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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 two constrictions observed in the crystal structure of C1C2.The two constrictions in the crystal structure of C1C2 (PDB ID 3UG9) and the electrophysiological analysis of the constrictions. (A) Overall structure of C1C2, viewed parallel to the membrane with the three key regions highlighted (magenta, blue, and red). The dashed area represents the putative ion-conducting pathway. (B) Magnified views of the highlighted regions in (A). Black dashed lines are hydrogen bonds, and orange dashed circles represent the putative conducting pathway.
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pone.0131094.g001: The two constrictions observed in the crystal structure of C1C2.The two constrictions in the crystal structure of C1C2 (PDB ID 3UG9) and the electrophysiological analysis of the constrictions. (A) Overall structure of C1C2, viewed parallel to the membrane with the three key regions highlighted (magenta, blue, and red). The dashed area represents the putative ion-conducting pathway. (B) Magnified views of the highlighted regions in (A). Black dashed lines are hydrogen bonds, and orange dashed circles represent the putative conducting pathway.

Mentions: In addition to these electrophysiological and spectroscopic analyses, the 2.3 Å resolution structure of the chimeric ChR of C. reinhardtii ChR1 and ChR2 (C1C2) provided detailed insights into the architecture of ChR, including its cation-conducting pathway [6]. ChR forms a dimer in this crystal structure, and the electronegative pore in each monomer, formed by TMs 1, 2, 3, and 7, probably functions as the cation-conducting pathway, consistent with the previous computational and electrophysiological studies [6,13–19] (Fig 1A). The crystal structure revealed that an extracellular vestibule, formed by the N-domain and extracellular loops 1 and 3, expands to a diameter of about 8 Å, and suggested that this passage would allow water molecules to move from the extracellular side to the middle of the pathway [6] (Fig 1B, panel a). In contrast to the extracellular half, the intracellular half of the pathway is occluded at two constrictions, revealing a closed state. This is consistent with the fact that C1C2 was crystallized and harvested in the dark. These two constrictions, called the intracellular and central constrictions (corresponding to Fig 1A 1b and 1c respectively), contain titratable residues, including Glu122 and Glu129, which are highly conserved in the ChR family, but not in other rhodopsin family members. These residues link the TM helices by hydrogen bonds and a salt bridge to occlude the conducting pathway, and these interactions are considered to be disrupted in the conducting state. Although the crystal structure of C1C2 identified certain residues that may be important for channel opening, it remains unknown how the residues in these constrictions prevent ion leakage in the ground state, and how the retinal isomerization induces the conformational change towards 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 two constrictions observed in the crystal structure of C1C2.The two constrictions in the crystal structure of C1C2 (PDB ID 3UG9) and the electrophysiological analysis of the constrictions. (A) Overall structure of C1C2, viewed parallel to the membrane with the three key regions highlighted (magenta, blue, and red). The dashed area represents the putative ion-conducting pathway. (B) Magnified views of the highlighted regions in (A). Black dashed lines are hydrogen bonds, and orange dashed circles represent the putative conducting pathway.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131094.g001: The two constrictions observed in the crystal structure of C1C2.The two constrictions in the crystal structure of C1C2 (PDB ID 3UG9) and the electrophysiological analysis of the constrictions. (A) Overall structure of C1C2, viewed parallel to the membrane with the three key regions highlighted (magenta, blue, and red). The dashed area represents the putative ion-conducting pathway. (B) Magnified views of the highlighted regions in (A). Black dashed lines are hydrogen bonds, and orange dashed circles represent the putative conducting pathway.
Mentions: In addition to these electrophysiological and spectroscopic analyses, the 2.3 Å resolution structure of the chimeric ChR of C. reinhardtii ChR1 and ChR2 (C1C2) provided detailed insights into the architecture of ChR, including its cation-conducting pathway [6]. ChR forms a dimer in this crystal structure, and the electronegative pore in each monomer, formed by TMs 1, 2, 3, and 7, probably functions as the cation-conducting pathway, consistent with the previous computational and electrophysiological studies [6,13–19] (Fig 1A). The crystal structure revealed that an extracellular vestibule, formed by the N-domain and extracellular loops 1 and 3, expands to a diameter of about 8 Å, and suggested that this passage would allow water molecules to move from the extracellular side to the middle of the pathway [6] (Fig 1B, panel a). In contrast to the extracellular half, the intracellular half of the pathway is occluded at two constrictions, revealing a closed state. This is consistent with the fact that C1C2 was crystallized and harvested in the dark. These two constrictions, called the intracellular and central constrictions (corresponding to Fig 1A 1b and 1c respectively), contain titratable residues, including Glu122 and Glu129, which are highly conserved in the ChR family, but not in other rhodopsin family members. These residues link the TM helices by hydrogen bonds and a salt bridge to occlude the conducting pathway, and these interactions are considered to be disrupted in the conducting state. Although the crystal structure of C1C2 identified certain residues that may be important for channel opening, it remains unknown how the residues in these constrictions prevent ion leakage in the ground state, and how the retinal isomerization induces the conformational change towards 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