Activation of the TRPML1 Ion Channel Induces Proton Secretion in the Human Gastric Parietal Cell Line HGT-1

Abstract

The lysosomal Ca²⁺ channel TRPML1 was found to be responsible for gastric acid secretion in murine gastric parietal cells by inducing the trafficking of H⁺/K⁺-ATPase containing tubulovesicles to the apical membrane. Therefore, we hypothesized a similar role of TRPML1 in regulating proton secretion in the immortalized human parietal cell line HGT-1. The primary focus was to investigate the involvement of TRPML1 in proton secretion using the known synthetic agonists ML-SA1 and ML-SA5 and the antagonist ML-SI3 and, furthermore, to identify food-derived compounds that target the channel. Proton secretion stimulated by ML-SA1 was reduced by 122.2 ± 22.7% by the antagonist ML-SI3. The steroid hormone 17β-estradiol, present in animal-derived foods, diminished the proton secretory effect of ML-SA1 by 63.4 ± 14.5%. We also demonstrated a reduction in the proton secretory effects of ML-SA1 and ML-SA5 on TRPML1 knock-down cells. The food-derived compounds sulforaphane and trehalose promoted proton secretion in HGT-1 cells but may act independently of TRPML1. Also, histamine- and caffeine-induced proton secretion were affected by neither the TRPML1 antagonist ML-SI3 nor the TRPML1 knock-down. In summary, the results obtained suggest that the activation of TRPML1 promotes proton secretion in HGT-1 cells, but the channel may not participate in canonical signaling pathways.

Keywords: Lamp1; MCOLN1; Mucolipin1; TRPML1; calcium.

Figure 1

Chemical structures of the synthetic TRPML1 agonists ML-SA1 and ML-SA5, the antagonist ML-SI3, and the potential food-derived TRPML1 activity modulators sulforaphane, trehalose, and 17β-estradiol.

Figure 2

TRPML1 is expressed in HGT-1 cells detected with super-resolution imaging. Fluorescent-labeled TRPML1 channels with anti-TRPML1 antibody and anti-Rabbit IgG with Alexa Fluor 488 fluorophore (green) are localized in the cell membrane, cytoplasm, and nucleus, in contrast to membrane staining with ConA and streptavidin-Alexa Fluor 633 (red) and nucleus staining with Hoechst-33342 (blue). The Zeiss LSM 780 microscope (Carl Zeiss AG, Munich, Germany) with airyscan detection (Carl Zeiss AG, Munich, Germany) and a 40×/1.2 Imm Korr DIC M27 objective lens (Carl Zeiss AG, Munich, Germany) was used for image acquisition. Scale bar: 5 μm.

Figure 3

Effect on proton secretion in HGT-1 cells incubated with 5 µM of the synthetic TRPML1 agonists ML-SA1 and ML-SA5 and the antagonist ML-SI3. Intracellular ΔH⁺ concentrations in nM are shown as mean ± SEM, n = 4–5, t. r. = 4–6. Statistics: Student’s t-test of untreated compared to treated cells (two-tailed, unpaired); significant differences are indicated with ns = not significant, * = p ≤ 0.05, and **** = p ≤ 0.0001. Student’s t-test of activator-only treated cells compared to cells treated with the co-incubation of ML-SI3 with ML-SA1 or ML-SA5 (two-tailed, unpaired); significant differences are indicated with ns = not significant; # = p ≤ 0.05.

Figure 4

Inhibitory effect of ML-SI3 on ML-SA1- and ML-SA5-stimulated HGT-1 cells in the calcium mobilization assay. (A): Co-incubation of ML-SA1 with ML-SI3 in the calcium mobilization assay. The ML-SA1 signal in TRPML1 kd cells is shown in green. (B): Co-incubation of ML-SA5 with ML-SI3 in the calcium mobilization assay. The ML-SA5 signal in TRPML1 kd cells is shown in green. A 5 µM ionomycin was applied as a positive control. Data are shown as relative fluorescence (RFU) normalized to baseline fluorescence n = 3, t. r. = 2.

Figure 5

Super-resolution image of 5 µM ML-SA1-incubated HGT-1 cells stained for TRPML1 (green) and the β-subunit of H⁺/K⁺-ATPase ATP4B (red) obtained with an airyscan detector: For image acquisition, a Zeiss LSM 780 microscope equipped with airyscan detection and a 40×/1.2 Imm Korr DIC M27 objective lens was used. Airyscan image processing was performed using the ZEN 2.3 SP1 black program. The scale bar represents 5 μm. The H⁺/K⁺-ATPase accumulation is indicated in the direction of the arrow. The vacuolar apical compartment formation is displayed with the star.

Figure 6

Co-localization of TRPML1 channels Lamp-1 and β-subunit of H⁺/K⁺-ATPase (ATP4B) in HGT-1 cells. Cells were incubated with either 1 mM histamine or 5 µM ML-SA1 for 10 min before staining with anti-TRPML1 antibody (green) and anti-human-CD107a (A in red) for Lamp-1. The β-subunit of H⁺/K⁺-ATPase was stained with anti-ATP4B antibody (B in red). Scale bar represents 5 μm. The co-localization was calculated for TRPML1 with Lamp-1 (C) and ATP4B (D) based on the pixel count. Data is presented as box plots with 10th and 90th percentiles. Data points outside of this range are indicated as dots, n = 1–2, t. r. = 10–20. Statistics: Student’s t-test of control compared treated cells (two-tailed, paired); ns: not significant.

Figure 7

Proton secretion-inducing effect of sulforaphane, trehalose, and 17β-estradiol on HGT-1 cells. Intracellular ΔH⁺ concentrations in nM are shown as a violin plot, n = 4–5, t. r. = 4–6. Statistics: Student’s t-test (two-tailed, unpaired); significant differences are indicated with ns = not significant; * = p ≤ 0.05; ** = p ≤ 0.01; **** = p ≤ 0.0001.

Figure 8

Effect of ML-SI3 on histamine-, caffeine-, trehalose-, and sulforaphane-induced proton secretion. Intracellular ΔH⁺ concentrations in nM are shown as mean ± SEM, n = 4–5, t. r. = 4–6. Statistics: Student’s t-test (two-tailed, unpaired); significant differences are indicated with ns = not significant; * = p ≤ 0.05. (A–D): Co-incubation of 5 µM ML-SI3 with 1 mM histamine, 3 mM caffeine, 1 µM sulforaphane, and 0.1 mM trehalose.

Figure 9

Inhibitory effect of 17β-estradiol on ML-SA1-stimulated proton secretion. Intracellular ΔH⁺ concentrations in nM are shown as mean ± SEM, n = 4–5, t. r. = 4–6. Statistics: Student’s t-test (two-tailed, unpaired); significant difference is indicated with ** = p ≤ 0.01.

Figure 10

Histamine, caffeine, and TRPML1-targeting compounds and the influence on the proton-secreting effect in TRPML1 kd cells compared to mock-transfected and non-transfected HGT-1 cells. Intracellular ΔH⁺ concentrations in nM are shown for the comparison of non-transfected (black bar), mock-transfected (dark grey bar), and TRPML1 kd (light grey bar) cells incubated with 1 mM histamine (A), 5 µM ML-SA1 (B), 5 µM ML-SA5 (C), 3 mM caffeine (D), 1 µM sulforaphane (E), 0.1 mM trehalose (F), and 5 µM 17β-estradiol (G). Data are shown as mean ± SEM, n = 3–4, t. r. = 6; statistics: Student’s t-test (two-tailed, paired [mock vs. TRPML1 kd], unpaired [non-transfected vs. mock]); significant differences are indicated with ns = not significant; * = p ≤ 0.05; ** = p ≤ 0.01.