All cells were cultivated at 37°C in humidified air with 5% CO₂ in McCoy’s 5A medium supplemented with 10% FCS.

To mediate TMEM206 knockdown, cells have been transfected with the Flexitube gene solution GS55248 (QIAGEN). Cells were transfected in a 4D Nucleofector system (Lonza) according to the manufacturer’s instructions and the recommended transfection solution for HCT116 cells.

With the help of the CRISPR/Cas9 system, TMEM206-knockout cell lines were generated. Guide RNAs were designed with the web tool CRISPOR (Concordet and Haeussler, 2018). By using two guide RNAS (gRNAs), 67 to 68 base pairs were deleted in exon 1 (Chr 1: 212′414′732 to 212′414′798 or 212′414′731 to 212′414′798). The oligonucleotides for the gRNAs were cloned into the pSpCas9(BB)-2A-GFP (PX458) (Addgene #48138) vector, according to the protocol from Addgene (Addgene, 2023). The transfection of the cells was performed as described in the transfection section with 2 µg of each gRNA plasmid (gRNA92rv/gRNA170fw). 24 h later cells were sorted in the ASTRIOS flow cytometry sorter for GFP positive cells. Single cells were seeded in four 96-wellplates. Approximately 2 weeks later, single cell clones could be transferred to 24-well plates. Around 100 clones were taken as single clones from the 96-well plates. The screening of the clones was done by genotyping. Clones showing the desired deletion in both alleles were amplified and then stocked in liquid N₂. Cell pellets were taken, and qPCR was done for further validation. The deletion was confirmed by Sanger sequencing.

All experiments were performed in whole-cell configuration at room temperature. Voltage ramps spanning from −150 mV to +150 mV for TMEM206 and from -100 mV to +100 mV for TRPM4 with a duration of 50 ms were applied every 2 s. Induced currents were acquired with an EPC-10 amplifier (HEKA), recorded and digitized with Patchmaster (v2x53, HEKA). Voltages were corrected for a liquid junction potential of 10 mV. Currents were filtered at 1 kHz and then sampled at 3 kHz. To evaluate current development, currents were extracted at −130 and +130 mV for TMEM206 and at −80 and +80 mV for TRPM4, normalized to the cell capacitance, and plotted versus time. To further analyze data IgorPro 6.37 (Wavemetrics) and GraphPad Prism 9 were used. Bath solutions contained: 140 mM NaCl, 3 mM MgCl₂, 0.5 mM CaCl₂, 10 mM HEPES and pH was adjusted to 7.2 with HCl. In the pH 4.5 bath solution, 10 mM HEPES was replaced by 5 mM Na₃-citrate and pH was adjusted to 4.5 with citric acid. Pipette solution contained: 120 mM Cs-glutamate, 10 mM HEPES, 3 mM MgCl_2, 20 mM Cs-BAPTA and pH was adjusted to 7.2 with CsOH (for TMEM206) and 140 mM Cs-glutamate, 10 mM EDTA, 10 mM HEPES, 8 mM NaCl, and final concentrations of 3 mM MgCl₂ and 10 µM CaCl₂ (for TRPM4 according to (Kappel et al., 2019)). If necessary, Osmolarity was adjusted to ∼310 mOsm with glucose.

Acid-induced cell death of HCT116 and HCT116 TMEM206 KO cells (clone 1-6) was assessed by staining with caspase 3/7 and SYTOX AADvanced Dead Cell stain. HCT116 and KO cells were incubated for 2.5 h at 37°C with DMEM medium at pH 4.5. For the quantification of caspase-3/7 activity, cells were stained with CellEvent caspase-3/7 green flow cytometry assay kit (ThermoFisher Scientific) according to the manufacturer’s protocol. Caspase activity was measured by flow cytometer using the BD LSR II SORP flow cytometer and analyzed using Flowjo software.

Total RNA from HCT116 cells was isolated with QIAshredder and RNAeasy kit (both QIAGEN). RNA was reversely transcribed with the High-Capacity cDNA Reverse Transcription kit (Thermo Fisher). For the TMEM206 TaqMan Gene Expression Assay, a 1:4 dilution of cDNA was used. The PCR conditions in the CFX system (Bio-Rad) were: 2-min activation at 50°C, then 10 min at 95°C; followed by 40 cycles of 15-s denaturation at 95°C and 1-min annealing at 60°C. TMEM206 expression was normalized to TATA box-binding protein and RNA-polymerase II expression.

Although database research already revealed that TMEM206 mRNA should be expressed in colonic tissue we performed qPCR to compare TMEM206 expression in four colorectal cancer cell lines with different dukes’ staging (HCT116, Dukes’ stage A, LS180, Dukes’ stage B, HCT15, Dukes’ stage C, Colo205, Dukes’ stage D) to the expression in the non-cancerous cell line CCD841 CoN. TMEM206 mRNA is expressed in all the tested cell lines with the highest expression in HCT116, similar expression levels in CCD841 CoN and LS180, and slightly lower expression in HCT15 and Colo205 that represent more advanced cancer stages (Figure 1A). Because HCT116 had the highest mRNA expression we used those cells for our experiments.

To test if TMEM206 is functionally expressed at the protein level in HCT116, we performed siRNA-mediated knockdown targeted at TMEM206. TMEM206 mRNA expression is reduced to 18% of the expression of the control transfected cells 24 h post transfection. The expression slightly increases to 26% and 40% after 48 and 72 h, respectively (Figure 1B). To determine if the reduction in mRNA expression is also reducing TMEM206 protein expression, whole-cell patch clamp experiments have been performed. In control-transfected HCT116 cells, pH 4.5 induced outward currents are at ∼46 pA/pF. Currents are reduced to ∼20 pA/pF after 24 h and are further decreased to ∼9 pA/pF and ∼7 pA/pF at 48 and 72 h after transfection, respectively (Figures 1C, D). Inward currents are reduced to a similar extent. The current-voltage relationships (Figure 1E) reveal a strong outward rectification reminiscent of TMEM206 currents.

TMEM206 was reported to contribute to acid-induced cell death. Therefore, we aimed to elucidate the contribution of TMEM206 to acid-induced cell death in colorectal cancer cells. First, we generated TMEM206 knockout cells by CRISPR/Cas9. None of the six obtained knockout clones shows mRNA expression for the targeted exon (Supplementary Figure S1) or acid-induced currents (Figures 2A, B) demonstrating that in HCT116 cells PAORAC/ASOR is exclusively mediated by TMEM206. We tested different commercially available antibodies against TMEM206 (Abcam ab99055, Alomone Labs ACL-031, Sigma-Aldrich ZRB1745, Novus Biologicals NBP2-85944, NBP1-88325 and NBP1-70729), but could not detect TMEM206 protein in HCT116 cells on Western Blots. Therefore, we cannot exclude expression of truncated TMEM206 protein. To test the contribution of TMEM206 to acid-induced cell death, we treated HCT116 and HCT116 TMEM206 KO cells with pH 4.5 DMEM medium for 2.5 h. Cell death was determined by SYTOX AADvanced Dead Cell staining and the mode of cell death by caspase 3/7 activity (Figure 2C). Cell death rates after low pH treatment were higher in TMEM206 KO clone 1 to 4 with 86%, 79%, 88%, and 82%, respectively versus 75% in parental HCT116 cells (Figure 2D). KO clones 5 and 6 showed lower cell death rates with 60% and 59% respectively. Caspase 3/7 activity rate ranged from 2.6% in clone 4 to 6.8% in clone 5, while in parental cells it was 6.6% (Figure 2E). None of the clones showed a significant change in cell death or apoptosis. In conclusion, in HCT116 cells, TMEM206 does not contribute to acid-induced cell death.

Both, flufenamic acid and niflumic acid inhibit PAORAC/ASOR (Lambert and Oberwinkler, 2005; Wang et al., 2007). To test if TMEM206 is inhibited by structurally similar compounds that arose from a LBVS performed earlier (Delalande et al., 2019), we applied them at a concentration of 100 µM and determined TMEM206 current inhibition. First, we activated the current by application of bath solution with pH 4.5, briefly deactivated by switching back to bath solution with pH 7.2 and activated again with pH 4.5 with the added compound or DMSO as solvent control (Figure 3A). The application of DMSO alone results in a reduction of the residual current to 93% (Figure 3B). The already-known TMEM206 blocker niflumic acid reduced residual currents to 38%. Flufenamic acid only slightly inhibited TMEM206 currents by 28%. The compounds LBA, NBA, and CBA, reduced residual currents to 53%, 74%, and 39%, respectively. Since all compounds fail to fully inhibit TMEM206 currents, we wanted to test if a longer application time leads to more effective current inhibition. Therefore, we tripled the application time from 60 s to 180 s (Figure 3C). Longer application of CBA results in lower currents (Figures 3C, D), 19% versus 39% for the short application. Notably, the reversal potential was shifted to more positive values upon application of 100 µM CBA pointing to a change in ion channel selectivity (Supplementary Figure S2). We also tested if a higher CBA concentration results in more efficient current inhibition. Doubling CBA concentration fails to further increase current inhibition, demonstrating that the inhibition to 19% is the maximal inhibition with CBA (Figure 3D).

To test for the CBA half maximal inhibitory concentration (IC₅₀) for TMEM206, we applied different concentrations ranging from 0.1 µM to 200 µM CBA and determined residual current densities. We found that CBA inhibits TMEM206 currents in a dose-dependent manner (Figure 4A, B). We fitted a non-linear Hill equation to calculate the IC₅₀ for CBA, which is 9.55 µM (Figure 4C).

The tumor microenvironment and inflammation are often characterized by tissue acidosis, and pH values drop below pH 6.0 (Justus et al., 2013; Riemann et al., 2015). Additionally, as already shown by Sato-Numata et al., TMEM206 activation threshold shifts to pH 6.0 at 37°C, while it is inactive at physiological pH. Thus, CBA needs to be a potent inhibitor at pH 6.0 to study TMEM206 in cancer and inflammatory processes.

Calculations show that at pH 6, CBA is 99% dissociated to its anionic form CBA_A- and H⁺, while at pH 4 only 88% of CBA is dissociated (Figure 5A). In addition, logD values indicate that CBA is a lipophilic substance with a more lipophilic character in low pH pointing to reduced solubility. Indeed, at pH 4.5, we observed some turbidity at higher CBA concentrations (≥30 µM), that might arise from precipitation.

We next tested if CBA blocks TMEM206 currents at pH 6.0, as our calculations show that at pH 6.0 CBA is 99% dissociated to CBA_A-, while the logD value is only half of the logD value at pH 4.0. Since TMEM206 is only slightly active at pH 6 at room temperature, we transiently overexpressed TMEM206 in TMEM206 knockout HCT116 cells to obtain higher current densities (Figure 5B). Under these conditions, 100 µM CBA only slightly inhibits TMEM206 currents. In contrast to a mean residual current of ∼19% at pH 4.5 when 100 µM CBA were applied, residual current is at ∼77% at pH 6 with the same CBA concentration. Interestingly, DMSO application increased TMEM206 currents at pH 6.0 by 32% (Figure 5C). In conclusion, at pH 6, CBA insufficiently blocks TMEM206 and is not feasible for cellular assays and can only be used in mechanistic studies at low pH.