Molecular determinants of peri-apical targeting of inositol 1,4,5-trisphosphate receptor type 3 in cholangiocytes

Isolation and culture of mouse bile duct units

Isolated bile duct units (IBDUs) were prepared from 8–10-week-old male C57BL/6J mice in the Cell Isolation Core Facility of the Yale Liver Center. Details about the methods used for isolation and phenotypic characterization have been described previously.^{[4, 14, 15]} All animal studies were approved by the Yale University Institutional Animal Care and Use Committee under protocol No. 2018–07602. Briefly, mice were anesthetized with isoflurane and livers were perfused first with Hanks A medium, and then with Hanks B medium containing collagenase (Cat. #C0130; Sigma-Aldrich) and trypsin inhibitor (Cat. #17,075,029; Thermo Fisher Scientific) through the portal vein. Portal tissue residue was separated, digested, and filtered, and fragments between 30-μm and 100-μm diameter were plated onto Matrigel-coated coverslips (Cat. No. 354,234; Corning Inc.) and incubated at 37°C in a 95% O₂/5% CO₂ atmosphere in Alpha Essential Medium Eagle media supplemented with insulin, fetal calf serum, L-glutamine, gentamycin, penicillin, and streptomycin (Thermo Fisher Scientific).

Immunofluorescence in IBDUs

Immunofluorescence was performed as described previously.^[4] Briefly, IBDUs were treated with or without 5 mm of methyl-beta-cyclodextrin (MβCD) (Cat. No. C4555; Sigma-Aldrich) for 30 min at 37°C, then fixed and permeabilized. After blocking, IBDUs were labeled with a rabbit polyclonal anti-ITPR3 (1:200; Cat. No. HPA003915;Sigma Aldrich), washed with phosphate-buffered saline (PBS), and incubated with an anti-mouse Alexa 488 (1:500; Thermo Fisher Scientific). For negative control, the primary antibody was omitted. IBDUs were double-labeled with TO-PRO-3 to observe nuclei (1:400; Cat. #T3605; Thermo Fisher Scientific). Specimens were excited at 488 nm and observed at 505–550 nm to detect Alexa 488 and excited at 633 nm and observed above 650 nm to detect TO-PRO-3. Images were collected using a Zeiss LSM 710 Confocal Microscope (Carl Zeiss).

Ca²⁺ measurements in IBDUs

Ca²⁺ experiments were performed 48 h after plating, using bile duct units with visible, sealed lumens. Cytosolic Ca²⁺ (Ca²⁺i) measurements were performed by time-lapsed confocal microscopy. To disrupt lipid rafts, IBDUs were treated with the cholesterol dispersing agent, 5 mm MβCD, for 30 min at 37°C, and controls were treated with vehicle (H₂O), then incubated with 6 μm Fluo-4/AM (Cat. #F23917; Thermo Fisher Scientific) for 30 min at 37°C. Coverslips containing the dye-loaded tissue were transferred to a chamber on the stage of a Zeiss LSM 710 Confocal Microscope, perfused with a HEPES-buffered solution with or without 50 μm adenosine triphosphate (ATP) (Cat. #A6419; Sigma-Aldrich) to induce Ca²⁺ release. Ca²⁺_i was monitored by excitation at 488 nm and detection at >515 nm at a rate of approximately 1 frame/s using a ×40 water immersion objective. Changes in fluorescence (F) were normalized by initial fluorescence (F₀) and are expressed as (F/F₀) × 100%. The rise time of the Ca²⁺ signal was calculated as the time in seconds between 25% and 75% of the maximal Ca²⁺ response.^{[4, 9]}

Ca²⁺ and apical secreted liquid in polarized cholangiocytes

Wild-type (WT) and CAV1KO cholangiocytes were cultured in Transwell inserts until fully polarized, and confirmed by transepithelial resistance measurements. For Ca²⁺ measurements, cells were transfected with an adenovirus vector encoding the fluorescent Ca²⁺ sensor GCamp6f (Cat #1910; Vector Biolabs). Two days following transfection, ATP-induced Ca²⁺ signals were imaged at three successive focal planes corresponding to the apical, midpoint, and basal pole of the cells. Cells were imaged on a Bruker Opterra Swept-Field confocal microscope at ×40 magnification at a frequency of 100 Hz. Cells were excited at 488 nm, and emission was collected at 5 > 05 nm by an EMCCD camera. Fluorescence was normalized as described previously.

Fluid secretion was measured by changes in ASL as described previously.^[16] Polarized WT and CAV1KO cells were loaded with the plasma membrane dye CellMask Orange (Cat. #C10045; Thermo Fisher Scientific) for 15 min at 37°C, followed by addition of 20 ml FITC Dextran-4000 MW (Cat. #D1844; Thermo Fisher Scientific) to the apical side, which was then layered with 80 ml of the inert oil Fluorinet FC70 to prevent evaporation (Cat. #F9880; Millipore Sigma) for 30 min. To induce cyclic adenosine monophosphate (cAMP) formation, Forskolin (FSK; Cat. #F6886; Sigma Aldrich) and 3-Isobutyl-1-methylxanthine (IBMX; Cat. #I5879; Sigma Aldrich) were added basolaterally for 30 min at 50 mm and 10 mm, respectively. Next, five z-stacks per insert were collected at ×40 magnification on a Zeiss LSM 710 confocal microscope, and the apical surface liquid (ASL) area was quantified in xz or yz slices using ImageJ.

MDCK and Cos-7 cell culture

MDCK II or Cos-7 cells (ATCC) were cultured at 37°C in 5% CO₂ in Dulbecco's modified Eagle's medium with high glucose (Thermo Fisher Scientific) containing 10% fetal bovine serum, 1 mm sodium pyruvate, 100 units/ml penicillin, and 100 μg/ml streptomycin (Thermo Fisher Scientific).

ITPR3 C-terminal constructs

The C-terminal fragment of the human ITPR3 (2357–2671 aa, accession No. Q14573) was ordered from IDT as a synthetic gene and then subcloned in pmCherry-C1 (Cat. #632524; Takara Bio USA, Inc.). Plasmids were digested with AgeI and BamH1, purified, ligated, and sequenced. This mCherry-ITPR3-C-term construct was used as a template to yield the C-terminal segment lacking the last five aa (mCherry-ITPR3-C-term-5aa).

Immunofluorescence

Confocal and superresolution immunofluorescence were performed as described previously.^[17] Briefly, MDCK cells were labeled with mouse monoclonal anti-ITPR3 (1:200; Cat. #610313; BD Biosciences), mouse monoclonal anti–zona occludens 1 (ZO-1) conjugated to Alexa-647 (1:200; Cat. #MA3-39100; Thermo Fisher Scientific), rabbit polyclonal anti–myosin heavy chain 9 (MYH9) (1:200, Cat. #PA5-17025; Thermo Fisher Scientific), and rabbit polyclonal anti-Caveolin 1 (1:100; Cat. #PA5-17447; Thermo Fisher Scientific). These antibodies were incubated with secondary antibodies conjugated to Alexa Fluor 488 or 546 (1:1000; Thermo Fisher Scientific). Hoechst 33342 (1 μg/ml; Cat. #H3570; Thermo Fisher Scientific) was used as a nuclear marker. Images were collected using a Zeiss 880 Airyscan microscope using a ×63, 1.4 NA objective lens. Images were processed using Zeiss ZEN software.

Transfection

MDCK cells were transfected with 1 μg or 2 μg mCherry-ITPR3-C-terminus or mCherry-ITPR3-C-terminus-5aa plasmids using Lipofectamine 2000 (1:3), according to the manufacturer's instructions (Cat. #11668019; Thermo Fisher Scientific). Then, cells were selected with 700 μg/ml of Geneticin (Cat. #10131027; Thermo Fisher Scientific) after 48 h. To select for cells with similar expression levels, cells were sorted by BD FACSAria II. For immunoprecipitation (IP), immunoblot, or immunofluorescence analysis, cells were plated in 6-well plates on collagen-coated coverslips (Cat. #354089; Corning). Cos-7 cells were co-transfected with BFP-KDEL plasmid (Plasmid #49150; Addgene) to confirm endoplasmic reticulum (ER) localization of the constructs.^{[18, 19]}

Primary mouse cholangiocyte isolation and culture

Mouse cholangiocytes were isolated from 8–10-week-old male C57BL/6J WT mice and cultured as previously described.^[16] Briefly, microdissected intrahepatic bile ducts were used to obtain WT or CAV1-knockout cholangiocytes.^[20] Cells were cultured on rat-tail collagen and subcultured when confluent. Cells were used up to the eighth passage.

IP and western blot

IPs were done as described previously.^[21] MDCK cells and primary mouse cholangiocytes were lysed in buffer containing 1% Triton X-100, 150 mm NaCl, 10 mm Tris–HCl pH 7.5, 1 mm ethylene diamine tetraacetic acid, 1 mm ethylene glycol tetraacetic acid, 0.5% NP-40, 10% sucrose, and with protease and phosphatase inhibitor cocktail (Cat. #78445; Thermo Fisher Scientific) at 4°C for 30 min. In the CAV1 IP, 60 mm of n-octyl-D-glucopyranoside (Cat. #O3757; Sigma Aldrich) was included in the lysis buffer. Lysates were centrifuged for 20 min at 18,000 g to pellet the insoluble fraction. Soluble fractions were collected, and 500 μg/ml of lysates was incubated with 5 μg/ml ITPR3 antibody (BD Biosciences) for 1 h at 4°C. Protein A/G magnetic agarose beads (Cat. #78609; Thermo Fisher Scientific) was added to each sample. The protein lysate and beads were incubated at 4°C under rotary agitation for 4 h. Beads were pelleted using a magnetic stand and washed in PBS. Proteins were resolved in 4%–20% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblots were performed^[17] using mouse monoclonal anti-ITPR3 (1:200; Cat. #610313; BD Biosciences), rabbit polyclonal anti-MYH9 (1:1000; Cat. #PA5-17025; Thermo Fisher Scientific), rabbit polyclonal anti-caveolin 1 (1:1000; Cat. #PA5-17447; Thermo Fisher Scientific), and mouse monoclonal anti-αTubulin (1:2000; Cat. #T6199; Sigma-Aldrich). Membranes were developed using ECL Western Blotting Substrate (Cat. #32209; GE Healthcare Life Sciences), and films were scanned and analyzed using ImageJ software (http://rsbweb.nih.gov/ij/). Antibodies for MYH9 and CAV1 were validated with at least two specific small interfering RNAs (siRNAs).

Mass spectrometry

Mass spectrometry (MS) was performed at Yale's Keck Laboratory, as described previously.^{[22, 23]} WT MDCK cells, primary mouse cholangiocytes, mCherry-ITPR3-C-term, and mCherry-ITPR3-C-term-5aa expressed in MDCK cells were immunoprecipitated using mouse anti-ITPR3 or rabbit anti-mCherry (Cat. #PA5-34974; Thermo Fisher Scientific). IPs were collected, fractionated by SDS-PAGE, and visualized with Coomassie blue (Cat. #B8647; Sigma Aldrich). Gel bands were cut and submitted to Gel Protein ID proteomic analysis. Products were digested and fractionated by high-performance liquid chromatography interfacing an electrospray ionization quadrupole time-of-flight mass spectrometer. Tandem mass spectrometry (MS-MS) spectra were searched using the Mascot algorithm (www.matrixscience.com).^[24] A protein was considered identified when there are two or more peptide matches, each with Mascot ion score > 30. The National Center for Biotechnology Information database was used. MS data were analyzed using Scaffold Q+/Q+S Software version 4.0 (www.proteomesoftware.com).^[23]

siRNA transfection

Polarized MDCK cells for 10 days in culture were transfected on day 7 using Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific) as described previously.^{[17, 25]} A total of 50 nm siRNAs were used for MYH9 or CAV1 knockdown. CAV1 sequence 13.6 5′-AAGAUGUGAUUGCAGAACCAGAAGG-3′ was from Integrated DNA Technologies (IDT; Cat. #210380101); myosin-9 sequence 13.5 5′-GGAGCCAACAUUGAGACUUAUCUTC-3′ was from IDT (Cat. #258431039); myosin-9 sequence 201 5′-CGUCAAGUCCAAGUACAAAtt-3′ was from Life Technologies (Cat. #s553201); and sequence 202 5′-GUAUCUCUAUGUGGAUAAAtt-3′ was from Life Technologies (Cat. #s553202). A total of 50 nm of the following nontargeting siRNAs were used as negative controls: Silencer Select Negative Control No. 2 siRNA (control siRNA 1; Cat. #4390846; Life Technologies) and IDT Scrambled negative control DsiRNA (control siRNA 2; 5′-CUUCCUCUCUUUCUCUCCCUUGUGA-3′; Cat. #51011908). Further analysis was performed 72 h after transfection.

RNA extraction and quantitative polymerase chain reaction analysis

Total RNA was extracted from liver tissues or cells using the RNeasy Mini Kit (Qiagen). The reverse-transcription reaction was performed with 1 μg of total RNA using iScript complementary DNA synthesis according to manufacturer's instructions (BioRad), and relative messenger RNA (mRNA) expression of selected genes was quantified with Taqman primers for mouse ITPR3 or canine MYH9 in an ABI 7500 real-time polymerase chain reaction (PCR) system (Thermo Fisher). Expression levels were normalized with reference genes 18S and HPRT (hypoxanthine-guanine phosphoribosyl transferase) and analyzed using the 2^(−ΔΔCt) method.^[26]

IBDU from CAV1 knockout mice

The caveolin-1 null (CAV1 KO) mouse model was generated by homologous recombination as previously described.^[20] IBDUs were prepared from 8–10-week-old male C57BL/6J control or CAV1 KO mice as described previously. The isolated IBDUs were used for immunofluorescence or immunoblot, as described previously.

Immunofluorescence in formalin-fixed, paraffin-embedded-tissue

Immunofluorescence in formalin-fixed, paraffin-embedded (FFPE) tissue was performed as previously described.^[27] Tissue sections from WT or CAV1 KO mice were dewaxed, rehydrated, and unmasked in trilogy solution (Cell Marque) using a steamer (95°C) for 20 min, then rinsed in PBS (137 mm NaCl, 2.7 mm KCl, and 10 mm phosphate buffer solution, pH 7.4) (Sigma-Aldrich), and then incubated in PBS containing 0.2% Triton X-100 (Cat. #T9284; Sigma-Aldrich) for another 20 min and blocked in PBS containing 1% bovine serum albumin (Sigma-Aldrich) for 30 min. Antibody incubation for anti-ITPR3, anti-cytokeratin-19 (Cat. #AB_2133570; DSHB), was performed as described previously. Primary antibodies were omitted for negative controls. Images were collected using a Zeiss LSM 880 confocal microscope using an oil ×40 1.3 NA objective. ZEN software was used to quantify fluorescence.

Statistical analysis

Results are presented as mean ± SD or ± SEM. Data were analyzed using GraphPad Prism 8 and Microsoft Excel. Differences between experimental groups were assessed for significance using Student's t test or one-way analysis of variance followed by Bonferroni post-hoc tests. Statistical significance was defined as p < 0.05.

ITPR3 is apical in cholangiocytes, and disruption of lipid rafts redistributes ITPR3 and impairs Ca²⁺ waves

Disruption of lipid rafts redistributes ITPR2 from the apical region in hepatocytes,^[28] so we examined whether lipid rafts play a similar role in targeting of ITPR3 in cholangiocytes. Lipid rafts were disrupted in IBDUs by treatment with the cholesterol-dispersing agent methyl-beta-cyclodextrin (MβCD) (5 mm; 30 min at 37°C). This led to the redistribution of ITPR3 from the apical region to a more diffuse cytoplasmic localization (Figure 1A). Analysis of the effect of MβCD treatment on Ca²⁺ signaling showed reduced amplitude of ATP-induced Ca²⁺ transients (Figure 1B,C). These results are consistent with the idea that lipid rafts in cholangiocytes are needed to maintain ITPR3 in proximity to the apical membrane, which in turn establishes Ca²⁺ waves in these cells.

ITPR3 is apical in MDCK cells, and the C-terminus is responsible for apical targeting

To investigate the molecular mechanisms responsible for apical localization, MDCK cells were used because this cell line has several characteristics in common with cholangiocytes. Both are epithelial cells that form adherens junctions, tight junctions, and primary cilia, and ITPR3 is the most abundant ITPR isoform in both cell types.^{[5, 29, 30]} MDCK cells were cultured in trans-wells, as this system allows for full polarization, which was confirmed by measurements of transepithelial resistance and visual microscopic inspection. The distribution of ITPR3 was observed by confocal immunofluorescence after 10 days in culture. It was mostly concentrated near the plasma membrane in the vicinity of the tight junction-associated protein ZO-1 and sparsely distributed along the rest of the apical membrane (Figure 2A). This clustering of ITPR3 at the apical pole and in proximity to ZO-1 in MDCK cells was also visualized in x-y and x-z projections (Figure 2B). The relative subcellular distribution of endogenous ITPR3 was measured by comparing ITPR3 in the apical and basolateral region. ITPR3 in the apical region accounted for most of the ITPR3 in polarized MDCK cells (98.36 ± 5.43%, p < 0.0001) (Figure 2C). There is significant variability in the C-terminal amino acids among the three ITPRs isoforms, especially in the five most c-terminal residues, so we hypothesized that this region contains isoform-specific targeting information. To test this hypothesis, we fused the C-terminus of mCherry to the two last transmembrane domains of ITPR3 to create the construct mCherry-ITPR3-C-term (Figure 2D). First, we investigated the distribution and subcellular localization of the mCherry-ITPR3-C-term. Cos-7 cells were used because this cell line has an expansive tubular ER network that facilitates live cell imaging of this organelle. This cell line was co-transfected with the mCherry-ITPR3-C-term construct and blue fluorescent protein (BFP) with a KDEL sequence peptide, thus functioning as an ER marker. mCherry-ITPR3-C-term and BFP-KDEL exhibited similar subcellular reticular localization, confirming ER targeting of the mCherry-ITPR3-C-term construct (Figure 2E). Next, a MDCK cell line that stably expresses mCherry-ITPR3-C-term was generated to examine whether this segment of the protein is responsible for apical targeting of ITPR3. The mCherry-ITPR3-C-term–fused protein showed a similar localization to the endogenous ITPR3 at the apical region that also coincided with ZO-1 localization, as observed by superresolution microscopy (Figure 2F). Orthogonal projections confirmed that endogenous ITPR3 and mCherry-ITPR3-C-term were similarly localized to the apical region (Figure 2F,G) and are mostly absent of the basolateral region. Together, these results demonstrate that the C-terminus of ITPR3 contains apical targeting information.

ITPR3 interacts with MYH9 and CAV1 in polarized epithelia

Next, proteomic analysis was performed to identify potential interacting partners of ITPR3 that determine its apical localization. First, endogenous ITPR3 was immunoprecipitated in polarized (10 days in culture) and semipolarized (5 days in culture) MDCK cells, as well as in nonpolarized (1 day in culture) cells, followed by western blot analysis. This method showed that the monoclonal antibody used successfully immunoprecipitated ITPR3 in MDCK cells (Figure 3A). No ITPR3 was observed in the control immunoglobulin G groups. IP products were subject to MS, which showed that ITPR3 in MDCK cells is more abundant after 10 days in culture than after 5 days (Figure 3B). MYH9 was the most enriched protein in the ITPR3 immunoprecipitant in fully polarized MDCK cells, when compared with either nonpolarized or partially polarized cells (Figure 3C). The top protein candidates comparing immunoprecipitant from MDCK cells in culture for 10 days versus 5 days or 1 day are listed in Tables 1 and 2, respectively. Next, co-IP of ITPR3 and MYH9 confirmed this interaction in MDCK cells (Figure 3D) as well as in mouse cholangiocytes (Figure 3E). CAV1 was also identified as an interacting partner of ITPR3 in the mass spectrometry results. Although no difference was observed in the amount of CAV1 in MDCK cells in culture for 5 or 10 days (data not shown), co-IP confirmed the interaction between CAV1 and ITPR3 in these cells (Figure 3F). Together these results demonstrate that MYH9 and CAV1 bind to ITPR3 in polarized epithelia and suggest that they may play a role in the targeting of this receptor.

The last five amino acids of ITPR3 C-terminus interact with MYH9

Certain proteins that are targeted to the apical membrane, such as those containing PDZ domains, typically recognize five residues of the C-terminal tail of its partners.^[31] Therefore, a stable MDCK cell line expressing the mCherry-ITPR3 C-term lacking the last five amino acids (mCherry-ITPR3-C-term-5 aa) was developed to investigate the importance of these residues for ITPR3 apical localization and binding to MYH9. The mCherry-ITPR3 C-term was most concentrated near the apical membrane, whereas the construct lacking the five last aa's was less concentrated there, as shown by superresolution imaging (Figure 4A), orthogonal projections (Figure 4B), and quantification of the plasma membrane over cytosolic fluorescence of these two constructs (Figure 4C). Similarly, endogenous ITPR3 co-localized more strongly with the full-length c-terminus construct (Figure 4D). Next, proteomic analysis of the immunoprecipitant was used to compare proteins that interact with mCherry-ITPR3-C-term and mCherry-ITPR3-C-term-5aa in polarized MDCK cells. More MYH9 was bound to mCherry-ITPR3-C-term than to the construct lacking the last five amino acids (Figure 4E). The normalized total spectra confirmed this finding (Figure 4F). The five leading protein candidates identified by the proteomic analysis comparing MDCK cells expressing mCherry-ITPR3-C-term with mCherry-ITPR3-C-term-5aa are listed in Table 3. Together these results suggest that the last five amino acids of the ITPR3 C-terminus are necessary for interaction with MYH9.

CAV1 or MYH9 knockdown displaces ITPR3 from the apical region of MDCK cells

The role of CAV1 and MYH9 in the apical localization of ITPR3 was investigated in several ways. First, CAV1 was knocked down with siRNAs (Figure S1A,B). ITPR3 protein expression displayed a trend toward reduction (~25%) after CAV1 knockdown on WT-MDCK cells cultured for 10 days by western blot; however, this decreased expression did not achieve statistical significance (p = 0.1744) (Figure S1C). However, CAV1 knockdown reduced ITPR3 in the apical region by 77.5 ± 20.5% (p = 0.0054) (Figure 5A,C). Next, the effect of MYH9 knockdown on ITPR3 localization was investigated. Confocal immunofluorescence showed that apical expression of endogenous ITPR3 was similarly reduced by 53.5 ± 12.2% (p = 0.0023) (Figure 6A,D). These results provide evidence that both MYH9 and CAV1 play a role in localizing ITPR3 to the apical region of MDCK cells.

Apical ITPR3 expression is reduced in bile ducts from CAV1 knockout mice

To understand the generalizability of these findings, IBDUs were isolated from WT and CAV1 KO mice. Only these KO mice were examined, because MYH9 KO mice die in utero due to defects in heart and brain development.^[32] The CAV1 knockout was confirmed in IBDUs by western blot (Figure 7A). ITPR3 expression was decreased by 98.4 ± 2.3% (p < 0.0001) in IBDUs isolated from CAV1 KO mice relative to WT IBDU (Figure 7B). Confocal imaging furthermore demonstrated reduced ITPR3 localization at the apical region of IBDUs from CAV1 KO, when compared with IBDUs from WT mice (Figure 7C,D). ITPR3 expression at the apical region was reduced by 46.9 ± 18.7% (p < 0.05) in CAV1 KO IBDUs (Figure 7E). This reduction in ITPR3 protein expression in CAV1KO mice was not likely due to transcriptional regulation, as quantitative PCR analysis revealed no difference in mRNA levels of all ITPR isoforms (Figure 7F). These results provide evidence that CAV1 is important for apical localization of ITPR3 in cholangiocytes, just as it is in MDCK cells.

Ca²⁺ signaling and secretion are impaired in mouse cholangiocytes lacking CAV1

Apical Ca²⁺ signals are important for Ca²⁺ waves ^[5] and secretion,^[4] so we investigated whether CAV1 KO cholangiocytes have impaired Ca²⁺ signaling. Polarized preparations of WT and CAV1 KO cholangiocytes expressing the fast Ca²⁺ sensor GCamp6f were imaged at three focal planes (apical, midsection, and basal) following stimulation with ATP. The amplitude of the Ca²⁺ signal in each focal plane was lower in CAV1 KO when compared with WT cells (Figure 8A,B). Additionally, Ca²⁺ signals in WT cells displayed a trend toward starting at the apical pole more frequently when compared with the CAV1 KO cells (Figure 8C). These results are consistent with the notion that apical to basolateral Ca²⁺ signaling in polarized cholangiocytes depends on CAV1.

Next, the functional role of CAV1 in cholangiocyte secretion was assessed in a modified fluid secretion assay.^[16] In these studies, the expansion of a layer of extracellular fluorescent dextran on the apical surface of polarized cholangiocytes was used to reflect fluid secretion (Figure 8D). The ASL area was significantly increased in WT cells treated with a cAMP cocktail (forskolin + IBMX), which promotes bicarbonate and fluid secretion in cholangiocytes.^[4] However, this treatment failed to promote ASL area expansion in CAV1 KO cholangiocytes (Figure 8E). These results support the idea that the presence of CAV1 to ensure the proper localization of ITPR3 plays a role in apical fluid secretion in cholangiocytes (Figure 8F).