Photo-activated Adenylate Cyclase Research Articles

ATP-dependent conformational dynamics in a photoactivated adenylate cyclase revealed by fluorescence spectroscopy and small-angle X-ray scattering

Structural insights into the photoactivated adenylate cyclases can be used to develop new ways of controlling cellular cyclic adenosine monophosphate (cAMP) levels for optogenetic and other applications. In this work, we use an integrative approach that combines biophysical and structural biology methods to provide insight on the interaction of adenosine triphosphate (ATP) with the dark-adapted state of the photoactivated adenylate cyclase from the cyanobacterium Oscillatoria acuminata (OaPAC). A moderate affinity of the nucleotide for the enzyme was calculated and the thermodynamic parameters of the interaction have been obtained. Stopped-flow fluorescence spectroscopy and small-angle solution scattering have revealed significant conformational changes in the enzyme, presumably in the adenylate cyclase (AC) domain during the allosteric mechanism of ATP binding to OaPAC with small and large-scale movements observed to the best of our knowledge for the first time in the enzyme in solution upon ATP binding. These results are in line with previously reported drastic conformational changes taking place in several class III AC domains upon nucleotide binding.

Single Amino Acid Mutation Decouples Photochemistry of the BLUF Domain from the Enzymatic Function of OaPAC and Drives the Enzyme to a Switched-on State

Photoactivated adenylate cyclases (PACs) are light-activated enzymes that combine a BLUF (blue-light using flavin) domain and an adenylate cyclase domain that are able to increase the levels of the important second messenger cAMP (cyclic adenosine monophosphate) upon blue-light excitation. The light-induced changes in the BLUF domain are transduced to the adenylate cyclase domain via a mechanism that has not yet been established. One critical residue in the photoactivation mechanism of BLUF domains, present in the vicinity of the flavin is the glutamine amino acid close to the N5 of the flavin. The role of this residue has been investigated extensively both experimentally and theoretically. However, its role in the activity of the photoactivated adenylate cyclase, OaPAC has never been addressed. In this work, we applied ultrafast transient visible and infrared spectroscopies to study the photochemistry of the Q48E OaPAC mutant. This mutation altered the primary electron transfer process and switched the enzyme into a permanent ‘on’ state, able to increase the cAMP levels under dark conditions compared to the cAMP levels of the dark-adapted state of the wild-type OaPAC. Differential scanning calorimetry measurements point to a less compact structure for the Q48E OaPAC mutant. The ensemble of these findings provide insight into the important elements in PACs and how their fine tuning may help in the design of optogenetic devices.

Time-resolved study on signaling pathway of photoactivated adenylate cyclase and its nonlinear optical response

Photoactivated adenylate cyclases (PACs) are multidomain BLUF proteins that regulate the cellular levels of cAMP in a light-dependent manner. The signaling route and dynamics of PAC from Oscillatoria acuminata (OaPAC), which consists of a light sensor BLUF domain, an adenylate cyclase domain, and a connector helix (α3-helix), were studied by detecting conformational changes in the protein moiety. Although circular dichroism and small-angle X-ray scattering measurements did not show significant changes upon light illumination, the transient grating method successfully detected light-induced changes in the diffusion coefficient (diffusion-sensitive conformational change (DSCC)) of full-length OaPAC and the BLUF domain with the α3-helix. DSCC of full-length OaPAC was observed only when both protomers in a dimer were photoconverted. This light intensity dependence suggests that OaPAC is a cyclase with a nonlinear light intensity response. The enzymatic activity indeed nonlinearly depends on light intensity, that is, OaPAC is activated under strong light conditions. It was also found that both DSCC and enzymatic activity were suppressed by a mutation in the W90 residue, indicating the importance of the highly conserved Trp in many BLUF domains for the function. Based on these findings, a reaction scheme was proposed together with the reaction dynamics.

Photocycle alteration and increased enzymatic activity in genetically modified photoactivated adenylate cyclase OaPAC

Photoactivable adenylate cyclases (PAC) are light activated enzymes that combine blue light sensing capacity with the ability to convert adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP) and pyrophosphate (PPi) in a light-dependent manner. In most of the known PACs blue light regulation is provided by a BLUF domain which undergoes a structural reorganization after blue-light absorption. This minor structural change then is translated towards the C-terminal of the protein, inducing a larger conformational change that results in the ATP conversion to cAMP. As cAMP is a key second messenger in numerous signal transduction pathways regulating various cellular functions, photoactivable adenylate cyclases are of great interest in optogenetic studies. The optimal optogenetic device must be "silent" in the dark and highly responsive upon light illumination. OaPAC is a very good candidate as its basal activity is very small in the dark and the conversion rates increase 20-fold upon light illumination. We studied the effect of replacing D67 to N, in the BLUF domain. This mutation was found to accelerate the primary electron transfer process in the photosensing domain of the protein, as has been predicted. Furthermore, it resulted in a longer lived signalling state, which was formed with a lower quantum yield. Our studies show that the overall effects of the D67N mutation lead to a slightly higher conversion of ATP to cAMP, which points in the direction that by fine tuning the kinetic properties more responsive PACs and optogenetic devices can be generated.

The role of electron transfer in the enzymatic activity of a photoactivable adenylate cyclase.

OaPAC is a photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata and belongs to a class of photoactive flavoenzymes which are involved in the conversion of ATP (adenosine triphosphate) to the important second messenger cAMP (cyclic adenosine monophosphate). The photoactivation is controlled by a BLUF (Blue Light Sensing Using Flavin) domain; a well-known light-triggered switch present in many biological photoreceptors and able to control photochemical energy into a diverse set of cellular responses (e.g. phototaxis, photophobia, enzyme activity, gene expression). Blue-light excitation of the BLUF domain of OaPAC results in a proton-coupled electron transfer process where the primary electron and proton donor is a tyrosine residue in the vicinity of the flavin. The electron transfer to the flavin is accompanied by a reorganization of its hydrogen bond network, and the induced structural changes are believed to propagate towards the AC domain enabling the conversion of ATP to cAMP. In this work we studied the relation between the electron transfer and the activity of the enzyme using ultrafast and fluorescence spectroscopy as well as differential scanning calorimetry. A.L. acknowledges funding from the Hungarian National Research and Innovation Office (K-137557).

Elucidating signal transduction pathway in light-activated adenylyl cyclase using unnatural amino acid mutagenesis.

There is extensive research which explores the photochemistry of Blue Light Using FAD photoreceptors, however there is limited literature available on how the signal is transduced from the BLUF domain to the functional/output domain. A detailed understanding of their operation is needed to direct applications such as the development of optogenetic devices. Here, we elucidate the signal transduction pathway of the photoactivated adenylate cyclase OaPAC, in which a BLUF domain is fused to an adenylate cyclase domain that catalyzes the light-dependent formation of cAMP from ATP. OaPAC has very low dark state activity and is therefore a promising starting point for optogenetic applications. We used unnatural amino acid mutagenesis to explore how light absorption by the N-terminal BLUF domain on the fs timescale leads to activation of the C-terminal adenylate cyclase domain. We incorporated the IR probe azidophenylalanine at F103, W90 and F131 using orthogonal aminoacyl-tRNA-synthetases. The FTIR light minus dark difference spectrum shows that the environment around F103 and W90 residues changes upon light activation, however, there is no change in the environment around F131 upon photoactaction. The enzymatic activity of these mutants was not affected due to the IR probe incorporation. We also replaced Y125 with fluorotyrosine analogues to increase the acidity of the phenolic hydroxyl group and probe the role of Y125 in signal transduction which forms an intersubunit hydrogen bond with N256 from the other monomer in the OaPAC dimer. OaPAC with 3-fluorotyrosine, 3,5-difluorotyrosine or 2,3,5-trifluorotyrosine incorporated at Y125 showed 10 fold higher activity in the dark compared to wild-type OaPAC, whereas Y125F and Y125S showed no dark state activity, implying that the intersubunit hydrogen bond between Y125 and N256 is critical for signal transduction.

Functional dynamics of the photoactivated adenylate cyclase OaPAC revealed by fluorescence spectroscopy and small-angle X-ray scattering

OaPAC is a photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata and belongs to a class of photoactive flavoenzymes which are involved in the conversion of ATP (adenosine triphosphate) to the important second messenger cAMP (cyclic adenosine monophosphate). The photoactivation is controlled by a BLUF (Blue Light Sensing Using Flavin) domain; a well-known light-triggered switch present in many biological photoreceptors and able to control photochemical energy into a diverse set of cellular responses (e.g.

Photoreaction of photoactivated adenylate cyclase from cyanobacterium Microcoleus chthonoplastes.

The photochemical reaction of photoactivated adenylate cyclase from cyanobacterium Microcoleus chthonoplastes PCC 7420 (mPAC), which consists of a Per-Arnt-Sim (PAS), a light‑oxygene-voltage (LOV), and an adenylate cyclase (AC) domain, was investigated mainly using the time-resolved transient grating method. An absorption spectral change associated with an adduct formation between its chromophore (flavin mononucleotide) and a cysteine residue was observed with a time constant of 0.66μs. After this reaction, a significant diffusion coefficient (D)-change was observed with a time constant of 38ms. The determined D-value was concentration-dependent indicating a rapid equilibrium between the dimer and tetramer. Combining the results of size exclusion chromatography and CD spectroscopy, we concluded that the photoinduced D-change was mainly attributed to the equilibrium shift from the dimer rich to the tetramer rich states upon light exposure. Since the reaction rate does not depend on concentration, the rate determining step of the tetramer formation is not the collision of proteins by diffusion, but a conformation change. The roles of the PAS and AC domains as well as the N- and C-terminal flanking helices of the LOV domain (A'α- and Jα-helices) were investigated using various truncated mutants. The PAS domain was found to be a strong dimerization site and is related to efficient signal transduction. It was found that simultaneous existence of the A'α- and Jα-helices in mPAC is important for the light-induced conformation change to lead the conformation change which induces the tetramer formation. The results suggest that the angle changes of the coiled-coil structures in the A'α and Jα-helices are essential for this conformation change. The reaction scheme of mPAC is proposed.

Optogenetic Modification of Pseudomonas aeruginosa Enables Controllable Twitching Motility and Host Infection.

Cyclic adenosine monophosphate (cAMP) is an important secondary messenger that controls carbon metabolism, type IVa pili biogenesis, and virulence in Pseudomonas aeruginosa. Precise manipulation of bacterial intracellular cAMP levels may enable tunable control of twitching motility or virulence, and optogenetic tools are attractive because they afford excellent spatiotemporal resolution and are easy to operate. Here, we developed an engineered P.aeruginosa strain (termed pactm) with light-dependent intracellular cAMP levels through introducing a photoactivated adenylate cyclase gene (bPAC) into bacteria. On blue light illumination, pactm displayed a 15-fold increase in the expression of the cAMP responsive promoter and an 8-fold increase in its twitching activity. The skin lesion area of nude mouse in a subcutaneous infection model after 2-day pactm inoculation was increased 14-fold by blue light, making pactm suitable for applications in controllable bacterial host infection. In addition, we achieved directional twitching motility of pactm colonies through localized light illumination, which will facilitate the studies of contact-dependent interactions between microbial species.

Radical Formation in the Photoactivated Adenylate Cyclase OaPAC Revealed by Ultrafast Spectroscopy

Adenylate cyclases (AC) form a class of enzymes which are involved in the conversion of ATP to cAMP, playing an important role in many regulatory processes as the cAMP produced is involved in signalling pathways controlled by cAMP binding proteins. There is a very special subgroup of AC enzymes called photoactivated adenylate cyclases (PAC) where the ATP to cAMP conversion is controlled by light. Interestingly the photoregulation is controlled by BLUF (Blue light sensing using FAD) domains which are responsible for light regulation in many processes like phototoaxis, photophobia etc. Light regulation of BLUF domains is governed by the reorganization of the hydrogen bond network around the flavin and it is reflected in a ∼10 nm red shift of the flavin absorption peak. The first discovered photo-activated adenylate cyclase was found in a unicellular flagellate Euglenagracilis where the flagellar movement is regulated via a photoactivated AC. Another photoactivated adenylate cyclase called bPAC was discovered in the sulfide-oxidizing bacterium Beggiatoa sp. where the light regulation is solved by the BLUF domain located at the N-terminal part of the protein, the cAMP conversion is accomplished by a class IIIb type adenylyl cyclase at the C-terminal. Another promising PAC was discovered recently in the photosynthetic cyanobacterium Oscillatoriaacuminata which shows substantial activation (20-fold increase in the cAMP level in light compared to the dark) upon light illumination. OaPAC is a 366-aa residue long homodimer having a class III adenylate cyclase domain at the C-terminal and a BLUF domain at the N-terminal. In this work we characterized the primary photochemistry of OaPAC by the means of ultrafast (femtosecond to nanosecond) spectroscopy. Ultrafast transient visible and transient infrared absorption spectroscopy reveals that neutral flavin radical is formed after the blue light absorption.

Cyclic Nucleotide-Specific Optogenetics Highlights Compartmentalization of the Sperm Flagellum into cAMP Microdomains.

Inside the female genital tract, mammalian sperm undergo a maturation process called capacitation, which primes the sperm to navigate across the oviduct and fertilize the egg. Sperm capacitation and motility are controlled by 3′,5′-cyclic adenosine monophosphate (cAMP). Here, we show that optogenetics, the control of cellular signaling by genetically encoded light-activated proteins, allows to manipulate cAMP dynamics in sperm flagella and, thereby, sperm capacitation and motility by light. To this end, we used sperm that express the light-activated phosphodiesterase LAPD or the photo-activated adenylate cyclase bPAC. The control of cAMP by LAPD or bPAC combined with pharmacological interventions provides spatiotemporal precision and allows to probe the physiological function of cAMP compartmentalization in mammalian sperm.

Molecular mechanism of photoactivation of a light-regulated adenylate cyclase

The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) detects light through a flavin chromophore within the N-terminal BLUF domain. BLUF domains have been found in a number of different light-activated proteins, but with different relative orientations. The two BLUF domains of OaPAC are found in close contact with each other, forming a coiled coil at their interface. Crystallization does not impede the activity switching of the enzyme, but flash cooling the crystals to cryogenic temperatures prevents the signature spectral changes that occur on photoactivation/deactivation. High-resolution crystallographic analysis of OaPAC in the fully activated state has been achieved by cryocooling the crystals immediately after light exposure. Comparison of the isomorphous light- and dark-state structures shows that the active site undergoes minimal changes, yet enzyme activity may increase up to 50-fold, depending on conditions. The OaPAC models will assist the development of simple, direct means to raise the cyclic AMP levels of living cells by light, and other tools for optogenetics.

Structural insight into photoactivation of an adenylate cyclase from a photosynthetic cyanobacterium

Cyclic-AMP is one of the most important second messengers, regulating many crucial cellular events in both prokaryotes and eukaryotes, and precise spatial and temporal control of cAMP levels by light shows great promise as a simple means of manipulating and studying numerous cell pathways and processes. The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) is a small homodimer eminently suitable for this task, requiring only a simple flavin chromophore within a blue light using flavin (BLUF) domain. These domains, one of the most studied types of biological photoreceptor, respond to blue light and either regulate the activity of an attached enzyme domain or change its affinity for a repressor protein. BLUF domains were discovered through studies of photo-induced movements of Euglena gracilis, a unicellular flagellate, and gene expression in the purple bacterium Rhodobacter sphaeroides, but the precise details of light activation remain unknown. Here, we describe crystal structures and the light regulation mechanism of the previously undescribed OaPAC, showing a central coiled coil transmits changes from the light-sensing domains to the active sites with minimal structural rearrangement. Site-directed mutants show residues essential for signal transduction over 45 Å across the protein. The use of the protein in living human cells is demonstrated with cAMP-dependent luciferase, showing a rapid and stable response to light over many hours and activation cycles. The structures determined in this study will assist future efforts to create artificial light-regulated control modules as part of a general optogenetic toolkit.

A LOV-domain-mediated blue-light-activated adenylate (adenylyl) cyclase from the cyanobacterium Microcoleus chthonoplastes PCC 7420

Genome screening of the cyanobacterium Microcoleus chthonoplastes PCC 7420 identified a gene encoding a protein (483 amino acids, 54.2kDa 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=450nm) and underwent conversion into the photoadduct form (λmax=390nm) upon BL-irradiation. The lifetime for thermal recovery into the parent state was determined as 16s at 20°C (25s 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 pH8 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 ~14s 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.

Photo-dynamics of the lyophilized photo-activated adenylate cyclase NgPAC2 from the amoeboflagellate Naegleria gruberi NEG-M strain

The absorption and emission spectroscopic behavior of lyophilized photo-activated adenylate cyclase NgPAC2 from the amoeboflagellate Naegleria gruberi NEG-M strain consisting of a BLUF domain (BLUF=Blue Light sensor Using Flavin) and a cyclase homology domain was studied in the dark, during blue-light exposure and after blue-light exposure at a temperature of 4°C. The BLUF domain photo-cycle dynamics observed for snap-frozen NgPAC2 was lost by lyophilization (no signaling state formation with flavin absorption red-shift). Instead, blue-light photo-excitation of lyophilized NgPAC2 caused sterically restricted Tyr–Tyr cross-linking (o,o′-ditysosine formation) and partial flavin cofactor reduction.

Optogenetic Modulation of an Adenylate Cyclase in Toxoplasma gondii Demonstrates a Requirement of the Parasite cAMP for Host-Cell Invasion and Stage Differentiation

cAMP research in intracellular parasites remains underappreciated, and it requires a specific method for cyclic nucleotide regulation. Optogenetic induction of cAMP in T. gondii affects host-cell invasion, stage-specific expression, and parasite differentiation. The underlying method allows a versatile control of parasite cAMP. Optogenetic parasite strains offer valuable tools for dissecting cAMP-mediated processes. The method is applicable to other gene-tractable intertwined systems. Successful infection and transmission of the obligate intracellular parasite Toxoplasma gondii depends on its ability to switch between fast-replicating tachyzoite (acute) and quiescent bradyzoite (chronic) stages. Induction of cAMP in the parasitized host cells has been proposed to influence parasite differentiation. It is not known whether the parasite or host cAMP is required to drive this phenomenon. Other putative roles of cAMP for the parasite biology also remain to be identified. Unequivocal research on cAMP-mediated signaling in such intertwined systems also requires a method for an efficient and spatial control of the cAMP pool in the pathogen or in the enclosing host cell. We have resolved these critical concerns by expressing a photoactivated adenylate cyclase that allows light-sensitive control of the parasite or host-cell cAMP. Using this method, we reveal multiple roles of the parasite-derived cAMP in host-cell invasion, stage-specific expression, and asexual differentiation. An optogenetic method provides many desired advantages such as: (i) rapid, transient, and efficient cAMP induction in extracellular/intracellular and acute/chronic stages; (ii) circumvention of the difficulties often faced in cultures, i.e. poor diffusion, premature degradation, steady activation, and/or pleiotropic effects of cAMP agonists and antagonists; (iii) genetically encoded enzyme expression, thus inheritable to the cell progeny; and (iv) conditional and spatiotemporal control of cAMP levels. Importantly, a successful optogenetic application in Toxoplasma also illustrates its wider utility to study cAMP-mediated signaling in other genetically amenable two-organism systems such as in symbiotic and pathogen-host models.

Photo-dynamics and thermal behavior of the BLUF domain containing adenylate cyclase NgPAC2 from the amoeboflagellate Naegleria gruberi NEG-M strain

The absorption and emission spectroscopic behavior of the photo-activated adenylate cyclase NgPAC2 from the amoeboflagellate Naegleria gruberi NEG-M strain was studied in the dark, during blue-light exposure and after blue-light exposure. The typical BLUF domain (BLUF=Blue Light sensor Using Flavin) flavin cofactor absorption and fluorescence photo-cycle dynamics was observed. For fresh samples a reversible concentration dependent protein oligomerization occurred showing up in free flavin binding and protein color center formation with increasing protein concentration. Thermal and temporal irreversible protein unfolding with loss of BLUF domain activity was investigated. Temperature dependent protein melting times and the apparent protein melting temperature were determined. The photodynamic behavior of the NgPAC2 is compared with the behavior of the previously investigated photo-activated cyclase NgPAC1 (nPAC) from the same N. gruberi NEG-M strain.

Thermal protein unfolding in photo-activated adenylate cyclase nano-clusters from the amoeboflagellate Naegleria gruberi NEG-M strain

The photo-activated adenylate cyclase (nPAC) protein from the amoeboflagellate Naegleria gruberi NEG-M strain consists of a BLUF domain (sensor of blue light using flavin) and a cyclase homology domain (CHD). The nPAC thermal stability is determined by its protein unfolding behavior which is quantified by the protein melting temperature and protein melting time. The protein unfolding in nPAC nano-clusters in aqueous solution at pH 7.5 is studied by light attenuation and fluorescence measurements. The temporal behavior of protein unfolding (denaturation) is monitored by observation of spectral changes of the first absorption band of the flavin cofactor. The nPAC unfolding occurs irreversible in a bi-exponential manner (different melting time constants for proteins at nano-cluster surface and in nano-cluster interior). The nPAC apparent melting temperature (there half of the proteins are unfolded) is determined by light attenuation measurement (light scattering increases due to coalescing of unfolded protein nano-clusters) in the non-absorbing spectral region of the protein. A measurement standard is developed employing a staircase temperature heating and cooling profile. High thermal stability of nPAC nano-clusters in pH 7.5 aqueous solution was found with an apparent melting temperature of 60 °C.

Absorption and fluorescence characteristics of photo-activated adenylate cyclase nano-clusters from the amoeboflagellate Naegleria gruberi NEG-M strain

The spectroscopic characteristics of BLUF (BLUF = sensor of blue light using flavin) domain containing soluble adenylate cyclase (nPAC = Naegleria photo-activated cyclase) samples from the amoeboflagellate Naegleria gruberi NEG-M strain is studied at room temperature. The absorption and fluorescence spectroscopic development in the dark was investigated over two weeks. Attenuation coefficient spectra, fluorescence quantum distributions, fluorescence quantum yields, and fluorescence excitation distributions were measured. Thawing of frozen nPAC samples gave solutions with varying protein nano-cluster size and varying flavin, tyrosine, tryptophan, and protein color-center emission. Protein color-center emission was observed in the wavelength range of 360–900 nm with narrow emission bands of small Stokes shift and broad emission bands of large Stokes shift. The emission spectra evolved in time with protein nano-cluster aging.

Stimulating PACα Increases Miniature Excitatory Junction Potential Frequency at the Drosophila Neuromuscular Junction

: Photoactivated adenylate cyclase alpha (PACα) is a light-activated adenylate cyclase that was originally cloned from the eye spot of the protozoan Euglena gracilis. PACα has been shown to rapidly increase intracellular cyclic adenosine monophosphate (cAMP) in vivo in Xenopus oocytes and HEK293 cells, increase the spike width in Aplysia sensory neurons, and modify behavior in Drosophila. Using the GAL4 UAS system, we heterologously expressed PACα in motorneurons and quantified the effects of its activation at the neuromuscular junction of the Drosophila third instar wandering larva, a well-characterized model synapse. By recording from body-wall muscle 6, we show that the presynaptic activation of PACα with blue light significantly increased miniature excitatory junction potential (mEJP) frequency in the presence of calcium with a delay of about 1 minute. Similar effects have been observed in previous studies that utilized adenylate cyclase agonists (Forskolin) or membrane-permeable cAMP analogs [dibutyryl cAMP and 4-chlorophenylthio-(CPT)-cAMP] to increase presynaptic cAMP concentrations. PACα′s efficacy in combination with its specificity make it an invaluable tool for the rapid regulation of cAMP in vivo and for investigating the mechanisms by which cAMP can modulate synaptic transmission and neuronal plasticity in Drosophila.