PACmn for improved optogenetic control of intracellular cAMP

Abstract

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.

Characterization and Modification of Light-Sensitive Phosphodiesterases from Choanoflagellates

“…Later, bPAC from soil bacterium Beggiatoa was characterized with a higher light/dark activity ratio [ 26 ]. Recently, bPAC was further engineered to show strongly reduced dark activity, and a new biPAC with reduced dark activity was characterized [ 27 , 28 ]. Be Cyclop from Blastocladiella emersonii is a green light-activated guanylyl cyclase opsin with a very high L/D ratio [ 10 ], ideal for cGMP manipulation.…”

Long-term in vivo application of a potassium channel-based optogenetic silencer in the healthy and epileptic mouse hippocampus

“…Our results suggest that the expression of bPAC affects the physiology of hippocampal principal cells in the absence of blue light. Functional dark activity of soluble bPAC has also been reported by others, who utilized a fluorescent PKA sensor to detect intracellular cAMP levels in bPAC-expressing CA1 cells in hippocampal slice cultures [ 65 ]. For reduced dark activity, the soluble bPAC from the Beggiatoa bacterium could be replaced by another photoactivated adenylyl cyclase (PAC).…”

“…Another approach would be targeting a PAC to the cell membrane. For instance, a recently developed membrane-anchored PAC has no detectable dark activity in hippocampal neurons [ 65 ]. Similarly, membrane-bound guanylyl cyclase rhodopsins, which have mutated to be adenylyl cyclases, virtually lack dark activity [ 67 , 68 ].…”

An Improved Genetically Encoded Fluorescent cAMP Indicator for Sensitive cAMP Imaging and Fast Drug Screening

“…A potent blue light-activatable optogenetic adenylyl cyclase bPACm (a bPAC variant with much lower dark activity) ( Yang et al, 2021 ), was also used to specifically increase the cytosolic cAMP concentration. Mito-G-Flamp2, G-Flamp2 or G-Flamp2 NC were co-expressed with bPACm-mCherry in HeLa cells and the 450 nm light was exploited to activate bPACm and excite the indicators simultaneously.…”