Structural insights into ion conduction by channelrhodopsin 2

The inner workings of an optogenetic tool Channelrhodopsins are membrane channel proteins whose gating is controlled by light. In their native setting, they allow green algae to move in response to light. Their expression in neurons allows precise control of neural activity, an approach known as optogenetics. Volkov et al. describe the high-resolution structure of channelrhodopsin 2, the most widely used optogenetics tool, as well as the structure of a mutant with a longer open-state lifetime (see the Perspective by Gerwert). Light activation perturbs an intricate hydrogen-bonding network to open the channel. The structures provide a basis for designing better optogenetic tools. Science, this issue p. 10.1126/science.eaan8862; see also p. 1000 Channelrhodopsin has an intricate hydrogen-bonding network that is perturbed by light activation, resulting in channel opening. INTRODUCTION Ion channels are integral membrane proteins that upon stimulation modulate the flow of ions across the cell or organelle membrane. The resulting electrical signals are involved in biological functions such as electrochemical transmission and information processing in neurons. Channelrhodopsins (ChRs) appear to be unusual channels. They belong to the large family of microbial rhodopsins, seven-helical transmembrane proteins containing retinal as chromophore. Photon absorption initiates retinal isomerization resulting in a photocycle, with different spectroscopically distinguishable intermediates, thereby controlling the opening and closing of the channel. In 2003, it was demonstrated that light-induced currents by heterologously expressed ChR2 can be used to change a host’s membrane potential. The concept was further applied to precisely control muscle and neural activity by using light-induced depolarization to trigger an action potential in neurons expressing ChR2. This optogenetic approach with ChR2 and other ChRs has been widely used for remote control of neural cells in culture and in living animals with high spatiotemporal resolution. It is also used in biomedical studies aimed to cure severe diseases. RATIONALE Despite the wealth of biochemical and biophysical data, a high-resolution structure and structural mechanisms of a native ChR2 (and other ChRs) have not yet been known. A step forward was the structure of a chimera (C1C2). However, recent electrophysiological and Fourier transform infrared data showed that C1C2 exhibits light-induced responses that are functionally and mechanistically different from ChR2. Given that ChR2 is the most frequently used tool in optogenetics, a high-resolution structure of ChR2 is of high importance. Deciphering the structure of the native channel would shed light on how the light-induced changes at the retinal Schiff base (RSB) are linked to the channel operation and may make engineering of enhanced optogenetic tools more efficient. RESULTS We expressed ChR2 in LEXSY and used in the meso crystallization approach to determine the crystal structure of the wild-type ChR2 and C128T slow mutant at 2.4 and 2.7 Å, respectively (C, cysteine; T, threonine). Two different dark-state conformations of ChR2 in the two protomers in the asymmetric unit were resolved. The overall structure alignment of the protomers does not show a visible difference in backbone conformation. However, the conformation of some amino acids and the position of water molecules are not the same. The dimerization is strong and provided mainly through the interaction of helices 3 and 4 and the N termini. In addition, the protomers are connected with a disulfide bond, C34/C36′. In both protomers, we identified ion conduction pathway comprising four cavities [extracellular cavity 1 (EC1), EC2, intracellular cavity 1 (IC1), and IC2] that are separated by three gates [extracellular gate (ECG), central gate (CG), and intracellular gate (ICG)] (figure, panel A). Arginines R120 and R268 are the cores of ECG and ICG, respectively, in all ChRs. The Schiff base is hydrogen-bond–connected to E123 and D253 amino acids (E, glutamic acid; D, aspartic acid) and is a key part of the CG that is further connected with two other gates through an extended H-bond network mediated by numerous water molecules (figure, panel B). The DC gate is separate from the gates in the channel pathway and is bridged by hydrogen bonds through the water molecule w5. Hydrogen bonding of the DC pair (C128 and D156) has two important consequences. It stabilizes helices 3 and 4 and provides connection from D156, a possible proton donor, to the RSB. The presence of the hydrogen bonds provides structural insights into how the DC gate controls ChR2 gating lifetime. CONCLUSION The determined structures of ChR2 and its C128T mutant present the molecular basis for the understanding of ChR functioning. They provide insights into mechanisms of channel opening and closing. A plausible scenario is that the disruption of the H-bonds between E123 and D253 and the Schiff base and the protonation of D253 upon retinal isomerization trigger rearrangements in the extended hydrogen-bonded networks, stabilizing the ECG and CG and also rearranging the H-bonding network in the cavities. Upon retinal isomerization, these two gates are opened and the network is broken. This leads to the reorientation of helix 2. Additional changes in helices 6 and 7 induced by the isomerization could help with opening the ICG and channel pore formation. General structure presentation of ChR2. (A) Four cavities and three gates forming the channel pore. (B) Extended hydrogen-bond network. The DC gate is shown in the red ellipse. The black arrows and gray horizontal lines show the putative ion pathway and position of hydrophobic/hydrophilic boundaries, respectively. The light-gated ion channel channelrhodopsin 2 (ChR2) from Chlamydomonas reinhardtii is a major optogenetic tool. Photon absorption starts a well-characterized photocycle, but the structural basis for the regulation of channel opening remains unclear. We present high-resolution structures of ChR2 and the C128T mutant, which has a markedly increased open-state lifetime. The structure reveals two cavities on the intracellular side and two cavities on the extracellular side. They are connected by extended hydrogen-bonding networks involving water molecules and side-chain residues. Central is the retinal Schiff base that controls and synchronizes three gates that separate the cavities. Separate from this network is the DC gate that comprises a water-mediated bond between C128 and D156 and interacts directly with the retinal Schiff base. Comparison with the C128T structure reveals a direct connection of the DC gate to the central gate and suggests how the gating mechanism is affected by subtle tuning of the Schiff base’s interactions.