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. 2011 Jan 14;286(2):1181-8.
doi: 10.1074/jbc.M110.185496. Epub 2010 Oct 28.

Light Modulation of Cellular cAMP by a Small Bacterial Photoactivated Adenylyl Cyclase, bPAC, of the Soil Bacterium Beggiatoa

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Light Modulation of Cellular cAMP by a Small Bacterial Photoactivated Adenylyl Cyclase, bPAC, of the Soil Bacterium Beggiatoa

Manuela Stierl et al. J Biol Chem. .
Free PMC article

Abstract

The recent success of channelrhodopsin in optogenetics has also caused increasing interest in enzymes that are directly activated by light. We have identified in the genome of the bacterium Beggiatoa a DNA sequence encoding an adenylyl cyclase directly linked to a BLUF (blue light receptor using FAD) type light sensor domain. In Escherichia coli and Xenopus oocytes, this photoactivated adenylyl cyclase (bPAC) showed cyclase activity that is low in darkness but increased 300-fold in the light. This enzymatic activity decays thermally within 20 s in parallel with the red-shifted BLUF photointermediate. bPAC is well expressed in pyramidal neurons and, in combination with cyclic nucleotide gated channels, causes efficient light-induced depolarization. In the Drosophila central nervous system, bPAC mediates light-dependent cAMP increase and behavioral changes in freely moving animals. bPAC seems a perfect optogenetic tool for light modulation of cAMP in neuronal cells and tissues and for studying cAMP-dependent processes in live animals.

Figures

FIGURE 1.
FIGURE 1.
Concept of light-activated cyclase. a, schematic arrangements of the photoreceptive BLUF domain (F) and the catalytic domain (C). b, part of the bPAC cyclase amino acid sequence aligned to the corresponding regions of other Type III cyclases; metal-binding Asps (Me) are shaded in red, essential adenine-binding Lys and Thr are in green, and transition state-stabilizing Asn and Arg are in blue. c, model of the dimeric bPAC with flavin-binding BLUF domain (F) in yellow and the catalytic domain (C) in blue. d, cyclase activity in an adenylate cyclase-deficient E. coli strain before (left) and after (right) transformation with bPAC on a MacConkey agar plate. Red color indicates rescue of maltose metabolism due to cAMP production.
FIGURE 2.
FIGURE 2.
bPAC activity in Xenopus oocytes. a, principle of the electrical assay. CFTR is activated by phosphorylation via an oocyte-endogenous PKA, whereas the CNG channel is directly activated by cAMP binding. I, current; V, voltage. b, photocurrents evoked by a 500-ms light pulse (450 nm) after coexpression of bPAC and CNG channel (dark red trace (trace 1)) and currents evoked by an 8-s (large arrow) or a 100-ms (small arrow) light pulse after co-injection of bPAC and CFTR (blue trace (trace 2)). Currents were measured at −40 mV for CFTR and CNG channels. In both test systems, the current reached values up to about −0.3 μA.
FIGURE 3.
FIGURE 3.
Spectral properties. a, absorption spectra of purified bPAC in its dark-adapted (trace 1) and light adapted (trace 2) state. The difference between the two is shown as a line (trace 3). OD, optical density. b, decay of the red-shifted intermediate that is considered as the signaling state. The fit is seen as a white line. The protein was excited for 3 s with a 455 nm LED, and the absorbance change was recorded at 489 nm. c, cAMP concentration at different time delays in the dark after a 4-s 475 nm light pulse; 300 mm KCl, 50 mm Tris-Hepes, pH 7.4, 21 °C, n = 3 with double determinations for each cAMP value. d, light (475 nm) intensity dependence of cAMP production by purified bPAC, conditions as in C, illumination for 60 s, and immediate quenching with 9-fold volume of 0.1 m HCl. n = 2 with double determinations for each cAMP value. Plotted are mean values with S.D. and a Michaelis-Menten fit curve, yielding a Km of 3.7 ± 0.4 μW mm−2.
FIGURE 4.
FIGURE 4.
Assessing bPAC function in hippocampal neurons. a, CA1 pyramidal cell expressing bPAC, CNG-A2, and RFP (two-photon imaging at 980 nm; scale bar, 30 μm). b, light-evoked cAMP-gated current at 0.14 milliwatt/mm (2). Arrow, 100-ms light pulse. The enlarged inset shows miniature excitatory postsynaptic currents. c, light-evoked cAMP-gated currents in one pyramidal cell at four different light doses (black traces, 0.14 milliwatt/mm2 for 50, 100, and 1000 ms; gray traces, 109 milliwatts/mm2 for 1 s). Traces were low pass-filtered at 10 Hz to remove miniature excitatory postsynaptic currents. At all stimulation intensities, currents were fully reversible and highly reproducible. CNG currents saturated at 0.14 milliwatt × s mm−2. d, light-evoked cAMP-gated currents before and after forskolin (100 μm) + IBMX (100 μm) wash-in. During forskolin/IBMX wash-in (dashed line, 5 min), holding current increased from −108 pA to −446 pA. Forskolin/IBMX application only partially occluded light-induced currents.
FIGURE 5.
FIGURE 5.
Comparing bPAC- and euPACα-induced currents in neurons. a, following a 100-ms light pulse (140 μW mm−2, blue arrow), cAMP elevation was much longer lasting in CA1 pyramidal cells expressing bPAC, CNG-A2, and RFP (black trace) when compared with cells expressing euPACα, CNG-A2, and RFP (red trace). Traces were low pass-filtered to remove miniature excitatory postsynaptic currents. b, time to half-peak current and current decay time constant were significantly longer in bPAC-expressing neurons when compared with euPACα-expressing neurons (bPAC, n = 8 conditions (three light doses, five cells); euPAC: n = 7 conditions (four light doses, three cells); ***, p < 0.001). c, under subsaturating conditions, light dose dependence of peak currents was similar for bPAC- and euPACα-expressing neurons. d, total charge transfer (integrated current) was ∼8 times higher in bPAC-expressing neurons when compared with euPACα-expressing neurons. nC, nano Coulomb.
FIGURE 6.
FIGURE 6.
Transgenic bPAC and euPACα exhibit different levels of dark activity and affect grooming behavior in freely moving Drosophila. a, expression of euPACα transgenes (elav::euPACα) resulted in distinctive dark activity, which was revealed by the phosphodiesterase blocker IBMX. Dark activity was not observed upon bPAC expression (elav::bPAC) or in wild type Canton-S control animals. Photoactivation of either PAC transgene resulted in a 10-fold increase in cAMP with no statistical difference between final concentrations of cAMP derived from either euPACα or bPAC (n = 11/group). 100 μm IBMX was used to block phosphodiesterase activity; light activation of cyclase transgenes was performed in 100 μm IBMX and irradiation (5 min, 455 nm, 40 milliwatts mm−2). b, photoactivation of pan-neuronally expressed PAC transgenes affects grooming activity, resulting in stereotypic freezing behavior (7). bPAC-expressing flies freeze significantly faster in blue light than euPACα flies (n = 11/group). They also take significantly longer to resume grooming behavior in the dark. Data represent means ± S.E.; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.

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