Hijacking of internal calcium dynamics by intracellularly residing viral rhodopsins

Abstract

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.

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 (BAPTA_in) 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 +BAPTA_in). 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 OLPVR1_HB compared to ChR2_HB 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 OLPVR1_HB and ChR2_HB 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, F_D (DFn 4, _DFd 250) = 71.2; F_E (DFn 2, DFd 143) = 68.8. Source data are provided as a Source Data file.

Fig. 5. Green light induces motion of Xenopus tadpoles expressing OLPVR1.

a Tadpoles from 3 independent batches of mRNA-injected embryos (Control injected with 250 pg lacZ mRNA, and OLPVR1 injected with 1 ng OLPVR1 mRNA) were subjected to 2-200-s green-light pulses and observed for light-triggered behaviour. No lacZ tadpole (n = 31) demonstrated green-light response. 51.4% of OLPVR1-injected tadpoles (n = 35) displayed green-light-dependent motion. b OLPVR1-injected light-responsive tadpoles from 1 batch (n = 5) were subjected to 0.5 mM of tubocurarine and 0.02% MS-222. 80% of tadpoles retained light-responsiveness as seen in middle panel of c. c Images of tadpoles 3-4 days after injection of embryos with LacZ (Control) or OLPVR1 mRNA before (Dark) and during illumination. d Light-induced responses of a single OLPVR1-expressing tadpole (n = 40 illuminations) were variable and consisted of a combination of tail flicking as in c, swimming or post-illumination body twitching (see Supplementary Note 3 & Supplementary Movies 1-5). OLPVR1-expressing tadpoles subjected to tubocurarine and MS-222 only showed tail flicking. e Delays of onset after light application, and offset after light switch-off, of behavioural response of OLPVR1 tadpoles. Numbers of observations are in parentheses (Error bars, SEM). Source data are provided as a Source Data file.