Pharmaco-Optogenetic Targeting of TRPC Activity Allows for Precise Control Over Mast Cell NFAT Signaling

Canonical transient receptor potential (TRPC) channels are considered as elements of the immune cell Ca2+ handling machinery. We therefore hypothesized that TRPC photopharmacology may enable uniquely specific modulation of immune responses. Utilizing a recently established TRPC3/6/7 selective, photochromic benzimidazole agonist OptoBI-1, we set out to test this concept for mast cell NFAT signaling. RBL-2H3 mast cells were found to express TRPC3 and TRPC7 mRNA but lacked appreciable Ca2+/NFAT signaling in response to OptoBI-1 photocycling. Genetic modification of the cells by introduction of single recombinant TRPC isoforms revealed that exclusively TRPC6 expression generated OptoBI-1 sensitivity suitable for opto-chemical control of NFAT1 activity. Expression of any of three benzimidazole-sensitive TRPC isoforms (TRPC3/6/7) reconstituted plasma membrane TRPC conductances in RBL cells, and expression of TRPC6 or TRPC7 enabled light-mediated generation of temporally defined Ca2+ signaling patterns. Nonetheless, only cells overexpressing TRPC6 retained essentially low basal levels of NFAT activity and displayed rapid and efficient NFAT nuclear translocation upon OptoBI-1 photocycling. Hence, genetic modification of the mast cells’ TRPC expression pattern by the introduction of TRPC6 enables highly specific opto-chemical control over Ca2+ transcription coupling in these immune cells.


INTRODUCTION
Photopharmacology and optogenetics have emerged as experimental strategies that allow for exceptionally precise interference with tissue functions (1,2). These technologies have proven particularly successful in elucidating basic principles of communication within complex signaling networks and are suggested as a prospective basis for light-mediated computer-cell/tissue interfaces in the context of synthetic biology (3). So far optogenetic and chemo-optogenetic have unequivocally made significant contributions to our understanding of neuronal circuits and provided important insights into the complex orchestration of immune reactions. Moreover, the feasibility of optogenetic manipulation of immune responses has repeatedly been demonstrated in a therapeutic context (4)(5)(6)(7).
We have recently developed a new photopharmacological tool that allows for specific, light-assisted control over TRPC3/6/7 conductances in native tissues (8). Here we set out to test the suitability of this new approach for modulation of immune responses, using the well-characterized RBL-2H3 mast cell model. Mast cells are tissue-resident and confer innate and adaptive immune reactions, thereby playing a pivotal role in allergic disorders, cancer, and autoimmune diseases (9). Mast cells mediate IgE-dependent allergic reactions by release of inflammatory mediators via degranulation, production of inflammatory lipids, and production of cytokines (10) and have long been recognized as a critical component of the tumor microenvironment in a number of cancer types (11)(12)(13). Based on the complex and essentially two-faced function of these tumor resident immune cells (12), therapeutic targeting of these cells requires uniquely specific approaches. High precision, local modulation of tumor-resident immune cells might represent a novel strategy for adjuvant immunotherapy in cancers (13). One approach to achieve sufficient specificity of immunomodulation is based on the finding that Ca 2+ downstream signaling is strictly dependent on temporal signaling features (14,15). Hence, the controlled sculpturing of immune cell Ca 2+ signals is expected to enable control over immune responses in a uniquely specific manner. So far, the therapeutic modulation of immune cell functions by light has focused mainly on the major player in immune cell Ca 2+ handling, the STIM/Orai Ca 2+ entry complex (7). Nonetheless, other Ca 2+ signaling elements may similarly serve as suitable targets for optical approaches. Although many other plasma membrane transporters and channels, including K + channels and TRP channels are reportedly critical for immune cell activation (16)(17)(18), these molecular players have so far not been considered and tested as targets.
For canonical transient receptor potential (TRPC) channels a contribution to Ca 2+ signaling in immune cells has repeatedly been suggested (19). While several studies provide evidence for a contribution of TRPC1 and TRPC5 proteins to storeoperated calcium signaling in rat (RBL-2H3) and mouse bone marrow-derived mast cells (BMMC), the exact function of TRPC isoforms in mast cells remains largely elusive (20)(21)(22). Interestingly, TRPC3/6/7 protein complexes were found to interact with fyn kinase during FcϵRI-mediated mast cell activation (23). However, a more recent study argues against the contribution of TRPC3/6 channels to FcϵRI-mediated Ca 2+ signaling in primary human lung mast cells as well as in LAD2 human mast cells, suggesting exclusively Orai but not TRPC may be considered as a target for the control of mast cells in allergic disease (24). Nonetheless, in clear contrast to a lack of linkage to FcϵRI stimulation, TRPC1, TRPC4, and TRPC6 proteins have been shown to confer downstream signaling in Mrgprb2-mediated mast cell activation of murine peritoneal mast cells (25). Thus, the efficacy and specificity of TRPC generated Ca 2+ signals in terms of their coupling to downstream effectors might be strictly dependent on the mechanism and mode of activation.
In the present study, we explored the functional consequences of direct, lipid-independent control of TRPC3/6/7 conductances in RBL-2H3 mast cells by a new photochromic benzimidazole agonist (OptoBI-1). We report that overexpression of the TRPC6 isoform, and thus specific modification of the TRPC expression pattern of RBL-2H3 cells, allows for efficient and temporally precise control over NFAT1 signaling by light. Our results provide the first proof of concept for efficient chemo-genetic targeting of mast cell Ca 2+ signaling and transcriptional regulation based on TRPC photopharmacology.

RNA Isolation and Quantitative Real-Time PCR
RNA was isolated from cell lysates using EXTRACTME TOTAL RNA KIT with 1% ß-Mercaptoethanol (Blirt) and reverse transcribed using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA) in a thermal cycler (Bio-Rad) according to the manufacturer's protocol. qPCR was performed using GoTaq ® qPCR and RT-qPCR Mix (Promega) in a LightCycler C1000 Thermal Touch, Thermal Cycler CFX96 ™ Real Time System (Bio Rad). Relative expression of the target gene was normalized to rat ß-Actin as a reference gene. Specific Primers used for rat Trpc genes are listed below.

NFAT Nuclear Translocation
Translocation of NFAT in mCherry-NFAT1 used as a control and YFP-TRPC3, YFP-TRPC6 and TRPC7-CFP overexpressed in RBL-2H3 cells was observed using an inverted microscope (Olympus IX71, Germany) equipped with a 40 × 1.3 NA oil immersion objective. During the recordings using Live Acquisition v2.6 software (TILL Photonics FEI Company, Gräfelfing Germany), the excitation of mCherry was achieved using 577/25 nm filter and fluorescent images were captured every 2 s at 632 nm (using 632/60 nm emission filter Chroma Technology, VT, USA) with an ORCA-05G digital CCD camera (Hamamatsu, Herrsching am Ammersee, Germany). ImageJ 1.51n software was used to measure the fluorescence intensity in the nucleus and cytoplasm before and after stimulation with 10 µM OptoBI-1. These values (nucleus/cytosol) were then plotted using the SigmaPlot 14.1 software (Systat Software Inc.

Statistical Analysis
Data analyses and graphical display were performed using Clampfit 11 (Axon Instruments) and SigmaPlot 14.1 (Systat Software Inc.). Data are presented as mean values ± S.E.M. Primarily, a Shapiro-Wilk test was conducted to test for normality of the value distribution. Whenever a normal distribution criterion was met, we used ANOVA to analyze the statistical significance. In general, differences were considered significant at p < 0.05 and indicated for individual comparisons in figures (* p < 0.05, ** p < 0.01, *** p < 0.001).

RESULTS
Benzimidazole (OptoBI-1)-Mediated Control Over RBL-2H3 Mast Cell Ca 2+ Signaling Requires Expression of Recombinant TRPC6 and TRPC7 Mammalian mast cells have been shown to express an array of TRPC gene products (27). Our initial experiments to explore the sensitivity of mast cell Ca 2+ signaling to a new photochromic TRPC ligand (OptoBI-1, Figure 1), clearly indicated that endogenous expression of benzimidazole TRPC target channels (8) is below the threshold for effective photopharmacological intervention in RBL-2H3 mast cells. Nonetheless, our analysis of the expression profile for TRPC subtypes in RBL-2H3 cells revealed that besides TRPC1 and TRPC4 also two potential benzimidazole targets, i.e. TRPC3 and TRPC7, were expressed at the mRNA level, while TRPC6 expression was not detectable (Supplementary Figure 1). Consistent with the lack of Ca 2+ signals generated in native RBL cells by OptoBI-1 photocycling (Figure 1; control), we failed to detect TRPC3 by immunoblotting. Unfortunately, a suitable antibody for TRPC7 detection is currently not available. Next, we attempted to reconstitute OptoBI-1 sensitive cation conductances in RBL-2H3 cells by overexpression of a single benzimidazole responsive TRPC proteins (TRPC3/6/7). Mast cells were genetically modified to express single OptoBI-1 target channels in combination with R-GECO as a reporter for cytosolic Ca 2+ changes (8,26). The overexpression of single TRPC proteins allowed us to investigate the consequences of photoactivation for reconstitution of each TRPC channel subtype in our mast cell model. Genetically modified RBL-2H3 cells were exposed to photocycling of OptoBI-1 to induce defined pattern of transient rises in cytosolic Ca 2+ as shown in Figure 1. These changes were modest in cells overexpressing TRPC3 but profound in cells expressing TRPC6 or TRPC7. OptoBI-1induced peak values of normalized R-GECO fluorescence in TRPC6 (3.29 ± 0.26; n = 23) and TRPC7 (4.4 ± 0.56; n = 21) overexpressing RBL cells remain below the maximum SOCEmediated signals achieved by thapsigargin (1 µM, 6.2 ± 0.14; n = 7), given as a reference stimulus. Of note, both "on" and "off" kinetic of light-controlled Ca 2+ changes were essentially fast allowing for precise control over signal frequency and duration. For TRPC6 the reversal of cellular Ca 2+ levels upon channel deactivation was incomplete and showed a tendency to remain elevated above control levels between consecutive photocycles. Importantly, TRPC6 as well as TRPC7 expression enabled the light-mediated generation of temporally defined Ca 2+ signaling pattern, with TRPC7 producing the largest UV light-induced rise in cytoplasmic Ca 2+ during the first illumination cycle as well as the most prominent desensitization during consecutive photocyling.

Recombinant TRPC3, TRPC6, and TRPC7 Are Similarly Targeted to the Plasma Membrane but Produce Divergent Levels of OptoBI-1-Sensitive Cation Conductances
To better understand the mechanistic basis of the observed differences in reconstitution of OptoBI-1 sensitive Ca 2+ signaling by the individual TRPC isoforms in RBL-2H3 cells, we continued with examination of the cellular localization and characterization of the membrane conductances generated with recombinant TRPC3/6/7. As shown in Figure 2A and Supplementary Figure 2A, recombinant TRPC channels fused to fluorescent markers (CFP or YFP) were similarly targeted to the plasma membrane. All cells expressing a TRPC fusion construct exhibited clear plasma membrane localized fluorescence. Of note, modification of RBL-2H3 cells to overexpress single TRPC-CFP fusion constructs did not affect cell morphology (Figure 2A) or promoted signs of degranulation. The OptoBI-1-induced TRPC conductances were quantified by electrophysiology applying the same OptoBI-1 photocycling protocol as in experiments measuring cytoplasmic Ca 2+ rises ( Figure 2B). All three TRPC isoforms reconstituted cation conductances with features  consistent with those recently described for OptoBI-1activated recombinant channels in HEK293 cells (8). These features included the I-V characteristics ( Figure 2C) and current inactivation/desensitization, consistent with previous reports on benzimidazole agonists as well as photochromic actuators (28,29). In full agreement with the OptoBI-1induced Ca 2+ signaling, recombinant TRPC3 produced the smallest conductance, while TRPC7 expressing cells showed the largest current density after photoactivation ( Figure  2C). Hence, the superior impact of TRPC6 and TRPC7 on mast cell Ca 2+ homeostasis corresponds to larger cation conductances generated by these isoforms in RBL-2H3 cells combined with the reported higher Ca 2+ permeability as compared to TRPC3 (30).

Genetic Modification of RBL-2H3 Cells to Overexpress TRPC6 Enables Control Over Cellular NFAT Activity by Light
In a next step we explored whether the generation of OptoBI-1 sensitivity in RBL-2H3 by overexpression of a single TRPC isoform is suitable to gain control over downstream Ca 2+dependent gene transcription. We set out to test the concept of opto-chemical control over NFAT1 activity by genetic modification of the mast cell's TRPC expression pattern.
NFAT1 nuclear translocation was recorded by expressing mCherry-NFAT1 fusion protein as a reporter, and applying the above-described protocol of repetitive photoactivation (three flashes of UV illumination). The extent of NFAT activation was quantified before and 12 min after initiation of the channel activation/deactivation cycles. Basal NFAT1 translocation was generally increased in all cells modified to overexpress TRPC channels. Nonetheless, this increase was modest with TRPC6 expression ( Figure 3A) and repetitive, transient activation of the TRPC6 conductance by light resulted in a robust and highly significant NFAT1 nuclear translocation (Figure 3 and Supplementary Video 1). By contrast cell expressing TRPC3 or TRPC7 displayed basal nuclear NFAT1 localization at levels comparable to that maximally achieved by TRPC6 activation. This phenomenon may be related to differences in basal channel activity, with an essentially low constitutive activity of TRPC6 (31), RBL cells expressing this channel subtype, displayed the lowest basal conductance measured immediately upon obtaining whole cell configuration in the absence of OptoBI-1 (Supplementary Figure 3). Importantly, only overexpression of TRPC6 channel in RBL-2H3 cells enabled precise control over mast cells Ca 2+ transcription coupling by the photochromic benzimidazole OptoBI-1.

DISCUSSION
With the present study, we provide evidence for practicability of chemo/pharmaco-optogenetic modulation of immune cells function. This strategy is based on targeting overexpressed TRPC6 channels by the photochromic benzimidazole OptoBI-1. We report proof of this concept by demonstrating control over NFAT1 activation in RBL-2H3 by light. RBL-2H3 cells were found to express TRPC proteins, specifically TRPC3 and TRPC7, which reportedly confer sensitivity to benzimidazole photopharmacology (8). However, this endogenous TRPC expression pattern was insufficient to generate significant cellular Ca 2+ signals and NFAT1 translocation in response to OptoBI-1 photocycling. Lack of benzimidazole sensitivity of native RBL-2H3 mast cells may be explained by essentially low TRPC expression at the protein level. TRPC3 protein was indeed barely detectable by immunoblotting (not shown), while a test for TRPC7 protein expression was hindered by the lack of an appropriate antibody. Nonetheless, it appears reasonable to assume that the expression of endogenous benzimidazole target channels (TRPC3/6/7) is below the threshold for coupling to the Ca 2+ /CaN/NFAT pathway. Of note, in certain cellular settings TRPC3-generated Ca 2+ signals failed to serve as an upstream trigger signal for NFAT activation (32). Nonetheless, the coupling of TRPC activity to downstream effectors may be strictly dependent on their mode of activation (33). In a recent study, we were able to demonstrate the linkage of recombinant TRPC channels to the calcineurin (CaN)/NFAT pathway in HEK293 cells (26). Expression of a TRPC3 gain-offunction mutant displaying enhanced benzimidazole sensitivity, was found to enable control of NFAT activity by OptoBI-1 (26). Consequently, we explored the option to achieve high precision control over mast cell NFAT signaling by combining photopharmacology and genetic modification. To do so, we altered mast cell TRPC expression by individual overexpression of benzimidazole-sensitive TRPC isoforms. Overexpression of recombinant TRPC3, TRPC6, or TRPC7 channels in RBL-2H3 cells reconstituted TRPC conductances with a clear order of efficacy with the largest OptoBI-1-induced current densities measured in TRPC7 expressing cells, whereas recombinant TRPC3 produced only a modest benzimidazole-sensitive conductance. All recombinant channel isoforms were found well targeted to the mast cell plasma membrane. The reason for the substantial difference observed upon reconstitution of TRPC3 and TRPC7 in RBL-2H3 cells remains unclear. Lightactivated TRPC conductances displayed marked inactivation/ desensitization as reported previously for benzimidazole agonists as well as lipid actuators (28,29). The prominent inactivation observed for TRPC7, may in part be explained by the high current density, considering a current-and/or Ca 2+ -dependent mechanism. Consistent with the higher Ca 2+ selectivity of TRPC6 and TRPC7 channels, surmounting that of TRPC3 (34), OptoBI-1-photocycling exerted a profound impact on mast cell Ca 2+ levels when cells expressed either TRPC6 or  TRPC7, but not with TRPC3 expression. Thus, genetic modification of mast cells to overexpress TRPC6 or TRPC7 channels generated OptoBI-1 sensitivity that allows temporal sculpturing of Ca 2+ signals in these immune cells. Notably, membrane currents and cytosolic Ca 2+ levels did not decline synchronously upon fast, light-induced deactivation as previously reported for TRPC3 mutants in the HEK293 expression system (26). Remarkably long-lasting Ca 2+ elevations were triggered by repetitive TRPC6 activation. This phenomenon may be related to cellular localization of these channels relative to major Ca 2+ extrusion systems. Since TRPC6 has repeatedly been found colocalized with NCX1 (35)(36)(37), it is tempting to speculate that Na + loading during TRPC6 activation might counteract NCX1mediated Ca 2+ extrusion thereby generating tonic elevation of basal Ca 2+ upon repetitive activation. Importantly, only the introduction of an expression pattern featuring TRPC6 as the prominent species, provided a basis for efficient optical control over NFAT activation in the mast cells. Of note, all three benzimidazole-sensitive isoforms were found to communicate with the CaN/NFAT pathway, albeit in a divergent manner. TRPC3 and TRPC7 overexpressing mast cells displayed significant constitutive levels of NFAT1 nuclear localization, indicating the generation of a tonic increase of NFAT dephosphorylation, due to Ca 2+ signals that arise from the constitutive activity of the overexpressed TRPC channels. Constitutive inward currents generated by TRPC3 and TRPC7 expression may results in tonic NFAT1 activation and preclude further control of NFAT transcriptional activity by photocycling of OptoBI-1. In RBL cells overexpressing TRPC6, which displays essentially low constitutive activity (31), basal NFAT1 translocation remained close to controls, and short pulsatile activationdeactivation of TRPC6 channels by OptoBI-1 photocycling resulted in rapid and significant NFAT1 nuclear translocation (within 10 min-see video in Supplementary Video 1). It is important to note that OptoBI-1 activation of TRPC6 may generate channel features and local Ca 2+ entry pattern different from those of TRPC channels activated in response to receptorphospholipase C pathways. The complex cascades inevitably linked to receptor-mediated activation of TRPC channels have been reported to modify the coupling between TRPC and NFAT signaling in cardiac muscle (38). To this end, the molecular basis of the observed efficient linkage between OptoBI-1-activated TRPC6 conductances and NFAT1 nuclear translocation in RBL-2H3 remains elusive. Nonetheless, our results demonstrate the general ability of TRPC6 to serve an important function, which is executed mainly by STIM/Orai channels in immune cells. Importantly, NFAT1 nuclear translocation triggered by TRPCmediated Ca 2+ entry, both by constitutive channel activity as well as short, transient pulses of channel activation using OptoBI-1 photocycling is potentially devoid of eliciting excessive exocytosis/degranulation, and may thereby enable a more specific modulation of immune responses as expected from interventions targeting the STIM/Orai machinery. This concept may be of relevance for the development of highly specific interventions to modify immune reactions of mast cells but also other immune cells, which play a complex, dual role in cancer pathology. It remains to be clarified if the here described concept of immunomodulation by TRPC photopharmacology can be adopted for control of other immune cells and as a basis of therapeutic strategies.