A unique choanoflagellate enzyme rhodopsin exhibits light-dependent cyclic nucleotide phosphodiesterase activity

Abstract

Photoactivated adenylyl cyclase (PAC) and guanylyl cyclase rhodopsin increase the concentrations of intracellular cyclic nucleotides upon illumination, serving as promising second-generation tools in optogenetics. To broaden the arsenal of such tools, it is desirable to have light-activatable enzymes that can decrease cyclic nucleotide concentrations in cells. Here, we report on an unusual microbial rhodopsin that may be able to meet the demand. It is found in the choanoflagellate Salpingoeca rosetta and contains a C-terminal cyclic nucleotide phosphodiesterase (PDE) domain. We examined the enzymatic activity of the protein (named Rh-PDE) both in HEK293 membranes and whole cells. Although Rh-PDE was constitutively active in the dark, illumination increased its hydrolytic activity 1.4-fold toward cGMP and 1.6-fold toward cAMP, as measured in isolated crude membranes. Purified full-length Rh-PDE displayed maximal light absorption at 492 nm and formed the M intermediate with the deprotonated Schiff base upon illumination. The M state decayed to the parent spectral state in 7 s, producing long-lasting activation of the enzyme domain with increased activity. We discuss a possible mechanism of the Rh-PDE activation by light. Furthermore, Rh-PDE decreased cAMP concentration in HEK293 cells in a light-dependent manner and could do so repeatedly without losing activity. Thus, Rh-PDE may hold promise as a potential optogenetic tool for light control of intracellular cyclic nucleotides (e.g. to study cyclic nucleotide-associated signal transduction cascades).

Keywords: biophysics; cyclic nucleotide; enzyme mechanism; membrane enzyme; optogenetics; phosphodiesterases; photoreceptor; rhodopsin.

Figure 1.

Architecture and amino acid composition of Rh-PDE. A, Rh-PDE is composed of the seven-transmembrane helical rhodopsin domain and the cytoplasmic PDE domain. B, phylogenetic tree of selected microbial rhodopsins based on the sequences of the transmembrane domains. C, conservation of important residues of the Rh domain. Red and blue colors represent acidic and basic amino acids, respectively. Residue numbers in Rh-PDE and BR are shown. D, conservation of important residues of the PDE domain. Residue numbers in Rh-PDE and PDE10A are shown, and sequences are grouped according to the substrate specificity.

Figure 2.

Enzymatic activity of Rh-PDE in HEK293 cells. A, luminescence signals representing cGMP levels, as observed in HEK293 cells with the empty vector (top), Rh-PDE (middle), and the PDE-inactive D605A mutant (bottom). HEK293 cells were preincubated in the culture medium with all-trans-retinal in the dark. The arrows and vertical light-blue line indicate sodium nitroprusside treatments and irradiation at 510 nm with 2.28 μW mm⁻² intensity, respectively, and the luminescence signals of non-irradiated cells are shown as a control (black lines). B, luminescence signals representing cAMP level, as observed in HEK293 cells with the empty vector (top), Rh-PDE (middle), and the PDE-inactive D605A mutant (bottom). HEK293 cells were preincubated in the culture medium with all-trans-retinal in the dark. The arrows and vertical light-blue line indicate forskolin treatments and irradiation at 510 nm with 2.28 μW mm⁻² intensity, respectively, and the luminescence signals of non-irradiated cells are shown as a control (black lines). Only HEK293 cells expressing Rh-PDE (red line in the middle panel) exhibited a decrease of the luminescence signal, indicating that cAMP concentration is lowered by light. C, changes of luminescence signals upon 2-min 510-nm irradiation (light-blue line) of HEK293 cells with the empty vector (top), Rh-PDE (middle), the PDE-inactive D605A mutant (bottom), expanded from B. Colored and black lines represent the data with and without irradiation, respectively. D, intensity dependence of the light-induced cAMP concentration decrease by Rh-PDE (cells were irradiated for 2 min with 510-nm light with 0–2.28 μW mm⁻² intensities). E, peak amplitudes of luminescence decrease plotted against the light intensity (n ≧ 5 cells). Error bars, S.D. The half-maximal light intensity (EC₅₀) was determined to be 0.244 μW mm⁻² by a single exponential decay fit (red dashed line).

Figure 3.

Rh-PDE as a potential optogenetic tool. A, comparison of in-cell hydrolytic activities of Rh-PDE (top) and mosquito Opn3 (bottom) toward cAMP. The top panel is reproduced from Fig. 2C. Mosquito Opn3 was expressed in HEK293 cells similar to Rh-PDE. During the time period shown in light blue, 510-nm light with 2.28 μW mm⁻² intensity was applied to the cells. B, effect of the addition of all-trans-retinal on the Rh-PDE activity. C, repeatability of the in-cell Rh-PDE activation. 2-min irradiation at 510 nm with 2.28 μW mm⁻² intensity was repeated every 15 min, and other conditions were as in Fig. 2C. D, irradiation wavelength dependence of the in-cell activity of Rh-PDE toward cAMP. All of the data were obtained from experiments at the same light intensity (0.4 μW mm⁻²).

Figure 4.

HPLC analysis of the enzymatic activity of Rh-PDE. A, HEK293 cell membranes expressing Rh-PDE were incubated with 100 μm cGMP for 2 min (blue), 5 min (light blue), 10 min (green), 15 min (orange), and 20 min (red) in the dark (top) and under the illumination (bottom), and the reaction products were analyzed by HPLC. Dotted black line, profile for 100 μm cGMP without Rh-PDE. B, time course of the decrease of cGMP concentration in the dark (black) and under illumination (blue) (n = 3). C, cGMP hydrolysis activity in the dark and under the illumination calculated from B. D, HEK293 cell membranes expressing Rh-PDE were incubated with 100 μm cAMP for 2 min (purple), 5 min (light blue), 10 min (green), and 20 min (red) in the dark (top) and under illumination (bottom), and the reaction products were analyzed by HPLC. Dotted black line, profile for 100 μm cAMP without Rh-PDE. E, time course of the decrease of cAMP concentration in the dark (black) and under illumination (red) (n = 3). F, cAMP hydrolysis activity in the dark and under illumination calculated from E. Error bars, S.D.

Figure 5.

Molecular properties of purified full-length Rh-PDE. A, absorption spectra of full-length Rh-PDE in the dark (black) and after irradiation with 510-nm light (red) at 4 °C. Inset, Coomassie Brilliant Blue-stained purified Rh-PDE on 12% SDS-PAGE. The arrow corresponds to about 70 kDa. B, absorption spectrum of purified full-length Rh-PDE (dotted line; reproduced from the black line in A) superimposed with the action spectrum of the light-induced enzymatic activity toward cAMP in HEK293T cells (the peak amplitudes in Fig. 3D are plotted versus irradiation wavelength with 0.4 μW mm⁻² intensity). C, difference absorption spectra of the photoactivation. D, M decay and the parent state recovery kinetics at 4 °C. E, temperature dependence of the M decay. F, Arrhenius plot for the M-decay kinetics. From the linear fit (dotted line), the decay time constant at 27 °C was estimated to be 6.78 s (red point). Error bars, S.D.

Figure 6.

Schematic representation of the Rh-PDE activation mechanism. The model shows a putative Rh-PDE dimer. A, in the dark, Rh-PDE exhibits constitutive activity toward cGMP and cAMP, suggesting that the catalytic region is somewhat exposed to the cytoplasm in mammalian cells. B, upon absorption of blue-green light by the Rh domain, photoisomerization of the retinal chromophore from all-trans-retinal (ATR) to the 13-cis form produces the M intermediate with deprotonated Schiff base (Rh domain switch on), but the PDE domain has not changed yet (PDE domain switch off). C, next, the M intermediate decays to the parent spectral state in 7 s (Rh domain switch off), accompanied by the structural changes in the PDE domain (PDE domain switch on). The resulting increased enzymatic activity is turned off within ∼70 s (PDE domain switch off).