Hisao Tsukamoto,Yoshihiro Kubo
A self-inactivating invertebrate opsin optically drives biased signaling toward Gβγ-dependent ion channel modulation
opsin,trimeric G protein,ion channel,optogenetics
Significance For optogenetics, animal opsins have been utilized to optically drive trimeric G protein-dependent signaling pathways. In comparison with channelrhodopsins, animal opsins show higher photosensitivity but cause more complicated responses, since they simultaneously drive Gα- and Gβγ-dependent signaling pathways. Thus, selective modulation of Gα-or Gβγ-signaling is necessary to precisely regulate cellular responses. We showed that an invertebrate opsin, Platynereis c-opsin1, having a self-inactivating property preferentially drives kinetically fast Gβγ-dependent ion channel modulation rather than Gα-dependent enzymatic responses. In addition, c-opsin1 can evoke photoresponses even under retinal-depleted conditions unlike vertebrate opsin. Furthermore, the Gβγ-biased signaling property of c-opsin1 was enhanced by fusion with RGS8 to accelerate G protein inactivation. Our findings pave the way for “Gβγ-biased” optogenetics.
A self-inactivating invertebrate opsin optically drives biased signaling toward Gβγ-dependent ion channel modulation

For optogenetics, animal opsins have been utilized to optically drive trimeric G protein-dependent signaling pathways. In comparison with channelrhodopsins, animal opsins show higher photosensitivity but cause more complicated responses, since they simultaneously drive Gα- and Gβγ-dependent signaling pathways. Thus, selective modulation of Gα-or Gβγ-signaling is necessary to precisely regulate cellular responses. We showed that an invertebrate opsin, Platynereis c-opsin1, having a self-inactivating property preferentially drives kinetically fast Gβγ-dependent ion channel modulation rather than Gα-dependent enzymatic responses. In addition, c-opsin1 can evoke photoresponses even under retinal-depleted conditions unlike vertebrate opsin. Furthermore, the Gβγ-biased signaling property of c-opsin1 was enhanced by fusion with RGS8 to accelerate G protein inactivation. Our findings pave the way for “Gβγ-biased” optogenetics.

Optical control tools are necessary for optogenetic studies, to regulate specific cellular responses in a light-dependent manner (1). In addition to channelrhodopsins, animal opsins, light-sensitive G protein-coupled receptors (GPCRs), are used as major optical control tools (2–4). Animal opsins drive G protein- and arrestin-dependent signaling pathways upon illumination. Among diversified opsins, Gi/o-coupled opsins are utilized as inhibitory tools via light-dependent Gi/oα-dependent inhibition of adenylyl cyclase and/or Gβγ-dependent modulation of G protein-activated inwardly rectifying potassium (GIRK) channels (Fig. 1A) as well as voltage-gated calcium channels (5–8). In particular, optical control of GIRK channels is valuable as the Gβγ-dependent activation of GIRK channels decreases or inhibits the cellular excitability. GIRK channels are involved in various physiological functions, such as opioid-induced analgesia and heartbeat regulation, and their dysregulation causes various diseases (9–11). Animal opsins exhibit much higher photosensitivity than channelrhodopsins, probably because of signal amplification via signaling cascades (6, 12, 13). Animal opsins have limitations as optical control tools. Most of the animal opsin-based optogenetic tools, such as opto-XRs, have been developed using vertebrate visual pigments such as rhodopsins (rod opsins) or cone opsins that are endogenously expressed in rod and cone photoreceptor cells in the retina, respectively (2–6). Vertebrate visual pigments selectively bind cis-retinal isomers that are scarce outside the eyes, and they are unidirectionally activated by light via cis to trans retinal isomerization (SI Appendix, Fig. S1A) (14, 15). Therefore, vertebrate visual pigments require continuous supply of cis-retinal molecules as in visual photoreceptor cells. Recent optogenetic studies have reported that bistable opsins can overcome the limitation (8, 16–18). Bistable opsins can bind trans- and cis- retinal isomers and are bidirectionally photointerconvertible between stable (cis-retinal bound) resting/dark and (trans-retinal bound) activated forms (SI Appendix, Fig. S1B) (3, 15, 19). Another disadvantage of animal opsins as optical control tools stems from the characteristics of GPCR signaling pathways. Activated GPCR promotes the dissociation of G protein α and βγ subunits, each of which drives its own downstream pathways. Thus, animal opsin-induced cellular responses tend to be complicated (20), but Gα- and Gβγ-dependent cellular responses do not seem to be independent due to the 1:1 stoichiometry of Gα and Gβγ subunits. In the case of opsin-mediated GIRK channel activation by Gβγ, the Gi/oα-dependent cAMP reduction is a sort of “side-effect” accompanied with the opsin activation. In this study, we aimed to selectively drive Gβγ-dependent ion channel modulation using opsins that transiently activate G proteins (Fig. 1B). We utilized the kinetic differences between major Gα- and Gβγ-dependent cellular responses for this purpose. Gβγ-mediated GIRK channel activation via the “membrane-delimited” process is much faster than Gα-mediated modulation of adenylyl cyclase regulating intracellular cAMP levels (Fig. 1A). Typically, Gβγ-induced GIRK activation occurs within a few seconds, whereas Gα-induced cAMP responses take several minutes (3, 10, 16, 21–25). This suggests that transient Gi/o activation can evoke significant and temporal GIRK activation, but not intracellular cAMP changes due to the insufficient temporal integration of G protein activity upon receptor activation. We previously reported that an invertebrate Gi/o-coupled opsin (Platynereis c-opsin1) (26) can be activated and deactivated by different colors of light like typical bistable opsins; however, the activated opsin is also spontaneously inactivated in a time-dependent manner (27) (Fig. 1C). This self-inactivating property (Fig. 1B) is suitable for the temporal activation of Gi/o. Based on these characteristics, we hypothesized that the self-inactivating c-opsin1 preferentially drives fast Gβγ-dependent GIRK channel responses rather than slow Gi/oα-dependent cAMP reduction (Fig. 1A). Therefore, we expected c-opsin1 to function as a Gβγ-biased optical control tool. We evaluated the light-dependent GIRK activation and adenylyl cyclase inhibition by c-opsin1 as well as other bistable and self-inactivating opsins. We found that c-opsin1 and a vertebrate visual pigment with self-inactivating properties predominantly drove Gβγ-dependent GIRK responses, rather than Gi/oα-dependent cAMP reduction, whereas a typical bistable opsin produced robust Gβγ- and Gα-dependent responses. Also, c-opsin1 required fewer retinal molecules to function than the vertebrate visual pigment. The Gβγ-biased nature of Platynereis c-opsin1 was enhanced by genetically combining with RGS8 to accelerate Gi/o inactivation. Taken together, we propose that the self-inactivating c-opsin1 and its fusion protein with RGS8 are suitable as optical control tools biased for kinetically fast Gβγ-dependent signaling, even in retinal-depleted conditions.

We previously reported that Platynereis c-opsin1 can activate GIRK channels via UV light-dependent Gi/o activation (27). The c-opsin1-induced GIRK current gradually decreased upon termination of the illumination or irradiation with visible light, indicating that the G protein-activating state of c-opsin1 is transiently formed upon UV absorption. The activated form was inactivated in a time-dependent (light-independent) manner, and deactivated by visible light illumination (Fig. 1 B and C). We expected the transient Gi/o activation to predominantly drive kinetically fast Gβγ-dependent GIRK activation, rather than the slow Gi/α-dependent cAMP reduction response (Fig. 1A and Introduction). To verify this, we quantified Gi/oα- and Gβγ-dependent cellular responses by c-opsin1 and other UV-sensitive opsins. We assessed Gi/oα-mediated adenylyl cyclase inhibition in mammalian cultured cells (COS-1 cells) using the GloSensor assay (28), and Gβγ-mediated GIRK activation in Xenopus oocytes using electrophysiological measurements (29, 30) (Materials and Methods). These cell-based analyses have been widely used to quantify Gi/oα- and Gβγ-mediated cellular responses by GPCRs including opsins, with high sensitivity and resolution. The Gi/oα- and Gβγ-dependent responses were compared between c-opsin1 and other UV-sensitive opsins, lamprey parapinopsin (LamPP), and mouse short-wavelength-sensitive opsin (SWO), which are commonly used as optogenetic tools (6, 12, 16, 18). LamPP is a typical bistable opsin (SI Appendix, Fig. S1B), and mouse SWO is a UV-sensitive vertebrate visual pigment (cone opsin, see SI Appendix, Fig. S1A).

In Xenopus oocytes, GIRK current was increased by c-opsin1 upon UV illumination, and the increased GIRK current decayed in a visible (yellow) light-dependent and time-dependent (light-independent) manner (Fig. 1C). The light-dependent and -independent current decays were caused by visible light-induced deactivation and self-inactivation of c-opsin1, respectively (Fig. 1B). From the recorded data, we calculated τoff (deact.) and τoff (inact.) values, which represent the decay kinetics of the GIRK currents due to visible light-induced deactivation and self-inactivation of c-opsin1, respectively (Fig. 1D). The respective τoff values are 13.4 ± 2.2 s [by light-induced deactivation, τoff (deact.)] and 122.5 ± 31.0 s [by self-inactivation, τoff (inact.)] (Fig. 1D). We previously reported that the photoproduct of c-opsin1 is spectroscopically stable for at least 25 min after UV absorption, indicating that the opsin holds the chromophore retinal (27). Therefore, the spontaneous inactivation of c-opsin1 was not caused by the (all-trans-) retinal release that occurs in vertebrate visual pigments (SI Appendix, Fig. S1A). The visible light-induced deactivation was reported to be caused by the photoisomerization of retinal from all-trans to 11-cis configurations (27) (Fig. 1B). In contrast to c-opsin1, a typical UV-sensitive bistable Gi/o-coupled opsin, LamPP (31), induced sustained GIRK activation upon UV illumination, and the GIRK current was turned off only by the visible light illumination (Fig. 1E), indicating that LamPP forms a stable G protein-activating state (SI Appendix, Fig. S1B). The sustained UV-induced GIRK activation by LamPP is consistent with the findings of previous optogenetic studies using LamPP (12, 16, 18, 32).

GloSensor assays of c-opsin1 and LamPP in COS-1 cells also demonstrated the transient nature of the G protein-activating state of c-opsin1 (Fig. 2). After 10 s of UV illumination, LamPP induced a larger decrease in intracellular cAMP levels (33) than c-opsin1 (Fig. 2A). The tiny cAMP reduction by c-opsin1 is consistent with the result of a previous study (34). Subsequent 1, 2, and 4 min of UV stimulations on c-opsin1 caused transient cAMP reductions, followed by recovery in several minutes, but the same UV stimulations on LamPP caused additional transient decreases in cAMP levels (Fig. 2A). The c-opsin1-induced transient cAMP responses can be explained by the temporal Gi/o activation (Fig. 1B). Owing to the transient nature of the G protein-activating state, short-term (10 s) UV illumination of c-opsin1 caused a tiny cAMP reduction, and long-term (several mins) continuous UV illuminations induced larger transient decreases in cAMP levels (Fig. 2A). In contrast, short-term UV illumination on LamPP led to the formation of the stable G protein-activating state that induced large and sustained cAMP reduction responses.

The K94T single mutation in the retinal binding site of c-opsin1, which causes a shift of absorption maximum from 383 to 491 nm (27), converted the opsin to produce sustained and larger cAMP responses upon UV or visible light illumination (Fig. 2 B and C). The results indicate that the mutation-induced spectral shift converts c-opsin1 so that it can be activated by visible and UV light. The K94T mutant cannot be deactivated by light due to overlapping absorption spectra of resting and activated forms (27). Interestingly, the K94T mutant evoked sustained GIRK current upon illumination (Fig. 2 D and E). These results show that the K94T mutation slows the self-inactivation of c-opsin1 (Fig. 2D), and increases cAMP responses (Fig. 2B), supporting that the limited cAMP responses by c-opsin1 WT are caused by its fast inactivation property.

We directly assessed the transient Gi/o activation by c-opsin1 using the NanoBiT G protein dissociation assay (35, 36). In this assay, the G protein subunits dissociation/association processes were monitored based on a luciferase (NanoLuc) luminescence. Fig. 3 shows that after UV illumination of c-opsin1 WT, dissociated Giα and Gβγ subunits rapidly (re-) associated; however, these subunits remained dissociated in the case of LamPP. These results are consistent with our data (Figs. 1 and 2) showing that the c-opsin1 transiently activate Gi/o upon UV illumination, whereas LamPP continuously activates Gi/o. Because of the dead-time (approximately 10 s) between illumination of the cells and luminescence measurement in our plate reader (Materials and Methods), we could not detect the maximal dissociation level of the Giα and Gβγ upon activation of the c-opsin1 WT. In addition, UV illuminated c-opsin1 K94T mutant caused a longer dissociation of Giα and Gβγ than WT (Fig. 3). These results are consistent with the differences in GIRK and cAMP responses induced by WT and the K94T mutant (Fig. 2 B–E).

The cAMP responses of c-opsin1 are consistent with our hypothesis that temporal Gi/o activation preferentially drives the fast Gβγ-dependent GIRK activation, rather than the slow Gi/oα-dependent reduction of cAMP levels. As photoactivated vertebrate visual pigments, particularly cone opsins, temporally activate G proteins and are spontaneously turned off by the retinal release (14, 37) (SI Appendix, Fig. S1A), we characterized mouse SWO, a typical vertebrate UV-sensitive visual pigment endogenously expressed in mouse short-wavelength-sensitive cone photoreceptors (6, 38). In Xenopus oocytes, mouse SWO activated GIRK channels in a UV-dependent manner, and the activated GIRK current decayed in a time-dependent manner (τoff = 18.9 ± 2.0 s) (Fig. 4 A and B). The GloSensor assay on the SWO showed that 10 s of UV irradiation caused a small transient cAMP reduction, and subsequent 1, 2, and 4 min of UV irradiations caused larger transient responses followed by recovery (Fig. 4C), similar to the case of c-opsin1 (Fig. 2A). The NanoBiT G protein dissociation assay on mouse SWO also revealed that mouse SWO, like Platynereis c-opsin1, induced transient Giαβγ dissociation upon UV illumination (Fig. 3). Our data indicate that both c-opsin1 and mouse SWO predominantly drive rapid GIRK activation via the temporal activation of Gi/o, although the mechanisms underlying the self-inactivating nature of these opsins are distinct from each other. Therefore, both Platynereis c-opsin1 and mouse SWO can act as optical control tools to preferentially drive Gβγ-dependent ion channel modulation.

Platynereis c-opsin1 and mouse SWO transiently activate Gi/o proteins. In contrast, c-opsin1 can directly bind cis- and trans-retinal isomers, unlike vertebrate visual pigments, including mouse SWO, and these retinal isomer-bound states are photointerconvertible (27) (Fig. 1B). The nonselective retinal-binding ability and photochemistry render c-opsin1 resistant to retinal depletion (Introduction). To verify this, we measured the light-induced cellular responses by c-opsin1 and mouse SWO under retinal-depleted conditions. We quantified the UV-induced GIRK current by c-opsin1 and mouse SWO in the absence or presence of exogenous 11-cis-retinal (1 μM) in Xenopus oocytes. Without exogenous retinal, both opsins only used the endogenous retinoids in oocytes. Platynereis c-opsin1 induced UV-dependent GIRK currents at similar levels, regardless of addition of exogenous retinal (Fig. 4 D, E, and H). In contrast, mouse SWO induced little GIRK current upon UV illumination in the absence of the exogenous retinal (Fig. 4 F–H). These results clearly support our hypothesis that c-opsin1 requires fewer exogenous retinal molecules to function. We also conducted the GloSensor assay with different concentrations of exogenous 11-cis-retinal (10 nM, 100 nM or 1 μM). A series of UV stimulations (10 s, 1 min, 2 min, and 4 min) were applied in this experiment, and the UV-dependent decreases in cAMP levels were compared between c-opsin1 and mouse SWO. Consistent with the electrophysiological data, the GloSensor assay data revealed that c-opsin1-induced reduction in cAMP levels was less affected by the amount of exogenous retinal (Fig. 4 I and K). Mouse SWO-induced cAMP responses drastically decreased in retinal-depleted conditions (Fig. 4J). Compared with retinal-abundant conditions, and in particular, responses to the last 4 min of UV illumination after 10 s, 1 min, and 2 min of UV irradiation were the most affected in retinal-depleted conditions (Fig. 4L). This is likely because mouse SWO, a typical vertebrate visual pigment, selectively binds cis-retinal isomers and releases isomerized (trans-) retinal after photoactivation (SI Appendix, Fig. S1A), and a series of activations by repeated and prolonged UV illumination consumes the available cis-retinal molecules. In contrast, c-opsin1 holds cis- and trans- retinal isomers, both of isomers are reversibly interconverted upon UV illumination (Fig. 1B). As c-opsin1 continuously uses the protein-bound retinal molecule, the opsin induces light-dependent responses even in a retinal-depleted environment, unlike vertebrate visual pigments including mouse SWO (Discussion).

As c-opsin1 can preferentially drive Gβγ-dependent GIRK activation (Figs. 1 and 2), we concluded that the Gβγ-biased signaling property of c-opsin1 is due to its self-inactivating nature. However, the opsin also exhibited some residual UV-dependent cAMP reduction (Fig. 2A). To minimize the residual Gi/oα-dependent responses by c-opsin1, we attempted to accelerate the turn-off of Gi/o signaling. RGS proteins accelerate GTP hydrolysis in trimeric G proteins and terminate G protein-mediated signal transduction (39). In particular, rat RGS8 efficiently accelerates both the turn-off as well as turn-on kinetics of GIRK activation by Gi/o-coupled receptors (30). Based on these insights, we engineered Platynereis c-opsin1 to be genetically fused with rat RGS8 at the C terminus of the opsin (Materials and Methods and SI Appendix, SI Data) to enhance the temporal activation of G proteins (Fig. 5A). cAMP responses typically occur over several minutes (Introduction and Fig. 2A). Thus, it is desirable to inactivate Gi/o signaling within several 10 s to minimize the residual Gi/oα-dependent responses. We expected that the fused protein to exhibit enhanced Gβγ-biased signaling properties. The c-opsin1/RGS8 fusion protein expressed in Xenopus oocytes exhibited faster turn-off of GIRK activation and smaller τoff (inact.) and τoff (deact.) values than those of c-opsin1 without RGS8 (Fig. 5 B–D). Upon termination of UV illumination, the fusion protein spontaneously stopped the GIRK activation with τoff (inact.) of 28.8 ± 9.9 s, and a visible light stimulation of the fusion protein quickly shut off the GIRK responses with τoff (deact.) of 2.9 ± 0.8 s (Fig. 5 C and D). These results suggest that the rate-limiting step in the decay of GIRK activation by c-opsin1 is the deactivation of G proteins via GTP hydrolysis. Notably, τoff (deact.) value of the c-opsin1/RGS8 fusion protein was lower than the τoff value of mouse SWO, whereas vertebrate cone opsins such as the SWO exhibited rapid self-inactivation via the retinal release (6, 14, 37) (Fig. 5C and SI Appendix, Fig. S1A). In other words, the c-opsin1/RGS8 fusion protein modulates GIRK channels faster than mouse SWO. The UV-induced activation and rapid visible light-induced deactivation of GIRK responses can be repeated, similarly to the function of intact c-opsin1 (Fig. 5 E and F). Electrophysiological experiments revealed that fusion with RGS8 enhances the characteristics of c-opsin1 to transiently and repeatedly activate Gi/o. The NanoBiT G protein dissociation assay on the c-opsin1/RGS8 fusion protein showed little UV-induced dissociation, with rapid diminishing of the signal (Fig. 5G). We speculate that during the approximately 10-s dead time between the UV illumination and luminescence measurement, the dissociated Gi trimer reassociated more rapidly in the case of the c-opsin1/RGS fusion than that of intact c-opsin1, leading to the decreased dissociation signal. We next performed the GloSensor assay on the c-opsin1/RGS8 fusion protein to confirm that the fusion with RGS8 minimizes residual Gα-dependent responses. Indeed, the c-opsin1/RGS8 fusion protein exhibited smaller residual cAMP reduction responses than the c-opsin1 WT (Fig. 5H). The fusion protein modulated GIRK channels in Xenopus oocytes in the absence of exogenous retinal, similar to the c-opsin1 WT (Fig. 4H), indicating that ligation with RGS8 does not interfere with the resistance of c-opsin1 to retinal depletion (Fig. 5 I–K). Taken together, the c-opsin1/RGS8 fusion protein is our best optical control tool biased for Gβγ-dependent ion channel modulation, with minimal residual activity of Gi/oα-dependent cAMP reduction responses (SI Appendix, SI Table).

In this study, we characterized Platynereis c-opsin1 as an optical control tool biased toward Gβγ-dependent ion channel responses, with small residual Gα-dependent enzymatic responses (Figs. 1 and 2). We showed that c-opsin1 can function in retinal-depleted environments (Fig. 4). Furthermore, we enhanced the Gβγ-biased property of c-opsin1 by fusion with RGS8 (Fig. 5). Here, we summarize the characteristics of c-opsin1-based optical control tools and their potential applications in optogenetics.

Platynereis c-opsin1 transiently forms G protein-activating state(s) followed by spontaneous (light-independent) inactivation (Figs. 1 C and D, 2A, and 3), although typical bistable opsins, such as LamPP (31), form stable G protein-activating states inducing sustained cellular responses (Figs. 1E, 2A, and 3). On the other hand, c-opsin1 can directly bind cis- and trans- retinal isomers, similar to typical bistable opsins, and the cis- and trans- retinal bound states are spectroscopically stable and photointerconvertible (27). Light-induced deactivation via trans to cis photoisomerization of retinal is faster than its (light-independent) self-inactivation (Figs. 1C and 5B). Based on these data, we summarized a potential scheme for the photoactivation, spontaneous inactivation, and light-dependent deactivation of c-opsin1 (Fig. 1B). These processes of c-opsin1 are remarkably different from those of typical bistable opsins with a stable G protein-activating form (SI Appendix, Fig. S1B) and vertebrate visual pigments, such as mouse SWO, involving trans-retinal release and cis-retinal binding (SI Appendix, Fig. S1A). Light-independent self-inactivation process in c-opsin1 is rather similar to the sequential transition from metarhodopsin-II (fully active) to metarhodopsin-III (partially active) in vertebrate visual pigments (40). Self-inactivating property of c-opsin is important to preferentially drive fast Gβγ-dependent GIRK response, rather than slow Gi/oα-dependent cAMP response. This is probably because upon a light-induced opsin activation, the amplitude of fast GIRK response depends on the momentary peak levels of G protein activity, whereas that of slow cAMP response depends on the time-integrated value of G protein activity over a longer time period. The light-dependent deactivation and time-dependent inactivation processes in c-opsin1 (Fig. 1B) can coordinately and effectively decrease the temporal integration of G protein activation. In particular, during illumination of activating UV light, some portion of c-opsin1 would be inactivated in a light-independent manner. These properties of c-opsin1 enable its suppression of the cAMP response. This is why self-inactivating c-opsin1 causes smaller cAMP response than bistable LamPP (Fig. 2A). The cAMP response is further decreased by the fusion of c-opsin1 with RGS8 (Fig. 5H). Light-independent self-inactivating property was also observed in another invertebrate Gi/o-coupled opsin, Anopheles Opn3 (41). In contrast to Platynereis c-opsin1, Anopheles Opn3 induces significant cAMP reduction responses (41), probably because its inactivation kinetics is much slower than that of Platynereis c-opsin1. Similarly, the K94T substitution in c-opsin1 slowed down the light-independent inactivation (Figs. 2 D and E and 3) leading to a larger light-dependent cAMP reduction than WT (Fig. 2 B and C). These results suggest that amino acid substitutions accelerating the self-inactivating kinetics enhance the Gβγ-biased signaling property of opsins.

As summarized in Figs. 4 H and K and 5K, Platynereis c-opsin1 and its fusion with RGS8 protein maintained their photosensitivity even under retinal-depleted conditions. This is probably because c-opsin1 possesses a nonselective binding ability for cis- and trans-retinal isomers and holds the retinal upon illumination, unlike vertebrate visual pigments such as mouse SWO (27) (Introduction). Notably, Anopheles Opn3 can directly bind 13-cis-retinal, which is easily generated from all-trans-retinal via thermal isomerization, and can function under retinal-depleted conditions, too (41). Taken together, the binding ability for all-trans and/or 13-cis retinal isomers and the continued use of the once-bound retinal molecules are beneficial for opsin to function in retinal-depleted environments. Many tissues/organs contain very limited quantities of retinal molecules. The pineal organ in mammals expresses visual pigments, but it is not photosensitive because of a lack of sufficient retinal supply (42, 43). Peripheral tissues, such as the outer ears and vibrissal pads of mice, cannot provide sufficient retinal molecules for endogenous opsin (Opn5) molecules ex vivo (44). In insects such as Drosophila, limited amounts of retinal are available outside the eyes (45). Although many neural cells in the mammalian brain contains sufficient retinal molecules for channelrhodopsins to function (1), exogeneous retinal molecules are used in some optogenetic studies (46, 47). Some invertebrate opsins, such as Platynereis c-opsin1 and Anopheles Opn3, can expand the applicability of optogenetic techniques to various tissues and organs containing limited retinal molecules. Future studies should identify other invertebrate opsins that can function in retinal-depleted conditions.

Gi/o-coupled bistable opsins are used as inhibitory optogenetic tools to evoke a sustained inhibitory GIRK current. In contrast, channelrhodopsin-based inhibitory tools require continuous illumination to generate a sustained inhibitory current (but see ref. 48). Self-inactivation of c-opsin1 seems to jeopardize the advantage, but here we proved that this self-inactivating property is rather beneficial for the selective control of Gβγ-dependent ion channel modulation. Vertebrate visual pigments including mouse SWO are used as optogenetic tools to drive Gi/o signaling pathways (5, 6). As c-opsin1 can be modulated using a single color (UV) light like mouse SWO, the two opsins would be applicable to optogenetics using a simple optical device with a single (UV) light source. Our data revealed that c-opsin1 and mouse SWO can preferentially drive Gβγ-dependent GIRK activation, and that c-opsin1 requires a smaller supply of retinal molecules than the SWO. Therefore, c-opsin1-based optical control tools are suitable for driving Gβγ-dependent ion channel responses in retinal-depleted tissues (SI Appendix, SI Table). As shown in Fig. 5, c-opsin1 fused with RGS8 enhanced the biased activity for Gβγ-dependent GIRK modulation by accelerating kinetics [smaller τoff (inact.) and τoff (deact.) values] to further decrease the time-integrated value of the G protein activity. The c-opsin1/RGS8 fusion protein maintained its resistance for retinal depletion. The c-opsin1/RGS8 fusion protein is our best optical control tool biased for Gβγ-dependent ion channel modulation with a minimal residual Gi/oα-dependent activity (SI Appendix, SI Table). Fusion with RGS proteins could change signaling properties of other animal opsins, helping development of valuable optical control tools.

Gi/o-coupled animal opsins are used as inhibitory optogenetic tools, and Gi-DREADD is used as an inhibitory chemogenetic tool (49). They suppress neural activities via Gi/oα-dependent cAMP reduction and/or Gβγ-dependent modulation of ion channels. To analyze some physiological functions, the Gα- and Gβγ-dependent responses should be separated. For example, opioids induce analgesia and cause various side effects linked to drug abuse, depending on the signaling pathways driven by Gi/o-coupled opioid receptors. Analgesia is mainly caused by Gβγ-dependent GIRK activation, and opioid withdrawal is linked to Gi/oα-dependent cAMP signaling pathways (50, 51). In addition, arrestin-dependent signaling pathways are responsible for various side effects of opioids related to the drug abuse (51, 52). G protein-biased agonists for opioid receptors have been developed in this context. Gβγ-biased drugs are desirable but not yet available. Our c-opsin1-based optical control tools have the potential to separate Gi/oα- and Gβγ-induced opioid effects. Due to the limited spatiotemporal resolution, selective activation of Gβγ-dependent pathways cannot be accomplished with chemogenetic tools or G protein-biased ligands for GPCRs. GIRK channels are targets of optogenetics in neural cells (7, 8) and in nonneural tissues such as cardiac atrial cells (46), and they are associated with various diseases such as Down syndrome, Parkinson’s disease, Keppen–Lubinsky syndrome, and epilepsy (9, 53). The c-opsin1-based optical control tools biased for Gβγ-dependent GIRK channel activation possess great potential in understanding various cellular functions and disease mechanisms in the future.

All animal experiments in this study were approved by the Animal Care Committee of the National Institutes of Natural Sciences (an umbrella institution of National Institute for Physiological Sciences, Japan), and were performed in accordance with its guidelines.

The cDNAs of Platynereis c-opsin1 and the K94T mutant with the coding sequence of the 1D4 tag (ETSQVAPA) on the C terminus were inserted into the EcoRI/NotI site in a mammalian expression vector pMT and into the EcoRI/HindIII site in the pGEMHE for expression in Xenopus oocyte (27). The plasmids of lamprey parapinopsin (LamPP) with the 1D4 tag in pCDNA3.1 (31), mouse UV-sensitive cone pigment (SWO) with the 1D4 tag in pCDNA3.1 (54) were kindly obtained from Drs. Akihisa Terakita and Mitsumasa Koyanagi (Osaka Metropolitan University), and Takahiro Yamashita (Kyoto University), respectively, and these cDNA were subcloned into the pMT and pGEMHE vectors. The plasmids of rat RGS8 in pGEMHE (30) were obtained from Osamu Saitoh (Nagahama Institute of Bio-Science and Technology), and the cDNA was fused with the sequence of Platynereis c-opsin1, a linker sequence (GGGSSGGG), and the 1D4 tag was added on the C terminus (SI Appendix, SI Data). The cDNA of c-opsin1/RGS8 fusion protein was inserted into the pMT and pGEMHE vectors. The cDNAs of Lg-BiT inserted mouse Giα2, human Gβ1, and the Sm-BiT fused human Gγ2 (C68S) were constructed according to Inoue et al. (35) and inserted into the pMT vector. The final amino acid sequences of the constructs are shown in SI Appendix, SI Data.

Xenopus oocytes were isolated from frogs as described previously (27, 29, 55). Briefly, Xenopus oocytes were surgically collected from frogs anesthetized in water containing 0.15% tricaine and treated with 2 mg/mL collagenase (Sigma-Aldrich) for 3 to 4 h to remove the follicular membrane. 5′-capped cRNA was prepared from the pGEMHE vector containing cDNA of opsin (see above) using an in vitro transcription kit (mMESSAGE mMACHINE Kit, Life Technologies). Typically, we injected the opsin cRNA (~500 pg/oocyte, but for mouse SWO, ~1.5 ng /oocyte) with rat GIRK1 and mouse GIRK2 cRNAs (~25 ng/oocyte and ~12.5 ng/oocyte, respectively) in 50 nL water/oocyte. The oocytes injected with cRNA were incubated in the standard frog Ringer solution (MBSH), a standard frog Ringer solution (88 mM NaCl, 1 mM KCl, 0.3 mM Ca(NO3)2, 0.41 mM CaCl2, 0.82 mM MgSO4, 2.4 mM NaHCO3, and 15 mM HEPES (pH 7.6) with 0.1% penicillin-streptomycin solution), at 17 °C in the dark chamber for 2 d.

We used a conventional two-electrode voltage-clamp technique (56, 57) to measure photoresponses caused by opsins. Before electrophysiological recording, oocytes injected with cRNAs were incubated in the standard frog Ringer solution (MBSH) containing 1 μM 11-cis-retinal (1/4,000 volume of 4 mM retinal in ethanol was added to MBSH) for ~1 h at 17 °C in the dark chamber to form photosensitive pigments. In Figs. 4 D and F and 5, 1/4,000 volume of ethanol solution instead of retinal solution was added to MBSH. All electrophysiological recordings were performed in a dark room, using only a dim red light which aids handling of oocytes and insertions of electrodes to the oocytes. The red light does not evoke any responses of opsins used in this study. Light-induced electrophysiological responses were recorded in a bath solution [96 mM KCl, 3 mM MgCl2, and 5 mM HEPES (pH 7.4)]. The tip resistance of the glass electrodes was 0.2 to 0.5 MΩ when filled with the pipette solution (3 M potassium acetate and 10 mM KCl). The increase in inward K+ current as a result of Gi/o activation by opsins was monitored by a two-electrode voltage-clamp technique using an OC-725C amplifier (Warner Instruments, Hamden, CT) at room temperature with continuous hyperpolarizing pulses of 0.2 s to −100 mV every 2 s from the holding potential of 0 mV and subsequent 0.2-s pluses of 40 mV. In Fig. 5B, hyperpolarizing pulses of 0.2 s to −100 mV every 1 s, because yellow-light-induced decrease in GIRK current was very fast and higher time resolution is required to precisely calculate τoff (deact.) value. Typically, after 15 repetitions of the hyperpolarizing pulses (~30 s), opsins were activated by illumination with UV light (395 nm LED, Sarspec, Portugal) (light intensity, ~0.7 mW/cm2) or blue light (470 nm LED, Sarspec) (light intensity, ~0.3 mW/cm2), and subsequently the opsin was deactivated by yellow (>480 nm) light (light intensity, ~30 mW/cm2; light source, 3-W LED light, OptoCode, Japan). Data acquisition was performed by a digital converter (Digidata 1440, Molecular Devices, Sunnyvale, CA) and pCLAMP 10 software (Molecular Devices).

Opsins, GloSensor assay sensor (coded by pGlo-22F), and NanoBiT-tagged G proteins were transiently expressed in COS-1 cells using polyethyleneimine as described previously (27, 58). For the GloSensor assay, each well of a 96-well assay plate (Corning, Kennebunk, ME) was transfected with 50 ng opsin plasmid, 50 ng pGlo-22F plasmid (Promega, Madison, WI), and 500 ng polyethyleneimine in 25 μL Opti-mem (Gibco, Waltham, MA) and 75 μL D-MEM (Wako, Japan) containing 10 % (v/v) FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin. For NanoBiT G protein dissociation assay, each well of a 96-well assay plate (Corning) was transfected with 50 ng opsin plasmid, 25 ng Lg-BiT inserted Giα2 plasmid, 125 ng Gβ1 plasmid, 125 ng Sm-BiT fused Gγ2 plasmid, and 500 ng polyethyleneimine in 25 μL Opti-mem (Gibco) and 75 μL D-MEM (Wako) containing 10 % (vol/vol) FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin.

The transfected COS-1 cells were incubated at 37 °C, 5 % CO2 for 2 d, and the medium was aspirated, followed by addition of HBSS (145 mM NaCl, 10 mM D-glucose, 5 mM KCl, 1 mM MgCl2, 1.7 mM CaCl2, 1.5 mM NaHCO3, 10 mM HEPES, pH 7.4) (59) containing 1 μM 11-cis-retinal, 2 % (vol/vol) GloSensor cAMP reagent stock solution, and 1 μM forskolin. In Fig. 4 I and J, the final exogenous retinal concentrations were changed to 100 nM or 10 nM. Then, the cells were incubated at room temperature for ~2 h to stabilize luminescence levels. Luminescence was measured using GM-2000 or GM-3510 microplate reader (Promega). Luminescence level of each well was measured every 30 s with integration time of 0.9 s. To illuminate opsins, luminescence measurement was interrupted, the plate was ejected, and the plate was illuminated by UV or blue light. UV and blue light source are 375 nm LED (OpotoCode) (light intensity, ~0.5 mW/cm2) and 500 nm light (Opto-spectrum generator L12194, Hamamatsu, Japan) (light intensity, ~0.5 mW/cm2), respectively. After illumination, luminescence measurement was resumed. The measured luminescence levels were normalized to the level at the starting point (time = 0 min).

The transfected COS-1 cells were incubated at 37 °C, 5 % CO2 for 1 d, and the medium was aspirated, followed by addition of HBSS containing 1 μM 11-cis-retinal and 5 μM coelenterazine h (Wako). Then, the cells were incubated at room temperature for ~2 h to stabilize luminescence levels. Luminescence was measured using a GM-2000 or GM-3510 microplate reader (Promega). Luminescence level of each well was measured every 6 s with integration time of 0.3 s. To illuminate opsins, luminescence measurement was interrupted, the plate was ejected, and the plate was illuminated by UV light (375 nm LED, OptoCode) (light intensity, ~0.5 mW/cm2). After illumination, luminescence measurement was resumed. The measured luminescence levels were normalized to the level at the starting point (time = 0 min).

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