Light-Mediated Control over TRPC3-Mediated NFAT Signaling

Abstract

Canonical transient receptor potential (TRPC) channels were identified as key players in maladaptive remodeling, with nuclear factor of activated T-cells (NFAT) transcription factors serving as downstream targets of TRPC-triggered Ca2+ entry in these pathological processes. Strikingly, the reconstitution of TRPC-NFAT signaling by heterologous expression yielded controversial results. Specifically, nuclear translocation of NFAT1 was found barely responsive to recombinant TRPC3, presumably based on the requirement of certain spatiotemporal signaling features. Here, we report efficient control of NFAT1 nuclear translocation in human embryonic kidney 293 (HEK293) cells by light, using a new photochromic TRPC benzimidazole activator (OptoBI-1) and a TRPC3 mutant with modified activator sensitivity. NFAT1 nuclear translocation was measured along with an all-optical protocol to record local and global Ca2+ pattern generated during light-mediated activation/deactivation cycling of TRPC3. Our results unveil the ability of wild-type TRPC3 to produce constitutive NFAT nuclear translocation. Moreover, we demonstrate that TRPC3 mutant that lacks basal activity enables spatiotemporally precise control over NFAT1 activity by photopharmacology. Our results suggest tight linkage between TRPC3 activity and NFAT1 nuclear translocation based on global cellular Ca2+ signals.

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

“…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.…”

“…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).…”

“…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.…”

“…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.…”

Mechanisms and significance of Ca2+ entry through TRPC channels

“…In agreement with others, our group found that TRPC3 exhibited ligand-dependent Ca 2+ selectivity. TRPC3 was found to have a carbachol (CCh)-induced P Ca /P Cs selectivity ratio of 3.7 [ 39 ], which was reduced when activated by a novel photoswitchable agonist OptoBI-1 (P Ca /P Cs = 3.06; [ 56 ]). Altogether, ligand-dependent Ca 2+ selectivity is likely a general feature of the TRP channel family, which might be subunit stoichiometry dependent, and should be considered as an important factor for governing signaling functions.…”