An optogenetic tool for intracellular cAMP production 

Since 2006, photo-sensitive ion channels have become an indispensable optogenetic tool in neuroscience. After forcibly expressing photo-sensitive ion channels on the specific neurons using genetic engineering techniques, these cells can be illuminated with a specific wavelength of light to excite or suppress each target neuron. Spatiotemporal accuracy of this technique is a feature not found in the conventional methods, such as pharmacological or electrical methods.


Incidentally, signaling factors, for instance, intracellular signal transduction pathways mediated by cyclic nucleotides such as cAMP and cGMP, are those that play essential roles for diverse and important biological functions. Activation of adenylate cyclase or inhibition of phosphodiesterase increases intracellular cAMP concentration and that’s what stimulates cyclic nucleotide-gated (CNG) channels, protein kinase A (PKA), and exchange protein directly activated by cAMP (Epac). These processes induce a variety of cellular responses, such as generation of receptor potentials, the axonal branching or elongation in neurons, increase in the heart rate, and glycogenolysis.


We will introduce an optogenetic tool "PAC" that can control cAMP production and induce these cellular responses triggered by various intensities of blue light.

■What is the optogenetics for controlling cell function via cAMP signaling pathway mediated by Photoactivated adenylyl cyclase (PAC)?

Function of PACs in microorganisms as a photosensor and its biomedical applications

PAC was first identified in photosynthetic flagellate Euglena glacilis as photosensitive molecule inducing intracellular cAMP production by light illumination (Ref. 1). Recently, homologous proteins of PAC have been widely discovered in several prokaryotes.

Control of cAMP signaling by PACs is simple to handle because this process is independent of signal cascade mediated by G protein-coupled receptor GPCRs. Since PACs produce cAMP just by light illumination, cAMP production can be temporally and spatially regulated with high precision. Therefore, it is highly expected that this method enables to control various physiological functions inside cells and tissues directly by using only light illumination (Ref. 2).

■Scheme: PAC expression in cells and activation by blue light illumination

Excerpted from Scientific Reports 5, 19679, doi:10.1038/srep19679 (2016)

C-terminal deletion modification of OaPAC yields optogenetic tools with different photosensitivity

Among the PACs discovered so far, the structures of OaPAC from cyanobacteria and bPAC from sulfur bacteria have been determined. Both PACs have a light-sensitive BLUF domain at the N-terminus and an adenylate cyclase (AC) domain at the C-terminus, and these domains are arranged next to each other. Although the sequence and the structure of OaPAC and bPAC are similar, there is a hundred-fold difference in their photo-sensitivities (Ref. 3). This difference should provide a clue for modifying the design of PACs as an optogenetic tool applied to a lot of variations. In a collaboration with the Graduate School for the Creation of New Photonics Industries (GPI), we built lots of deletion mutants on the C-terminus and compared their enzymatic activities. We found that the longer the C-terminal region was ascribed the lower the enzymatic activity, and that the variation in their photoactivities was more than fifty-fold (Ref. 4).

■C-terminal amino acid sequence of various PACs

Amino acid sequence of OaPAC from Oscillatoria acuminata (NCBI, WP_015149803.1) and its deletion variants (Oa-363, Oa-360, Oa-357, Oa-354, Oa-351, Oa-348), bPAC from soil bacterium Beggiatoa (GenBank: GU461306.2), PACα from Euglena gracilis (GenBank: BAB85619.1) amino acid sequences in the C-terminal region. Yellow highlighting: conserved amino acids.

Excerpted from Scientific Reports 9, 20262 (2019)

User friendly PAC variants for researches

Low photosensitivity of wild-type OaPAC had been a bottleneck for practical use because of its susceptibility to phototoxicity due to strong light illumination. We have solved this problem and made it possible to use OaPACs for a wide range of applications. Two types of user-friendly OaPAC mutants with different photosensitivities, namely OaPAC360 and OaPAC348, are now commercially available as our products.


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Excerpted from Scientific Reports 9, 20262 (2019)


  1. Iseki, M. et al. ”A blue-light-activated adenylyl cyclase mediates photoavoidance in Euglena gracilis. ”Nature 415, 1047-1051, doi:10.1038/4151047a (2002).
  2. Zhou, Z. et al. Photoactivated adenylyl cyclase (PAC) reveals novel mechanisms underlying cAMP-dependent axonal morphogenesis. Scientific Reports 5, 19679, doi:10.1038/srep19679 (2016).
  3. Ohki, M. et al. ”Structural insight into photoactivation of an adenylate cyclase from a photosynthetic cyanobacterium.” Proc Natl Acad Sci U S A 113, 6659-6664, doi:10.1073/pnas.1517520113 (2016).
  4. Hirano et al. “The C-terminal region affects the activity of photoactivated adenylyl cyclase from Oscillatoria acuminata.” Scientific Reports 9, Article number: 20262 (2019).

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