Channelrhodopsin-2, a directly light-gated cation-selective membrane channel

Key findings

• Nagel et al. expressed ChR2 (Chop2) in Xenopus laevis oocytes and mammalian cells, establishing it as a directly light-switched cation-selective ion channel. Large photocurrents were observed with both full-length and truncated constructs (aa 1–315), confirming that channel function resides in the seven-transmembrane helix domain. The channel generated passive cation conductance that varied with membrane potential, distinct from the proton-selective ChR1.
• ChR2 showed broad monovalent cation permeability (H+, Na+, K+, Ca2+) while excluding NMG+ and Mg2+, suggesting a pore diameter larger than voltage-gated Na+ channels. In continuous light, the channel desensitized to a smaller steady-state conductance; recovery was accelerated by extracellular H+ and negative membrane potential, while closing was decelerated by intracellular H+. Direct photon-gating required no G protein or second messenger intermediaries.
• Nagel et al. showed that ChR2 expression in mammalian HEK293 and BHK cells enabled light-induced membrane depolarization, establishing its potential as a genetically encoded optogenetic actuator. ChR2 was preferentially expressed in Chlamydomonas reinhardtii under low-light conditions, consistent with a role in photoreception in dark-adapted cells. Its novel seven-transmembrane topology defined a new class of directly light-gated ion channels.

Abstract

Microbial-type rhodopsins are found in archaea, prokaryotes, and eukaryotes. Some of them represent membrane ion transport proteins such as bacteriorhodopsin, a light-driven proton pump, or channelrhodopsin-1 (ChR1), a recently identified light-gated proton channel from the green alga Chlamydomonas reinhardtii. ChR1 and ChR2, a related microbial-type rhodopsin from C. reinhardtii, were shown to be involved in generation of photocurrents of this green alga. We demonstrate by functional expression, both in oocytes of Xenopus laevis and mammalian cells, that ChR2 is a directly light-switched cation-selective ion channel. This channel opens rapidly after absorption of a photon to generate a large permeability for monovalent and divalent cations. ChR2 desensitizes in continuous light to a smaller steady-state conductance. Recovery from desensitization is accelerated by extracellular H+ and negative membrane potential, whereas closing of the ChR2 ion channel is decelerated by intracellular H+. ChR2 is expressed mainly in C. reinhardtii under low-light conditions, suggesting involvement in photoreception in dark-adapted cells. The predicted seven-transmembrane alpha helices of ChR2 are characteristic for G protein-coupled receptors but reflect a different motif for a cation-selective ion channel. Finally, we demonstrate that ChR2 may be used to depolarize small or large cells, simply by illumination.