1. Volume 25, Issue 2, Pages 215-223.e3 (February 2018)
PhoDAGs Enable Optical Control of Diacylglycerol-Sensitive Transient Receptor Potential Channels 
Trese Leinders-Zufall, Ursula Storch, Katherin Bleymehl, Michael Mederos y Schnitzler, James A. Frank, David B. Konrad, Dirk Trauner, Thomas Gudermann, Frank Zufall 
Cell Chemical Biology 
Volume 25, Issue 2, Pages e3 (February 2018)
DOI: /j.chembiol
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2. Cell Chemical Biology 2018 25, 215-223. e3DOI: (10. 1016/j. chembiol
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3. Figure 1
Rapid Photoactivation and -Deactivation of Native Trpc2 Currents in Mouse VSNs
(A) Chemical structures of photoswitchable PhoDAG-3 in its less active form (trans-PhoDAG-3) at visible light (λ > 460 nm) and its active form (cis-PhoDAG-3) after exposure to UV light (λ < 370 nm).
(B) UV exposure (365 nm) of a freshly dissociated VSN preincubated with PhoDAG-3 (5 μM) causes rapid activation of sustained channel activity that can be terminated upon exposure to 470 nm light. Whole-cell voltage-clamp recording; holding potential, −70 mV. No such currents could be induced in VSNs not preincubated with PhoDAG-3 (lower trace).
(C) Repeatability of current photoactivation and -deactivation with PhoDAG-3 (5 μM) after switching between 365 and 470 nm light.
(D) Dose-dependence of average light-activated currents (at −70 mV, 365 nm) from multiple VSNs (magenta bars). Mean ± SEM: 5 μM PhoDAG-3, −16.6 ± 3.7 pA; 10 μM PhoDAG-3, −112.0 ± 40.6 pA; 50 μM PhoDAG-3, −392.2 ± 131.7 pA. No currents were observed in the dark (black bars). The number of cells is indicated above each bar.
(E) Current activation and deactivation times after photoswitching with 5 and 10 μM PhoDAG-3 recorded from individual VSNs (number of cells indicated above each bar). Current activation was concentration independent (t test: t(5) = 0.75, p = 0.49), whereas current deactivation increased strongly with higher a PhoDAG-3 concentration (t test: t(6) = 4.18, **p < 0.01). Onset time, mean ± SEM: 5 μM PhoDAG-3, −0.8 ± 0.1 s; 10 μM PhoDAG-3, 2.4 ± 1.8 s. Offset time, mean ± SEM: 5 μM PhoDAG-3, 3.4 ± 0.3 s; 10 μM PhoDAG-3, 21.0 ± 4.2 s.
(F) Representative families of whole-cell currents to a series of depolarizing and hyperpolarizing voltage steps (as indicated in the figure) recorded from a VSN. Cells were exposed successively to no light (dark) in the absence of PhoDAG-3 and 365 nm light or 470 nm light in the presence of 10 μM PhoDAG-3 in the bath. Experiments were performed in the presence of 1 μM tetrodotoxin to block voltage-gated Na+ channels; voltage-activated K+ channels were blocked by using a Cs+-based pipette solution. Dotted line, zero current level.
(G and H) Plots of steady-state current-voltage relationships of photoactivated currents as shown in (E), plotted either as raw data (G) or after digital subtraction of the currents obtained before PhoDAG-3 treatment (no PhoDAG-3, dark) (H).
(I and J) Absence of photoactivated currents in Trpc2−/− VSNs (n = 5). Shown are current families to a series of depolarizing and hyperpolarizing voltage steps (10 μM Pho-DAG3) (I) and plots of steady-state current-voltage relationships (J). Same voltage protocol as in (F). Concentration of PhoDAG-3 was 10 or 50 μM, respectively (J).
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4. Figure 2
Combined Optical Photoswitching and Ca2+ Imaging Reveals DAG-Stimulated Ca2+ Transients
(A) Photostimulation (355 nm, magenta) of a freshly dissociated VSN loaded with PhoDAG-3 (5 μM) and fluo-4/AM (9 μM) caused repeatable activation of Ca2+ transients that decayed rapidly back to baseline (upper traces). No such responses occurred in the absence of PhoDAG-3 (lower traces). Results are based on recordings from seven VSNs for each condition. Average peak amplitude of Ca2+ responses was 9.9% ± 2.4%, with an average decay time constant of 1.9 ± 0.3 s (n = 7).
(B) Kinetics of photostimulated VSN Ca2+ transients at enhanced temporal resolution (5 μM PhoDAG-3).
(C and D) Transmitted light (C) and confocal fluorescence (D) images acquired at rest (pseudocolor scale) showing subcellular structures of a dissociated VSN. The dashed circle indicates the VSN dendritic ending containing the microvilli and dendritic knob (MV/Knob). Scale bar, 5 μm.
(E) Spatially localized photoswitching (355 nm, magenta) at the dendritic knob and microvilli (SK) produced a transient Ca2+ responses at the microvilli/knob region (MV/Knob) but not at the soma. Localized photoswitching at the soma (SS) produced no Ca2+ responses at all. Results are based on four independent experiments.
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5. Figure 3
PhoDAG Photoswitching Enables Mapping of Diacylglycerol-Evoked Neuronal Activity in Acute Tissue Slices
(A) Transmitted light and confocal fluorescence images (top) acquired at rest (pseudocolor scale) in the sensory epithelium of an acute VNO slice (C57BL/6). Tissue was loaded with 5 μM PhoDAG-3 and 9 μM fluo-4/AM. White box indicates area of ΔF/F0 images shown below. Dashed area shows the boundaries of laser scanning-mediated light stimulation. ΔF/F0 images (bottom) show Ca2+ fluorescence (1) before stimulation, (2) after exposure to 355-nm light, and (3) after recovery back to baseline. The numbers refer to the time points indicated in (B). Scale bar, 50 μm.
(B) Time dependence of somatic VSN Ca2+ responses (C57BL/6) after photostimulation (355 nm, magenta). Responses from 21 individual VSN somata in a given optical section are plotted. Arrows with numbers indicate the time points of the ΔF/F0 images shown in (A).
(C) Time course of somatic Ca2+ transients in VNO slices (C57BL/6) evoked by elevated KCl (60 mM).
(D) Absence of photo-evoked VSN Ca2+ responses (355 nm, magenta) in VNO slices obtained from Trpc2−/− mice.
(E) Elevated KCl (60 mM) evoked somatic Ca2+ transients in Trpc2−/− VNO slices, demonstrating cell viability and excitability. Same cells as those shown in (D).
(F) Bar histograms comparing peak fluorescence values (Fpeak) of somatic VSN Ca2+ responses in C57BL/6 versus Trpc2−/− mice to various stimuli as indicated. Data are represented as means ± SEM.
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6. Figure 4
Photoswitching Reveals Diacylglycerol-Mediated Ca2+ Responses in an Oxygen Sensor of the Main Olfactory Epithelium
(A) Photostimulation (355 nm, thin magenta line) of freshly dissociated type B and type A cells co-loaded with PhoDAG-3 (5 μM) and fluo-4/AM (9 μM) caused repeatable activation of Ca2+ responses in these two cell types. Responses were characterized by strikingly slow decay time courses. No Ca2+ responses occurred in OSNs and without switching to 355 nm (dashed gray line, no light exposure). Cells were dissociated from the MOE of Trpc2-IRES-taumCherry mice in which all Trpc2-expressing cells can be identified on the basis of their intrinsic red fluorescence of mCherry.
(B) Bar histograms summarizing peak fluorescence values (Fpeak) of photoactivated Ca2+ responses to repeated light stimulation in type B and type A cells. No responses were observed in canonical OSNs. ***p <
(C) Analyses of decay time courses of photoactivated Ca2+ responses from type B and type A cells revealed average time constants of τ = 139.6 ± 14.9 s (n = 15) in type B cells and τ = 208.7 ± 46.9 s (n = 5) in type A cells. Decay time constants of photoactivated Ca2+ responses from VSNs are shown for comparison, with τ = 1.9 ± 0.3 s (n = 7). Data are represented as means ± SEM.
(D) Recording example of a type B cell loaded with PhoDAG-1 (10 μM). Photoswitching to 355 nm (thin magenta line) evoked a Ca2+ transient with virtually identical properties (n = 3) as that observed in (A) after PhoDAG-3 stimulation.
(E) Chemical structures of photoswitchable PhoDAG-1 in its inactive form (trans-PhoDAG-1) at visible light (λ > 450 nm) and in its active form (cis-PhoDAG-1) after exposure to UV light (λ < 370 nm).
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7. Figure 5
PhoDAG Enables Photoswitchable Activation and Deactivation of Human TRPC6 Cation Channels
(A–F) Electrophysiological whole-cell measurements with untransfected HEK293 cells (B, E, and F) and HEK293 cells overexpressing human TRPC6 (A, C, and D). (A and B) Representative current time courses at holding potentials of ±60 mV of TRPC6-expressing (A) and of untransfected HEK293 cells (B). Application of fluorescence light of λ = 350 nm or 450 nm is indicated. (C–F) Representative current-voltage relations and summaries of current densities of TRPC6-overexpressing (C and D) and untransfected HEK293 cells (E and F). Basal currents (control) were determined in the presence of 100 μM PhoDAG-1 in the dark before first application of fluorescence light. n indicates the number of measured cells from at least three independent experiments. Statistical differences were calculated using paired Student's t test between control and 350 nm-induced current densities (mean ± SEM; p < 0.01 at −60 mV and p < 0.01 at +60 mV for TRPC6-expressing cells and p = 0.87 at −60 mV and p = 0.41 at +60 mV for untransfected HEK293 cells), between 350 nm- and 450 nm-induced current densities (p < 0.01 at −60 mV and p < 0.05 at +60 mV for TRPC6 expressing HEK293 cells and p = 0.85 at −60 mV and p = 0.31 at +60 mV for untransfected HEK293 cells), and between control and 450 nm-induced current densities (p = 0.24 at −60 mV and p = 0.11 at +60 mV for TRPC6 expressing HEK293 cells and p = 0.35 at −60 mV and p = 0.89 at +60 mV for untransfected HEK293 cells). *p < 0.05; **p < Data are normally distributed (Shapiro-Wilk test).
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