Pharmaco-Optogenetic Targeting of TRPC Activity Allows for Precise Control Over Mast Cell NFAT Signaling - PubMed (original) (raw)

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

Bernadett Bacsa et al. Front Immunol. 2020.

Abstract

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.

Keywords: NFAT nuclear translocation; OptoBI-1; canonical transient receptor potential channels; mast cells; opto-chemical immunomodulation; photopharmacology.

Copyright © 2020 Bacsa, Graziani, Krivic, Wiedner, Malli, Rauter, Tiapko and Groschner.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a past co-authorship with one of the authors, RM.

Figures

Figure 1

Figure 1

OptoBI-1 photocycling induced Ca2+ signaling triggered by overexpressed TRPC3, TRPC6 or TRPC7 channels in RBL-2H3 cells. Comparison of responses in cells overexpressing YFP-TRPC3, YFP-TRPC6 or TRPC7-CFP together with R-GECO as a Ca2+ reporter. Cells expressing R-GECO was only used as a control. Time courses (mean ± SEM) of R-GECO fluorescence intensity (red, 577 nm) during photoactivation of TRPC3 (black, n = 8 cells), TRPC6 (red, n = 23 cells), TRPC7 (gray, n = 21 cells), and controls (white, n = 10 cells). Cis-trans OptoBI-1 photocycling was repeated three times, by illuminating cells with UV (365 nm, 10 s; violet) followed by blue light (430 nm, 10 s, blue).

Figure 2

Figure 2

Overexpressed TRPC3/6/7 proteins are targeted to the plasma membrane of RBL-2H3 cells and generate cation conductances with divergent efficiency. (A) Representative epifluorescence images of RBL-2H3 cells expressing CFP-TRPC3 (n = 57), CFP-TRPC6 (n = 26) and TRPC7-CFP (n = 45), respectively. Scale bar = 10 µm. (B) Time courses of control (sham transfected, white, n = 6 cells), YFP-TRPC3 (black, n = 6 cells), YFP-TRPC6 (red, n = 8 cells) and TRPC7-CFP (grey, n = 8 cells) conductances recorded at −90 to +70 mV during repetitive photoconversion of OptoBI-1. Light illumination cycling is indicated as violet (365 nm) and blue (430 nm). (C) Representative net I–V relations (_I_max − _I_basal) of OptoBI-1 induced currents in YFP-TRPC3 (black), YFP-TRPC6 (red), TRPC7-CFP (dark grey) transfected RBL-2H3 cells applying voltage-ramp protocols. Insert: Current density of net, maximum responses obtained at −90 to +70 mV (mean ± SEM). Statistical significance was tested by two tailed t-test (normally distributed values) or Mann–Whitney tests (non-normally distributed values), *p < 0.05, if not indicated differences are not significant (p > 0.05).

Figure 3

Figure 3

TRPC6 expression enables OptoBI-1-mediated control of NFAT1 activity in RBL-2H3 cells by light. (A) NFAT1 nuclear translocation in RBL-2H3 cells overexpressing either mCherry-NFAT1 only (control; white, n = 30) or mCherry-NFAT1 along with YFP-TRPC3 (black, n=17), YFP-TRPC6 (red, n = 31) or with TRPC7-CFP (gray, n = 21). Mean ± SEM nucleus/cytosol fluorescence intensity ratio. The translocation of mCherry-NFAT1 was monitored during OptoBI-1 photoconversion, applying three photocycles of illumination (three Flashes) as indicated. Statistical significance was tested by One-Way ANOVA (Holm–Sidak test for normally distributed values) or One-Way ANOVA for Ranks (Dunn’s test for non-normally distributed values), *p < 0.05, ***p < 0.001, if not indicated—not significant (p > 0.05). (B) Images of NFAT1 translocation in RBL-2H3 cells expressing mCherry-NFAT1 only (control; white) or coexpressing mCherry-NFAT1 and YFP-TRPC3 (black), YFP-TRPC6 (red) or TRPC7-CFP (grey) before (0 min) and after (12 min) three cycles of UV (365 nm, 10 s) and blue light (430 nm, 10 s) illuminations. Scale bar represents 5 µm.

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