1. Volume 90, Issue 5, Pages 1016-1027 (June 2016)
Melanopsin-Encoded Response Properties of Intrinsically Photosensitive Retinal Ganglion Cells 
Ludovic S. Mure, Megumi Hatori, Quansheng Zhu, James Demas, Irene M. Kim, Surendra K. Nayak, Satchidananda Panda 
Neuron 
Volume 90, Issue 5, Pages (June 2016)
DOI: /j.neuron
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2. Figure 1
Photoactivated Melanopsin Is Phosphorylated at C Terminus S/T Residues
(A) Schematic diagram shows mammalian rhodopsin, Drosophila rhodopsin, and mammalian melanopsin.
(B) Melanopsin residues 381–397 are important for desensitization upon light stimulation. Xenopus oocytes injected with mRNAs encoding mouse Opn4, β-arr1 (or β-arr2), Gq, and TrpC3 and perfused with 11-cis retinal showed light-activated membrane depolarization. After lights off, the photocurrent returned to baseline in oocytes expressing Opn4WT and arrestin, but remained depolarized if arrestin was not co-injected. Sustained depolarizing photocurrents also were observed with C-terminal truncated versions of melanopsin (Opn4380Δ). A version of Opn4 with 397 aa (Opn4397Δ) exhibited arrestin-dependent deactivation.
(C) Putative phosphorylation sites in mouse melanopsin between residues 380 and 397 are conserved across species.
(D) WT or C terminus truncations (380Δ or 364Δ) were expressed in HEK cells and metabolically labeled with P32. Western blotting revealed that full-length melanopsin was phosphorylated, but not the truncated versions.
(E) Phosphopeptide-digested products from light-activated melanopsin separated by thin-layer chromatography showed phosphorylation primarily at S and, to a lesser extent, at T sites.
Neuron  , DOI: ( /j.neuron )
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3. Figure 2 Melanopsin Phosphorylation Contributes to Response Properties
(A) Mouse Opn4 mutants carrying alanine (A) aa substitution at candidate S/T phosphorylation sites. The aa sequence and coordinates for mouse melanopsin are shown on the top.
(B) Deactivation of melanopsin photoresponse in CHO cells transduced with different mutant versions of Opn4. Average time (s ±SEM) for the peak response to return to 30% of maximal response is shown.
(C) Representative calcium responses for CHO cells expressing Opn4WT or Opn4 variants are shown.
(D–F) Comparison of arbitrary clusters of S/T mutations. Representative traces (D), design (E), and average peak amplitude and time (s ±SEM) for the peak response to return to 30% of peak amplitude (F) are shown.
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4. Figure 3
Alterations to the Melanopsin C Terminus Tail Results in Altered Response Properties
(A) Circadian wheel-running activity of rd;Opn4Cre/Cre mice intravitreally transduced with AAV expressing Opn4WT or Opn4 variants. A few days after transduction (red arrows), functional expression of Opn4 mediates photoentrainment of daily activity to the LD cycle. rd;Opn4−/− and rd;Opn4cre/cre mice retinas transduced with melanopsin are recorded on MEA.
(B and C) Proportion of recorded cells responding to 100-ms or 10-s stimulations (B). Data for 100 ms, 1 s, 10 s, and 60 s are shown in (C). Light responses are shown for rd;Opn4−/− retinas expressing WT (Opn4WT, n = 57) and altered melanopsins, including truncated (Opn4380Δ, n = 33; Opn4397Δ, n = 45), phospho-null mutations (Opn42A, n = 103; Opn44A, n = 100; Opn49A, n = 125), and phosphomimetic mutations (Opn42D, n = 61).
(D–I) Average traces (D and E), response latency (F and G), and response duration (H and I) are shown. Recordings (MEA and PLR) in response to monochromatic light stimulations of increasing duration (480 nm, photons/cm2/s, 100 ms, 1 s, 10 s, and 60 s in MEA and 480 nm, photons/cm2/s, 1 s, and 60 s in PLR) are shown (average values ± SEM, F–I). For detailed statistics see Table S1.
Neuron  , DOI: ( /j.neuron )
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5. Figure 4
Altered Response Properties Translate In Vivo ipRGCs Responses to Light
(A) rd;Opn4cre/cre mice conditionally infected with floxed Opn4WT (n = 54) or altered melanopsins (Opn4397Δ, n = 38; Opn49A, n = 36; and Opn49A397Δ, n = 29) are mirrored by pupillary constriction in animal transduced with the same type of mutants.
(B) PLR average traces are shown (Opn4WT, n = 4; Opn4397Δ, n = 5; Opn49A, n = 2; and Opn49A397Δ, n = 6).
(C) Representative pictures show the different phases of PLR.
Neuron  , DOI: ( /j.neuron )
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6. Figure 5
Response Sensitivity to Light Is Affected by Alterations to the Melanopsin C Terminus Tail
rd;Opn4cre/cre mice retinas conditionally expressing melanopsin variants are recorded on MEA.
(A–C) Average responses of ipRGCs to 1-min stimulations of increasing irradiance are shown (A, Opn4WT, n = 35; B, Opn4397Δ, n = 47; and C, Opn49A, n = 42).
(D and E) Dose-response curves associated with response duration (D) and the number of spikes €. Data have been fit with the appropriate function (sigmoidal or linear). Correlation coefficients for duration and number of spikes are as follows: 0.9 and 0.96 for Opn4WT, 0.95 and 0.96 for Opn49A, and 0.94 and 0.91 for Opn4397Δ. Recordings (MEA) were performed in response to monochromatic light stimulations of increasing irradiance (480 nm, 60 s, photons/cm2/s to  photons/cm2/s; average values ± SEM).
Neuron  , DOI: ( /j.neuron )
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7. Figure 6
Melanopsin C-Terminus Tail Contributes to Its Unique Response Properties
(A) Distribution of the persistence (delay between light off and return to baseline) through increasing irradiances. At all irradiances, Opn49A displays a large range of persistence durations while Opn4397Δ-transduced cells display very little persistence of the response irrespective of the stimulation irradiance (in italic, number of responses out of boundaries of the recording).
(B) Deactivation speed (plateau/tonic response (last 10 s before light off) to the baseline discharge rate divided by the delay between the light off and the return to baseline) after 1-min stimulations of increasing irradiance. Opn4WT deactivation speed remained constant independent of the irradiance of the stimulation (Opn4WT linear regression slope not different from 0, p = ), whereas it increased for Opn4397Δ (MANOVA, F = (2,8), p < 0.001).
Neuron  , DOI: ( /j.neuron )
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8. Figure 7 Proposed Melanopsin Deactivation Model
Active melanopsin (Ma, and/or extramelanopsin [E]) is photoisomerized into activated melanopsin (M∗), the signaling form, upon photon absorption. Phosphorylation of M∗ induces its binding by arrestins and subsequent deactivation (Mi, inactive melanopsin) and recycling/regeneration. Absorption of photon from another wavelength may photo regenerate directly M∗ into Ma/E forms.
Neuron  , DOI: ( /j.neuron )
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