The human promonocytic cell line THP-1 was maintained in RPMI 1640 medium supplemented with 10 mM HEPES, 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine, 1% sodium pyruvate, 1% non-essential amino acids solution and 50 μM β-mercaptoethanol at 37°C in a 5% CO₂ humidified incubator. For differentiation to a macrophage phenotype, the cells were treated with 25 ng/ml PMA for 24 h, after which they were left to rest for 48 h (8).

HEK293 cells expressing human TLR4/MD2/CD14 (HEK-TLR4, Invivogen, Catalog #293-htlr4md2cd14) were cultured in DMEM supplemented with 2 mM glutamine, 10% fetal bovine serum (FBS), 1% sodium pyruvate, 1% non-essential aminoacid solution, 100 U/ml penicillin, 100 μg/ml streptomycin, 5 μg/ml blasticidin and 25 μg/ml hygromycin B at 37°C in a 5% CO₂ humidified incubator. The cells were passaged twice a week by detachment with 1 mM EDTA in PBS.

Human TRPC3 (eYFP-hTRPC3) and the N-terminal fragment of hTRPC3 (hTRPC3 (1– 321), eYFP-N-ter-TRPC3) expression plasmids were kindly provided by Dr. K. Groschner (University of Graz, Austria). Wild-type hTRPC3 was mutagenized to modify S712 phosphorylation by replacing Ser 712 with Ala (S712A) using the Quick-Change XL site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) with the following oligonucleotides: forward primer 5´-ATTACCTCCACCTTTCAGTCTAGTTCTgcTCCAAAATCATTTGTTTATTT-3´ and reverse primer 5´-AAATAAACAAATGATTTTGGAgcAGGAACTAGACTGAAAGGTGGAGGTAAT-3´. Mutagenesis was confirmed by sequencing. eYFP-TRPC3 variants were amplified by PCR adding 5´Xba-I and Sal-I 3´specific restriction sites and which removes copGFP gene in the plasmid and inserted into lentiviral vector pCDH-CMV-MCS-EF1α-copGFP. pBudCE4.1-vYC4er (vYC4er cameleon) was provided by Dr. M.T. Alonso from our institute (originally from Dr. W. Graier, University of Graz, Austria). Constructs expressing mCherry-PKCs were provided by Dr. Alexandra Newton, UCSD, USA. Positive control FRET plasmid pmCherry-eYFP was kindly provided by Dr. Johannes Schmid (University of Vienna, Austria) (14).

Gene silencing was performed using INTERFERin transfection reagent (Polyplus-transfection, France) according to the manufacturer’s protocol. Briefly, THP-1 cells were seeded at a density of 2 × 10⁵ cells/mL in antibiotic-free RPMI-1640 medium supplemented with 10% FBS and allowed to equilibrate for 24 hours. 50 nM small interfering RNA (siRNA) targeting PRKCE was diluted in OPTIMEM and mixed with INTERFERin reagent at a ratio of 3 µL per 10 nM siRNA. The mixture was incubated for 10 minutes at room temperature to allow complex formation.

The siRNA-INTERFERin complexes were then added dropwise to the cells and gently mixed. Cells were incubated under standard culture conditions for 24 h, then 25 ng/ml PMA was added to differentiate cells to macrophages for another 24 h and they were rested for final extra 24 h. Knockdown efficiency was assessed by Western blot analysis 72 h after transfection. MISSION siRNA Universal Negative Control (SIC001-SIGMA) was used as a negative control to assess off-target effects and baseline expression.

Plasmid delivery (eYFP-TRPC3, eYFP-N-ter-TRPC3, eYFP-S712A- TRPC3, mCherry-PKCs and vYC4er cameleon) in HEK293-TLR4 cells was done using Lipofectamine 2000 (Thermo) or jetPRIME in THP-1 cells complexed with DNA in a 2:1 ratio respectively for 24–48 h.

Trpc3 gene (gene ID:7223) in THP-1 cells was removed taking advantage of Synthego Express knockout cell pools high quality synthetic multiguide sgRNA technology (5′-UCCAGCAUCUUGCGCACCAC-3′; 5′-CGGCACCAGCCUCACGCCG-3′; and 5′-CUGUCAUGCGUCUCAGGGAU-3′) and transfected SpCas9. Editing efficiency was determined by T7 Endonuclease I (NEB) mismatch detection assay and confirmed by Sanger sequencing followed by ICE (Inference of CRISPR Edits) analysis (Synthego ICE tool). After limiting dilution selection of different clones, knockout of TRPC3 protein was validated by Western blot using anti-TRPC3 antibody (e.g., Alomone Labs, cat# ACC-016) and anti-β-actin as loading control. Macrophage function, including cytokine production, and phagocytic activity, was assessed post-editing to evaluate the impact of TRPC3 loss.

VSV-G–pseudotyped lentiviral particles were produced by co-transfection of 293FT cells with transfer constructs (eYFP-TRPC3 mutants) and the compatible packaging plasmids pMD2.G and psPAX2 in the presence of Lipofectamine 2000. Viruses were harvested at 48 and 72 hours after transfection. Lentiviral transduction of THP-1 macrophages was carried out using concentrated lentiviral particles in the presence of 8 μg/ml Polybrene (Sigma-Aldrich), and infected cells were selected by FACS sorting and subjected to qPCR.

Cells were loaded with 3 mM Fluo-4-AM for 20 min in culture medium at 37°C in a 5% CO₂ incubator. Cells were then washed in indicator-free medium to remove any dye that was nonspecifically associated with the cell surface and then incubated for a further 20-min period to allow for complete hydrolysis of the acetoxymethyl esters. Live cell fluorescence was monitored by Leica Dmi8 system with an HC PL FLUOTAR 63X/1.3 NA oil immersion lens and an infrared focus maintenance system. For Fluo-4 imaging fluorescence was obtained using an excitation filter at 470/40 nm and an emission filter at 525/54 nm. Before imaging started, medium was replaced by HBSS supplemented with 10 mM HEPES, with 0 CaCl₂ and 1.3 mM MgCl₂. We recorded a 5 min baseline before adding LPS acquiring images each 5 s. and then restore extracellular calcium concentration by adding 1.3 mM CaCl₂. Fluorescence data were analyzed using a combination routine of ImageJ and Cellpose semiautomated segmentation detection (15).

Cells expressing FRET-based sensors (vYC4er or CKAR) were cultured on glass-bottom dishes and stimulated with LPS (1µg/mL). Imaging was performed at 37°C using a Leica Dmi8 system. Sensor localization was verified by YFP fluorescence. Ratiometric FRET/CFP and YFP signals were acquired with standard filter sets, and ratio images were generated using ImageJ as previously described (8, 14, 16).

FRET efficiency between TRPC3 and PKC isoforms was assessed by acceptor photobleaching using a Leica confocal system as previously described (14). Briefly, mCherry was selectively photobleached in defined ROIs, and changes in eYFP fluorescence were quantified with ImageJ (AccPbFRET plugin). FRET efficiency was calculated using standard formulas, and appropriate positive and negative controls were included to confirm specificity (17).

Total RNA was extracted using TRIzol reagent (Ambion). cDNA was obtained using Verso cDNA kit Reverse Transcription for RT-PCR (Thermo Fisher Scientific), following the manufacturer’s instructions. Quantitative PCR analysis was performed with an ABI7500 machine (Applied Biosystems) as previously described (18).

Cells were lysed in Triton X-100 buffer containing protease and phosphatase inhibitors. Proteins were separated by SDS–PAGE, transferred to nitrocellulose membranes, and probed with specific primary antibodies followed by IRDye-conjugated secondary antibodies. Detection and quantification were performed using a LiCor Odyssey system as previously described (19, 20).

Statistical details of experiments are indicated in the figure legends. All data analyses were performed with Prism software (GraphPad). All data are presented as means ± standard error of the mean (SEM), indicating individual biological replicates. No statistical analysis was used to predetermine sample size. All datasets were analyzed by unpaired two-tailed Student’s t test. Welch’s correction was performed when the variances were significantly different.

It has been proposed that pharmacological stimulation with PMA induces PKC-dependent phosphorylation of TRPC3 at S712 (7), leading to channel inactivation, we aimed to investigate whether specific PKC isoforms are responsible for this modification under more physiological conditions. As a first step, we assessed whether LPS stimulation induces PKC activity in HEK-TLR4 cells. To this end, we expressed the FRET-based PKC activity reporter CKAR (16), which is a pan PKC activity reporter composed by a monomeric FHA2 domain (forkhead-associated) from the yeast protein rad53p as a phospho-serine binding domain and a substrate consensus sequence predicted to be an excellent substrate for all PKC isoforms but suboptimal for other kinases (21, 22). Treatment of CKAR expressing HEK-TLR4 cells with LPS resulted in a ~35% rapid increase in PKC activity within 5 min, which was reduced to baseline by treatment with Gö6983 (Figure 1A), confirming PKC involvement in the response (23).

We next investigated the subcellular localization dynamics of PKC isoforms during LPS stimulation using live-cell confocal imaging of mCherry-tagged 7 out of 9 PKC isoforms. As shown in Figures 1B–D, only two isoforms exhibited stimulus-dependent translocation to membranous compartments. Specifically, PKCα translocated to the plasma membrane, whereas PKCϵ localized to intracellular endomembranes following LPS treatment (Figure 1B). The remaining PKC isoforms did not display detectable changes in subcellular distribution under these conditions.

PKC activation is tightly regulated and typically short-lived, leading to rapid but transient phosphorylation of its substrates. This phosphorylation is often quickly reversed by cellular phosphatases, making it difficult to capture kinase/substrate interactions using traditional immunoprecipitation assays. In the case of TRPC3 and PKC, this transient nature—combined with the importance of subcellular localization—suggests that immunoprecipitation may not be the most suitable method for studying their interaction. As shown in Figure 1, subcellular redistribution of PKC isoforms appears to be a key regulatory mechanism during LPS stimulation. Then, we employed FRET- assays to monitor the interaction between TRPC3 and specific PKC isoforms in live cells. We co-expressed TRPC3-eYFP with either PKCα-mCherry or PKCϵ-mCherry, treated cells with PMA for 10 minutes to induce maximum membrane translocation of PKCs, and then analyzed FRET efficiency by acceptor photobleaching to assess proximity and potential interaction. The results showed that PKCα interacts with TRPC3 at the plasma membrane, whereas PKCϵ interacts with TRPC3 at endomembranes as compared with a FRET positive control consisting in an eYFP-mCherry fusion protein and a negative control of separately coexpressed eYFP and mCherry (Figure 2).

As previous results confirmed a role in signaling for PKCα and ϵ during LPS stimulation, we next investigated ER Ca²⁺ dynamics under conditions of PKC inhibition. To distinguish between the roles of conventional and novel PKC isoforms, we utilized the selective inhibitors Gö6976, which targets conventional PKCs, and Gö6983, which inhibits both conventional and novel PKCs but not atypical isoforms (23–25). We employed a FRET-based calcium cameleon indicator (vYC4er), specifically engineered to localize within the endoplasmic reticulum (ER) lumen. This biosensor enables the detection of dynamic changes in ER [Ca²⁺] during cellular stimulation (26). HEK-TLR4 cells expressing the ER-targeted calcium sensor vYC4er were pretreated with either inhibitor or vehicle prior to LPS stimulation. Inhibition of conventional PKCs with Gö6976 had no significant effect on ER Ca²⁺ release following LPS stimulation. In contrast, treatment with Gö6983 caused ~35% higher basal ER Ca²⁺ and ~45% reduction in LPS-induced ER Ca²⁺ release compared to vehicle, similar to S712A cells (Figure 3A). Under extracellular Ca²⁺-free conditions, analysis of cytosolic Ca²⁺ dynamics revealed that cells pretreated with the pan-PKC inhibitor Gö6983 exhibited higher initial cytosolic Ca²⁺ levels compared to those treated with the classical PKC inhibitor Gö6976 or vehicle control. This elevated Ca²⁺ levels gradually declined during stimulation with LPS, suggesting active sequestration into endoplasmic reticulum (ER) stores (Figure 3B). These findings suggest that a novel PKC isoform, rather than a conventional one, mediates TRPC3 inactivation via phosphorylation. Among the novel PKCs examined by live-cell imaging, only PKCϵ exhibited stimulus-dependent translocation upon LPS treatment (Figure 1).

To further explore the contribution of PKCϵ, we performed immunoblot analysis in HEK-TLR4 cells co-transfected with TRPC3-WT and PKCϵ. Using an anti-phospho-Ser substrate PKC antibody, we detected an increased phosphorylation signal in a protein band whose molecular weight is coincident with that of TRPC3 (Figures 4A, B). However, when TRPC3-S712A mutant and PKCϵ were co-expressed this phosphorylation increase was extremely reduced indicating that PKCϵ would be the isoform responsible for TRPC3-S712 phosphorylation. Based on this observation, we selectively silenced PKCϵ in human macrophages and monitored ER Ca²⁺ dynamics (Figures 4C, D). Consistent with pharmacological inhibition, PKCϵ knockdown resulted in a constitutive Ca²⁺ influx into the ER since the signal in the absence of PKCϵ is consistently much higher than in the control, indicating a greater Ca²⁺ content in the ER. Together, these results strongly implicate PKCϵ as the key isoform responsible for TRPC3 regulation via phosphorylation at serine 712 in response to LPS stimulation.

Previous work suggested that TRPC3 is negatively regulated via phosphorylation at Ser 712 by PKC (7). To investigate the functional relevance of this post-translational modification, we expressed either eYFP-tagged TRPC3 wild-type (eYFP-TRPC3-WT) or a non-phosphorylatable mutant (eYFP-S712A-TRPC3), in which Ser 712 is substituted with Ala, in HEK-TLR4 cells (27). Live-cell confocal imaging revealed that, consistent with prior reports (8, 28) TRPC3-WT localized to both the plasma membrane and intracellular compartments. In contrast, the S712A mutant was exclusively localized to the plasma membrane (Figure 5). Following LPS stimulation, TRPC3-WT-expressing cells exhibited vesicular trafficking events, indicative of internalization or intracellular redistribution, which were absent in cells expressing the S712A mutant (Figure 5). These findings support the notion that phosphorylation at Ser 712 modulates TRPC3 subcellular localization and trafficking dynamics.

To further explore the role of S712 phosphorylation of the TRPC3 channel, HEK-TLR4 cells were transfected with eYFP-TRPC3 or eYFP-S712A-TRPC3 and the production of proinflammatory cytokines was measured using qPCR. We observed that when TRPC3 could not be phosphorylated at S712, signaling effects are stronger, resulting in ~2.5-fold higher TNF and ~2-fold higher PTGS2 expression compared to TRPC3-WT stimulated with LPS (Figure 6A). To confirm this effect in an immune relevant context, we took advantage of CRISPR/Cas9 technology and specifically deleted TRPC3 gene in THP-1 macrophages (Figure 6B). In THP-1 macrophages, S712A reconstitution increased TNF by ~2.8-fold and PTGS2 by ~2.2-fold relative to WT (Figure 6C).

Previous research work from our lab has demonstrated the direct involvement of TRPC3 in the production of proinflammatory cytokines by LPS, a process that requires Ca²⁺ release from the ER (8). Therefore, we analyzed the dynamics of Ca²⁺ in the ER when TRPC3 could not be phosphorylated at S712. To that end, we co-expressed the ER targeted calcium cameleon vYC4er together with either TRPC3-WT or S712A-TRPC3. We observed that cells expressing TRPC3-WT exhibited Ca²⁺ release from the ER in response to LPS. This did not occur when cells expressed only the N-terminal fragment (302 aa), which acts as a dominant-negative preventing proper channel assembly. However, S712A-expressing cells showed ~30% higher basal ER Ca²⁺ levels and ~40% smaller LPS-induced ER Ca²⁺ release compared to WT (Figure 7A).

Consistent with previous findings, under extracellular Ca²⁺-free conditions, cells expressing the S712A mutant exhibited ~25% initial cytosolic Ca²⁺ compared to TRPC3-WT, which progressively declined over time, suggesting active sequestration into ER stores (Figure 7B) similar to the behavior observed in the Gö6983 treated cells in Figure 3B. Collectively, these findings suggest that phosphorylation of TRPC3 at S712 is necessary to contribute to the correct localization of the channel and to ensure the adequate Ca²⁺-mediated signaling responsible to produce proinflammatory cytokines.