Mitochondrial Calcium Uniporter (MCU) deficiency reveals an alternate path for Ca2+ uptake in photoreceptor mitochondria

Rods and cones use intracellular Ca2+ to regulate many functions, including phototransduction and neurotransmission. The Mitochondrial Calcium Uniporter (MCU) complex is thought to be the primary pathway for Ca2+ entry into mitochondria in eukaryotes. We investigate the hypothesis that mitochondrial Ca2+ uptake via MCU influences phototransduction and energy metabolism in photoreceptors using a mcu-/- zebrafish and a rod photoreceptor-specific Mcu-/- mouse. Using genetically encoded Ca2+ sensors to directly examine Ca2+ uptake in zebrafish cone mitochondria, we found that loss of MCU reduces but does not eliminate mitochondrial Ca2+ uptake. Loss of MCU does not lead to photoreceptor degeneration, mildly affects mitochondrial metabolism, and does not alter physiological responses to light, even in the absence of the Na+/Ca2+, K+ exchanger. Our results reveal that MCU is dispensable for vertebrate photoreceptor function, consistent with its low expression and the presence of an alternative pathway for Ca2+ uptake into photoreceptor mitochondria.

Maintaining proper intracellular Ca 2+ homeostasis is essential for cellular function. Mitochondria have the ability to sequester Ca 2+ via the Mitochondrial Calcium Uniporter complex (MCU) 1,2 . Mitochondrial Ca 2+ uptake via MCU can modulate both cytosolic and mitochondrial Ca 2+ levels, meaning that Ca 2+ -sensitive reactions that occur in both compartments can be affected by MCU activity. In the mitochondrial matrix, increasing Ca 2+ can stimulate tricarboxylic (TCA) cycle dehydrogenases and enhance ATP production, although Ca 2+ overload in the matrix can trigger cell death 3,4 . Mitochondrial Ca 2+ uptake can also regulate cytosolic Ca 2+ levels and thus influence Ca 2+ -sensitive cytosolic reactions. Since these Ca 2+ -sensitive cytosolic reactions vary widely among cell-types depending on each cell's specific function, disruptions in mitochondrial Ca 2+ -uptake can have tissuespecific consequences 5,6 .
Photoreceptors, the light-sensitive neurons in the retina, rely on spatially distinct changes in cytosolic Ca 2+ to regulate both phototransduction and neurotransmission. At the outer segment, intracellular Ca 2+ changes in response to light stimulation and this is critical for timely shut-down of the phototransduction cascade and light adaptation 7 . At the synapse, changes in intracellular Ca 2+ modulate vesicle release and neurotransmission [8][9][10] . The outer segment and synaptic Ca 2+ pools that control these functions are separated by a cell body filled with a dense cluster of mitochondria, and evidence suggests that these mitochondria may help isolate these pools from each other. Mitochondria from zebrafish cones can prevent Ca 2+ in the outer segment from reaching the rest of the cell and conversely, they can prevent Ca 2+ in the synapse from reaching the outer segment 11 . Increasing mitochondrial Ca 2+ uptake in zebrafish cones by overexpressing MCU also accelerates cytosolic Ca 2+ clearance, which causes cones to recover from light exposure faster 12 .
Efficient uptake of Ca 2+ into mitochondria could also enhance the ability of photoreceptors to meet their high energy demands. Darkness is the most energetically demanding state of the photoreceptor, and intracellular Ca 2+ is at its highest levels in darkness 13,14 . If this Ca 2+ were to enter mitochondria, it could enhance ATP production by stimulating TCA cycle dehydrogenases 3 . In line with this hypothesis, overexpressing MCU in zebrafish Scientific RepoRtS | (2020) 10:16041 | https://doi.org/10.1038/s41598-020-72708-x www.nature.com/scientificreports/ cones alters the distribution of TCA cycle metabolites in a way which is consistent with enhanced activity of Ca 2+ -sensitive dehydrogenases 12 .
These observations suggested to us that mitochondrial Ca 2+ uptake could play an important role in modulating photoreceptor function. Given this, it is surprising that zebrafish cone photoreceptors express extremely low levels of MCU 12 . MCU has been widely thought to be the sole route of Ca 2+ entry into mitochondria in eukaryotes, as loss of MCU completely inhibits mitochondrial Ca 2+ uptake in skeletal muscle, liver, heart, brown adipose tissue, and a wide variety of cell lines [15][16][17][18][19] . Despite this, it is possible that certain specialized cell types might not rely solely on MCU for mitochondrial Ca 2+ uptake, since it also has been observed that brain mitochondria lacking MCU expression do not have a complete loss of mitochondrial Ca 2+ uptake 20 . Thus, the role that MCU plays in vertebrate photoreceptors remains an open question.
To resolve the role MCU-mediated mitochondrial Ca 2+ uptake plays in modulating photoreceptor function, we evaluated the morphological, biochemical and physiological consequences of loss of MCU expression using both a global mcu -/zebrafish model and a rod photoreceptor-specific Mcu -/mouse model. Our study demonstrates that MCU is remarkably dispensable for photoreceptor function, which is consistent with our discoveries of very low levels of MCU expression and evidence for an additional mechanism for Ca 2+ uptake into photoreceptor mitochondria.

Results
Mcu contributes to clearance of cytosolic Ca 2+ in zebrafish cones. We first generated global mcu -/zebrafish so that we could take advantage of well-established ex vivo retinal Ca 2+ -imaging tools and techniques to determine the role Mcu plays in modulating cytosolic and mitochondrial Ca 2+ dynamics in cone photoreceptors 21 . Global mcu -/zebrafish were generated using a CRISPR-Cas9 strategy outlined previously 22 . A founder carrying a 12-nucleotide deletion in exon 5, which introduces a premature stop codon in exon 6 of mcu was used in this study (mcu w249 ; notated as mcu -/-, Fig. 1A). We used a custom zebrafish-specific Mcu antibody to probe immunoblots of mitochondria isolated from retinas and brains of WT and global mcu -/zebrafish 12 .
Mcu expression is entirely ablated in global mcu -/tissues (Fig. 1B). Similar to the global Mcu -/mouse model, a smaller than expected ratio of homozygous mcu -/zebrafish survive to adulthood from crosses of mcu +/parents (Supplemental Fig. 1A) 15,23 . There are no obvious effects of Mcu-deficiency on retinal or photoreceptor morphology (Fig. 1C).
To determine what role Mcu plays in clearing cytosolic Ca 2+ , we crossed mcu -/zebrafish with zebrafish expressing the cytosolic Ca 2+ sensor GCaMP3 (gnat2:GCaMP3) 11 . Retinal slices from mcu -/-gnat2:GCaMP3 and wild-type (WT) gnat2:GCaMP3 siblings were incubated in Krebs-Ringer Bicarbonate (KRB) buffer containing 0 mM CaCl 2 and 0.4 mM EGTA for 10 min. Cones were then imaged to establish a baseline fluorescence reading prior to delivery of a 5 mM bolus of CaCl 2 (Fig. 1D). Cones from global mcu -/zebrafish exhibit a higher maximum fold change in cytosolic GCaMP3 fluorescence compared to WT cones (Fig. 1E, 1.192 ± 0.024-fold greater than WT, mean ± SEM reported). A single exponential decay was fit to each curve and the decay constant was calculated to determine if WT and mcu -/cones clear cytosolic Ca 2+ at different rates (Fig. 1F). The rate of decay calculated for mcu -/-(0.01781 s −1 ) was significantly smaller compared to WT (0.02433 s −1 ), indicating that mcu -/cones clear cytosolic Ca 2+ at a slower rate compared to WT cones. This indicates that Mcu plays a role in mediating mitochondrial uptake of cytosolic Ca 2+ in zebrafish cones.
Mitochondrial ca 2+ uptake in cones from global mcu -/zebrafish is diminished, but not ablated. To directly observe mitochondrial Ca 2+ levels in mcu -/photoreceptors, we next crossed mcu -/zebrafish with zebrafish expressing the mitochondrially-targeted Ca 2+ sensor GCaMP3 in cones (gnat2:mito-GCaMP3) 11 . Eyes from live larvae were imaged and we found that there is no difference in basal mito-GCaMP3 fluorescence between WT and mcu -/cones ( Fig. 2A). Although we analyzed mito-GCaMP3 fluorescence in sibling larvae, which had only a single insertion of the mito-GCaMP3 transgene, it is possible that differences in transgene expression between fish could obscure differences in WT and mcu -/basal mitochondrial Ca 2+ . So, we next determined basal mitochondrial Ca 2+ levels in mature cones from WT and mcu -/fish using an ex vivo imaging method that reports mito-GCaMP3 fluorescence independent of mito-GCaMP3 probe concentration. We compared baseline mito-GCaMP3 fluorescence (F 0 ) to the maximum fluorescence (F max , obtained by addition of ionomycin to media containing 2 mM Ca 2+ ) and minimum fluorescence (F min , obtained by the addition of 5 mM EGTA to chelate Ca 2+ ). Similar to larvae, we found that baseline mito-GCaMP3 fluorescence was not significantly different in adult WT and mcu -/zebrafish cones (Fig. 2B,C).
We next tested whether mitochondrial Ca 2+ uptake is altered in cones from global mcu -/fish. It has been previously established that the treatment of photoreceptors with the PDE inhibitor sildenafil in the presence of the Na + /Ca 2+ exchanger inhibitor KB-R7943 causes significant increases in cytoplasmic Ca 2+ levels and a coincident increase in mitochondrial Ca 2+ levels 11 . We used the same strategy here to assess changes in mitochondrial Ca 2+ uptake in mcu -/cones. We imaged WT and global mcu -/-gnat2:mito-GCaMP3 cones after a 10 min KB-R7943 preincubation to obtain baseline measurements of mito-GCaMP3 fluorescence, and continued to image after exposing the retinal slices to sildenafil. In WT cones, mito-GCaMP3 fluorescence increases after sildenafil treatment (Fig. 2D, top left panel). The average response of all WT mitochondrial clusters is shown in the dark trace, and the individual responses of each individual mitochondrial cluster is shown in semi-transparent traces. We determined the number of WT mitochondrial clusters which increase mitochondrial Ca 2+ levels in response to sildenafil and found that 82% of mitochondrial clusters respond ( Fig. 2E; in order for a mitochondrial cluster to have considered to have responded, it must have exhibited a 1.2-fold or greater increase above baseline in Scientific RepoRtS | (2020) 10:16041 | https://doi.org/10.1038/s41598-020-72708-x www.nature.com/scientificreports/ mito-GCaMP3 fluorescence at any time during imaging). The population of mitochondrial clusters which did not respond to sildenafil can be visualized by identifying the traces that do not cross the dotted line on the graph indicating 1.2-fold above baseline.
In mcu -/retinal slices, we found that mito-GCaMP3 fluorescence increases after sildenafil treatment in many cones (Fig. 2D, top right panel). However, we observed that there was a greater proportion of mcu -/cone mitochondrial clusters which never respond to sildenafil (Fig. 2E, 41% of mcu -/mitochondrial clusters never increase 1.2-fold above baseline compared to 18% of WT mitochondrial clusters). The mcu -/cone mitochondrial clusters which do respond to sildenafil treatment exhibited a smaller maximum fold change in mito-GCaMP3 fluorescence compared to WT mitochondrial clusters which respond to sildenafil (Fig. 2F). We also observed showing Mcu expression in retina and brain from global mcu -/zebrafish. 20 µg of protein from mitochondrial lysate from 6 pooled retinas and 1 brain was analyzed. The custom Mcu antibody detects a faint non-specific band at a slightly higher molecular weight than Mcu. (C) Scanning electron microscopy (SEM) images of WT and mcu -/zebrafish cone mitochondria (top panel) and retinas (bottom panel) from 11-month old sibling fish. Retinal and mitochondrial morphology appear unchanged by loss of Mcu (n = 8 retinas from WT and mcu /zebrafish were examined, representative images from 1 WT and 1 mcu -/retina are shown). (D) Traces of relative cyto-GCaMP3 fluorescence of cone cell bodies in adult retinal slices of WT or mcu -/fish expressing gnat2:cyto-GCaMP3. Baseline mitochondrial fluorescence was determined in KRB buffer containing 0 mM CaCl 2 and 0.4 mM EGTA, then a bolus of CaCl 2 was delivered in order to bring the [Ca 2+ ] free to 5 mM. The mean is reported and shaded region = 95% CI. (n = 110 cells (four fish) for WT and n = 112 cells (four fish) for mcu -/-). (E) Maximum fold change in cyto-GCaMP3 fluorescence for each cell body after exposure to 5 mM [Ca 2+ ] free . WT: 3.345 ± 0.085, mcu -/-: 3.985 ± 0.082, mean ± SEM reported, p < 0.0001 using Welch's t test. (n = 110 cells (from four fish) for WT and n = 112 cells (from four fish) for mcu -/-). (F) Decay constants calculated using a single exponential decay fit. WT: 0.02433 s −1 (0.02384 to 0.02483), mcu -/-: 0.01781 s −1 (0.01743 to 0.01821), decay constant with 95% CI reported, p < 0.0001 using Welch's t test.

Scientific RepoRtS
| (2020) 10:16041 | https://doi.org/10.1038/s41598-020-72708-x www.nature.com/scientificreports/ that mcu -/cone mitochondrial clusters take longer to respond to sildenafil treatment compared to WT mitochondrial clusters (Fig. 2G, T Response is defined as the time mito-GCaMP3 fluorescence first increased 1.2-fold above baseline). There is heterogeneity in the timing of the mito-GCaMP3 fluorescence increase from within seconds to many minutes after sildenafil addition in both WT and mcu -/cone mitochondrial clusters. However, since mcu -/mitochondrial clusters respond later on average, this suggests that a different biological threshold (such as the size of the cytosolic Ca 2+ load or the amount of time it is sustained) must be met in order for an mcu -/mitochondria to begin taking up Ca 2+ .
To assess whether the residual Ca 2+ uptake exhibited by mcu -/cone mitochondrial clusters in this assay could be attributed to an Mcu-independent mechanism, we repeated this experiment in the presence of the Mcu inhibitor Ru360 (Fig. 2D, bottom two panels). Ru360 treatment increases the proportion of WT mitochondrial clusters which never respond (Fig. 2E, 64% of WT + Ru360 mitochondrial clusters do not respond vs 18% of WT mitochondrial clusters without Ru360). WT + Ru360 cone mitochondrial clusters also exhibit a significantly smaller maximum fold change in fluorescence compared to WT mitochondrial clusters without Ru360 (Fig. 2F). Ru360 treatment also slightly increases the proportion of mcu -/mitochondrial clusters which never respond (Fig. 2E, 53% of mcu -/-+ Ru360 mitochondrial clusters fail to respond versus 41% of mcu -/mitochondrial clusters without Ru360). Compared to untreated mcu -/mitochondrial clusters, mcu -/-+ Ru360 mitochondrial clusters do not have a significantly different maximum fold change in fluorescence or T Response (Fig. 2F,G). Notably, a large number of both WT + Ru360 and mcu -/-+ Ru360 mitochondrial clusters still exhibit an apparent increase in mitochondrial Ca 2+ in this assay.
Since GCaMP3 is a GFP derivative, its fluorescence can be sensitive to changes in pH. The fluorescence of cpYFP (another GFP derivative) is not sensitive to Ca 2+ but has been shown to be extremely sensitive to changes in pH 24 . To test if the increases in mito-GCaMP3 fluorescence observed in mcu -/cone mitochondria and cone mitochondria treated with Ru360 could be due to changes in pH and not mitochondrial Ca 2+ uptake, we treated retinal slices from gnat2:mito-cpYFP zebrafish with KB-R7943 and sildenafil (Supplemental Fig. 2A). We observed no increases in cp-YFP fluorescence over the time course, indicating that the increases in fluorescence we observe with mito-GCaMP3 are due to changes in mitochondrial Ca 2+ and not pH.
Taken together, these results show that Mcu contributes to some mitochondrial Ca 2+ uptake in cones, but that Ca 2+ can also enter cone mitochondria through an alternative pathway.
Retinas from mcu -/zebrafish have normal morphology, metabolism, and photoresponses. We next determined if the diminished ability of mcu -/mitochondria to take up Ca 2+ in cones might lead to metabolic or electrophysiological defects. To assess potential changes in metabolism, we measured total metabolite levels from freshly dissected dark-adapted retinas from mcu -/zebrafish. We detected no changes in total metabolite levels in mcu -/retinas, although α-ketoglutarate levels trend slightly but not significantly higher after loss of Mcu (Fig. 3A). Previous studies of MCU -/tissues describe an increase in the amount of phosphorylated pyruvate dehydrogenase (PDH), which is attributed to diminished activity of the Ca 2+ -sensitive phosphatase PDP1c 15,16,[25][26][27] . We assessed the P-PDH/PDH ratio and found that it is not different between WT and mcu -/retinas (Fig. 3B). This result is consistent with the unaltered resting Ca 2+ levels we observed in mcu -/cones.
Photoreceptors rely on efficient clearance of cytosolic Ca 2+ from the outer segment to stimulate recovery of the photoresponse 28 . Photoresponses of zebrafish cones that overexpress Mcu recover faster following a flash of light, suggesting that uptake of Ca 2+ through Mcu could contribute to clearing outer segment Ca 2+ in the time Figure 2. Mitochondrial Ca 2+ uptake in cones from global mcu -/zebrafish is diminished, but not ablated. (A) Total cone mitochondrial fluorescence in gnat2:mito-GCaMP3 larval zebrafish eyes. The mean is reported with bars indicating standard error. (n = 8 WT fish, 4 mcu +/fish, and 5 mcu -/fish. ns = not significant using one-way ANOVA with Tukey's multiple comparisons test). (B) Relative mito-GCaMP3 fluorescence of cone mitochondrial clusters in adult retinal slices of WT or mcu -/fish expressing gnat2:mito-GCaMP3. Baseline mitochondrial fluorescence was determined in KRB buffer containing 2 mM CaCl 2 , then ionomycin (5 µM) was added to allow 2 mM Ca 2+ entry into the mitochondria to saturate the probe. Next, EGTA (5 mM) was added to the solution (holding 5 µM ionomycin constant) to chelate Ca 2+ and determine minimum mito-GCaMP3 fluorescence. The mean is reported and shaded region = 95% CI. (n = 55 mitochondrial clusters (three fish) for WT and n = 51 mitochondrial clusters (three fish) for mcu -/-).  Total TCA cycle metabolite levels in mcu -/zebrafish retinas relative to WT. Zebrafish were dark-adapted for 18 h and retinas were dissected under red light. α-ketoglutarate levels trend higher in mcu -/zebrafish retinas, although they are not significantly different than WT (1.4 ± 0.5-fold higher in mcu -/retinas, p = 0.09 using Welch's t test, mean ± standard deviation is reported, n = 6 WT and 6 mcu -/retinas from 3 different fish each).

Rod photoreceptors express low levels of MCU.
We did not detect any metabolic or physiological consequences caused by Mcu deficiency in zebrafish retinas. However, phenotypes can vary significantly between chronic, induced, and tissue-specific Mcu -/animal models 6 . For example, the cardiac phenotype from the global Mcu -/mouse is surprisingly mild, while constitutive and inducible heart-specific Mcu knockdown models have both different and more severe phenotypes 15,18,26,30,31 . This indicates that some tissues have both the capacity and the necessity to adapt to chronic loss of MCU. Since we observed that a smaller than expected number of mcu -/fish reach adulthood from mcu +/crosses, it is likely that the mcu -/fish that survived to adulthood adapted to loss of Mcu in some way. In order to ensure that the mild phenotypes we observed in global mcu -/zebrafish retinas were truly due to the dispensable nature of MCU in photoreceptors and not due to adaptations made in response to chronic loss of MCU, we also generated a rod photoreceptor specific Mcu  To determine if MCU expression in mouse rods is similarly low, we analyzed MCU expression in WT and Rod Mcu -/retinas using immunoblot (Fig. 4C). MCU expression is not significantly altered in Rod Mcu -/retinas relative to WT, despite rods being by far the most abundant cell type in the mouse retina 33 . This indicates that MCU expression is extremely low in rods relative to other cell types in the retina. Overall retinal and photoreceptor morphology appears to be unaltered by loss of MCU expression in rods: we observed no defects in our immunohistochemistry images and scanning electron microscopy (SEM) analysis of Rod Mcu -/retinas at 6-months old revealed no changes in retinal morphology (Fig. 4D).
Rod Mcu -/retinas accumulate α-ketoglutarate. Overexpressing MCU in zebrafish cone photoreceptors leads to changes in the steady-state concentrations of TCA cycle metabolites, likely due to increased Ca 2+ binding lowering the K m of α-ketoglutarate dehydrogenase (α-KGDH) and isocitrate dehydrogenase 12 . To test if the reduced mitochondrial Ca 2+ uptake ability of Mcu -/photoreceptors causes a subsequent increase in enzyme K m , we used gas chromatography-mass spectrometry (GC-MS) to evaluate the influence of MCU on metabolic flux in retinas. We incubated retinas from WT and Rod Mcu -/light-adapted mice in 5 mM U-13 C-glucose for 0, 5, and 30 min and quantified accumulation of unlabeled and labeled metabolites using GC-MS (an isotopomer diagram is included in Supplemental Fig. 5B). When we measured total metabolite levels at each time point, we observed that the TCA cycle metabolite α-ketoglutarate was the only metabolite which was consistently elevated in Rod Mcu -/retinas across all time points (α-ketoglutarate levels are elevated 1.99 ± 1.09-fold at 0 min; 1.46 ± 0.36-fold 5 min, and 1.90 ± 0.77-fold at 15 min; mean ± standard deviation propagated to include variation of WT samples reported) (Fig. 5A). Labeled α-ketoglutarate (m2 α-ketoglutarate made in the first round and m3 α-ketoglutarate made in the second round) also accumulate to slightly higher steady state levels in Rod Mcu -/retinas (Fig. 5B, Supplemental Fig. 5A). Other metabolites do not accumulate consistently differently in Rod Mcu -/retinas compared to WT. We also measured the lactate/pyruvate ratio at the 0 min timepoint as a proxy for the cytosolic NADH/NAD + ratio in freshly dissected retinas and found it to be unaltered in Rod Mcu -/retinas (Supplemental Fig. 5C).
Photoreceptor energy demands are significantly higher in darkness, and intracellular Ca 2+ levels in photoreceptors can increase 10-to 60-fold in darkness relative to light 14,34,35 . So, it is possible that MCU plays a larger role in modulating photoreceptor metabolism in darkness. We first determined how darkness normally alters steady-state metabolite levels by comparing metabolite abundance between light-and dark-adapted WT retinas (for dark-adapted retinas, mice were dark-adapted for 18 h and retinas were dissected and snap-frozen under infrared light). In WT retinas, steady-state levels of many metabolites are slightly but significantly lower in darkness relative to light, with α-ketoglutarate and pyruvate levels being the most reduced (α-ketoglutarate: 0.61 ± 0.12-fold lower; pyruvate: 0.62 ± 0.23-fold lower; mean ± standard deviation propagated to include variation of "light" samples reported) (Fig. 5C). We repeated this comparison using light-and dark-adapted Rod Mcu -/retinas. We observed the same trend in that the steady-state levels of many metabolites are slightly but significantly lower in dark-adapted Rod Mcu -/retinas compared to light-adapted Rod Mcu -/retinas (Fig. 5D). Similar to what was observed in WT retinas, both pyruvate and α-ketoglutarate levels are the most reduced in Rod Mcu -/retinas (α-ketoglutarate: 0.71 ± 0.20-fold lower; pyruvate: 0.82 ± 0.15-fold lower; mean ± standard deviation propagated to include variation of "light" samples reported). When comparing the fold change in metabolite abundance in darkness relative to light, it appeared as though the influence of darkness in Rod  5E). While the influence of darkness on pyruvate and α-ketoglutarate is slightly different between WT and Rod Mcu -/retinas, we found that this difference is not statistically significant. This indicates that while MCU might play a small role in modulating steady-state metabolite levels in darkness, it is likely not the primary effector of change. We next assessed if loss of MCU expression altered metabolic flux in dark-adapted retinas differently than in light-adapted retinas. WT and Rod Mcu -/mice were dark-adapted for 18 h and retinas were dissected, incubated in U-13 C-glucose, and snap-frozen all under infrared light. Once again, we found that steady-state α-ketoglutarate levels from freshly-dissected dark-adapted retinas are consistently higher in Rod Mcu -/retinas compared to www.nature.com/scientificreports/ WT (α-ketoglutarate levels are elevated 1.56 ± 0.20-fold at 0 min, 1.93 ± 0.45-fold at 5 min, and 1.41 ± 0.17-fold at 30 min; mean ± standard deviation propagated to include variation of WT samples reported) (Fig. 5F). We also observed a similar increase in Rod Mcu -/-m2 and m3 α-ketoglutarate levels throughout the time course of U-13 C-glucose incubation (Fig. 5G, Supplemental Fig. 5D). We also assessed the levels of lactate, pyruvate, and the lactate/pyruvate ratio (as a proxy for cytosolic NADH/NAD + ) in freshly dissected and snap-frozen darkadapted retinas and found it was unchanged in Rod Mcu -/retinas (Supplemental Fig. 5E). Overall, we found that metabolic flux in dark-adapted Rod Mcu -/retinas matches flux in dark-adapted WT retinas with the exception of a consistent accumulation of α-ketoglutarate. Finally, we determined if loss of MCU expression in rods might alter the P-PDH/PDH ratio. Our immunoblot analysis showed that the P-PDH/PDH ratio in light-adapted Rod Mcu -/retinas was not different than in WT (Supplemental Fig. 5F). Accordingly, we did not observe a decrease in citrate production in our isotopic labeling experiments in either light-or dark-adapted Rod Mcu -/retinas.
MCU-deficiency does not influence mouse rod photoresponses. We determined how much MCU normally contributes to the rod photoresponse recovery by recording ex vivo transretinal ERG responses from Rod Mcu -/mice and WT controls in scotopic conditions. Dark-adapted flash responses of Mcu -/rods are slightly but not significantly larger than controls (Fig. 6A-C). The normalized flash response family plots superimposed over each other, indicating that the intensity to produce half-maximum response (a measure of sensitivity) of Mcu -/rods is not significantly different from controls (Fig. 6C, inset). A summary of the response parameters in the control and Mcu -/rods is given in Table 1. There are no notable differences in the time to peak (T p ), integration time (T int ), and recovery time constant τ rec of the flash response.
We next tested if Ca 2+ sequestering through MCU affects light adaptation of rods. When illuminated, photoreceptors must adapt to the light exposure in order to retain the ability to respond. Ca 2+ levels in the outer segment of photoreceptors modulate the activity of several proteins to mediate this light adaptation [36][37][38] . To test if Ca 2+ sequestering through MCU has an effect on light adaptation of rods, we exposed retinas to a series of background light steps of increasing intensity. The peak response to background light of Mcu -/rods was comparable to controls (Fig. 6D,E). Notably, the background response plateau of Mcu -/rods was lower than the controls and was significant for the two highest background light steps tested (Fig. 6F). However, the light adapted sensitivity (S f ) remained largely unaffected in Rod Mcu -/compared to the controls across all background light levels tested (Fig. 6G).
MCU activity is controlled by multiple regulatory proteins that confer cooperativity to the channel so that it can robustly respond to changes in cytoplasmic Ca 2+39-46 . In the previous experiments, all responses were in either the completely dark-adapted state or in steady-state light adaptation, with cytoplasmic Ca 2+ being stable at high or low concentrations, respectively. We next examined if MCU modulates responses when photoreceptor intracellular Ca 2+ levels are rapidly changing and when Ca 2+ flux through MCU may be changing more dynamically. To do this, we presented test flashes to the retinas that were in the process of dark adapting. After turning off the background light, two test flashes were presented at 2 and 4 s, followed by a saturating light flash (Fig. 6H). This allowed us to assess both flash response kinetics (Fig. 6I) and sensitivity ( Table 2). Both parameters were unaltered by the rod MCU deficiency.
McU-mediated mitochondrial ca 2+ uptake does not contribute to the small photoresponse seen in Nckx1 -/mice. The rod Na + /Ca 2+ , K + exchanger (NCKX1) is the only known route for Ca 2+ clearance from the outer segment plasma membrane, and it is thought to be the driver of Ca 2+ clearance from this compartment. Since MCU expression in mouse rods is extremely low, its contribution to cytosolic Ca 2+ clearance may be small compared to that of NCKX1. In an effort to unmask any possible modulation of the rod photoresponse by MCU, we investigated the effect of MCU loss on the rod photoresponse in mice lacking NCKX1. NCKX1-deficiency compromises Ca 2+ extrusion in the rod outer segments and it delays photoresponse recovery following a flash stimulus 29 . If the residual Ca 2+ extrusion in NCKX1-deficient rods is mediated by MCU, then it would be expected that the subsequent deletion of MCU would further suppress or completely block Ca 2+ extrusion, causing a further delay in photoresponse recovery and suppressing light adaptation.
We recorded transretinal ERG responses from Nckx1 -/control and Nckx1 -/-Rod Mcu -/mice (Fig. 7A,B). As previously shown, deletion of NCKX1 results in a significant reduction in the photoresponse generated by mouse rods (compare Fig. 7A with Fig. 6A) 29 . The responses of Nckx1 -/-Mcu -/double knockout rods are significantly smaller compared to Nckx1 -/controls (Fig. 7C). However, the sensitivity and kinetics of the flash response are comparable in Nckx1 -/-Mcu -/rods and control Nckx1 -/rods (Fig. 7C inset and D, respectively). This indicates that Ca 2+ sequestration by MCU does not modulate the rod photoresponse recovery even in the absence of the dominant NCKX1 Ca 2+ extrusion mechanism. We wondered if the smaller responses of the Nckx1 -/-Mcu -/double knockout rods might be due to accelerated retinal degeneration, since fewer rods would explain the observed reduction in the photoresponse. However, when we measured the thickness of the different retinal layers of hematoxylin and eosin stained Nckx1 -/control and Nckx1 -/-Mcu -/ -eyes we found no differences, indicating that early retinal degeneration is not responsible for the decreased response (Supplemental Fig. 7A). Table 3 summarizes the rod response properties of Nckx1 -/controls and Nckx1 -/-Rod Mcu -/mice. Overall, these results indicate that Ca 2+ uptake mediated by MCU does not significantly influence the rod photoresponse.

Discussion
Photoreceptors have abundant mitochondria in the compartment of the cell between the nucleus and the outer segment that can influence cytosolic Ca 2+ pools 11 . Photoreceptors also depend on Ca 2+ homeostasis for proper function and viability 9,10 . Despite this, we find that loss of the presumptive primary Ca 2+ channel for Scientific RepoRtS | (2020) 10:16041 | https://doi.org/10.1038/s41598-020-72708-x www.nature.com/scientificreports/ mitochondrial Ca 2+ uptake (MCU) is tolerated surprisingly well by photoreceptors. MCU-deficient photoreceptors appear healthy, their TCA cycle activity is largely unaltered, and the photoresponse is preserved. In cones from global mcu -/zebrafish, cytosolic Ca 2+ is cleared more slowly and mitochondrial Ca 2+ uptake is reduced in a significant population of mitochondria. However, some mitochondria still display robust increases in mito-GCaMP3 fluorescence in the absence of MCU. Ca 2+ uptake in these mitochondria appears to occur more slowly and with a lower magnitude than in WT mitochondria. Consistent with this result, we found that both WT and mcu -/cones have a population of mitochondrial clusters which still appear to take up Ca 2+ even in the presence of the MCU inhibitor Ru360, and the characteristics of this population match those of the responding mitochondria in mcu -/cones. This is similar to what has been observed in non-synaptic brain mitochondria isolated www.nature.com/scientificreports/ from Mcu -/mice, in which uptake of Ca 2+ into mitochondria was not blocked but instead occurred at a slower rate compared to controls 20 . Taken together, these results suggest that that neuronal tissue like retina and brain may have an alternative mitochondrial Ca 2+ uptake pathway with different response kinetics and Ca 2+ uptake capacity. Future experiments which use other strategies for measuring mitochondrial Ca 2+ uptake will be critical for confirming, identifying, and assessing the role of MCU-independent mechanisms for mitochondrial Ca 2+ uptake in photoreceptors. Table 1. Dim flash response sensitivity and kinetics parameters. R max saturated response amplitude measured at the plateau, I 1/2 intensity required to produce half of the saturated response, S fD dark adapted sensitivity, t p time to peak of a dim flash response, t int integration time of the response, τ rec recovery time constant during response shut off, I o intensity required to decay the sensitivity to one half. R max (μV) I 1/2 (phot/µm 2 ) S fD (µV/phot/µm 2 ) t p (ms) t int (ms) τ rec (ms) I o * (phot/µm 2 /s) n*  Table 2. Normalized sensitivity at 2 s and 4 s time points after turning off the background light step. S f /S fD normalized fractional sensitivity, I BD intensity of the background illumination. www.nature.com/scientificreports/ Other candidates for Ca 2+ entry have been described but are not as well-characterized as MCU, as MCU is thought to be primarily responsible for mitochondrial Ca 2+ influx. However, the viability of Mcu -/mice and zebrafish, and the extremely mild consequences of MCU loss in photoreceptors we describe here, suggest that there is an alternative uptake mechanism in at least some cell types 15,47 . Letm1 is a Ca 2+ /H + exchanger on the inner mitochondrial membrane that links Ca 2+ influx and efflux to electron transport chain activity and mitochondrial pH 48 . Letm1 appears to act primarily as a Ca 2+ extrusion mechanism, but it is also capable of Ca 2+ influx. Knockdown of Letm1 impairs Ca 2+ transport into the mitochondria of Flp-In-293 cells and patient-derived fibroblasts, and Letm1 +/mice have impaired ATP production and PDH activity specifically in neuronal tissue 49,50 . There is also some evidence that mitochondrial ryanodine receptors may exist in neurons and cardiac cells, but they have yet to be unambiguously identified 51,52 . Another candidate is the mitochondrial Na + /Ca 2+ exchanger, which is associated with Ca 2+ efflux but can run in reverse to promote Ca 2+ entry into mitochondria 53 .
A possible advantage of relying on these other mechanisms for Ca 2+ transport is that it would link mitochondrial Ca 2+ influx to factors other than cytosolic Ca 2+ levels. The mitochondrial Na + /Ca 2+ exchanger would couple Ca 2+ entry to cytosolic Na + , which fluctuates in photoreceptors in response to light. Letm1 would tie Ca 2+ entry to mitochondrial pH and ATP production, and Letm1 has even been shown to be upregulated in cancer tissue, which is highly glycolytic like photoreceptors 54 . Whatever the alternative pathway of mitochondrial Ca 2+ uptake might be in photoreceptors, the low expression of MCU in photoreceptors raises the possibility that this alternative pathway could play a significant role in controlling photoreceptor mitochondrial Ca 2+ uptake.
Loss of MCU has very mild metabolic consequences for photoreceptors. Evidence continues to suggest that tissue-specific metabolic specialization causes the metabolic consequences of MCU loss to vary from to tissue. For example, the liver has the important role of synthesizing fatty acids for the body and can also store lipids. In mouse and zebrafish hepatocytes, loss of MCU disrupts lipid metabolism and leads to hepatic lipid accumulation due to delayed cytosolic Ca 2+ clearance and subsequent disrupted AMP-activated protein kinase dephosphorylation 47 . Conversely, in skeletal muscle, loss of MCU causes a metabolic shift towards increased lipid oxidation due to PDH inhibition restricting the availability of pyruvate-derived acetyl-CoA 17,27 . These examples suggest that the basal metabolic specialization of a tissue will determine in large part the metabolic consequences of MCU loss. Since photoreceptors rely heavily on aerobic glycolysis to generate ATP, it is not entirely surprising that Rod Mcu -/mouse retinas are metabolically quite normal and exhibit only a mild accumulation of α-ketoglutarate that does not appear to affect photoreceptor function (Fig. 5).
α-ketoglutarate participates in multiple mitochondrial and cytosolic reactions, making it difficult to pinpoint the exact reaction that is altered by loss of MCU in rods which leads to this accumulation. This is not the first time an accumulation of α-ketoglutarate has been observed to accompany MCU loss, as a similar accumulation of α-ketoglutarate occurs in Mcu -/fibroblasts. These fibroblasts have defects in glucose metabolism upstream of α-ketoglutarate, and the accumulated α-ketoglutarate was attributed to upregulated glutaminolysis 55 . However, Mcu -/photoreceptors exhibit no defects in glucose metabolism upstream of α-ketoglutarate and we see no evidence for altered synthesis of glutamate or glutamine in retinas supplied with U-13 C-glucose, which suggests a different mechanism is responsible.
An alternative possibility is that α-ketoglutarate levels increase after loss of MCU due to decreased Ca 2+ stimulation of α-KGDH. When α-KGDH is bound to Ca 2+ , its K m for α-ketoglutarate is lowered 3,56 . We previously observed that increased matrix Ca 2+ in zebrafish cone photoreceptors lowers the steady-state concentration of α-ketoglutarate, presumably because less substrate is needed to activate α-KGDH due to its lowered K m 12 . Since steady-state α-ketoglutarate levels increase in MCU-deficient retinas, it is reasonable to hypothesize that the opposite occurs: more substrate is needed to activate α-KGDH due to its increased K m when one route of Ca 2+ entry into the matrix (MCU) is shut down (Fig. 5). However, our measurements of matrix Ca 2+ show that the overall basal matrix Ca 2+ levels are unaltered in cone mitochondria lacking MCU (Fig. 2). We suggest the following possible explanations for elevated α-ketoglutarate levels in the absence of a detectable change in matrix Ca 2+ : 1. Matrix Ca 2+ levels are decreased in rods lacking MCU but not in cones from global mcu -/zebrafish. Genetically encoded, mitochondrially-targeted Ca 2+ -sensors with rod-specific expression do not exist in mice, and we are unable to selectively purify rod mitochondria from the rest of the inner retinal mitochondria, so we were unable to directly measure matrix Ca 2+ levels in rod photoreceptors. 2. Loss of MCU results in subtle changes in matrix Ca 2+ microdomains which influence α-KGDH activity but that we are unable to detect using our imaging methods 3. α-ketoglutarate levels are affected by an unknown, matrix Ca 2+ -independent process (such as altered malateaspartate shuttle activity or changes in another metabolic reaction involving α-ketoglutarate) Table 3. Dim flash response sensitivity and kinetics parameters. R max saturated response amplitude measured at the plateau, I 1/2 intensity required to produce half of the saturated response, S fD dark adapted sensitivity, t p time to peak of a dim flash response, t int integration time of the response, τ rec recovery time constant during response shut off.
R max (μV) I 1/2 (phot/µm 2 ) S fD (µV/phot/µm 2 ) t p (ms) T int (ms) τ rec (ms) www.nature.com/scientificreports/ Mcu -/rods exhibit a normal photoresponse with no change in the sensitivity and kinetics of the flash response and light adaptation (Fig. 6). However, mitochondrial Ca 2+ uptake via MCU is regulated cooperatively by several regulatory proteins [39][40][41][42] . So, we also investigated if MCU might play a role in modulating the photoresponse only when intracellular Ca 2+ levels are more rapidly changing, such as when photoreceptors are adapting to background light or during the subsequent recovery back to their dark adapted state. However, Mcu -/rods did not display a change in photoresponse parameters even while dark adapting following exposure to background light. This lack of phenotype may reflect the extremely low expression of MCU in mouse rods relative to the more dominant Ca 2+ clearance pathways such as NCKX1.
To determine if MCU might play a small role in clearing Ca 2+ that is difficult to detect in the presence of other Ca 2+ clearance pathways, we compared photoresponses of single knockout Nckx1 -/mice and double knockout Nckx1 -/-Rod Mcu -/mice. Nckx1 -/mice maintain a small photoresponse and are remarkably slow to degenerate, which indicates that they are able to clear a small amount of outer segment Ca 2+ through a not yet understood pathway 29 . We hypothesized that if the small photoresponse observed in Nckx1 -/mice were due to Ca 2+ sequestering via MCU, that this response would be ablated in the double knockout Nckx1 -/-Rod Mcu -/mice. However, the sensitivity, flash response kinetics and even light adaptation were unchanged in Nckx1 -/-Rod Mcu -/mice compared to Nckx1 -/controls, which indicates that mitochondrial Ca 2+ uptake via MCU does not contribute substantially to outer segment Ca 2+ clearance or photoresponse recovery (Fig. 7). It remains a possibility that mitochondrial Ca 2+ uptake through an alternative pathway contributes to the small photoresponse that is seen in Nckx1 -/-Rod Mcu -/mice. We did observe a reduction in the flash response amplitude in Nckx1 -/-Rod Mcu -/as compared to Nckx1 -/rods. The reason for this remains unclear. Although we confirmed that there was no obvious degeneration of rods, it is still possible that there is a subtle shortening of the rod outer segments that we were unable to detect in our sections. Overall, we find that MCU does not play a significant role in Ca 2+ feedback mechanisms during the photoresponse.
Maintaining intracellular Ca 2+ homeostasis is vital for many cell types, including photoreceptors. While most cells are thought to facilitate mitochondrial Ca 2+ uptake primarily through MCU, we find that both rod and cone photoreceptors have limited MCU expression. In the absence of MCU, cone mitochondrial Ca 2+ uptake is diminished but not entirely ablated. We found that this fraction of MCU-mediated mitochondrial Ca 2+ uptake has a very limited effect on modulating metabolism and no role in modulating the photoresponse. However, this does not mean that mitochondria do not play a role in modulating photoreceptor function by buffering intracellular Ca 2+ , as we find evidence for MCU-independent mitochondrial Ca 2+ uptake in photoreceptors. Overall, we find that MCU is surprisingly dispensable for photoreceptor function, possibly in favor of an alternative mitochondrial Ca 2+ uptake pathway. Animal care. At Washington University in St. Louis, mice were kept under a 12 h light/dark cycle and given free access to food and water. At the University of Washington, mice were housed in the UW Medicine SLU 3.1 vivarium, where they experienced a 6 AM-9 PM (fall-winter) and 7 AM-9 PM (spring-summer) light/dark cycle and had free access to food and water. In Figs. 4 and 5, "WT" animals are Mcu fl/fl iCre-negative littermates. In Fig. 6, "Control" animals are iCre-positive mice in which Mcu is not floxed. In Fig. 7, "Control" animals are Nckx1 -/single knockouts. In Figs. 5 and 6, the control and experimental mice were obtained from separate lines derived from common parents. iCre-75 mice were a gift from the lab of Ching Kang (Jason) Chen 32 . MCU fl mice (B6;129S-Mcu tm1.1Jmol /J) were obtained from Jackson Labs. The Nckx1 -/mice used in this study were generated as described in a previous publication 29 . Mice were maintained on a C57BL/6 J background. Rod Mcu -/mice used for histology were 6 and 11 months of age (6 months of age is shown in Fig. 1), mice used for metabolic analysis were between 5 and 9 months of age, mice used for ERG analysis were 6 to 8 weeks of age, and Nckx1 -/and Nckx1 -/-Rod Mcu -/mice used for histology were 6 to 8 weeks of age. Mice were genotyped to confirm the absence of the Rd8 mutation. Equal numbers of male and female mice were used in this study.

Methods
All fish used in this analysis were maintained in the University of Washington South Lake Union aquatics facility or the Centralized Zebrafish Animal Resource (CZAR) at the University of Utah at 27.5 °C on a 14/10 h light/dark cycle and were maintained in the Roy -/genetic background. All wild-type fish (WT) used in analysis were age-matched siblings to CRISPR-generated mcu -/zebrafish. Fish used for mito-GCaMP3 slice preparation were between 7 and 11 months of age, fish used for cyto-GCaMP3 slice preparation were 18 months of age, fish used for metabolic analysis were 11 months of age, fish used in ERG analysis were 7 months of age, and fish used for retinal morphology analysis were 11 months of age. Equal numbers of male and female fish were used.
Immunoblotting. Protein was extracted by homogenizing in RIPA buffer (150 mM NaCl, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0) and run on 14% polyacrylamide gels. After running, gels were transferred onto PVDF membranes (Millipore, IPFL00010) and briefly washed with PBS. Primary antibodies were diluted in blocking buffer (LI-COR, 927-40,000) and incubated overnight on blots at 4 °C. Membranes were washed twice with PBS containing 0.1% Tween-20 and once with PBS, then incubated with secondary antibodies diluted 1:5000 in blocking buffer for 1 h at RT and washed again before imaging. Membranes Mass spectrometry sample preparation. Metabolites were extracted from retinas using ice-cold 80% MeOH. 150 μL extraction buffer was added to each sample and tissue was disrupted by sonication. Samples were then spun at maximum speed, the supernatant transferred to a new tube, and the pellet saved for protein quantification. The supernatant was lyophilized at room-temp until dry. Extracted metabolites were derivatized using a two-step process: (1) 10 μL of 20 mg/mL Methoxyamine HCl (Sigma, Cat#: 226904) dissolved in pyridine (Sigma, Cat#: 270970) was added and samples were incubated at 37 °C for 90 min, then (2) 10 μL of tert-butyldimethylsilyl-N-methyltrifluoroacetamide (Sigma, Cat#: 394882) was added and samples were incubated at 70 °C for 90 min. Metabolites were analyzed on an Agilent 7890/5975C GC-MS using selected-ion monitoring methods described in previous work [7][8][9][10] . Peaks were manually integrated using MSD ChemStation software (Agilent), and correction for natural isotope abundance was performed using Isocor software 58 . Raw signals for each metabolite were converted to molar amounts using metabolite standard curves which were run alongside each experiment. Molar amounts were normalized to the total amount of protein (determined using a BCA assay) for each sample to determine the molar amount per μg of cellular protein. Live larval imaging of mito-GCaMP3. Larvae were imaged as described previously 12 . Larvae were maintained in embryo media containing 0.0003% 1-phenyl 2-thiourea (PTU, Sigma-Aldrich P7629) starting at 20 h postfertilization for confocal imaging. Larvae were analyzed at 6 days postfertilization (dpf) by embedding in 0.5% low melting point agarose containing embryo media with 0.02% (w/v) Tricaine (Sigma-Aldrich, E10521). The agarose was submerged in embryo media containing 0.0003% PTU and 0.02% (w/v) tricaine. Imaging was performed using an Olympus FV1000 with a 40 × water objective in conjunction with Olympus FluoView Scientific RepoRtS | (2020) 10:16041 | https://doi.org/10.1038/s41598-020-72708-x www.nature.com/scientificreports/ Mouse electrophysiology. Mice were dark adapted overnight prior to the day of experiment and were euthanized by CO 2 incubation. Eyes were enucleated under dim red light immediately after euthanasia followed by dissection under infrared illumination. The retinas were gently detached from posterior eye cups and were stored in dark in a dish containing oxygenated Ames medium at room temperature until recording. Recordings were conducted using previously described methods 61 . The retinas were mounted photoreceptors facing up in a closed chamber and were continuously superfused with oxygenated Ames medium (Sigma) at a flow rate of 3-5 mL/min. For isolating the a-wave of ERG, 50 μM DL-AP 4 (Tocris) and 100 μM BaCl 2 (Sigma) were included in the Ames medium. The recording chamber was maintained at 35-36 °C and retinas were allowed to adapt to the chamber temperature for at least 15 min before experiments. Ex-vivo transretinal ERG recordings were made in scotopic conditions by presenting light flashes produced by LEDs (Thor Labs). The ERG signals were amplified using a differential amplifier (Warner Instruments), low-pass filtered at 300 Hz (Krohn Hite Corp.), digitized using digidata 1440 (Molecular Devices), and were recorded at a sampling frequency of 10 kHz using pClamp 10 software.
Zebrafish electrophysiology. Zebrafish were approximately 7 months of age for all electrophysiology experiments. Zebrafish were briefly dark adapted (~ 30 min), before euthanasia by ice water immersion. Eyes were enucleated into Modified Salamander Ringer's solution (110 mM NaC, 2.5 mM KCl, 1.0 mM CaCl 2 , 1.6 mM MgCl 2 , 10.0 mM HEPES, 10.0 mM Glucose) with pH adjusted to 7.8 with NaOH. The eyes were hemisected and retinas isolated from the eyecup. All procedures after the dark adaptation were performed under dim red light. To ensure ex vivo ERG signal was predominantly cone responses, dark adaption was limited to ~ 30 min to allow cone photopigment regeneration but not provide enough time for full rod photopigment regeneration, and experiments were carried out during the day (between 11 AM and 4 PM) when rod contributions to retinal responses are at their lowest due to the circadian regulation of photoreceptor biology in the zebrafish retina. Ex vivo ERG recordings were performed as described previously 61,62 . Isolated retinas were mounted photoreceptor side up onto the specimen holder and perfused with Modified Salamander Ringer's solution, supplemented with 40 µM DL-AP4 (Tocris Bioscience) and 40 µM CNQX (Tocris Bioscience) to isolate the photoreceptor component of the ERG signal (a-wave). The rate of perfusion was ~ 5 mL/min and the experiments were conducted at room temperature (~ 23 °C). ERG signal was first amplified (100 ×) and low-pass filtered at 300 Hz by a differential amplifier (DP-311, Warner Instruments), and data was further amplified (10 ×) and acquired at 10KHz using an integrated amplifier/digitizer (IPA, Sutter Instrument, CA). A High Power LED light source (Solis-3C, Thorlabs, Newton, NJ), with filter for red light (630 nm, FWHM bandwidth 69 nm, FF01-630/69-25, Semrock, Rochester, NY) and LED driver (DC2200, Thorlabs) were used to provide the flashes of light stimuli, durations ranged from 5 to 100 ms. The SutterPatch software (SutterPatch v1.1.2, Sutter Instrument, CA) drove both stimulus generation and data acquisition via the IPA amplifier's analogue output and input, respectively. Light stimuli were calibrated before experiments using a calibrated photodiode (FDS100-CAL, Thorlabs, Newton, NJ) and flash intensities converted to photons/µm 2 . Data analysis, including statistical analysis and figure preparation, was performed with GraphPad v 8.0.0 (for Windows, GraphPad Software, San Diego CA, USA, www.graph pad.com). Normalized responses were calculated for each retina by dividing the response amplitude data by the maximal amplitude measured at the peak/plateau of the response to the brightest flash. To quantify the gain of phototransduction activation, we fitted the Lamb-Pugh model to the initial leading edge of the dim flash response for each retina, and compared the average amplification constant (A) between WT and mcu -/siblings 63 . www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.