Significance: Controlling neuronal activity in live tissue is a long sought-after goal in the neurosciences. Channelrhodopsin-2 (ChR2) is a microbial-type rhodopsin that can be genetically expressed to depolarize neurons with light. Thereby, this “optogenetic tool” delivers cellular specificity and elegant options for studying the neuronal basis of behavior in intact organisms. Unfortunately, low-light transmission through pigmented tissue greatly complicates light delivery to target cells and curtails experiments in freely moving animals. This study introduces a ChR mutant, ChR2-XXL, that gives rise to the largest photocurrents of all ChRs published so far and increases light sensitivity more than 10,000-fold over wild-type ChR2 in Drosophila larvae. As a result, behavioral photostimulation is evoked in freely moving flies using diffuse, ambient light.

Adhesion-type G protein-coupled receptors (aGPCRs), a large molecule family with over 30 members in humans, operate in organ development, brain function and govern immunological responses. Correspondingly, this receptor family is linked to a multitude of diverse human diseases. aGPCRs have been suggested to possess mechanosensory properties, though their mechanism of action is fully unknown. Here we show that the Drosophila aGPCR Latrophilin/dCIRL acts in mechanosensory neurons by modulating ionotropic receptor currents, the initiating step of cellular mechanosensation. This process depends on the length of the extended ectodomain and the tethered agonist of the receptor, but not on its autoproteolysis, a characteristic biochemical feature of the aGPCR family. Intracellularly, dCIRL quenches cAMP levels upon mechanical activation thereby specifically increasing the mechanosensitivity of neurons. These results provide direct evidence that the aGPCR dCIRL acts as a molecular sensor and signal transducer that detects and converts mechanical stimuli into a metabotropic response.

Cyclic GMP (cGMP) signalling regulates multiple biological functions through activation of protein kinase G and cyclic nucleotide-gated (CNG) channels. In sensory neurons, cGMP permits signal modulation, amplification and encoding, before depolarization. Here we implement a guanylyl cyclase rhodopsin from Blastocladiella emersonii as a new optogenetic tool (BeCyclOp), enabling rapid light-triggered cGMP increase in heterologous cells (Xenopus oocytes, HEK293T cells) and in Caenorhabditis elegans. Among five different fungal CyclOps, exhibiting unusual eight transmembrane topologies and cytosolic N-termini, BeCyclOp is the superior optogenetic tool (light/dark activity ratio: 5,000; no cAMP production; turnover (20 °C) ∼17 cGMP s⁻¹). Via co-expressed CNG channels (OLF in oocytes, TAX-2/4 in C. elegans muscle), BeCyclOp photoactivation induces a rapid conductance increase and depolarization at very low light intensities. In O₂/CO₂ sensory neurons of C. elegans, BeCyclOp activation evokes behavioural responses consistent with their normal sensory function. BeCyclOp therefore enables precise and rapid optogenetic manipulation of cGMP levels in cells and animals.

Guard cells control the aperture of plant stomata, which are crucial for global fluxes of CO₂ and water. In turn, guard cell anion channels are seen as key players for stomatal closure, but is activation of these channels sufficient to limit plant water loss? To answer this open question, we used an optogenetic approach based on the light-gated anion channelrhodopsin 1 (GtACR1). In tobacco guard cells that express GtACR1, blue- and green-light pulses elicit Cl⁻ and NO₃⁻ currents of −1 to −2 nA. The anion currents depolarize the plasma membrane by 60 to 80 mV, which causes opening of voltage-gated K⁺ channels and the extrusion of K⁺. As a result, continuous stimulation with green light leads to loss of guard cell turgor and closure of stomata at conditions that provoke stomatal opening in wild type. GtACR1 optogenetics thus provides unequivocal evidence that opening of anion channels is sufficient to close stomata.

Genome screening of the cyanobacterium Microcoleus chthonoplastes PCC 7420 identified a gene encoding a protein (483 amino acids, 54.2 kDa in size) characteristic of a BL (blue light)-regulated adenylate (adenylyl) cyclase function. The photoreceptive part showed signatures of a LOV (light, oxygen, voltage) domain. The gene product, mPAC (Microcoleus photoactivated adenylate cyclase), exhibited the LOV-specific three-peaked absorption band (λmax=450 nm) and underwent conversion into the photoadduct form (λmax=390 nm) upon BL-irradiation. The lifetime for thermal recovery into the parent state was determined as 16 s at 20°C (25 s at 11°C). The adenylate cyclase function showed a constitutive activity (in the dark) that was in-vitro-amplified by a factor of 30 under BL-irradiation. Turnover of the purified protein at saturating light and pH 8 is estimated to 1 cAMP/mPAC per s at 25°C (2 cAMP/mPAC per s at 35°C). The lifetime of light-activated cAMP production after a BL flash was ~14 s at 20°C. The temperature optimum was determined to 35°C and the pH optimum to 8.0. The value for half-maximal activating light intensity is 6 W/m2 (at 35°C). A comparison of mPAC and the BLUF (BL using FAD) protein bPAC (Beggiatoa PAC), as purified proteins and expressed in Xenopus laevis oocytes, yielded higher constitutive activity for mPAC in the dark, but also when illuminated with BL.