PACmn for improved optogenetic control of intracellular cAMP.

BACKGROUND: Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger that transduces extracellular signals in virtually all eukaryotic cells. The soluble Beggiatoa photoactivatable adenylyl cyclase (bPAC) rapidly raises cAMP in blue light and has been used to study cAMP signaling pathways cell-autonomously. But low activity in the dark might raise resting cAMP in cells expressing bPAC, and most eukaryotic cyclases are membrane-targeted rather than soluble. Our aim was to engineer a plasma membrane-anchored PAC with no dark activity (i.e., no cAMP accumulation in the dark) that rapidly increases cAMP when illuminated. RESULTS: Using a streamlined method based on expression in Xenopus oocytes, we compared natural PACs and confirmed bPAC as the best starting point for protein engineering efforts. We identified several modifications that reduce bPAC dark activity. Mutating a phenylalanine to tyrosine at residue 198 substantially decreased dark cyclase activity, which increased 7000-fold when illuminated. Whereas Drosophila larvae expressing bPAC in mechanosensory neurons show nocifensive-like behavior even in the dark, larvae expressing improved soluble (e.g., bPAC(R278A)) and membrane-anchored PACs exhibited nocifensive responses only when illuminated. The plasma membrane-anchored PAC (PACmn) had an undetectable dark activity which increased >4000-fold in the light. PACmn does not raise resting cAMP nor, when expressed in hippocampal neurons, affect cAMP-dependent kinase (PKA) activity in the dark, but rapidly and reversibly increases cAMP and PKA activity in the soma and dendrites upon illumination. The peak responses to brief (2 s) light flashes exceed the responses to forskolin-induced activation of endogenous cyclases and return to baseline within seconds (cAMP) or ~10 min (PKA). CONCLUSIONS: PACmn is a valuable optogenetic tool for precise cell-autonomous and transient stimulation of cAMP signaling pathways in diverse cell types.

Synthetic Light-Activated Ion Channels for Optogenetic Activation and Inhibition.

Optogenetic manipulation of cells or living organisms became widely used in neuroscience following the introduction of the light-gated ion channel channelrhodopsin-2 (ChR2). ChR2 is a non-selective cation channel, ideally suited to depolarize and evoke action potentials in neurons. However, its calcium (Ca2+) permeability and single channel conductance are low and for some applications longer-lasting increases in intracellular Ca2+ might be desirable. Moreover, there is need for an efficient light-gated potassium (K+) channel that can rapidly inhibit spiking in targeted neurons. Considering the importance of Ca2+ and K+ in cell physiology, light-activated Ca2+-permeant and K+-specific channels would be welcome additions to the optogenetic toolbox. Here we describe the engineering of novel light-gated Ca2+-permeant and K+-specific channels by fusing a bacterial photoactivated adenylyl cyclase to cyclic nucleotide-gated channels with high permeability for Ca2+ or for K+, respectively. Optimized fusion constructs showed strong light-gated conductance in Xenopus laevis oocytes and in rat hippocampal neurons. These constructs could also be used to control the motility of Drosophila melanogaster larvae, when expressed in motoneurons. Illumination led to body contraction when motoneurons expressed the light-sensitive Ca2+-permeant channel, and to body extension when expressing the light-sensitive K+ channel, both effectively and reversibly paralyzing the larvae. Further optimization of these constructs will be required for application in adult flies since both constructs led to eclosion failure when expressed in motoneurons.

Rhodopsin-cyclases for photocontrol of cGMP/cAMP and 2.3 Å structure of the adenylyl cyclase domain.

The cyclic nucleotides cAMP and cGMP are important second messengers that orchestrate fundamental cellular responses. Here, we present the characterization of the rhodopsin-guanylyl cyclase from Catenaria anguillulae (CaRhGC), which produces cGMP in response to green light with a light to dark activity ratio >1000. After light excitation the putative signaling state forms with τ = 31 ms and decays with τ = 570 ms. Mutations (up to 6) within the nucleotide binding site generate rhodopsin-adenylyl cyclases (CaRhACs) of which the double mutated YFP-CaRhAC (E497K/C566D) is the most suitable for rapid cAMP production in neurons. Furthermore, the crystal structure of the ligand-bound AC domain (2.25 Å) reveals detailed information about the nucleotide binding mode within this recently discovered class of enzyme rhodopsin. Both YFP-CaRhGC and YFP-CaRhAC are favorable optogenetic tools for non-invasive, cell-selective, and spatio-temporally precise modulation of cAMP/cGMP with light.