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. 2017 May 8;41(3):262-273.e6.
doi: 10.1016/j.devcel.2017.04.003.

Gastric Acid Secretion From Parietal Cells Is Mediated by a Ca 2+ Efflux Channel in the Tubulovesicle

Free PMC article

Gastric Acid Secretion From Parietal Cells Is Mediated by a Ca 2+ Efflux Channel in the Tubulovesicle

Nirakar Sahoo et al. Dev Cell. .
Free PMC article


Gastric acid secretion by parietal cells requires trafficking and exocytosis of H/K-ATPase-rich tubulovesicles (TVs) toward apical membranes in response to histamine stimulation via cyclic AMP elevation. Here, we found that TRPML1 (ML1), a protein that is mutated in type IV mucolipidosis (ML-IV), is a tubulovesicular channel essential for TV exocytosis and acid secretion. Whereas ML-IV patients are reportedly achlorhydric, transgenic overexpression of ML1 in mouse parietal cells induced constitutive acid secretion. Gastric acid secretion was blocked and stimulated by ML1 inhibitors and agonists, respectively. Organelle-targeted Ca2+ imaging and direct patch-clamping of apical vacuolar membranes revealed that ML1 mediates a PKA-activated conductance on TV membranes that is required for histamine-induced Ca2+ release from TV stores. Hence, we demonstrated that ML1, acting as a Ca2+ channel in TVs, links transmitter-initiated cyclic nucleotide signaling with Ca2+-dependent TV exocytosis in parietal cells, providing a regulatory mechanism that could be targeted to manage acid-related gastric diseases.

Keywords: Ca(2+) release; TRPML1; cAMP; exocytosis; membrane trafficking; tubulovesicle.

Conflict of interest statement

Author contributions:

N.S. and H.X. designed the study; N.S., M.G., X.Z., J.Y., M.B., T.K., and S.S. performed the laboratory experiments; R.C., Q.G., W.W., S.P., X.H., M.F., G.K., S.S., M.S., L.S., J.M., and J. L. M. contributed the reagents; N.S., M.G., X.Z., N.R., L.S., J. L. M., X.Z and H.X. analyzed and interpreted the data; N.S. and H.X. wrote the paper with inputs and final approval from all authors.


Fig. 1
Fig. 1. ML1 involvement in histamine-stimulated acid secretion
(A) Corpus gland immunoblots. (B, C) Whole-endolysosomal IML1 was activated by TRPML agonists (ML-SA1/3/5; 1–20 μM) and inhibited by ML1 antagonists ML-SI3/4 (10–20 μM) in WT (B), but not ML1 KO parietal cells (C). (D–G) Histamine (100 μM + 20 μM IBMX) and ML-SI4 (10 μM) effects on proton secretion, indexed by NIPR observed while cytoplasmic pH (pHc) was under H+/K+-ATPase control (see also Fig. S1O) relative to responses in 0 Na+ (red dotted lines). (H) NIPR rates (n = 10–30 cells) under resting and histamine-stimulated conditions. (I, J) Whole-stomach acid contents following histamine administration (1 mg/kg, IP). (K) Histamine-induced acid increases in the presence of ML-SI3 (20 μM) or ML-SI4 (10 μM). n = 3–4 mice/group. (L) Effect of ML-SIs on histamine-stimulated [14C] aminopyrine accumulation in gastric glands (n = 4 mice per experiment; normalized to basal output). ML-SI3 (20 μM) and ML-SI4 (10 μM) were applied 30 min before histamine (100 μM + 20 μM IBMX). Panels H, K, and L show means ± SEMs from ≥3 experiments. *P < 0.05, **P < 0.01, ***, P < 0.001 one-way ANOVA, Bonferroni’s post-hoc analysis.
Fig. 2
Fig. 2. ML1 activation induces gastric acid secretion
(A, B) ML-SA5 (10 μM, applied 1 h prior) potentiation of NIPR in WT (A), but not ML1 KO (B) parietal cells. (C) NIPR of ML-SA5-treated WT and ML1 KO parietal cells. (D, E) Representative traces of ML-SA5 effects on whole-stomach acid contents in vivo. (F) Average acid secretory rates (n = 3–4 mice/group). (G) ML-SA5 effects on [14C]-aminopyrine incorporation into isolated glands. (H) Genotype verification of ML1 Rosa loxSTOPlox (lSl), ATP4BCre, and ML1 Rosa lSl: ATP4B-Cre (ML1PC) mice. Three DNA bands, each amplified by specific PCR primers, represent WPRE (upper), ROSA (middle), and ATP4BCre (lower) (see Fig. S2A). (I) Immunohistochemical detection of GCaMP3-ML1 and H+/K+-ATPase α subunit in corpus tissues. Scale bar, 100 μm. (J) Immunoblot demonstration of GCaMP3-ML1 expression in ML1PC corpus glands. (K) Whole-endolysosome IML1 in ML1PC parietal cells. (L) IML1 current densities in parietal cells. (M, N) ML-SI4 (10 μM) annulment of constitutive NIPR in ML1PC parietal cells. (O) ML-SI4 effects on ML1PC -cell NIPR. (P) Plasma gastrin levels of overnight-fasted WT (control, histamine, and ML-SA5), ML1 KO, ML1PC, and ML1 KO: ML1PC mice. Quantitative data are means ± SEM from ≥3 experiments. *P < 0.05, **P < 0.01, ***, P < 0.001 one-way ANOVA, Bonferroni’s post-hoc analysis (G, P) or Student’s t-test (C, F, O).
Fig. 3
Fig. 3. TV-localized ML1 mediate Ca2+ release from TVs
(A) Dual-STED images of WT corpus tissues immuno-labeled with anti-HK-α and anti-ML1. A pre-incubation of anti-ML1 with ML1 epitope peptide confirmed the specificity of the ML1 antibody. Scale bar, 5 μm. (B) Dual-STED images of ML1PC corpus tissues immuno-labeled with anti-HK-α and anti-GFP (recognizing GCaMP3-ML1). Note that constitutive TV exocytosis in ML1PC cells was blocked by ML-SI4 for this particular experiment. (C) Quantitative co-localization analysis of ML1 and H/K-ATPase based on randomly-selected images, as shown in A & B. (D) Immuno-gold electron microscopy images of WT and ML-SI4-treated (to prevent constitutive TV exocytosis) ML1PC parietal cells. Scale bar, 0.1 μm. (E) Gradient centrifugation purification of TVs from WT parietal cells. P1 (3,200 ×g), P2 (20,000 ×g), and P3 (100,000 ×g) pellets represent plasma membrane (PM)-rich, lysosome/mitochondria (LY), and H+/K+-ATPase-rich fractions (TV), respectively. (F) The expression of ML1, H+/K+-ATPase, and VAMP2 in TVs that were immunoisolated by anti-HK-α from P3 TV-derived membranes in E. IgG was used as a negative control. Western blot analyses were performed in both bound (B) immunoisolated vesicles and unbound (U) supernatants using P3 (see E) as a comparison. Shown were representative blots of three separate experiments. (G, H) Effects of GPN pretreatment on ML-SA5-induced Ca2+ release (GCaMP3 fluorescence, F480, in zero Ca2+ solution) from ML1PC parietal cells (G) or GCaMP3-ML1-transfected HEK293 cells (H). Cells were pretreated with ML-SI4 for 30 min to prevent TV exocytosis. (I) Quantitation of ML-SA5-induced GCaMP3 Ca2+ responses under control and GPN-pretreated conditions (mean ± SEM, n = 30–50 cells from ≤4 coverslips/experiment). ***P < 0.001 Student’s t-test. (J) TEM images of free TVs and apical canaliculi (A) in parietal cells. Scale bar, 0.5 μm.
Fig. 4
Fig. 4. ML1 is necessary and sufficient to trigger TV exocytosis
(A) Anti-HK-α immunocytochemistry with phalloidin-labeled F-actin. Cells were treated with ML-SA5 (10 μM) or histamine (50 μM+ 10 μM IBMX) in the presence or absence of ML-SI3 (20 μM) or ML-SI4 (10 μM) for 30 min at 37 °C. Scale bar, 10 μm. Blue and white arrows indicate basolateral and apical membranes, respectively. (B) VAC total surface area, an index of TV exocytosis associated with gastric acid secretion, under various treatment conditions (average of 3–6 z-plane sections/cell). (C) ML-SI4 (10 μM) effects on constitutive VAC formation in ML1PC parietal cells. Scale bar, 10 μm. (D) ML-SI effects on VAC total surface area. Bar graphs (B, D) show means ± SEM from ≥3 experiments. ***P < 0.001 one-way ANOVA, Bonferroni’s post-hoc analysis. (E) Summary of the percentages of large TVs as determined from the G2 gate (> 1 μm) of FSC/SSC dot plots. Mean ± SEMs from 4 experiments were shown. *P < 0.05, **P < 0.01; Student’s t-test.
Fig. 5
Fig. 5. ML1 promotes polarized TVs trafficking towards apical membranes
(A) Patch-clamp recording from basolateral (standard whole-cell recording) or apical (lyse cell to expose VACs before whole-VAC recording) membranes. Note that the extracellular side of the apical membrane is facing the VAC lumen. (B, C) Representative whole-VAC (apical membrane) recordings of ML1-like currents in histamine-stimulated WT (B) and ML1PC (C) parietal cells. (D, E) Representative whole-cell (basolateral membrane) detection of IML1 in WT and ML1PC cells. (F) Group averages of data from experiments shown in panels BE (n = 3–5 patches/condition). Bar graphs show means ± SEM from ≥3 experiments. *P < 0.05, **P < 0.01, ***P < 0.001 one-way ANOVA, Bonferroni’s post-hoc analysis.
Fig. 6
Fig. 6. Histamine evokes ML1-mediated TV Ca2+ release through cAMP/PKA signaling
(A, B) Effects of cAMP (A) and forskolin (B) on NIPR in WT parietal cells. (CE) PKA inhibitor H89 (20 μM) (C) or Ca2+-chelator BAPTA-AM (20 μM) (D) block histamine-stimulated NIPR. Data were summarized in (E). (F) H89 and BAPTA-AM effects on VAC formation induced by histamine (50 μM + 10 μM IBMX). Scale bar, 10 μm. (G) Forskolin (50 μM) or 8-Br-cAMP (20 μM) stimulate VAC formation, measured as total surface area. (H–K) Histamine (50 μM) induced TV Ca2+ release in GCaMP3-ML1-expressing ML1PC cells (See also Movie S4), was blocked by ML-SI4 (10 μM) (I) or H89 (20 μM) (J); experiment averages in (K). (L, M) ML-SI4 (10 μM) blocked forskolin (50 μM)-induced Ca2+ oscillations in ML1PC parietal cells (see also Movie S5). (N and O) ML-SI4 (10 μM) blocked 8-Br-cAMP (20 μM)-induced Ca2+ oscillations in ML1PC cells (See also Movie S6). (P) Whole-endolysosomal IML1 in WT parietal cells with and without 8-Br-cAMP (20 μM) and H89 (20 μM). (Q) Mean current densities under treatments in P. Mean ± SEMs from ≥3 experiments are shown. *P < 0.05, **P < 0.01, ***P < 0.001 one-way ANOVA, Bonferroni’s post-hoc analysis (E) or Student’s t-test (K, M, O, Q).
Fig. 7
Fig. 7. Signaling pathways that mediates histamine-stimulated ML1-dependent TV exocytosis and acid secretion
Activation of type 2 histamine receptor (H2Rs) in parietal cells by histamine induces cAMP-dependent PKA activation. Localized on TVs, ML1 may mediate a PKA-sensitive Ca2+ release pathway to trigger Ca2+-dependent fusion of TVs with each other and with apical membranes. Histamine-induced exocytosis of H+/K+-ATPase-enriched TVs toward lumen-facing apical membranes in a cAMP/PKA-dependent manner.

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