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Spontaneous activation of visual pigments in relation to openness/closedness of chromophore-binding pocket

  1. Wendy Wing Sze Yue
  2. Rikard Frederiksen
  3. Xiaozhi Ren
  4. Dong-Gen Luo
  5. Takahiro Yamashita
  6. Yoshinori Shichida
  7. M Carter Cornwall
  8. King-Wai Yau  Is a corresponding author
  1. Johns Hopkins University School of Medicine, United States
  2. Boston University School of Medicine, United States
  3. Peking University, China
  4. Peking-Tsinghua Center for Life Sciences, China
  5. Kyoto University, Japan
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Cite this article as: eLife 2017;6:e18492 doi: 10.7554/eLife.18492

Abstract

Visual pigments can be spontaneously activated by internal thermal energy, generating noise that interferes with real-light detection. Recently, we developed a physicochemical theory that successfully predicts the rate of spontaneous activity of representative rod and cone pigments from their peak-absorption wavelength (λmax), with pigments having longer λmax being noisier. Interestingly, cone pigments may generally be ~25 fold noisier than rod pigments of the same λmax, possibly ascribed to an ‘open’ chromophore-binding pocket in cone pigments defined by the capability of chromophore-exchange in darkness. Here, we show in mice that the λmax-dependence of pigment noise could be extended even to a mutant pigment, E122Q-rhodopsin. Moreover, although E122Q-rhodopsin shows some cone-pigment-like characteristics, its noise remained quantitatively predictable by the ‘non-open’ nature of its chromophore-binding pocket as in wild-type rhodopsin. The openness/closedness of the chromophore-binding pocket is potentially a useful indicator of whether a pigment is intended for detecting dim or bright light.

https://doi.org/10.7554/eLife.18492.001

eLife digest

At the back of our eyes is a thin layer of cells that contain light-absorbing pigment molecules. These cells convert light energy into electrical signals that the brain then interprets to allow us to see. In this cell layer, the so-called cone cells work in bright light and provide us with the sense of color, whereas rod cells are for vision in dim light. Each visual pigment consists of a protein with a pocket-like space that holds a compound called a chromophore. Light causes the chromophore to change shape inside the pocket, which in turn activates the pigment. However, the pigments can also become activated at random, even in darkness. These false signals, nicknamed “dark light”, are caused by heat instead of light and essentially create a kind of visual noise that can interfere with vision.

In 2011, researchers found that pigments that are most sensitive to the longer wavelengths of light (that is, light redder in color) tend to be noisier. The researchers also found that cone pigments are noisier than rod pigments even if they are most sensitive to the same wavelengths of light.

To understand what causes this difference between cone and rod pigments, Yue, Frederiksen et al. – who include many of the researchers involved in the 2011 study – made use of mice with a mutated pigment in their rod cells. The mutant pigment was more sensitive to light of shorter wavelengths and, importantly, it behaved like a cone pigment in some ways but kept the closed pocket that is found in rod pigments. Indeed,Yue, Frederiksen et al. showed that the noise level of this mutant pigment could be accurately predicted from the wavelength it was most sensitive to and how closed its pocket was (in other words, the pocket's “closedness”). Further analyses revealed that an open pocket seems to be common to cone pigments from different species. So, it appears that cone pigments are noisier because they have a more open pocket, and the extra space might allow the chromophore to move around and change shape more easily.

Going forward, more visual pigments need to be tested to confirm the relationship between the openness of the chromophore-binding pocket and spontaneous activity. If confirmed, it might be possible to one day predict whether a pigment is intended for dim- or bright-light vision simply by knowing whether its chromophore-binding pocket is more open or closed.

https://doi.org/10.7554/eLife.18492.002

Introduction

Retinal rod and cone photoreceptors, although having similar phototransduction mechanisms, elaborate different morphological and molecular features for functioning in dim and bright light, respectively. At the pigment level, rod pigments have a low rate of spontaneous activation in darkness (Baylor et al., 1980), thus offering a good signal-to-noise ratio for dim-light vision. Spontaneous activation originates from internal thermal energy of the pigment molecule, generating an electrical event indistinguishable from that triggered by an absorbed photon (Baylor et al., 1980), thus interfering with real-light detection. Recently, Luo et al. (2011) have developed a macroscopic physicochemical theory about pigment noise based on the notion that a pigment’s spontaneous activity originates from thermal isomerization, with an energy barrier closely related to the pigment’s λmax. By using multi-vibrational-mode statistical mechanics (Ala-Laurila et al., 2004; Hinshelwood, 1940St George, 1952), the theory was able to explain quantitatively the λmax-dependence of pigment noise, with the noise increasing by 107-fold from blue (short-wavelength-sensitive, or SWS) cone pigment to red (long-wavelength-sensitive, or LWS) cone pigment (Fu et al., 2008; Kefalov et al., 2003; Luo et al., 2011). This theory clarifies the decades-long uncertainty about whether the spontaneous pigment activity arises from canonical isomerization of the pigment’s chromophore (as in photoisomerization) or from some different, unknown chemical reaction.

Very interestingly, noise measurements in conjunction with the theory indicate that, for a given λmax, a cone pigment may be generally ~25 fold more spontaneously active than a rod pigment (Luo et al., 2011). The simplest interpretation is that a cone pigment has a higher molecular frequency of attempting to cross the isomerization barrier (Luo et al., 2011). Concurrently, unlike rod pigment, a number of cone pigments show observable dark chromophore-exchange without isomerization when exposed to another chromophore (Kefalov et al., 2005; Matsumoto et al., 1975), suggesting a tendency of spontaneous dissociation between apo-cone-opsin and 11-cis-retinal by Schiff-base hydrolysis, in turn implicating the binding pocket being accessible – or ‘open’ – to external water. It was hypothesized that this ‘openness’ of cone pigments’ chromophore-binding pocket – defined by the property of dark chromophore-exchange – imposes less constraint on the chromophore’s attempts to isomerize spontaneously, resulting in a higher thermal noise compared to rod pigments for a given λmax (Luo et al., 2011).

Considering the fundamental success of the above theory in explaining the spontaneous activities of several representative rod and cone pigments, it is important to test the theory’s overall predictive power more generally. However, this test is non-trivial, requiring in each case a separate genetic mouse line expressing a test pigment for stringent interrogation in vivo. As such, the already-available RhoE122Q/E122Q knock-in mouse (Imai et al., 2007) offers an unusual opportunity. Its rods express a mutant rhodopsin with its Glu122 residue (conserved in rhodopsin) in the chromophore-binding pocket replaced by Gln, which is common in cone pigments. This E122Q mutation causes a blue-shift in λmax to ~480 nm from ~500 nm in wild-type (WT) rhodopsin (Imai et al., 2007), substantial enough for validating the quantitative connection between pigment noise and λmax. Equally interestingly, this mutant rhodopsin has acquired some cone-pigment-like properties such as faster decays of the meta-II and meta-III states as well as a shift of the meta-I/meta-II equilibrium (Imai et al., 2007), although retaining the indication of a closed chromophore-binding pocket as gleaned from in vitro experiments (Sakurai et al., 2007). Thus, we can also check in this ‘hybrid’ pigment the correlation between pigment noise and openness/closedness of the chromophore-binding pocket as we hypothesized.

Results and discussion

To examine spontaneous activation, we used RhoE122Q/E122Q mice in a Guca1a-/-;Guca1b-/- (more commonly known as Gcaps-/-, and will be referred to as such) background, which removes the Ca2+-dependent negative feedback on the guanylate cyclase via GCAP proteins in phototransduction (Mendez et al., 2001) and boosts the spontaneous event’s amplitude by ~5 fold for easy identification over background noise. RhoE122Q/E122Q;Gcaps-/- rods had broadly similar morphology as RhoWT/WT;Gcaps-/- rods (Figure 1A). Expression levels of the pigment and other phototransduction components were also normal in mutant retinae based on Western blot analysis (Figure 1B). A normal expression level of the mutant pigment was further supported by electrophysiological measurements from single rods and by optical-density measurements by microspectrophotometry (Materials and methods). To measure pigment noise, we obtained ~10 min recordings in darkness from RhoWT/WT;Gcaps-/- and RhoE122Q/E122Q;Gcaps-/- rods (Figure 1C) and extracted the spontaneous-activation rate by two methods. The first was to count quantal events based on a criterion amplitude of >30% of the single-photon-response amplitude measured in the same cell and also on a criterion integration time (reflecting its overall kinetics) of being within 50–200% of that of the average dim-flash response (Fu et al., 2008). Collective data at 37.5°C gave 0.015 ± 0.010 s−1 cell−1 (mean ± SD, n = 12) for RhoWT/WT;Gcaps-/- rods, and 0.0024 ± 0.0025 s−1 cell−1 (n = 20) for RhoE122Q/E122Q;Gcaps-/- rods. The measurements from RhoWT/WT;Gcaps-/- rods matched previous estimates (Burns et al., 2002; Fu et al., 2008). A variation of this method (Luo et al., 2011) allows us to also validate the Poisson occurrence of spontaneous events, by dividing the dark records into 100 s epochs and counting the number of epochs containing no event, one event, two events, etc. Indeed, the resulting probability histogram fits the Poisson distribution (red lines in Figure 1D), with the probability, p(u), of observing u events in each epoch being given by p(u)=wuew/u!, where w is the average number of events per epoch. From altogether 118 epochs from 20 rods, we obtained the w value, giving a thermal rate of 0.0023 s−1 cell−1 for RhoE122Q/E122Q;Gcaps-/- rods at 37.5°C, very similar to the above measurement.

Measurement of spontaneous-activation rate of E122Q-rhodopsin.

(A) Paraffin sections of 2.5-month-old RhoWT/WT;Gcaps-/- (left) and RhoE122Q/E122Q;Gcaps-/- (right) retinas stained by haematoxylin and eosin showing normal rod morphology. Similar results were found in altogether 3 sets of experiments. (B) Western blots from retinal extracts of RhoWT/WT;Gcaps-/-(different animal in each of the left two columns) and RhoE122Q/E122Q;Gcaps-/-mice (different animal in each of right two columns) showing normal expression of various phototransduction protein components. RHO: rhodopsin; G: α subunit of transducin; PDE6: phosphodiesterase isoform 6; CNGA1: A1 subunit of cyclic nucleotide-gated (CNG) channel; CNGB1: B1 subunit of CNG channel; ARR1: Arrestin 1; RGS9: regulator of G protein signaling isoform 9; GAPDH: glyceraldehyde 3-phosphate dehydrogenase (control for protein amount). (C) Sample 10 min recordings from a RhoWT/WT;Gcaps-/- rod (left) and a RhoE122Q/E122Q;Gcaps-/- rod (right) in darkness. Traces (continuous from top to bottom) were low-pass filtered at 3 Hz. Quantal events were identified based on amplitude and kinetics (see Text) and are marked by asterisks. (D) Poisson analysis of dark recordings collected from all RhoE122Q/E122Q;Gcaps-/- rods. Bars indicate the measured probabilities of observing 0, 1, 2 and 3 events in 100 s epochs. A total of 118 epochs were analyzed. Red lines give the fit by the Poisson distribution with a mean event rate of 0.0023 s−1 cell−1. (E) Difference power spectrum (square symbols) of a RhoE122Q/E122Q;Gcaps-/- rod fitted with the power spectrum (curve) of the single-photon-response function.

https://doi.org/10.7554/eLife.18492.003

In the second method (Fu et al., 2008; Kefalov et al., 2003), we computed the power spectrum of the spontaneous events for each cell by subtracting the power spectrum of a segment in the dark recording with no obvious events from the power spectrum of the entire ~10 min recording. This ‘difference spectrum’ was then fitted with a scaled power spectrum of the same cell’s average single-photon response (Figure 1E). From the scaling factor, we obtained a spontaneous-activation rate of 0.0025 ± 0.005 s−1 cell−1 (n = 11) for RhoE122Q/E122Q;Gcaps-/- rods at 37.5°C, very similar to that from the first method.

For a total of 6.5 × 107 rhodopsin molecules in a mouse rod (Luo et al., 2011), the overall molecular rate constant of spontaneous activation from either method above was 3.69 × 10−11 s−1, ~6-fold lower than WT (2.31 × 10−10 s−1). From our theory (Luo et al., 2011), the predicted spontaneous-activation rate constant as a function of λmax is given by Ae0.84hcRTλmax1m1(m1)!(0.84hcRTλmax)m1, where h is Planck’s constant, c is velocity of light, R is universal gas constant, T is absolute temperature, and m is the nominal number of vibrational modes in the pigment molecule contributing thermal energy to pigment isomerization. The pre-exponential factor A, taken to represent the frequency at which a pigment molecule attempts to isomerize thermally, was found in previous work (Luo et al., 2011) to be 7.19 × 10−6 s−1 for rod pigments (with a ‘closed’ binding pocket) and 1.88 × 10−4 s−1 for cone pigments (with an ‘open’ binding pocket), a 26-fold difference. Inserting T = (37.5 + 273) oK = 310.5 oK, m = 45 [see (Luo et al., 2011)], and λmax = 481 nm for E122Q-rhodopsin (Figure 2A), we predict a molecular thermal-rate constant of 3.68 × 10−11 s−1 for a ‘closed’, and 9.63 × 10−10 s−1 for an ‘open’, binding pocket. Thus, the predicted rate for a ‘closed’ pocket matched the measurement very well. Recently, one of the authors here (Y.S.) has found biochemically a higher instead of lower thermal rate constant of E122Q-rhodopsin over WT rhodopsin (Yanagawa et al., 2015). This discrepancy may arise from using detergent-solubilized samples in the biochemical method.

Chromophore-exchange experiment for probing the openness/closedness of chromophore-binding pocket.

Absorption spectra (normalized to peak optical density) were obtained from dark-adapted RhoWT/WT;Gcaps-/- rods (left) and RhoE122Q/E122Q;Gcaps-/- rods (right) that were incubated in darkness in (A) Ames solution for 3 hr, (B) Ames solution with 15 μM 9-cis-retinal in darkness for 3 hr and (C) Ames solution with 15 μM 9-cis-retinal in darkness for 6 hr. (D) Absorption spectra from rods 99%-bleached followed by 3 hr 9-cis incubation in darkness. In all panels, curves are mean (black) ± SD (gray). Black dashed lines indicate the λmax of dark-adapted RhoWT/WT;Gcaps-/- (left) and RhoE122Q/E122Q;Gcaps-/- rods (right) not exposed to exogenous chromophore. Red dashed lines indicate the λmax of rods of the respective genotypes after the respective experimental treatment. The black dashed lines in (A) are replotted in (B), (C) and (D) for comparison with the red lines. For RhoWT/WT;Gcaps-/- rods, λmax’s are (A) 499.9 ± 4.8 nm (n = 33 recordings), (B) 495.6 ± 3.0 nm (n = 5 recordings, p=0.06), (C) 501.3 ± 4.5 nm (n = 6 recordings, p=0.52) and (D) 481.6 ± 4.1 nm (n = 5 recordings, p<0.0001), with p values from Student’s t-test comparing with (A). For RhoE122Q/E122Q;Gcaps-/- rods, λmax’s are (A) 480.9 ± 5.4 nm (n = 7 recordings), (B) 479.6 ± 3.6 nm (n = 5 recordings, p=0.65), (C) 479.2 ± 3.9 nm (n = 5 recordings, p=0.56) and (D) 469.3 ± 3.2 nm (n = 8 recordings, p=0.0002), with p values from Student’s t-test comparing with (A).

https://doi.org/10.7554/eLife.18492.005

To investigate whether E122Q-rhodopsin indeed has a closed binding pocket, we checked its capability of chromophore-exchange in darkness. We incubated dark-adapted RhoE122Q/E122Q;Gcaps-/-rods in Ames solution with or without 15 μM exogenous 9-cis-retinal (which, for a given opsin, forms a pigment with shorter λmax than does 11-cis-retinal) in darkness for 3 hr, then measured their absorption spectrum by microspectrophotometry (Materials and methods). No spectral shift was detected in the 9-cis-exposed rods, suggesting no dark chromophore-exchange, which is similar in behavior to RhoWT/WT;Gcaps-/-rods (Figure 2A,B). We found no apparent exchange even with 6 hr of dark incubation (Figure 2C). As control, we delivered a 99%-bleach by 500 nm light to the rods prior to dark incubation with 9-cis. In this case, a spectral shift occurred in RhoE122Q/E122Q;Gcaps-/- rods as in RhoWT/WT;Gcaps-/- rods (Figure 2D), indicating normal hydrolysis and formation of the Schiff-base between E122Q-opsin and chromophore.

Chromophore-exchange experiments in live cells have previously been done only in salamander red cones (Kefalov et al., 2005). Given our hypothesis that the openness of a cone pigment’s chromophore-binding pocket explains its higher spontaneous activity than that of rhodopsin of the same λmax, we would like to check whether dark chromophore-exchange is indeed common also to other cone types. To avoid potential complications from in vitro conditions, we confined our question to live cells with microspectrophotometry, as described earlier and used previously on salamander red cones (Kefalov et al., 2005). We decided to examine zebrafish, which has reasonably large cones of multiple spectral types. Figure 3A shows their single-cell absorption spectra: red (LWS), green (medium-wavelength-sensitive, or MWS), blue (SWS), and ultraviolet-sensitive (UVS). After dark incubation with 15 µM 9-cis-retinal for 3 hr, the absorption spectra of dark-adapted, native (11-cis) LWS, MWS and SWS cones all shifted to shorter wavelengths, indicating incorporation of 9-cis-retinal (Figure 3B, with native spectra shown in black and spectra after 3 hr incubation with 15 µM 9-cis-retinal shown in red). Because pigments with shorter λmax’s show smaller λmax-differences between their 11-cis- and 9-cis-conjugated forms (the latter obtained by a full bleach followed by dark incubation with 9-cis; indicated in green in Figure 3B), the absolute amount of spectral shift due to dark chromophore-exchange was smaller for MWS and SWS cones than for LWS cones and was too small to be resolved for UVS cones. Figure 3C shows the time course of dark chromophore-exchange for LWS cones, quantified by the degree of spectral shift (Materials and methods). A single-exponential fit to the collected data (mean ± SD, 20–22°C) gives a time constant of 37 min, ~4 fold faster than previously found for salamander red cones (Kefalov et al., 2005), although the exchange in zebrafish did not appear to have reached completion (i.e.,<100%, see Figure 3C), possibly because of some 11-cis-retinal released from the pigment staying around and competing with the exogenous 9-cis-retinal. The time courses of dark chromophore-exchange for other zebrafish cone pigments were not well-resolved owing to the smaller spectral shift, but appeared to be faster at least for MWS and SWS cones, in that we found a significant shift to have occurred within the first 10 min of dark incubation (not shown). In contrast, zebrafish rhodopsin showed no obvious dark chromophore-exchange even after incubation with 9-cis-retinal for 6 hr (blue trace in Figure 3B, rod panel), same as mouse rhodopsin. Thus, at least for salamander and zebrafish, dark chromophore-exchange appears to be a general property for cone pigments with a time constant of the order of an hour, whereas rhodopsins behave quite differently. As such, we favor at present the simple approach of referring to these two collective groups of pigments as having ‘open’ versus ‘closed’ chromophore-binding pockets, respectively. If, in the future, the noise property and the pocket openness/closedness of a larger number of native and mutant pigments could be quantitatively measured, a finer sub-division may become more pertinent.

Chromophore-exchange experiment with zebrafish photoreceptors.

(A) Absorption spectra of dark-adapted zebrafish long-wavelength-sensitive (LWS), medium-wavelength-sensitive (MWS), short-wavelength-sensitive (SWS) and ultraviolet-sensitive (UVS) cones. Spectra are mean (intense traces) ± SD (faint traces), normalized to peak optical density. n = 41 (LWS), 32 (MWS), 17 (SWS) and 5 (UVS) cells. (B) Absorption spectra of dark-adapted zebrafish rods and cones that were incubated with 15 μM 9-cis-retinal in darkness for 3 hr (red) and 6 hr (blue, only for rods). Spectra of dark-adapted cells after dark incubation for 3 hr in Ames solution without 9-cis (black), and of cells 99%-bleached followed by dark incubation for 3 hr in Ames solution with 9-cis (green), are given as reference. Spectra are mean (intense traces) ± SD (faint traces), normalized to peak optical density. For LWS cones, n = 24 (red), 41 (black) and 6 (green) cells. For MWS cones, n = 15 (red), 32 (black) and 7 (green) cells. For SWS cones, n = 10 (red), 17 (black) and 4 (green) cells. For rods, n = 13 (red), 16 (blue), 25 (black) and 20 (green) cells. (C) Time course of dark chromophore-exchange in LWS cones. The percentage of 9-cis-conjugated pigment (Materials and methods) is plotted (mean ± SD, n = 9 cells) against time in chromophore incubation. Curve is a saturating exponential function, 0.72 (1- e-t/τ), with an asymptote of 0.72 and a time constant, τ, of 37 min fitted to the data.

https://doi.org/10.7554/eLife.18492.007

In summary, the E122Q-rhodopsin with hybrid rod- and cone-pigment-like properties has provided a quantitative validation and generalization of our macroscopic physicochemical theory of pigment noise previously proposed based on representative rod and cone pigments. At the same time, our hypothesized correlation between closedness/openness of the chromophore-binding pocket and pigment noise level also continues to hold. A low level of pigment noise is without exception beneficial for dim-light vision. The successful application of our theory to E122Q-rhodopsin as a non-canonical pigment underscores the potential usefulness of evaluating a pigment as being intended for dim-light or bright-light function based on its noise level and, by extension, on the closedness/openness of its chromophore-binding pocket, provided the correlation between the two features continues to hold for other pigments. Such a functional criterion may be more informative than amino-acid-sequence comparison, especially for pigments with ambiguous evolutionary origin, such as the Gecko P467-pigment (Kojima et al., 1992).

Note added in proof

A paper just appeared (Tian et al., 2017) reporting that rhodopsin purified from bovine rod outer segments dissociates into opsin and 11-cis-retinal in darkness, with a half life for holo-rhodopsin of the order of days. This rhodopsin behavior is not incompatible with our findings here because it is clearly still very different in time scale from the chromophore exchange in cone pigments, which occurs within hours.

Materials and methods

Animals

All animal experiments were carried out according to protocols approved by the Institutional Animal Care and Use Committee at Johns Hopkins University (MO14M199 for mouse) and Boston University (AN15427 for both mouse and zebrafish). Animals used in this study include RhoWT/WT;Gcaps-/- (RRID:MGI:3586516) and RhoE122Q/E122Q;Gcaps-/- mice as well as zebrafish (AB Danio rerio; RRID:ZIRC_ZL1).

Histology

An eyeball of an acutely-euthanized animal was fixed in an alcohol-based zinc-formalin solution (Z-fix, Anatech, Battle Creek, MI) at room temperature overnight. The eyeball was then sent to the Johns Hopkins Medical Laboratories, where it was dehydrated through a series of increasing concentrations of ethanol, embedded in paraffin, and sectioned at a thickness of 5–8 µm. Sections close to the plane of the optic disc were collected, then de-paraffinized and rehydrated by passing through Xylene and a series of ethanol solutions of decreasing concentrations. After rinsing with water, the sections were stained with haematoxylin for 3 min. Following a wash with water, the sections were cleared, rinsed and blued. The sections were then rinsed again and stained with eosin for 1 min. Finally, the slides were rinsed, dehydrated through graded alcohols, cleared by Xylene and mounted.

Western blotting

Retinas were isolated from euthanized mice into RIPA lysis buffer (140 mM NaCl, 0.1% Na-deoxycholate, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100 and 0.1% SDS). Proteins were extracted by grinding the tissues with plastic pestles and vortexing every 5 min over a total of 30 min of incubation. Protein concentrations were determined using the bicinchoninic acid (BCA) Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA). Subsequently, protein extracts (20 μg) were separated on 3–20% continuous SDS-PAGE gels (Bio-Rad, Hercules, CA) and transferred to polyvinylidene difluoride (PVDF) membrane. The membranes were blocked with 5% normal non-fat milk in TBST (500 mM NaCl, 20 mM Tris-HCl, pH 7.4, 0.1% Tween-20) for 1 hr and then incubated with different primary antibodies at 4°C overnight. Primary antibodies included a mouse anti-bovine rhodopsin (RHO) monoclonal antibody (1D4; 1:50; gift from Dr. Robert Molday, University of British Columbia), a rabbit anti-human transducin (G) polyclonal antibody (RRID:AB_2294749; 1:500; Santa Cruz, Dallas, TX), a mouse anti-bovine phosphodiesterase-6 (PDE6) monoclonal antibody (1: 1000; gift from Dr. Theodore Wensel, Baylor College of Medicine), a mouse anti-bovine cyclic-nucleotide channel subunit A1 (CNGA1) monoclonal antibody (PMc1D1; 1:100; gift from Dr. Robert Molday), a mouse anti-bovine CNG channel subunit B1 (CNGB1) monoclonal antibody (GARP4B1; 1:1000; gift from Dr. Robert Molday), a rabbit anti-mouse arrestin-1 (ARR1) polyclonal antibody (1:2500; gift from Dr. Jason C.-K. Chen), a rabbit anti-mouse regulator of G protein signaling isoform 9 (RGS9) polyclonal antibody (1:1000; gift from Dr. Jason C.-K. Chen), and a chicken anti-human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) polyclonal antibody (RRID:AB_10615768; 1:500; Millipore, Germany). After being washed with TBST, the blots were incubated with the appropriate HRP-conjugated secondary antibodies (1:10,000; Bio-Rad) at room temperature for 1 hr. Finally, the proteins on the membranes were detected by using the Enhanced Chemiluminescence (ECL) system (Thermo Fisher Scientific).

Suction-pipette recording

One- to three-month-old mice were dark-adapted overnight, euthanized and their eyes removed under dim red light. The eyes were hemisected and the retinas were removed under infrared light in Locke’s solution [112.5 mM NaCl, 3.6 mM KCl, 2.4 mM MgCl2, 1.2 mM CaCl2, 3 mM Na2-succinate, 0.5 mM Na-glutamate, 0.02 mM EDTA, 10 mM glucose, 0.1% vitamins (Sigma-Aldrich, St. Louis, MO), 0.1% amino-acid supplement (Sigma-Aldrich), 10 mM HEPES, pH 7.4 and 20 mM NaHCO3]. Retinas were stored in Locke’s solution bubbled with 95% O2/5% CO2 at room temperature until use over not longer than 6 hr. When needed, a fraction of the retina was chopped into small pieces with a razor blade in the presence of DNase I (~20 U/ml) and was transferred to the recording chamber perfused with bubbled Locke’s solution at 37.5°C ± 0.5°C. Temperature was monitored by a thermistor situated close to the recorded cell.

Single-cell recordings were made under infrared light by drawing the outer segment of a rod projecting from a fragment of retina into a tight-fitting glass pipette containing the following pipette solution: 140 mM NaCl, 3.6 mM KCl, 2.4 mM MgCl2, 1.2 mM CaCl2, 0.02 mM EDTA, 10 mM glucose and 3 mM HEPES, pH 7.4. In most experiments with light stimulation, 10- to 30-msec monochromatic flashes were used. Signals were sampled at 1 kHz through an Axopatch 200B amplifier and low-pass filtered at 20 Hz (RC filter, Krohn-Hite, Brockton, MA), unless specified otherwise.

Measurements of the rates of spontaneous activation

The average single-photon response function [f(t)] was computed by first obtaining the average response profile of a rod to 80–100 identical dim flashes and then scaling it to the amplitude of the single-photon response, which was calculated as the ensemble variance-to-mean amplitude ratio at the transient peak of these dim-flash responses.

Continuous 10 min recordings were obtained from rods in complete darkness, and the rate constant of spontaneous activation were measured by two methods. In the direct counting method, traces were usually low-pass filtered at 3 Hz for identifying and counting quantal events. Two criteria were imposed during identification: (1) the amplitude of the event should be >30% of the single-photon response amplitude of the same cell, and (2) the integration time of the event should be within 50–200% of that of the average dim-flash response. The cellular rate constant of thermal activation was given by the total number of spontaneous events divided by the total recording time for each cell. Alternatively, the dark-recording traces were divided into 100 s epochs. The frequency of observing no event, one event, two events, etc. in an epoch was plotted and fitted with the Poisson distribution p(u)=wuew/u!, where p(u) was the probability of observing u events in each epoch and w was the average number of spontaneous activation event per 100 s epoch.

In the second method, power spectra were computed from the entire dark-recording trace and from a segment of it containing no obvious spontaneous events (based on visual inspection) for each cell by using Clampfit 9 (Molecular Devices, Sunnyvale, CA) in 8.192 s segments with 50% overlap. The difference spectrum between these two spectra constituted the spectrum for the spontaneous events. This difference spectrum was fitted with a scaled power spectrum of the average single-photon response function [f(t); see above] of the same cell. The rate of spontaneous isomerization is given by the scaling factor divided by the acquisition time (8.192 s).

The molecular rate constant was obtained by dividing the measured cellular rate by the number of pigment molecules (6.5 × 107) per rod. The expression of rhodopsin appeared normal in RhoE122Q/E122Q;Gcaps-/- retinas based on Western blotting (Figure 1B). Another way to assess pigment content in a rod outer segment is to measure the probability (ps) of successfully eliciting electrical responses in an experiment using repeated dim-flash trials of known intensity. This probability is related to the rod outer segment’s effective collecting area (Ae) and the flash intensity (I) by ps = 1  eAeI. In turn, Ae is directly proportional to the pigment content. As such, we found Ae to be 0.44 ± 0.09 µm2 (mean ± SD, n = 10) for RhoWT/WT;Gcaps-/- rods and 0.35 ± 0.10 µm2 (mean ± SD, n = 18) for RhoE122Q/E122Q;Gcaps-/- rods. Thus, the E122Q/WT pigment-content ratio is 1/1.26. Meanwhile, microspectrophotometry (see below) measured an average relative peak optical density of 0.37 ± 0.07 unit (mean ± SD, n = 44 recordings from seven experiments) for RhoE122Q/E122Q;Gcaps-/- rods and 0.30 ± 0.09 unit (n = 84 recordings from 33 experiments) for RhoWT/WT;Gcaps-/- rods, giving a E122Q/WT pigment-content ratio of 1.23/1. The mild discrepancy between methods may reflect measurement uncertainties. Taken together, the pigment levels appear similar between RhoWT/WT;Gcaps-/- and RhoE122Q/E122Q;Gcaps-/- rods.

Pigment noise prediction

The rate of spontaneous activation (k) is given by:

(1) k=Ae0.84hcRTλmax1m1(m1)!(0.84hcRTλmax)m1,

where A is the pre-exponential factor, h is Planck’s constant (1.58 × 10−37 kcal sec), c is speed of light (3.00 × 1017 nm sec−1), R is universal gas constant (1.99 × 10−3 kcal oK−1 mol−1), T is absolute temperature (310.5 oK) and m is the nominal number of vibrational modes contributing thermal energy to pigment activation. Based on previous work (Luo et al., 2011), m is 45 for rhodopsin and is taken to be the same for cone pigments, given the same chromophore. The average A-values were empirically determined to be 7.19 × 10−6 s−1 for rod pigments and 1.88 × 10−4 s−1 for cone pigments (Luo et al., 2011). Predictions were made by substituting these parameters and λmax = 481 nm (for E122Q-rhodopsin) into Equation 1.

Microspectrophotometry

For experiments on mouse rods, mice were dark-adapted for 12 hr before experiment. After euthanization, eyes were removed under dim red light. Under infrared illumination, the eyes were hemisected and the retinas were isolated in HEPES (10 mM, pH 7.4)-buffered Ames medium (Sigma-Aldrich). Each retina was divided in half, yielding altogether four pieces of tissues to be subjected to different treatments. Two pieces of retina were kept dark-adapted and incubated for 3 hr in darkness in HEPES-buffered Ames medium containing 1% fatty-acid-free bovine serum albumin (BSA) with or without 15 μM 9-cis-retinal; the other two pieces of retina were subjected to a 99%-bleach (see below) and then incubated in the same HEPES-buffered, BSA-supplemented Ames medium as above with or without 15 μM 9-cis-retinal.

After their respective treatments, the absorbance spectra of the retinal pieces were measured using a custom-built microspectrophotometer. A retinal piece was gently flattened by forceps and a slice anchor (Warner Instruments, Hamden, CT) on a quartz cover-slip window in the bottom of a 2 mm-deep Plexiglass recording chamber with the photoreceptors facing up. The recording chamber was placed on a microscope stage located in the beam path of the microspectrophotometer. The retinal tissue was superfused at a rate of 4 ml/min with Ames medium (Sigma-Aldrich) buffered with sodium bicarbonate and equilibrated with 95% O2/5% CO2. Temperature was maintained at 35–37°C. Absorption spectra were obtained from a region of the retina along its edge where outer segments could be seen protruding perpendicular to the light beam, with tens of outer segments in the light path. The measured area contained predominantly rod instead of cone photoreceptors, as evinced by the λmax. Measurements were made over the wavelength range of 300–700 nm with 2 nm resolution, with the polarization of the incident beam parallel to the plane of the intracellular disks (T-polarization). The absorbance spectrum was calculated from Beers’ Law OD = log(Ii/It), where OD is the optical density or absorbance, Ii is the light transmitted through a cell-free space adjacent to the outer segments, and It is the light transmitted through the tissue. Generally, 10 complete sample scans and 10 baseline scans were averaged to increase the signal-to-noise ratio. All absorbance spectra were baseline-corrected.

For experiments on zebrafish photoreceptors, wild-type (AB) zebrafish (Danio rerio), obtained from the colony held by the Animal Science Department at Boston University School of Medicine, was dark-adapted for 12 hr prior to experimentation. Euthanasia, dissection and tissue manipulation were performed in darkness with the aid of infrared image converters. Fishes were euthanized by exposure to cold (0°C) water followed by decapitation. The eyes were removed and hemisected in recording solution containing 104 mM NaCl, 2.5 mM KCl, 1.2 mM MgCl2, 1.6 mM CaCl2, 0.1 mM NaHCO3, 1 mM-NaH2PO4, 1 mM sodium pyruvate, 15 mM glucose, 15 mM HEPES (acid), 5 mM HEPES (base, Na-salt), 0.5 µg/ml insulin, 5 µg/ml d-biotin, 70 µl/ml fetal bovine serum, 10 µl/ml penicillin streptomycin, 150 µg/ml L-glutamine, 10 µL/ml 50× MEM amino acids, 5 µl/ml 100× MEM vitamins, pH = 7.8. The retinas were then isolated from the eyecups and the retinal pigment epithelium. Retinal tissues not immediately used for experiment were stored in the above solution in a dark container on ice.

For experiments in Figure 3B, a retina was treated off-stage in one of the following four ways: (1) directly used for microspectrophotometric measurements to obtain dark spectra, (2) incubated in recording solution with additional 1% bovine serum albumin (fatty-acid-free) and 15 µM 9-cis-retinal in darkness for 3 hr, (3) incubated with 1% bovine serum albumin and 15 µM 9-cis-retinal in the same way for 6 hr, and (4) bleached (see below) and incubated with 1% bovine serum albumin and 15 µM 9-cis-retinal in the same way for 3 hr. After treatment, the retina was cut into small (~50 µm×50 µm) pieces and then triturated in solution, producing isolated photoreceptors. Cells were transferred to a recording chamber containing recording solution maintained at 20–22°C. Different types of photoreceptors were identified by their morphology and confirmed by spectral absorbance, which was recorded similarly as in mouse experiments except from single zebrafish photoreceptors.

To measure the time course of chromophore-exchange in zebrafish LWS cones (Figure 3C), dark-adapted photoreceptors were dissociated as above directly after retina isolation and were transferred to the MSP recording chamber. A LWS cone was identified and its dark spectrum measured. The solution in the recording chamber was then replaced by recording solution containing 1% bovine serum albumin and 15 µM 9-cis-retinal. Measurements of spectral absorbance were made periodically over 3 hr, at 20–22°C. The light for probing the spectrum at a given time point was at an intensity that would bleach less than 0.1% of the pigment content per scan. To quantify the degree of chromophore-exchange, we used the spectrum of dark-adapted LWS cones (Figure 3B, black) and that of bleached LWS cones regenerated with 9-cis-retinal (Figure 3B, green) as the spectra for 11-cis- and 9-cis-pigment, respectively. A polynomial with degree 10 was fitted into each of these spectra. For each LWS cone, the spectrum acquired at each time point during 9-cis incubation was fitted in the 510–750 nm range with a linear combination of the two polynomials to obtain the percentage of 9-cis-conjugated pigment. Data from all cells were then averaged.

Pigment-bleaching

For mouse rods, bleaching was performed off-stage on a portable optical bench consisting of a tungsten/halogen lamp, a set of neutral density filters, a 500 nm interference filter and a small aperture (3.25 mm). The retinal tissue was placed in Ames medium in a 35 mm petri dish under the focused circular light spot. The onset of light was controlled by a manual shutter. The bleached fraction, F, was estimated from the relation F = 1 eIPt, where I was the bleaching light intensity (1.33 × 106 photons µm−2 s−1), P was the photosensitivity [5.7 × 10−9 µm2; see (Woodruff et al., 2004)] of mouse rhodopsin measured in situ at its λmax and t was the duration of light exposure; the retinal tissue was typically light-exposed for 16 min to achieve a > 99% bleach. For zebrafish photoreceptors, bleaching was done as above except for using recording solution instead of Ames solution, and using white light of the same source intensity (i.e., not including the 500 nm interference filter).

Chromophore-preparation

9-cis-retinal was handled in dim red light. A stock solution of 30 mM 9-cis-retinal was prepared by dissolving 9-cis-retinal in ethanol. The peak absorbance (OD) of retinoid in the stock solution was measured using a conventional spectrophotometer, and its concentration was calculated as c = (OD373l)/ε373, where l was a 1 cm path length and ε373 = 36,100 M−1 cm−1 was the extinction coefficient of 9-cis-retinal in ethanol. Working solutions containing 9-cis-retinal were prepared by first adding 1 µl of stock solution to a conical vial. HEPES-buffered Ames medium containing 1% delipidated BSA was then added in multiple times in increasing amounts (9 × 5 μl, 1 × 50 μl, 2 × 450 μl, 1 × 1000 μl) until the final volume was 2 ml; the concentration of 9-cis-retinal in the working solution was 15 µM.

References

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    The Kinetics of Chemical Change
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Decision letter

  1. Richard Aldrich
    Reviewing Editor; The University of Texas at Austin, United States

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "Spontaneous Activation of Visual Pigments In Relation To Openness of Chromophore-Binding Pocket" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Richard Aldrich as the Senior and Reviewing Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Theodore G Wensel (Reviewer #3).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

The reviewers are generally enthusiastic about the work, but they offer several criticisms and suggestions for improvement. Ordinarily we excerpt the most critical comments for the focused attention of the authors, but in this case the reviewers and editor felt you should see their full reviews to help in your revisions.

Reviewer #1:

Yue et al. here attempt to test an earlier "macroscopic physicochemical theory" (Luo et al., 2011) concerning the basis for thermal noise in visual photoreceptor cells based on an analysis of molecular pigment type (rod versus cone) and wavelength of maximal absorption (λmax). The primary experimental system is a knockin/knockout mouse (RhoE122Q/E122Q, Gcaps-/-) in which the usual wild-type rhodopsin (Rho) is replaced by the mutant Rho E122Q, which has been characterized in some detail earlier using a variety of methods. Much of the paper seems to be a reiteration of the earlier 2011 paper and the authors repeatedly refer to "our" theory or model even though it appears that only 2 of the 8 current authors were associated with the earlier work. Although this paper ascribes to being quantitative in presenting Rho E122Q is a definitive experimental example to validate the earlier theory paper, the claims of the paper are either too vague (for example, using ill-defined terms like "openness" of the retinal binding pocket), or too overreaching (for example, claiming that a single test case proves the earlier theory. In fact, it's not clear that the Rho E122Q mouse is a logical or sufficient model system for a variety of reasons. However, the data set is interesting and valuable and there are no major issues, but the authors should consider rewriting sections of the paper and should address a number of questions/concerns as outlined below:

1) Abstract. "…spontaneously activated by internal thermal energy" – what does that mean, actually? Isn't is just thermal activation? "Confusion in the field"? What is the confusion? "Quantitatively correlated with the closedness of its chromophore-binding pocket"? How is closedness actually quantitated? It isn't, so any correlation cannot be quantitative. Is it the "closedness" or the λmax value in the end that's the most important factor. The Abstract is extremely confusing in and of itself.

2) Introduction. Of course the ground-state isomerization energy barrier is closely related to the λmax. That's trivial, isn't it? Why is that a part of a unifying theory? The trivial definition of "open" pocket refers to accessibility of the Schiff base to hydrolysis, for example by hydroxylamine (with a 1955 Wald reference), but the actual chemistry of the hydrolysis reaction is a very complicated pH dependent mechanism that is only partly dependent on accessibility or hydration state. It is not stated why the E122Q pigment is cone-like, or "non-canonical." Some detail is required to justify its use, other than the mouse model happened to exist. What other mutants might be useful and why weren't they used or considered?

3) Results, third paragraph. What is the total number of E122Q pigment molecules per cell? Can it be assumed that it is the same as the total Rho in WT? If so, why? The scaling factor related to the number of molecules is very important in the equation used to a spontaneous activation rate constant. Also at the end of the subsection “Measurements of the rates of spontaneous activation”, it is stated that the "molecular rate constant could be obtained by further dividing the measured cellular rate by the number of pigment molecules per rod." Again, how is it assumed that E122Q is the same as Rho?

4) Results, last paragraph. The competition experiment seems problematic. Why was only a single time point of 3 hours in darkness chosen? Does the retinal bind to the 1% BSA predominantly (subsection “Chromophore-preparation”, Methods)?

5) Results, end of last paragraph. It is now stated that E122Q is resistant to hydroxylamine? What? Isn't the λmax of 9-cis Rho 490 nm, not 482 nm? The data actually show Rho at about 498 nm or so, not 500. Why not use the actual data values in the text?

6) Discussion. The main criticism is that the authors need to justify how one example case can provide a justification for the earlier theory, and why and how the E122Q Rho is a legitimate test system. Does it really have hybrid rod-cone properties when expressed in a mouse rod cell?

7) Subsection “Pigment noise prediction”. m is taken to be the same for Rho and cone pigments? That's really strange and some additional explanation seems warranted.

8) Figure 1B. Again, referring to the issue of E122Q quantitation, the Rho band looks much heavier than the E122Q knockin band.

Reviewer #2:

This manuscript entitled "Spontaneous Activation of Visual Pigments In Relation To Openness of Chromophore-Binding Pocket" by Yue, Frederiksen, Ren, Luo, Yamashita, Shichida, Cornwall & Yau tests the validity of a previously proposed model (Luo et al., 2011) correlating the spectral sensitivity of visual pigments with their thermal stability. This model is currently the dominant unifying theory correlating the two key functional properties of visual pigment molecules: their spectral sensitivity characterized by the peak-absorption wavelength (λmax) and their susceptibility to thermal noise characterized by the rate of thermal isomerizations. The theory by Luo et al. proposes a similar dependency between the dark event rate and λmax for rod and cone pigments. However, cone pigments with the same λmax as rod pigments have overall 25-fold higher dark even rates according to measurements. In the model formulation, this has been implemented by using a pre-exponential factor that is 25-fold larger for cone pigments compared to rod pigments. This difference has been earlier hypothesized to relate to the closeness/openness of the chromophore-binding pocket (Ala-Laurila et al., 2004 & Luo et al., 2011). The validity of this hypothesis has not been tested directly experimentally. The current paper tests the validity of this interpretation. The current paper uses a mutant rhodopsin (E122Q-rhodopsin) with some cone-like pigment properties (e.g. faster decay of rhodopsin intermediates, meta-II and meta-III) but with a rod-like chromophore exchange rate (correlated with the openness of the binding pocket). The paper shows that the dark-event rate of E122Q-rhodopsin is in accordance with the model prediction for a typical rod pigment. This result is in line with the proposed interpretation that the pre-exponential factor of the theory correlates with the accessibility of the chromophore binding pocket rather than some other features typical of cone pigments.

Although the paper provides only one data point to test the theory, this can be seen as a very important contribution to the field. This is because of the nature of the model relying on multi-vibrational-mode statistical mechanics of visual pigment activation. Direct experimental testing of such molecular properties on a selection of pigments with different spectral properties seems very difficult. Instead, an approach testing the key predictions of the model by careful selections of pigments seems effective. This is exactly what has been done. In addition, the physiological experiments have been carefully carried out with the highest technical standards. Overall, this is a very important contribution to the field. I have only one important technical concern that needs to be addressed by the authors (see below).

Justification of the chromophore exchange rate argument: The authors use microspectrophotometry (MSP) and 9-cis retinal to estimate the exchange rate of the chromophore in E122Q- and WT rods. The authors claim that within the measured times there is no spectral shifts in E122Q-rods that have been exposed to 9-cis retinal. This data is used to conclude that the exchange rate is in line with rod pigments in E122Q-rods. The conclusion is central for the paper. It would be important to validate this argument by giving an estimate of the expected spectral shifts in MSP measurements for a chromophore exchange rate that is in line with that expected for cone-pigments in a similar time interval and relate this predicted shift to the accuracy of the λmax measurements. This is needed to assess to which extent the MSP with its current measurement accuracy for λmax is sensitive to the chromophore exchange rates representing rod and/or cone type pigments on these time scales. This is especially true for E122Q rods where the bleached native rods that have been regenerated with 9-cis retinal have a smaller spectral shift (λmax shift) compared to the WT rods.

Reviewer #3:

This paper provides confirmation for a previously published and validated theory of the relationship between the frequency of spontaneous isomerization of a vertebrate photopigment and it wavelength of maximum absorbance- also the peak wavelength of its photoisomerization action spectrum. Although it provides a single data point for this theory, it is important because the theory is more challenging to test for rods (for which spontaneous isomerization is arguably of much greater biological significance than for cones) than for cones because of the limited range of λmax values found in naturally occurring rod pigments. In addition, the frequency is so low for rods that it can only be measured in intact rods, making use of their ability to use extremely efficient amplification mechanisms to allow measurement of single-molecule isomerization events. Making use of an existing mouse model with a knock-in of a rod pigment with a single point mutation that results in a significant blue shift, the authors have quantitatively confirmed that the spontaneous rate is predicted by the theory. Secondarily, they verify that the binding pocket for the chromophore is very similar to that in WT rhodopsin, based on its extremely slow chromophore exchange kinetics.

https://doi.org/10.7554/eLife.18492.011

Author response

[…] Reviewer #1:

Yue et al. here attempt to test an earlier "macroscopic physicochemical theory" (Luo et al., 2011) concerning the basis for thermal noise in visual photoreceptor cells based on an analysis of molecular pigment type (rod versus cone) and wavelength of maximal absorption (λmax). The primary experimental system is a knockin/knockout mouse (RhoE122Q/E122Q, Gcaps-/-) in which the usual wild-type rhodopsin (Rho) is replaced by the mutant Rho E122Q, which has been characterized in some detail earlier using a variety of methods. Much of the paper seems to be a reiteration of the earlier 2011 paper and the authors repeatedly refer to "our" theory or model even though it appears that only 2 of the 8 current authors were associated with the earlier work. Although this paper ascribes to being quantitative in presenting Rho E122Q is a definitive experimental example to validate the earlier theory paper, the claims of the paper are either too vague (for example, using ill-defined terms like "openness" of the retinal binding pocket), or too overreaching (for example, claiming that a single test case proves the earlier theory. In fact, it's not clear that the Rho E122Q mouse is a logical or sufficient model system for a variety of reasons. However, the data set is interesting and valuable and there are no major issues, but the authors should consider rewriting sections of the paper and should address a number of questions/concerns as outlined below:

We thank this reviewer for commenting that “our data set is interesting and valuable and there are no major issues”. We have now tightened the writing by defining more explicitly what we mean by the “openness” of the retinal-binding pocket. Regarding the choice of the RhoE122Q/E122Qmouse and only this line for study, the simple reason is that this mouse line happens to be especially interesting by having an exceptionally large λmax shift as well as having hybrid rhodopsin/cone- pigment properties in its mutant rhodopsin.

1) Abstract. "…spontaneously activated by internal thermal energy" – what does that mean, actually? Isn't is just thermal activation? "Confusion in the field"? What is the confusion? "Quantitatively correlated with the closedness of its chromophore-binding pocket"? How is closedness actually quantitated? It isn't, so any correlation cannot be quantitative. Is it the "closedness" or the λmax value in the end that's the most important factor. The Abstract is extremely confusing in and of itself.

We like the phrase "spontaneously activated by internal thermal energy" because it introduces the word “spontaneous”, which in the field of visual pigments and phototransduction is understood to refer to the activity of single rhodopsin molecules in the absence of light stimulation, leading to electrical events identical to single-photon responses in both waveform and amplitude. At the same time, we would like to equate the concepts of “spontaneous activity” and “thermal activity”, which may not be obvious to everyone.

"Confusion in the field". The confusion for 30 years is whether the spontaneous activity of a pigment comes from thermal isomerization or from some other irrelevant chemical reaction. This confusion is now more explicitly stated in the revised manuscript. We now define operationally “openness” and “closedness” of the chromophore-binding pocket in a pigment simply by whether the pigment in live cells allows dark-chromophore exchange or not within an experimentally manageable yet appropriately long time frame (3-6 hours). The new data on live zebrafish cones and rods (with the long time required for acquiring these data explaining the delay in submitting the revised manuscript) suggest that, as in the case of salamander red cone pigment previously studied by us (Kefalov et al., Neuron, 2005), there is a clear difference between native rhodopsin and cone pigments with respect to dark chromophore-exchange – namely, either no observable exchange up to 6 hours for rod pigment or >50% exchange already in 2 hr or less for cone pigments. Thus, we have taken the simple approach of referring to these pigments as having “closed” and “open” chromophore-binding pocket, respectively. Should we later find that the openness/closedness of the chromophore-binding pocket ought to be quantified in a finer fashion, we shall refine our theory accordingly. Finally, we would like to emphasize that, in our theory, both λmax and the openness/closedness of the chromophore-binding pocket contribute to the absolute rate of spontaneous activity – with relative importance depending on the absolute λmax value – albeit difficult to elaborate in the Abstract given the required brevity.

2) Introduction. Of course the ground-state isomerization energy barrier is closely related to the λmax. That's trivial, isn't it? Why is that a part of a unifying theory? The trivial definition of "open" pocket refers to accessibility of the Schiff base to hydrolysis, for example by hydroxylamine (with a 1955 Wald reference), but the actual chemistry of the hydrolysis reaction is a very complicated pH dependent mechanism that is only partly dependent on accessibility or hydration state. It is not stated why the E122Q pigment is cone-like, or "non-canonical." Some detail is required to justify its use, other than the mouse model happened to exist. What other mutants might be useful and why weren't they used or considered?

According to classical physics, a photon with energy whether equal to or exceeding the ground-state isomerization energy barrier should have an equal probability of being absorbed. Thus, the existence of a λmax – namely, the probability of light absorption actually goes down at wavelengths shorter than λmax [though of even higher energy] – does not strictly speaking arise in classical physics, but is a quantum- mechanical concept. In Luo et al., Science, 2011, we have found experimentally that a pigment’s λmax is related to its isomerization barrier height, Ea, by Ea ≈ 0.84hc/λmax, thus allowing calculations in our theory.

Regarding the chromophore-binding pocket, please refer to our answer to Point 1 above. In this revision of the manuscript, we have decided not to use the susceptibility of a pigment to attack by external hydroxylamine as another criterion for the binding pocket’s openness because, from reading the literature, this property appears to depend on experimental conditions and possibly hydroxylamine concentration, thus not a reliable indicator.

The E122Q-rhodopsin is labeled “cone-pigment-like” because it has photochemical kinetics much faster than that of rhodopsin (therefore resembling cone pigments). In this revision, we have dropped the word “canonical” in most instances when describing a rod or cone pigment because we agree that the word is ambiguous. Finally, if pigment mutations could lead easily to substantial shifts in λmax or changes in the binding pocket’s openness, and if mouse lines associated with such mutations were available, we would have loved to measure the spontaneous noise of each and every pigment mutant and check against our theory. However, the truth of the matter is that mutations leading to substantial λmax shifts are rare, based on existing literature. Even more significantly, a large number of rhodopsin mutations lead to rod degeneration, rendering them difficult to study. Thus, it is indeed a good fortune that E122Q-rhodopsin produces a substantial λmax shift and causes no degeneration, and the associated mouse line is available to us for study.

3) Results, third paragraph. What is the total number of E122Q pigment molecules per cell? Can it be assumed that it is the same as the total Rho in WT? If so, why? The scaling factor related to the number of molecules is very important in the equation used to a spontaneous activation rate constant. Also at the end of the subsection “Measurements of the rates of spontaneous activation”, it is stated that the "molecular rate constant could be obtained by further dividing the measured cellular rate by the number of pigment molecules per rod." Again, how is it assumed that E122Q is the same as Rho?

E122Q-rhodopsin is expressed at normal level in RhoE122Q/E122Qmice (Imai et al., J. Biol. Chem., 2006). In the Gcaps-/-background, we have confirmed the same by Western blotting (Figure 1B, please see also response to comment8). Additionally, we have found with single-cell recordings and microspectrophotometry that the pigment contents of RhoWT/WT;Gcaps-/-and RhoE122Q/E122Q;Gcaps-/-rods indeed roughly agree (see revised manuscript).

4) Results, last paragraph. The competition experiment seems problematic. Why was only a single time point of 3 hours in darkness chosen? Does the retinal bind to the 1% BSA predominantly (subsection “Chromophore-preparation”, Methods)?

In Figure 2, a single time point of 3 hours in darkness was chosen just for simplicity because, based on previous work from salamander, we think 3 hours should already approach the end point of chromophore exchange if exchange indeed takes place. We have now also added 6 hours in darkness, and saw no difference in result for E122Q- rhodopsin.

Regarding BSA, yes, it is commonly used as a carrier for introducing retinal into rods and cones. Retinal delivery by 1% BSA has been used successfully to restore the sensitivity of bleached photoreceptors within a couple of minutes.

5) Results, end of last paragraph. It is now stated that E122Q is resistant to hydroxylamine? What? Isn't the λmax of 9-cis Rho 490 nm, not 482 nm? The data actually show Rho at about 498 nm or so, not 500. Why not use the actual data values in the text?

We have now decided not to include the phenomenon of susceptibility of a pigment to attack by external hydroxylamine as another criterion for the binding pocket’s openness because, from reading the literature, this property appears to depend on experimental conditions and possibly hydroxylamine concentration, thus not a reliable indicator.

From the literature, the λmax of 9-cis-rhodopsin is in the range of 481-489 nm. The λmax for WT rhodopsin (with 11-cis retinal) is about 500 nm, but can also vary a bit in different hands and also in the literature. Our measured λmax’s of both 11-cis- and 9- cis-rhodopsin fall within these ranges. The key point of the experiment here is that there is either a shift or no shift in λmax after dark incubation with 9-cis. Thus, the exact λmax value is not critical. We have, however, followed the reviewer’s suggestion of using our own measured λmax (481 nm for E122Q-rhodopsin) instead of a previously reported value (480 nm from Imai et al., J. Biol. Chem., 2006) for theoretical noise prediction.

6) Discussion. The main criticism is that the authors need to justify how one example case can provide a justification for the earlier theory, and why and how the E122Q Rho is a legitimate test system. Does it really have hybrid rod-cone properties when expressed in a mouse rod cell?

Please see the final paragraph in our response to point 2 above.

We would like to emphasize that E122Q-rhodopsin represents an important experimental model because: i) its λmax is considerably blue-shifted, thus providing a valuable data point for rod pigments, which normally have a very limited range of λmax, and ii) it has fast photochemical kinetics characteristic of cone pigments (Imai et al., J. Biol. Chem., 2006) but, on the other hand a rod-pigment-like chromophore-binding pocket, thus allowing the teasing apart of the contributions of pocket openness and of other cone-pigment properties to the higher noise of cone pigments. We have now modified the text to make these points clearer.

7) Subsection “Pigment noise prediction”. m is taken to be the same for Rho and cone pigments? That's really strange and some additional explanation seems warranted.

We used the same m value for both rhodopsin and cone pigments, as we did previously (Luo et al., Science, 2011) because they have the same chromophore. Given that the isomerization reaction occurs in the chromophore, it is not unreasonable to think that the energy for thermal isomerization does come mostly, if not all, from the chromophore itself.

8) Figure 1B. Again, referring to the issue of E122Q quantitation, the Rho band looks much heavier than the E122Q knockin band.

In Figure 1B, the left two lanes are duplicates of RhoWT/WT;Gcaps-/-retinas and the right two lanes are duplicates of RhoE122Q/E122Q;Gcaps-/-retinas. On the whole, there is no significant difference between the two genotypes. Moreover, single-cell recordings and microspectrophometry suggest that the rhodopsin content is comparable between the two genotypes (see text of revised manuscript).

Reviewer #2:

[…] Justification of the chromophore exchange rate argument: The authors use microspectrophotometry (MSP) and 9-cis retinal to estimate the exchange rate of the chromophore in E122Q- and WT rods. The authors claim that within the measured times there is no spectral shifts in E122Q-rods that have been exposed to 9-cis retinal. This data is used to conclude that the exchange rate is in line with rod pigments in E122Q-rods. The conclusion is central for the paper. It would be important to validate this argument by giving an estimate of the expected spectral shifts in MSP measurements for a chromophore exchange rate that is in line with that expected for cone-pigments in a similar time interval and relate this predicted shift to the accuracy of the λmax measurements. This is needed to assess to which extent the MSP with its current measurement accuracy for λmax is sensitive to the chromophore exchange rates representing rod and/or cone type pigments on these time scales. This is especially true for E122Q rods where the bleached native rods that have been regenerated with 9-cis retinal have a smaller spectral shift (λmax shift) compared to the WT rods.

Prompted in part by the comments of this reviewer, we have taken the time to study another animal species, zebrafish, which has multiple cone types with distinct pigments. The zebrafish data are now included in the revised manuscript. The bottom line is that, to the extent resolvable, the property of dark chromophore-exchange does appear to be common across cone pigments but negligible in rhodopsin. Regarding the chromophore-binding pocket, we now simply define it operationally as being “open” if the pigment shows the property of dark chromophore exchange with an exogenous chromophore without isomerization. Rhodopsin shows no sign of any exchange up to 6 hours, whereas cone pigments show substantial exchange in the order of an hour, whether in salamander (Kefalov et al., Neuron, 2005) or zebrafish (in this paper). We would like to emphasize here (and have stated as such in the paper) that our work, which uses one value for the pre-exponential factor A for rod pigments and another for cone pigments (i.e., a binary situation), is not necessarily intended to be the final word. If it turns out in the future that the A value has more than two possible values, we may have to fine-tune the theory.

Reviewer #3:

This paper provides confirmation for a previously published and validated theory of the relationship between the frequency of spontaneous isomerization of a vertebrate photopigment and it wavelength of maximum absorbance- also the peak wavelength of its photoisomerization action spectrum. Although it provides a single data point for this theory, it is important because the theory is more challenging to test for rods (for which spontaneous isomerization is arguably of much greater biological significance than for cones) than for cones because of the limited range of λmax values found in naturally occurring rod pigments. In addition, the frequency is so low for rods that it can only be measured in intact rods, making use of their ability to use extremely efficient amplification mechanisms to allow measurement of single-molecule isomerization events. Making use of an existing mouse model with a knock-in of a rod pigment with a single point mutation that results in a significant blue shift, the authors have quantitatively confirmed that the spontaneous rate is predicted by the theory. Secondarily, they verify that the binding pocket for the chromophore is very similar to that in WT rhodopsin, based on its extremely slow chromophore exchange kinetics.

We thank this reviewer for the general compliments about our work.

https://doi.org/10.7554/eLife.18492.012

Article and author information

Author details

  1. Wendy Wing Sze Yue

    1. Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, United States
    2. Center for Sensory Biology, Johns Hopkins University School of Medicine, Baltimore, United States
    3. Biochemistry, Cellular and Molecular Biology Graduate Program, Johns Hopkins University School of Medicine, Baltimore, United States
    Present address
    Department of Physiology, University of California, San Francisco, United States
    Contribution
    WWSY, Conceptualization, Resources, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing
    Contributed equally with
    Rikard Frederiksen
    Competing interests
    The authors declare that no competing interests exist.
  2. Rikard Frederiksen

    Department of Physiology and Biophysics, Boston University School of Medicine, Boston, United States
    Contribution
    RF, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—review and editing
    Contributed equally with
    Wendy Wing Sze Yue
    Competing interests
    The authors declare that no competing interests exist.
  3. Xiaozhi Ren

    1. Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, United States
    2. Center for Sensory Biology, Johns Hopkins University School of Medicine, Baltimore, United States
    Contribution
    XR, Data curation, Formal analysis, Investigation, Methodology
    Competing interests
    The authors declare that no competing interests exist.
  4. Dong-Gen Luo

    1. State Key Laboratory of Membrane Biology, Peking University, Beijing, China
    2. McGovern Institute for Brain Research, Peking University, Beijing, China
    3. Center for Quantitative Biology, Peking University, Beijing, China
    4. Peking-Tsinghua Center for Life Sciences, Beijing, China
    5. College of Life Sciences, Peking University, Beijing, China
    Contribution
    D-GL, Resources, Supervision, Methodology
    Competing interests
    The authors declare that no competing interests exist.
  5. Takahiro Yamashita

    Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto, Japan
    Contribution
    TY, Resources, Validation, Investigation
    Competing interests
    The authors declare that no competing interests exist.
  6. Yoshinori Shichida

    Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto, Japan
    Contribution
    YS, Resources, Validation, Investigation
    Competing interests
    The authors declare that no competing interests exist.
  7. M Carter Cornwall

    Department of Physiology and Biophysics, Boston University School of Medicine, Boston, United States
    Contribution
    MCC, Resources, Data curation, Formal analysis, Supervision, Funding acquisition
    Competing interests
    The authors declare that no competing interests exist.
  8. King-Wai Yau

    1. Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, United States
    2. Center for Sensory Biology, Johns Hopkins University School of Medicine, Baltimore, United States
    3. Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, United States
    Contribution
    K-WY, Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Validation, Writing—original draft, Project administration, Writing—review and editing
    For correspondence
    kwyau@jhmi.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8880-3091

Funding

National Eye Institute (EY06837)

  • King-Wai Yau

National Eye Institute (EY001157)

  • M Carter Cornwall

National Natural Science Foundation of China (31471053)

  • Dong-Gen Luo

Antonio Champalimaud Foundation, Portugal (Antonio Champalimaud Vision Award)

  • King-Wai Yau

Howard Hughes Medical Institute (International Student Research Fellowships)

  • Wendy Wing Sze Yue

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Dr. Jeannie Chen (University of Southern California) for the Gcaps-/- line. We also thank Drs. Robert Molday (University of British Columbia), Jason C-K Chen and Theodore Wensel (Baylor College of Medicine) for antibodies. This work was supported by NIH Grants EY06837 (K-WY) and EY001157 (MCC); National Natural Science Foundation of China Grant 31471053 (D-GL); the António Champalimaud Vision Award, Portugal (K-WY); and a Howard Hughes Medical Institute International Predoctoral Fellowship (WWSY).

Ethics

Animal experimentation: All animal experiments were carried out according to protocols approved by the Institutional Animal Care and Use Committee at Johns Hopkins University (MO14M199 for mouse) and Boston University (AN15427 for both mouse and zebrafish).

Reviewing Editor

  1. Richard Aldrich, The University of Texas at Austin, United States

Publication history

  1. Received: June 5, 2016
  2. Accepted: January 23, 2017
  3. Version of Record published: February 10, 2017 (version 1)

Copyright

© 2017, Yue et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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