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 seventransmembrane ␣ helices of ChR2 are characteristic for G proteincoupled 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.voltage clamp ͉ patch clamp ͉ light sensitive ͉ Chlamydomonas reinhardtii ͉ Xenopus laevis oocyte

For studying the function of specific neurons in their native circuitry, it is desired to precisely control their activity. This often requires dissection to allow accurate electrical stimulation or neurotransmitter application , and it is thus inherently difficult in live animals, especially in small model organisms. Here, we employed channelrhodopsin-2 (ChR2), a directly light-gated cation channel from the green alga Chlamydomonas reinhardtii, in excitable cells of the nematode Caenorhabditis elegans, to trigger specific behaviors, simply by illumination. Channelrhodopsins are 7-transmembrane-helix proteins that resemble the light-driven proton pump bacteriorhodopsin , and they also utilize the chromophore all-trans retinal, but to open an intrinsic cation pore. In muscle cells, light-activated ChR2 evoked strong, simultaneous contractions, which were reduced in the background of mutated L-type, voltage-gated Ca2+-channels (VGCCs) and ryanodine receptors (RyRs). Electrophysiological analysis demonstrated rapid inward currents that persisted as long as the illumination. When ChR2 was expressed in mechanosensory neurons, light evoked withdrawal behaviors that are normally elicited by mechanical stimulation. Furthermore, ChR2 enabled activity of these neurons in mutants lacking the MEC-4/MEC-10 mechanosensory ion channel . Thus, specific neurons or muscles expressing ChR2 can be quickly and reversibly activated by light in live and behaving, as well as dissected, animals.

The field of optogenetics uses channelrhodopsins (ChRs) for light-induced neuronal activation. However, optimized tools for cellular inhibition at moderate light levels are lacking. We found that replacement of E90 in the central gate of ChR with positively charged residues produces chloride-conducting ChRs (ChloCs) with only negligible cation conductance. Molecular dynamics modeling unveiled that a high-affinity Cl(-)-binding site had been generated near the gate. Stabilizing the open state dramatically increased the operational light sensitivity of expressing cells (slow ChloC). In CA1 pyramidal cells, ChloCs completely inhibited action potentials triggered by depolarizing current injections or synaptic stimulation. Thus, by inverting the charge of the selectivity filter, we have created a class of directly light-gated anion channels that can be used to block neuronal output in a fully reversible fashion.

Phototaxis and photophobic responses of green algae are mediated by rhodopsins with microbial type chromophores, i.e. all-trans-retinal in the ground state. The green alga Chlamydomonas reinhardtii was recently completely sequenced and the EST (expressed sequence tag) database was made public. We and others detected overlapping partial cDNA sequences that encode two proteins which we termed channelopsins (Chops). The N-terminal half of chop1 (approximately 300 of 712 amino acids) comprises hypothetical seven-transmembrane segments with sequence similarity to the proton pump bacteriorhodopsin and the chloride pump halorhodopsin. Even though the overall sequence homology is low, several amino acids are conserved that define the retinal-binding site and the H+-transporting network in BR (bacteriorhodopsin). Expression of Chop1, or only the hydrophobic core, in Xenopus laevis oocytes, enriched with retinal, produced a light-gated conductance (maximum at approx. 500 nm), which shows characteristics of a channel [ChR1 (channelrhodopsin-1)] that is selectively permeable for protons. Also ChR2 (737 amino acids) is an ion channel that is switched directly by light and also here the hydrophobic N-terminal half of the protein is sufficient to enable light-sensitive channel activity. The action spectrum is blue-shifted (maximum at approx. 460 nm) with respect to ChR1. In addition to protons, ChR2 is permeable to univalent and bivalent cations. We suggest that ChRs are involved in phototaxis of green algae. We show that heterologous expression of ChR2 is useful to manipulate intracellular pCa or membrane potential of animal cells, simply by illumination.

Previously we have described a cGMP‐activated Ca2+‐dependet Cl− channel (ICl(cGMP,Ca)) which has markedly different properties to the classical Ca2+‐activated Cl− channel. In the present study we investigated the role of PKC in modulating ICl(cGMP,Ca) in freshly dispersed rat mesenteric artery myocytes at the single channel level using patch clamp techniques. In cell‐attached patches bath application of 300 μM 8‐bromo‐cGMP with 5 mM caffeine activated ICl(cGMP,Ca) channel activity which was markedly increased by co‐application of either 1 μM PDBu or 100 nM PMA, both PKC activators, by about 80 %. Moreover PDBu‐evoked increases in ICl(cGMP,Ca) channel activity were reduced by about 90 % following bath application of PKC inhibitors chelerythrine (3 μM) and RÖ‐31‐8220 (1 μM). We also tested whether norepinephine‐induced increases in ICl(cGMP,Ca) were mediated through a PKC‐dependent pathway. In cell‐attached patches bath application of 1 μM norepinephine in the presence of 300 μM 8‐bromo‐cGMP activated ICl(cGMP,Ca) channel activity which was significantly inhibited by co‐application of 3 μM chelerythrine by over 80 %. These data provide strong evidence that ICl(cGMP,Ca) channel activity in rat mesenteric artery myocytes is positively regulated by PKC and moreover these results suggest that a PKC‐dependent pathway has an important role in modulating ICl(cGMP,Ca) by the physiological agonist norepinephine.