Formation Mechanism of Ion Channel in Channelrhodopsin-2: Molecular Dynamics Simulation and Steering Molecular Dynamics Simulations

Abstract

Channelrhodopsin-2 (ChR2) is a light-activated and non-selective cationic channel protein that can be easily expressed in specific neurons to control neuronal activity by light. Although ChR2 has been extensively used as an optogenetic tool in neuroscience research, the molecular mechanism of cation channel formation following retinal photoisomerization in ChR2 is not well understood. In this paper, studies of the closed and opened state ChR2 structures are presented. The formation of the cationic channel is elucidated in atomic detail using molecular dynamics simulations on the all-trans-retinal (ChR2-trans) configuration of ChR2 and its isomerization products, 13-cis-retinal (ChR2-cis) configuration, respectively. Photoisomerization of the retinal-chromophore causes the destruction of interactions among the crucial residues (e.g., E90, E82, N258, and R268) around the channel and the extended H-bond network mediated by numerous water molecules, which opens the pore. Steering molecular dynamics (SMD) simulations show that the electrostatic interactions at the binding sites in intracellular gate (ICG) and central gate (CG) can influence the transmembrane transport of Na⁺ in ChR2-cis obviously. Potential of mean force (PMF) constructed by SMD and umbrella sampling also found the existing energy wells at these two binding sites during the transportation of Na⁺. These wells partly hinder the penetration of Na⁺ into cytoplasm through the ion channel. This investigation provides a theoretical insight on the formation mechanism of ion channels and the mechanism of ion permeation.

Keywords: Steering Molecular Dynamics Simulations; channelrhodopsin-2; hydrogen bond network; ion channel; photoisomerization.

Figure 1

Overall structure presentation of the channelrhodopsin-2 (ChR2) dimer. (PDB ID: 6EID). (A) ChR2 side view. (B) ChR2 top view. The yellow and blue helixes represent different monomer respectively.

Figure 2

All-trans to 13-cis isomerization of the retinal chromophore in ChR2.

Figure 3

Time evolutions of root mean square difference (RMSD) of backbone atoms of ChR2-trans (black) and ChR2-cis (red).

Figure 4

Cluster analysis of ChR2-cis systems. The first column shows the stable structures of the intracellular gate (ICG), central gate (CG), and extracellular gate (ECG), in the equilibrium trajectory of ChR2-trans. The second, third, and fourth columns are the representative conformations of the top three clusters in the ICG, CG, and ECG of ChR2-cis. The cluster percentages are also shown in the figure.

Figure 5

Structure comparison of ChR2-trans and ChR2-cis. (A) Structure of ChR2-trans in equilibrium trajectory. (B) The first cluster representative structure of ChR2-cis. In the figure, the black solid arrow represents the potential ion channel, the red dotted circle highlights the ICG, CG and the ECG in the pores of the protein channel. The hydrogen bonding between key residues is indicated by a black dotted line.

Figure 6

(A) The probability of contact among the key residues surrounding the channel in the equilibrium trajectories of ChR2-trans and ChR2-cis. (B) The hydrogen bond distance between E90 and N258 (black), as well as E90 and K93 (red) during the whole trajectory of ChR2-cis, the distance between E90 and N258 increases and the hydrogen bonding disappears; the distance between E90 and K93 is close to form a new hydrogen bond interaction.

Figure 7

The number of hydrogen bonds between the residues and residues surrounding the channel (e.g., S63, E82, E83, E90, K93, E97, E101, Q117, R120, E123, T246, D253, N258, R268), as well as between residues and water molecules in the last 50 ns of equilibrium trajectory of ChR2-trans and ChR2-cis. It is apparent that ChR2-cis (red) has fewer hydrogen bonding interactions than ChR2-trans (black).

Figure 8

The static structure of the ion channel in the ChR2-cis. On the left is the overall view of the ion channel; the right side respectively is the side view of the channel at the ICG and the top view of the channel at CG.

Figure 9

(A) The simulated trajectory of ion permeation through channels. Na⁺ is represented by the yellow sphere. (B) The force received varies with the reaction coordinates during ion pulling in the ChR2-cis system. The vertical arrow indicates the Z position of the force local minima.

Figure 10

(A) PMF reconstructed used the Jarzynski equality and SMD trajectory for Na⁺ permeation across ions channel in ChR2-cis system. (B) PMF reconstructed use umbrella sampling for Na⁺ permeation across ions channel in ChR2-cis system.