XB-ART-60533
Nat Commun
2024 Jan 02;151:65. doi: 10.1038/s41467-023-44548-6.
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Hijacking of internal calcium dynamics by intracellularly residing viral rhodopsins.
Eria-Oliveira AS
,
Folacci M
,
Chassot AA
,
Fedou S
,
Thézé N
,
Zabelskii D
,
Alekseev A
,
Bamberg E
,
Gordeliy V
,
Sandoz G
,
Vivaudou M
.
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Rhodopsins are ubiquitous light-driven membrane proteins with diverse functions, including ion transport. Widely distributed, they are also coded in the genomes of giant viruses infecting phytoplankton where their function is not settled. Here, we examine the properties of OLPVR1 (Organic Lake Phycodnavirus Rhodopsin) and two other type 1 viral channelrhodopsins (VCR1s), and demonstrate that VCR1s accumulate exclusively intracellularly, and, upon illumination, induce calcium release from intracellular IP3-dependent stores. In vivo, this light-induced calcium release is sufficient to remote control muscle contraction in VCR1-expressing tadpoles. VCR1s natively confer light-induced Ca2+ release, suggesting a distinct mechanism for reshaping the response to light of virus-infected algae. The ability of VCR1s to photorelease calcium without altering plasma membrane electrical properties marks them as potential precursors for optogenetics tools, with potential applications in basic research and medicine.
???displayArticle.pubmedLink??? 38167346
???displayArticle.pmcLink??? PMC10761956
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???displayArticle.grants??? [+]
ANR-15-CE11-0029-01 Agence Nationale de la Recherche (French National Research Agency), ANR-19-CE11-0026 Agence Nationale de la Recherche (French National Research Agency), ANR-11-LABX-0015-01 Agence Nationale de la Recherche (French National Research Agency)
Species referenced: Xenopus laevis
Genes referenced: ano1 ano2 clca1.3 il6 papss1 rho
GO keywords: ion transport [+]
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Fig. 1: Photoactivation of OLPVR1 elicits CaCC currents in Xenopus oocytes. a Responses to a 10-s pulse of light of decreasing intensity. Current records were taken every 50 s from the same oocyte injected with 30 ng OLPVR1 RNA clamped at +40 mV. Bath solution was ND96. b Average peak current, normalized to current at 100% intensity (75 µW mm-2), vs. light intensity obtained with the protocol of a, applied to 4 oocytes (Error bars, SEM). c Photocurrents at different holding voltages from oocytes expressing OLPVR1 (30 ng RNA) or ChR2 (7.5 ng), in the specified different bath solutions. d OLPVR1 current-voltage relationships obtained from records as in c, in different extracellular ionic conditions. Control refers to currents recorded in non-injected oocytes. Currents were measured after 10-s illumination. (Error bars, SEM; n = 15, 11, 9, and 8 for 94 K+ 100 Cl-, 94 Na+ 100 Cl-, 94 K+ 10 Cl-, and control, respectively). e OLPVR1 photocurrents recorded before (Black) and after (Red) 60’ incubation in ND96 solution containing 30 µM Ani9 or MONNA, inhibitors of TMEM16A CaCCs. Statistics are shown in Supplementary Fig. 1. f Photocurrents from OLPVR1-expressing oocytes (7.5 ng RNA) with and without intracellular injection of BAPTA (BAPTAin) in ND96 bath solution. g Average peak photocurrent vs voltage in ND96 solution with and without injected BAPTA. (Error bars, SEM; n = 9 for ND96, n = 3 for +BAPTAin). Source data are provided as a Source Data file. | |
Fig. 2: OLPVR1 is expressed intracellularly and activates surface CaCCs through release of intracellular Ca2+. a Surface expression of HiBit-tagged OLPVR1HB compared to ChR2HB measured using XenoGlo technique. OLPVR1, in contrast with ChR2, is not expressed at the surface membrane of oocytes. Mean luminescence recorded in oocytes injected with 7.5 ng RNA coding for OLPVR1HB and ChR2HB before (blue) and after (red) membrane permeabilization. The OLPVR1 surface value is 2360 ± 700 RLUs. The numbers of oocytes batches are in parentheses (Error bars, SEM). **P = 0.007, ****P < 0.0001 two-way ANOVA with Sidak’s post hoc test (DF = 60). b Photocurrents elicited by successive 10-s illuminations separated by a 40-s dark interval. Oocytes coexpressing OLPVR1 (30 ng RNA) and Gq-coupled muscarinic M3 receptor (2.5 ng) were bathed in ND96 0Ca solution. c, d Between the 2 illuminations, activation of M3 by ACh (5 µM; 30 s) induced Ca2+ release and large transient CaCC currents. Panel d is an enlarged version of c, showing a drastic reduction of the second photocurrent. e Average ratios of OLPVR1 peak current induced by the second illumination (Peak 2) over that of the first (Peak 1) with and without ACh application in between. Numbers of oocytes are in parentheses (Error bars, SEM). f Photocurrents at +60 mV in ND96 solution from oocytes expressing OLPVR1 (7.5 ng RNA) before (Control) and after 10’ incubation with 10 µM YM-254890. g, h Representative photocurrents at +60 mV in ND96 (g) or 49 Ca2+(h) solution from oocytes expressing OLPVR1 (7.5 ng RNA) before (Control) and after 60’ incubation with 100 µM 2-APB. Statistics are shown in Supplementary Fig. 1. Source data are provided as a Source Data file. | |
Fig. 3: In mammalian cells, OLPVR1 localizes to the ER and activates surface Ca2+-activated channels through release of intracellular Ca2+. a Confocal images of HEK293T cells cotransfected with plasmids for expression of the ER-marker DsRed2-ER (red) and GFP-fused OLPVR1 (green). Merge panel shows in yellow, overlapping ER (red) and OLPVR1 (green) signals. The nucleus is in blue and the plasma membrane in magenta. Image is representative of 18 cells from 3 independent transfections. b-d Whole-cell recordings were obtained from HEK293T cells transfected with OLPVR1 alone (b; n = 7) or with OLPVR1 and, either TMEM16A (c; n = 5) or SK1 (d; n = 8). Left panels: Current responses to illumination (green bars, 505 nm light). Cells were held at the indicated voltages. Right panels: Light-induced current (peak current elicited by first illumination – current before illumination) vs. voltage (Error bars, SEM). Currents were elicited by 400-ms voltage ramps from −100 to +100 mV repeated every second. The pipette solution had 10 µM EGTA, except for the current-voltage curve drawn in red (right panel of d) where it had 1 mM EGTA (n = 7). Source data are provided as a Source Data file. | |
Fig. 4: A fused Calcium sensor shows OLPVR1-delimited Ca2+ influx from the ER. a Schematic representation of the calcium-imaging conditions tested. HEK293T cells were transfected with plasmids encoding for wild-type OLPVR1 and GCaMP6s (1&2), the fusion construct OLPVR1-GCaMP6s (3&4) or loss-of-function OLPVR1(K204Q)-GCaMP6s (5) and imaged with an epifluorescence microscope under 470-nm light in the absence (1,3&5) or presence (2&5) of 15 µM BAPTA-AM. b Time courses of fluorescence changes upon light application in the conditions described in a. c Confocal images of cells transfected with a plasmid coding for OLPVR1-GCaMP6s after 2 h incubation in 15 µM BAPTA-AM at T = 0, 15 and 30 s after light application. T = 0 is the first frame acquired after light is turned on. d Average maximal fluorescence changes upon light application of cells from at least 3 independent transfections in the conditions shown in a. Numbers of cells are in parentheses. (Error bars, SEM) e Half times of rise in fluorescence during illumination for conditions 1, 3 and 4. Numbers of cells are in parentheses (Error bars, SEM). **** p < 0.0001, one-way ANOVA, Tukey’s multiple comparisons test, FD (DFn 4, DFd 250) = 71.2; FE (DFn 2, DFd 143) = 68.8. Source data are provided as a Source Data file. | |
Supplementary Figure 1. Comparison of the effects of inhibitors on OLPVR1 photocurrents and M3- mediated currents. a Representative currents elicited by 5 µM ACh in oocytes injected with 2.5 ng RNA coding for the Gq-coupled muscarinic M3 receptor. Bath solution = ND96, voltage = +40 mV. Oocytes were incubated in ND96 solution containing no inhibitor (Control), 30 µM Ani9, 30 µM MONNA, 100 µM 2-APB, or 10 µM YM-254890. Duration of incubation was 10’ for YM-254890, 60’ otherwise. BAPTA designates oocytes injected with 50 nl of a 40-mM BAPTA solution and left 60’ in ND96 solution prior to recording. The expected intracellular BAPTA concentration of BAPTA-injected oocytes is 3-4 mM for oocytes of diameter 1-1.1 mm. b Normalized average currents measured in absence (Control) and in presence of the specified inhibitors in the same conditions as panel a for M3 and OLPVR1 injected oocytes. For M3, all compounds caused a significant inhibition (Student t-test; p<1x10-7 ). For OLPVR1, Ani9, MONNA, and BAPTA caused a significant inhibition (p<5x10-4 ), YM-254890 had no effect (p=0.64), and 2-APB had intermediate effect (p=0.012). Numbers of oocytes tested are in parentheses. The value of 1 corresponds to 9.95 µA for M3, and 0.53 µA for OLPVR1. c Estimates of inhibition of M3-induced currents in the conditions of panel a (M3; Black bars) and of OLPVR1 photocurrents measured at +40 mV in ND96 solution (OLPVR1; Blue bars) calculated from the data in panel b. Values, specified in or above the bars, are calculated using solely the mean currents in Control and those in the presence of the indicated compounds. Tests in other solutions and/or at different voltages gave similar results. Source data are provided as a Source Data file. | |
Supplementary Figure 2. Unlike ChR2, OLPVR1 photocurrents are abrogated by intracellular injection of BAPTA. a Representative photocurrents from OLPVR1-expressing oocytes (7.5 ng RNA) with and without intracellular injection of BAPTA (BAPTAin) in ND96 bath solution (which includes 1.8 mM Ca2+). b Same in 49 Ca2+ bath solution. c Average peak photocurrent vs voltage in ND96 solution with (n=3) and without (n=9) injected BAPTA. d Same in 49 Ca2+ bath solution with (n=4) and without (n=5) injected BAPTA. e, f Same as panels a and b, but with an oocyte expressing ChR2 (7.5 ng RNA). All traces are from the same oocyte. g, h Same as panels c and d, but with ChR2 in ND96 solution with (n=5) and without (n=4) injected BAPTA, and in 49 Ca2+ bath solution with (n=5) and without (n=4) injected BAPTA. (Error bars, SEM). Source data are provided as a Source Data file. | |
Supplementary Figure 3. OLPVR1 photocurrents activates slowly upon illumination. a Representative currents elicited by illumination of oocytes expressing OLPVR1 (blue trace) or ChR2 (black trace), or by application of 5 µM ACh to an oocyte expressing M3 receptors (brown trace; break in the trace represents 22 s of omitted data). Superimposed traces were scaled vertically to the same size, vertical scale bars for each trace are indicated. Bath solution = ND96 solution, voltage = +40 mV. b Average half-times of current activation upon light or ACh application, and deactivation upon light switch-off. Conditions were as in panel a; one data point/oocyte. The values for M3 were calculated as the time interval between the onset of the increase in current after ACh application and the time where current reached 50% of its peak value. They therefore do not include the latency inherent to bath application of agonists to oocytes. The time resolution of the recordings is ~50 ms so that the measured activation and deactivation T½ of ChR2 of 65±6 and 135±9 ms, respectively, are upper estimates of the actual values. Numbers of oocytes are in parentheses (Error bars, SEM). Source data are provided as a Source Data file. | |
Supplementary Figure 4. HiBit tag does not modify the function of rhodopsins. Representative photocurrents of HiBit-tagged OLPVR1 (OLPVR1HB; 30 ng RNA/oocyte) and ChR2 (ChR2HB; 7.5 ng RNA/oocyte) in ND96 solution. Such currents were recorded in 7 (OLPVR1) and 3 (ChR2) oocytes. | |
Supplementary Figure 5. OLPVR1 activates surface Ca2+ -activated TMEM16B channels in mammalian cells through release of intracellular calcium. Data were obtained from HEK293T cells transfected with OLPVR1 alone (a) or with OLPVR1 and TMEM16B (b). Left panels : Representative current responses to illumination (green bars, 505 nm, 10 s). Cells were held at the indicated voltages. Right panels: Light-induced current (peak current elicited by first illumination – current before illumination) vs. voltage. Currents were elicited by voltage ramps from -100 to +100 mV (400 ms duration) repeated every second. n=7 for panel a, n=6 for panel b (Error bars, SEM). The bath solution contained (in mM): 150 NaCl, 5 KCl, 2 CaCl2, and 10 HEPES (pH 7.4). The pipette solution contained (in mM): 155 KCl, 3 MgCl2, 10 HEPES (pH 7.3) and 10 µM EGTA. Source data are provided as a Source Data file. | |
Supplementary Figure 6. Surface expression of various OLPVR1 constructs. Mean luminescence (left: surface values, right: values after lysis) recorded in control conditions (no oocyte and non-injected oocytes) or from RNA-injected oocytes. Luminescence values were not blank corrected and averages were calculated from all oocytes tested. Amounts of RNA coding for specified proteins, in ng per oocyte, are indicated in parentheses. HB suffix indicated the presence of a HiBiT tag at the N-terminus. Oocytes were maintained in ND96 solution supplemented with 1 µM all-trans-retinal and tested 24-72h after injection. Increasing the amount of OLPVR1 RNA injected per oocyte from 7.5 to 30 ng did not affect surface expression. Modifying the protein by inserting signal sequences did not improve surface expression (MT, Golgi export trafficking signal of Kir2.1 potassium channel; SS, Signal sequence of human nicotinic acetylcholine alpha 7 receptor subunit; IL6, Interleukin 6 secretion signal sequence). Coexpressing OLPVR1 with viral rhodopsin OLPVR2 did not improve surface expression. However, the fusion DOR=OLPVR1 (30 ng) had a surface expression 15-fold higher than OLPVR1 (30 ng), but still 25-fold lower than DOR alone (DOR, -opioid receptor). **** P<0.0001; ns (not significant), P>0.9999; one-way ANOVA followed by Dunnett’s multiple comparison test against the control OLPVR1 (7.5 ng). Numbers of oocytes are in parentheses (Error bars, SEM). Source data are provided as a Source Data file. | |
Supplementary Figure 7. GCaMP6s and OLPVR1-GCaMP6s have distinct cell expression patterns. a, b Confocal images of HEK293T cells transfected with plasmids for expression of (a) GCaMP6s and wtOLPVR1, (b) OLPVR1-GCaMP6s, and (c) OLPVR1(K204Q)-GCaMP6s incubated with 30 µM ionomycin. d Time course of fluorescence change from a OLPVR1(K204Q)-GCaMP6s–transfected cell upon application of 30 µM ionomycin. Images are representative of 12 cells from 2 independent transfections. e Average change in fluorescence of OLPVR1(K204Q)-GCaMP6s–transfected cells after application of 30 µM ionomycin. Results are pooled from 2 independent transfections. Number of experiments is in parentheses (Error bars, SEM). Blue point indicates the value from trace in d. GCamP6s has a cytoplasmic distribution while OLPVR1-GCaMP6s expresses at the ER. The K204Q mutation eliminates the formation of the Schiff base between lysine K204 and retinal, impeding the function of the rhodopsin. The mutation did not change the expression pattern of OLPVR1-GCaMP6s or the function of the GCaMP6s sensor, which still displayed fluorescence upon the increase of intracellular Ca2+ elicited by ionomycin. Source data are provided as a Source Data file. | |
Supplementary Figure 8. BAPTA-AM chelates Ca2+ released from neighboring proteins. a Schematic representation of the calcium-imaging conditions tested. HEK293T cells were transfected with OLPVR1(K204Q)-GCaMP6s, alone (1) or together with wild-type OLPVR1, and imaged in an epifluorescence microscope under 470-nm light in the absence (2) or presence (3) of 15 µM BAPTA-AM. b Time courses of fluorescence changes upon light application of cells corresponding to the conditions in a. c Average maximal fluorescence changes upon light application of cells from at least 3 independent transfections corresponding to the conditions in a. Number of experiments is in parentheses (Error bars, SEM). One-way ANOVA, Dunnett’s multiple comparisons test **** p<0.0001, F (DFn 2, DFd 120) = 153. The K204Q mutation eliminates OLPVR1 light sensitivity without altering its expression (Fig. S7). Incubation of the cells with 15 µM BAPTA-AM for 2h eliminated the fluorescence signal due to Ca2+ influx from nearby OLPVR1 proteins, demonstrating that the conditions tested allow to identify the site of Ca2+ entry from the ER, i.e., whether Ca2+ is released by nearby proteins (chelated by BAPTA) or directly through the protein attached to the calcium sensor (not chelated by BAPTA). Source data are provided as a Source Data file. | |
Supplementary Figure 9. Photo-induced currents of members of the VR1 family have a similar profile which is distinct from that of ChR2. a Representative recordings in 94 K+ 100 Cl- solution from oocytes expressing, from left to right, ChR2 (7.5 ng RNA), and the viral rhodopsins OLPVR1, TARA150 (construct SS-TARA150), and VirChR1 (construct SS-VirChR1-MT) (30 ng). b Current-voltage relationships of currents measured after 10 seconds of illumination, in KCl solution. (n=10 for OLPVR1, n=3 otherwise; Error bars, SEM). c Representative whole-cell patch clamp recordings from a HEK293T transfected with HA-VirChR1-MT. Standard bath and pipette solutions. d Idem from a cell co-transfected with VirChR1 (construct HA-VirChR1-MT) and TMEM16A. Source data are provided as a Source Data file. | |
Supplementary Figure 10. ChR2ER localizes at the ER where it induces a change in voltage upon illumination, but does not increase intracellular Ca2+ enough to activate TMEM16A channels. a Confocal images of HEK293T cells transfected with plasmids for expression of ChR2ER fused to a fluorescent protein (green). The nucleus is in blue and the plasma membrane in magenta. Image is representative of 12 cells from two independent transfections. b Average maximal fluorescence changes of cells transfected with fluorescent voltage sensor ASAP3ER alone or with ChR2ER upon 470 nm-light application. Numbers of experiments are in parentheses (Error bars, SEM). Results are pooled from 3 independent transfections. Two-tailed t-test with Welch’s correction (t=2.7, df=33.5, p=0.011), Variance F test (F=41.9, DFn=32, Dfd=33, p=<0.0001). c Representative whole-cell patch clamp recordings from a HEK293T co-transfected with ChR2ER and TMEM16A under illumination with different wavelengths. Standard bath and pipette solutions (10 µM EGTA). Dashed lines represent the baseline at 0 current. d Average current induced by 470-nm light measured at +40 mV as in panel c. Results are pooled from 2 independent transfections. Number of experiments is in parentheses (Error bars, SEM). Source data are provided as a Source Data file. |
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