Koyama M, Ito J, Kamiya M, Hayashi S, Maturana AD, Deisseroth K, Ishitani R, Nureki O - PLoS ONE (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

The movements of TM helices upon retinal isomerization.(A) Structural comparison between the snapshots from the ATR-bound (grey) and 13-cisR-bound (green) simulations. (B) Magnified cytoplasmic view of the red-highlighted region in the left panel. (C-E) The RMSD values of (C) TM6, (D) TM7 and (E) TM2, compared between the ATR-bound and 13-cisR-bound forms. (F) Distance between Glu121-Arg07 in the intracellular constriction.
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pone.0131094.g006: The movements of TM helices upon retinal isomerization.(A) Structural comparison between the snapshots from the ATR-bound (grey) and 13-cisR-bound (green) simulations. (B) Magnified cytoplasmic view of the red-highlighted region in the left panel. (C-E) The RMSD values of (C) TM6, (D) TM7 and (E) TM2, compared between the ATR-bound and 13-cisR-bound forms. (F) Distance between Glu121-Arg07 in the intracellular constriction.

Mentions: The initial event of the photocycle, upon the absorption of blue light, is the isomerization of ATR to 13-cis retinal (13-cisR), which occurs on a femto-second time scale [8,10]. To investigate this initial conformational change in the photocycle, we performed the MD simulation of C1C2 with 13-cisR. The initial structure was modeled by simply replacing the ATR moiety of the crystal structure with 13-cisR, and subsequent energy minimization. We assumed that Glu122 was deprotonated in the ground state, considering the results of the ATR-E122Δp-E129p simulation, as described above. In this 13-cisR bound simulation (13-cisR-E122Δp-E129p; Table 1), conformational changes in the TM domains were observed. The 13-methyl group of 13-cisR shifted toward the cytoplasmic side and pushed out the indole ring of Trp262 (Trp223 in ChR2) on TM6 (Fig 5A–5C). The local steric conflict of the 13-methyl group with Trp262 caused the subsequent movement of the cytoplasmic half of TM6 (Fig 6A–6C). This movement was consistent with previous studies of other microbial rhodopsins, such as bacteriorhodopsin (BR) and sensory rhodopsin II (SRII), which indicated that the retinal isomerization and the steric collision between the 13-methyl group of 13-cisR and the tryptophan residue on TM6 (Trp182 in BR, Trp171 in SRII and Trp262 in C1C2) cause the movements of TM6 and TM7 [25–28]. To verify the functional importance of Trp262, we measured the photocurrents of the W262A mutant of C1C2 in HEK293 cells, and found that this mutant completely abolished the photocurrent, despite its robust membrane expression (Fig 5D, 5E and 5F). These results suggested that the presence of a bulky side chain adjacent to the 13-methyl group of 13-cisR is important for triggering the channel opening.


Molecular Dynamics of Channelrhodopsin at the Early Stages of Channel Opening.

Takemoto M, Kato HE, Koyama M, Ito J, Kamiya M, Hayashi S, Maturana AD, Deisseroth K, Ishitani R, Nureki O - PLoS ONE (2015)

The movements of TM helices upon retinal isomerization.(A) Structural comparison between the snapshots from the ATR-bound (grey) and 13-cisR-bound (green) simulations. (B) Magnified cytoplasmic view of the red-highlighted region in the left panel. (C-E) The RMSD values of (C) TM6, (D) TM7 and (E) TM2, compared between the ATR-bound and 13-cisR-bound forms. (F) Distance between Glu121-Arg07 in the intracellular constriction.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482709&req=5

pone.0131094.g006: The movements of TM helices upon retinal isomerization.(A) Structural comparison between the snapshots from the ATR-bound (grey) and 13-cisR-bound (green) simulations. (B) Magnified cytoplasmic view of the red-highlighted region in the left panel. (C-E) The RMSD values of (C) TM6, (D) TM7 and (E) TM2, compared between the ATR-bound and 13-cisR-bound forms. (F) Distance between Glu121-Arg07 in the intracellular constriction.
Mentions: The initial event of the photocycle, upon the absorption of blue light, is the isomerization of ATR to 13-cis retinal (13-cisR), which occurs on a femto-second time scale [8,10]. To investigate this initial conformational change in the photocycle, we performed the MD simulation of C1C2 with 13-cisR. The initial structure was modeled by simply replacing the ATR moiety of the crystal structure with 13-cisR, and subsequent energy minimization. We assumed that Glu122 was deprotonated in the ground state, considering the results of the ATR-E122Δp-E129p simulation, as described above. In this 13-cisR bound simulation (13-cisR-E122Δp-E129p; Table 1), conformational changes in the TM domains were observed. The 13-methyl group of 13-cisR shifted toward the cytoplasmic side and pushed out the indole ring of Trp262 (Trp223 in ChR2) on TM6 (Fig 5A–5C). The local steric conflict of the 13-methyl group with Trp262 caused the subsequent movement of the cytoplasmic half of TM6 (Fig 6A–6C). This movement was consistent with previous studies of other microbial rhodopsins, such as bacteriorhodopsin (BR) and sensory rhodopsin II (SRII), which indicated that the retinal isomerization and the steric collision between the 13-methyl group of 13-cisR and the tryptophan residue on TM6 (Trp182 in BR, Trp171 in SRII and Trp262 in C1C2) cause the movements of TM6 and TM7 [25–28]. To verify the functional importance of Trp262, we measured the photocurrents of the W262A mutant of C1C2 in HEK293 cells, and found that this mutant completely abolished the photocurrent, despite its robust membrane expression (Fig 5D, 5E and 5F). These results suggested that the presence of a bulky side chain adjacent to the 13-methyl group of 13-cisR is important for triggering the channel opening.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus