Myenteric plexus interstitial cells of Cajal (ICC-MY) in the small intestine are Kit+ electrical pacemakers that express the Ano1/TMEM16A Ca2+-activated Cl– channel, whose functions in the gastrointestinal tract remain incompletely understood. In this study, an inducible Cre-LoxP-based approach was used to advance the understanding of Ano1 in ICC-MY of adult mouse small intestine. KitCreERT2/+;Ano1Fl/Fl mice were treated with tamoxifen or vehicle, and small intestines (mucosa free) were examined. Quantitative RT-PCR demonstrated ~50% reduction in Ano1 mRNA in intestines of conditional knockouts (cKOs) compared with vehicle-treated controls. Whole mount immunohistochemistry showed a mosaic/patchy pattern loss of Ano1 protein in ICC networks. Ca2+ transients in ICC-MY network of cKOs displayed reduced duration compared with highly synchronized controls and showed synchronized and desynchronized profiles. When matched, the rank order for Ano1 expression in Ca2+ signal imaged fields of view was as follows: vehicle controls>>>cKO(synchronized)>cKO(desynchronized). Maintenance of Ca2+ transients’ synchronicity despite high loss of Ano1 indicates a large functional reserve of Ano1 in the ICC-MY network. Slow waves in cKOs displayed reduced duration and increased inter-slow-wave interval and occurred in regular- and irregular-amplitude oscillating patterns. The latter activity suggested ongoing interaction by independent interacting oscillators. Lack of slow waves and depolarization, previously reported for neonatal constitutive knockouts, were also seen. In summary, Ano1 in adults regulates gastrointestinal function by determining Ca2+ transients and electrical activity depending on the level of Ano1 expression. Partial Ano1 loss results in Ca2+ transients and slow waves displaying reduced duration, while complete and widespread absence of Ano1 in ICC-MY causes lack of slow wave and desynchronized Ca2+ transients.
NEW & NOTEWORTHY The Ca2+-activated Cl− channel, Ano1, in interstitial cells of Cajal (ICC) is necessary for normal gastrointestinal motility. We knocked out Ano1 to varying degrees in ICC of adult mice. Partial knockout of Ano1 shortened the widths of electrical slow waves and Ca2+ transients in myenteric ICC but Ca2+ transient synchronicity was preserved. Near-complete knockout was necessary for transient desynchronization and loss of slow waves, indicating a large functional reserve of Ano1 in ICC.
View this article's corresponding video summary at https://youtu.be/cyPtDP0KLY4.
- slow wave
- Ca2+ transients
- ion channel
- genetic knockout
over the last two decades, interstitial cells of Cajal (ICC) emerged as key determinants of gastrointestinal physiology. Primary physiological functions attributed to ICC include pacemaker slow-wave generation, smooth muscle membrane potential regulation, mediation of neuromuscular transmission, mechanosensation, and mechanotransduction (12, 19, 46). ICC are specialized cells found in specific locations in gut musculature very close to smooth muscle cells, neurons, macrophages and other interstitial cells such as fibroblast-like cells, also known as platelet-derived growth factor receptor-α cells (32, 47, 49). Among the various cellular markers of ICC, the Kit tyrosine kinase receptor is the most commonly recognized at present (10, 22, 51, 60). The Kit receptor and its ligand, Steel Factor, are essential regulators of ICC development and maintenance, with the loss of Kit expression occurring in aging and in gastrointestinal dysfunction states such as gastroparesis and slow-transit constipation (reviewed in Refs. 12, 19, 46, 47).
The pacemaker electrical slow wave, a regular oscillation in the membrane potential, originates in ICC and spreads to the smooth muscle regulating its contractility. During the slow-wave plateau phase, enhanced excitability facilitates the opening of L-type Ca2+ channels and generation of contractions in smooth muscle cells. Ever since the discovery of the electrical slow wave in the gut (1, 7) and realization of its importance for regulation of gastrointestinal function (54), elucidation of underlying mechanisms and regulation of the slow wave have been targets of primary scientific inquiry. They still remain incompletely understood today. Several ion channels and plasma membrane transporters have been proposed to play a role in slow-wave generation (32, 45–47, 49). Among them, evidence points to a central role of the Ano1 Ca2+-activated chloride channels expressed in ICC.
Ano1 (Anoctamin type 1) channels belong to a family of related ion channels sharing a proposed 8 or 10 transmembrane segment topology. In addition to Ano1, this family also includes Ano2–10/TMEM16B–H, J, and K. Ano proteins can function as Ca2+-activated anion channels (e.g., Ano1), nonselective cation channels, or a lipid scramblases (e.g., Ano6) although precise functions of many members still remain unclear. The most studied and understood member of the family, Ano1, has widespread distribution and diverse physiological roles including mucosal secretion, insulin release, airway and vascular muscle contractility, nociception, cell proliferation, tumorigenesis, and signal transduction (reviewed in Refs. 38, 41, 42). In the gastrointestinal tract, a subset of epithelial (mucosal) cells regulating secretion (50), and all types of ICC (13, 24) express Ano1, supporting regulation of gastrointestinal function. Ano1 has also been reported in anal sphincter smooth muscle (64) but not found in intestinal smooth muscle (18). The precise roles of Ano1 in ICC remain to be elucidated. Ano1 is robustly expressed in ICC subtypes at both transcript and protein levels (13, 24). In vitro cell culture studies revealed a role of Ano1 in ICC proliferation (37, 52). Chloride currents sharing some biophysical and pharmacological properties of recombinant Ano1 currents have been recorded in freshly isolated and cultured ICC (66). Rhythmic chloride currents or voltage oscillations have been measured in ICC in culture or in situ acute muscle preparations (26, 56, 67). Studies using constitutive genetic Ano1 knockouts provided the strongest evidence up-to-date for a role of Ano1 in ICC regulating gastrointestinal function. In the small intestine, constitutive Ano1 knockouts (Ano1KOs) showed loss of slow-wave activity, depolarization of the resting membrane potential, attenuation of contractility in vitro, and abnormal motility (24, 51). Importantly, while myenteric plexus ICC (ICC-MY) still showed Ca2+ transients in Ano1KO small intestine, they were not coordinated implicating a role of Ano1 in coordination or synchronization of Ca2+ transients in ICC-MY network (51). A number of open questions still remained: How precisely does Ano1 regulate the Ca2+ transient activity ensuring synchronization of Ca2+ transients in adjacent ICC in the ICC-MY network? How does Ano1 contribute to electrical slow-wave activity under partial Ano1 knockdown? What degree of Ano1 expression in the network is needed to sustain normal functions given that dysregulation of Ano1 is associated with human disease (36)?
Constitutive genetic knockout models suffer from a number of limitations. The global nature of the knockout lacks specificity and involves all cell types throughout the body contributing to undesired complications. Indeed, constitutive Ano1 knockouts die shortly after birth due to a number of complications including abnormal tracheal development limiting their usefulness in the study of Ano1 functions in adults (44). To study the role of Ano1 in ICC of adult mice, we employed an inducible conditional knockout of Ano1 (icAno1KO) using the Cre-LoxP system where treatment with tamoxifen activated Cre fused to mutated hormone-binding domains of the estrogen (ERT) receptor expressed under control of the ICC-specific Kit promoter (KitCreERT2). Cre then caused excision of Ano1 exon8 flanked by LoxP sites, in-frame deletion, and loss of Ano1 protein expression in affected cells. Such inducible genetic models provided insights for the function of Ano1 in cells other than ICC (2, 8, 9, 16, 64). They also have been utilized in ICC but not yet for targeting Ano1 or any other ion channel (3, 27, 29). We used the inducible conditional Ano1 knockout (icAno1KO) mice to elucidate the role of Ano1 in mediating the functions of ICC-MY, specifically Ca2+ transients and electrical slow waves, and to reveal the effect of moderate-to-severe loss of Ano1 on ICC. The data indicate dependence of the ICC-MY-mediated functions on the level of Ano1 expression. Partial loss of Ano1 results in Ca2+ transients and slow waves both showing reduced duration. Complete absence of Ano1 links to desynchronized ICC-MY Ca2+ transients, absence of slow waves, and depolarization in the musculature. Existence of significant functional reserve of Ano1 in the ICC-MY network ensures synchronicity of the pacemaker activities (Ca2+ transients and slow wave) across the network.
Mouse Strain Generation and Dosing Regimen
The mice used in this study were maintained and the experiments were done with approval from the Institutional Animal Care and Use Committee of Mayo Clinic and cared for in accordance of the guidelines of National Institutes of Health. To generate KitCreERT2/+;Ano1Fl/Fl transgenic mouse strain, two previously established mouse strains, KitCreERT2/+ (27) and Ano1Fl/Fl (9) on a C57/BL6 background, were crossed to establish founders. Genotyping was done on offsprings to confirm genetic linage (KitCreERT2/+;Ano1Fl/Fl). Phenotypically all Ano1Fl/Fl; KitCreERT2/+ mice, just like KitCreERT2/+;Ano1+/+ mice, showed a white spot on the chest or abdomen. At the age of 2–6 mo, KitCreERT2/+;Ano1Fl/Fl mice of either sex were injected intraperitoneally (total injection volume of 120–170 μl) with either vehicle (90% peanut oil and 10% 200 proof ethanol) or tamoxifen (75 mg·kg−1·day−1) dissolved in vehicle for 5–7 days and then left to recover for at least 2 days allowing sufficient time for elimination of tamoxifen and its active metabolite 4-OH-tamoxifen from their bodies (43). Mice were tested on the 3rd to 70th day (~2 mo) following the last dose. There was no obvious difference in the degree of Ano1 protein knockdown over this time scale, indicating that recombination was sustained, and therefore the data from mice were pooled. In 2 of 16 tamoxifen-treated mice, the dose on the first day was 145 mg·kg−1·day−1 and then 75 mg·kg−1·day−1 for the remaining days. In separate experiments to test for possible effects of having one knockout allele for Kit (the KitCreERT2 is a null) and to control for effects of tamoxifen treatment, Kit+/+;Ano1Fl/Fl mice (i.e., expressing only the floxed allele and not Cre recombinase) were treated with tamoxifen for 6–7 days and tested on the 3rd to 9th day following the last delivered dose.
Mice of either sex were killed by carbon dioxide inhalation and jejunum was dissected as a tube and placed immediately in cold oxygenated Krebs-Ringer buffer (KRB; see Solutions and Chemicals section below for constituents) until further dissection. Next a small jejunum segment, ~20–100 mm in length, was cut and placed into a 100- or 150-mm dish coated with Sylgard elastomer (Dow Corning, Midland, MI) and filled with cold KRB. The tube was then cut open along the mesenteric border and pinned out flat with the serosa facing upward. The external muscle layers were carefully peeled off the mucosa. The mucosa was then discarded and whole thickness preparation with intact ICC-MY and deep muscular plexus ICC (ICC-DMP) networks of appropriate size processed for quantitative RT-PCR (qRT-PCR), immunohistochemistry (IHC), Ca2+ imaging, and sharp microelectrode electrophysiology as described previously (51) and outlined below.
The method followed the protocol detailed elsewhere (51). Briefly, total RNA was extracted from snap-frozen muscularis propria with attached serosa and part of submucosa (in liquid nitrogen) using RNA-Bee isolation solvent (Tel-test, Friendswood, TX) according to the manufacturer’s instructions. Reverse transcription was done with a Superscript VILO cDNA synthesis kit (Life Technologies) using 500 ng of RNA and the reaction protocol consisted of annealing at 25°C for 10 min, followed by cDNA synthesis at 42°C for 60 min and the termination of reaction by incubation at 85°C for 5 min. The cDNA (~12.5 ng) was then used for real-time PCR using LightCycler 480 SYBR Green I Master Mix (Roche, Indianapolis, IN) with 120 nM of primers using the Roche LightCycler II. Primers for mouse Ano1 (PPM26917B detecting 97 bp on exon 23) and β-actin (PPM02945A detecting 154 bp on exon 4) were purchased from Qiagen (Valencia, CA). All PCR reactions were done in a total volume of 25 μl with the following sequence: one cycle of initial denaturation for 5 min at 95°C and 45 amplification cycles (denaturation for 10 s at 95°C, annealing for 10 s at 60°C followed by extension for 10 s at 72°C). Expression of Ano1 was normalized to the endogenous control, β-actin, using the formula ΔΔCt = [(CtAno1(tamoxifen/vehicle) − Ctβ-actin(tamoxifen/vehicle)] − [(CtAno1(vehicle) − Ctβ-actin(vehicle)] and the fold change is given by 2e−ΔΔCt, where Ct indicates the cycle number at which the fluorescence signal of the PCR product crosses an arbitrary threshold set within the exponential phase of the PCR amplification.
Whole mount IHC for Kit and Ano1 was carried out according to the method described in Singh et al. (51) with modifications explained below. Tissues were fixed with cold acetone (−20 to 4°C) for 15–30 min and in most cases stored in cold PBS (~4°C) before exposure to antibodies. After a brief washing step in PBS, tissues were blocked with 5% BSA (Sigma) and 0.3% Triton X-100 (Sigma) in PBS for 4–5 h and then incubated with primary antibodies, rat anti-Kit at 1:1,000 (catalog no. 14-1172-85, eBioscience, San Diego, CA) and rabbit anti-Ano1 at 1:1,500 (catalog no. ab53212, Abcam, Cambridge, MA) in 5% BSA and 0.3% Triton X-100 overnight at ~4°C. The next day, tissues were washed with cold PBS (5×5 min) and treated with secondary antibodies (1:500), donkey anti-rat AF488 (catalog no. 712-545-153, Jackson ImmunoResearch Laboratories), and donkey anti-rabbit Cy3 antibody (catalog no. AP182C, Millipore) in the same buffer (5% BSA, 0.3% Triton X-100 in PBS) overnight at 4°C protected from light. The next day, tissues were washed with sterile water (2×5 min), incubated with DAPI for 45 min in the cold (~4°C) protected from light, and washed 5×6 min with cold PBS. Following a brief washing step with water, the labeled tissues were mounted using Slowfade mounting medium (ThermoFisher) and either immediately imaged or stored at −20°C until doing so. Whole mounts were imaged on an upright Olympus confocal FV1000 microscope using a ×20 water objective (numerical aperture 0.95) with laser light excited at 488 or 543 nm.
Calcium Imaging and Data Analysis
Cal520AM dye (AAT Bioquest, Sunnyvale, CA) was used to visualize Ca2+ transients in ICC-MY following the protocol described previously (51). Briefly, small tissue segments were pinned to 50-mm dishes covered with Sylgard and filled with cold KRB solution and incubated with KRB containing 1.8–2.4 μM Cal520AM in 0.02% Cremophor-EL (Sigma) and 0.2% DMSO (Sigma) with or without rat anti-mouse ACK2 (catalog no. 14-1172-85; eBioscience, San Diego, CA) conjugated to Alexa Fluor 555 using an IgG labeling kit (catalog no. A20187, Life Technologies, Carlsbad, CA) for 25–40 min at 30–32°C in the dark. A washout with KRB at 34–36°C was done in the perfusion chamber for at least 20 min to allow deesterification of Cal520AM. Imaging was carried out on an upright Olympus confocal FV1000 microscope using a ×20 water immersion objective (numerical aperture 0.95) with laser light excited at 488 nm to image Cal520AM and 543 nm for AF555-labeled rat anti-mouse ACK2 if included. Single image frames were collected from fields of view (FOVs), ranging from ~160 × 160 μm to ~320 × 320 μm, using the confocal aperture fully open at 34−36°C in the presence of 1–3 μM nicardipine (Sigma) to minimize contractility at the sampling rate of 187 to 553 ms per frame. In a series of experiments, following collection of Ca2+ transients, the same FOVs were continuously exposed to UV laser (405 nm) for at least 1 min to induce an increase in background fluorescence in FOVs. The marked spots were confirmed at reduced magnification and processed for IHC as described above but without DAPI. The presence of Ano1 relative to Kit in ICC-MY network in marked spots was visually quantified by an examiner blinded to the treatment and any other identifiable mouse information other than a randomly generated ID matching the marked spot to Ano1 and Kit immunoreactivities. The areas within FOVs received a score on a scale from 0 (no Ano1 expression in Kit+ cells/pixels) to 10 (maximum indicating full colocalization of both Ano1 and Kit immunoreactivities in ICC-MY). The scoring data were then processed by another investigator and matched with Ca2+ transient activity.
Data were obtained by measuring emitted fluorescence from regions of interest (ROIs) over single ICC. The image files (16-bit, .tif) were analyzed with ImageJ (version 1.46r, https://imagej.nih.gov/ij/) as described previously (51). Movements of the preparations were corrected for, if needed, using an Image J Stabilizer plug-in (http://www.cs.cmu.edu/~kangli/code/Image_Stabilizer.html), and were further reduced by applying a walking average plug-in in ImageJ with the processing of two consecutive frames. Individual ROIs representing Ca2+-induced fluorescence “hot spots” were manually selected using the ROI Manager tool in ImageJ. The average intensity value over time in each ROI was calculated for every frame and expressed as ΔF/Fo before determination of Ca2+ transient synchronicity indexes and other parameters. Plots of ΔF/Fo vs. time were made using the Interactive 3D Surface Plot plugin in Image J (https://imagej.nih.gov/ij/plugins/surface-plot-3d.html). Ca2+ transient synchronicity indexes were determined on the scale of 0 (no synchronicity) to 1 (full synchronicity) among ROIs (from ICC-MY) in the field-of-view (FOV) as explained elsewhere (51). Briefly, for each ROI (an ICC) peaks of Ca2+ transients were detected using a Gaussian derivative-based peak detector in MATLAB (version R2011a). The time value corresponding to each peak value was collated and compared with the activation times in every other ROI in a round-robin manner in the same image stack using an event synchronization measurement. In an image stack with M ROI, M*(M − 1)/2 synchronization values were determined and the average of these values was reported as the final synchronization index of the image stack. Furthermore, for each ROI (defining an ICC) the following parameters of Ca2+ transients were determined: the frequency, coefficient of variability (CV) for frequency (CV = standard deviation/mean*100%), amplitude, CV for amplitude, duration (at half maximum amplitude), and CV for duration. Duration and CV for duration were analyzed when the sampling interval was less than 225 ms per frame. For each FOV, these six parameters were averaged and represent single data points (see Fig. 3). The synchronicity indexes were also included (Fig. 3A).
Electrophysiology and Data Analysis
The protocol followed the procedure steps described elsewhere (51). Briefly, jejunum tissue strips were pinned (serosa down) using fine wire pins (California Fine Wire, Grover Beach, CA or Fine Science Tools, Foster City, CA) to Sylgard-coated dishes (30 or 50 mm in diameter) filled with cold KRB and then transferred to a microscope stage to allow constant perfusion with oxygenated KRB at 35–37°C. The incubation period before microelectrode recordings was at least 1 h in the presence of nicardipine (1–3 μM). Sharp microelectrode recordings were made from smooth muscle cells in the intact musculature. Recordings were most likely made from circular muscle since muscle strips were placed with circular muscle facing upward and approached first with microelectrodes. Glass capillary microelectrodes (borosilicate glass tube, 1.2 mm OD, 0.6 mm ID, 75 mm length, FHC, Bowdoin, ME) were pulled using a P-97 micropipette puller (Sutter Instruments, Novato, CA). Microelectrodes filled with 3 M KCl had tip resistances ranging between 30 and 90 MΩ. Transmembrane potentials were measured using a Duo 773 Electrometer (World Precision Instruments, Sarasota, FL) and a Digidata 1440A (Molecular Devices, Sunnyvale, CA) acquisition system and were stored on a computer running AxoScope 10.0/10.3 software (Molecular Devices). Signals were recorded at a sampling rate of 1 kHz (interval of 1,000 μs). Electrical recordings were carried out in the presence of 1–3 μM nicardipine (Sigma) at 35–37°C to minimize muscle contractility and to aid in obtaining stable impalements. Prior studies have shown that L-type Ca2+ channel blockade does not affect slow-wave generation in mouse small intestine (34, 35). Hence, the inclusion of nicardipine, an L-type Ca2+ channel inhibitor, provided the conditions to compare electrical activities of small intestinal slow waves in vehicle controls and icAno1KO mice.
Data were analyzed offline for resting membrane potential, peak amplitude, rate of rise of upstroke (10–90% slope), frequency, and duration (at half-amplitude) using either template search function in Clampfit 10 (Molecular Devices) as described elsewhere (10) or MiniAnalysis 6 (Synaptosoft, Fort Lee, NJ). The use of both methods gave comparable values when the same set of recordings was analyzed. MiniAnalysis was used to determine the inter-slow-wave interval according to the following determination. For each slow-wave event, the MiniAnalysis 6 software provided the time of the peak amplitude of the upstroke (P), 10–90 rise time (R), and decay time (D), describing the period from the peak amplitude to the point at which the voltage decayed to 5% (user selected) of the peak amplitude (hence near the base of the slow wave at the resting membrane potential). The start time for a slow-wave event denoted x (Sx) corresponded to Sx = Px−Rx, and the whole slow-wave duration (Ux = Rx + Dx). The time interval (Tx) between two consecutive starting points of the slow waves (Tx = Sx+1 − Sx) was calculated and the inter-slow-wave interval (Ix) was obtained from Ix = Tx − Ux. The above calculations were made in Excel. For each recording from a single cell, average inter-slow-wave interval was calculated by averaging individual inter-slow-wave events. Recordings were accepted if they were stable for at least 20 s. For each cell, the six slow-wave parameters were obtained by averaging each of the slow-wave events and then representing the parameters as single data points included in Fig. 6.
Solutions and Chemicals
The Ca2+ imaging bath chamber was constantly perfused with oxygenated KRB having the following composition (mM): NaCl 120.3, KCl 5.9, MgCl2 1.2, NaHCO3 15.5, NaH2PO4 1.2, glucose 11.5, CaCl2 2.5. The pH of the KRB was 7.3–7.4, when bubbled with 97% O2/3% CO2 at 34–37°C. For the Ca2+ imaging and microelectrode experiments, nicardipine (Sigma, St. Louis, MO) was dissolved in ethanol at the stock concentration of 10 mM and added at the indicated concentrations. Cal520AM dye was dissolved in 100% DMSO (Sigma) at 1.8–2.3 mM and used freshly or stored at −20°C. The stock solution of 20% Cremophor EL was prepared fresh daily in DMSO and diluted in the loading solution as indicated. Tamoxifen was dissolved in vehicle (90% peanut oil, 10% ethanol) at 12.5 mg/ml. The sources for other reagents used were as described above in each appropriate section or from Sigma.
Data are reported as means ± SE (normal distribution) or median (25th and 75th percentiles) (nonnormal distribution). Statistical analyses and the distribution pattern (normality test using an appropriate algorithm) were determined in GraphPad Prism ver. 4 or 5. Values were compared using ANOVA followed by Dunn’s multiple-comparison test (if at least one of the data sets failed the normality test) or Tukey's multiple-comparison test (for data sets showing normal distribution), two-tailed Student’s t-test, or Mann-Whitney U-test as appropriate and specified in the text for each comparison made. The n values describe the number of FOVs in Ca2+ imaging experiments or the number of cells impaled in sharp microelectrode electrophysiology studies, and the N defines the number of mice used. A P value of < 0.05 was considered statistically significant.
Attenuation of Ano1 mRNA and Protein Expressions by Tamoxifen-Induced Cre-loxP Recombination of the Ano1 Gene Driven by the ICC-Specific Kit Promoter
In this study, KitCreERT2/+;Ano1Fl/Fl transgenic mice were generated by crossing Kit+/+;Ano1Fl/Fl (9) and KitCreERT2/+;Ano1+/+ (27) strains (see methods for details), and treated with either vehicle or tamoxifen, resulting in controls and ICC-specific inducible conditional Ano1KOs (icAno1KOs), respectively. Quantitative RT-PCR (Fig. 1A) of mucosa-free musculature confirmed reduction of Ano1 expression in ICC networks of the small intestine of icAno1KOs. When normalized to vehicle controls and β-actin levels, the mRNA expression of Ano1 was reduced by ~50% (P < 0.05, Student’s t-test, Fig. 1A). To further confirm the cell-specific loss of Ano1 protein in ICC, whole mount IHC was done for Ano1 and Kit immunoreactivities in the jejunum preparations devoid of mucosa. In vehicle-treated transgenic small intestines, both networks of ICC, ICC-MY (associated with myenteric plexus) and ICC-DMP (associated with deep muscular plexus), robustly coexpressed Ano1 and Kit (Fig. 1, B and C) as described previously (13, 24, 51). In contrast, the small intestines of icAno1KOs showed loss of Ano1 in ICC-MY and ICC-DMP networks (Fig. 1, B and C). The loss, however, was not complete or uniform throughout both networks and included sites displaying severe loss of Ano1 in ICC-MY and ICC-DMP networks and those with minimal loss in both networks in the same mice. Collectively, quantitative RT-PCR and IHC experiments revealed establishment of an inducible conditional knockdown of Ano1 in ICC of the adult small intestine.
ICC-MY Ca2+ Transients in Control Mice
Vehicle-treated KitCreERT2/+;Ano1Fl/Fl transgenic mice showed highly synchronized Ca2+ transients in the ICC-MY network as depicted in Fig. 2A, Supplemental Video S1 (Supplemental Material for this article is available online at the Journal website), and summary graph in Fig. 3A. When pooled together, individual FOVs had a synchronicity index of 0.85 ± 0.014 (mean ± SE; n = 43, N = 9; Fig. 3A) and the two standard deviation range from the mean of 0.66–1.04. Therefore, the value of 0.66 defines the lower limit of normal distribution for synchronicity. The frequency was 30.0 ± 3.5 (mean ± SE) cycles per minute (cpm), the amplitude (expressed as ratio of ΔF/Fo) 0.55 [0.43, 0.79] [median (25th, 75th percentiles)], and duration (at half-amplitude) 738.6 ± 10.3 (mean ± SE) ms. Ca2+ transients showed low degrees of variability for their amplitudes, duration intervals, and frequencies. The median coefficients of variability for frequency, amplitude, and duration were, respectively, 11.7% (25th and 75th percentiles: 9.7 and 13.0%), 26.0% (25th and 75th percentiles: 20.4 and 30.2%), and 14.7% (25th and 75th percentiles: 12.5 and 17.3%) (Fig. 3). Collectively, these data illustrate that FOVs imaged in jejuna of vehicle-treated mice showed highly synchronized regular Ca2+ transients of relatively constant frequencies, amplitudes, and duration intervals.
ICC-MY Ca2+ Transients in ICC-Specific Inducible Conditional Ano1KOs
In contrast to control mice, tamoxifen-induced icAno1KOs displayed highly variable Ca2+ transients in the jejunum ICC-MY network as illustrated by representative traces in Fig. 2B, summary graphs in Fig. 3, A–G, and Supplemental Videos S2–S4. For all icAno1KO mice, the synchronicity indexes ranged from 0.003 to 1.0, the frequency values from 6.0 to 48.5 cpm, CV for frequency from 4.6 to 54.9%, amplitude 0.06 to 1.85 (ΔF/Fo), CV for amplitude from 8.0 to 96.0%, half-width duration 354.0 to 798.0 ms, and CV for duration 6.3 to 42.3% (n = 73–83, N = 16). Therefore, the profile of Ca2+ transients included those comparable to vehicle controls and others distinct from them. For purposes of additional analyses of Ca2+ transients, the data from tamoxifen-treated (icAno1KO) mice were divided into two subgroups. The first contained data from FOVs where the synchronicity indexes were in the normal range (0.66–1.00) based on the lower limit of the normal range for the synchronicity index in vehicle-treated controls. This subclassification differentiated high and low Ca2+ transient synchronicity subgroups referred herein as icAno1KO-synchronized (normal range) and icAno1-desynchronized (synchronicity indexes < 0.66), respectively (Figs. 2B and 3).
Desynchronized profile of Ca2+ transients in ICC-MY network of ICC-specific inducible conditional Ano1 KOs.
Seventeen (N = 9) of 83 FOVs (~20%) were in the icAno1KO-desynchronized subgroup. The Ca2+ transients lacked regularity and synchronicity of vehicle-treated data points (see Fig. 2Ba for a representative recording and Supplemental Video S2). As summarized in Fig. 3, the low Ca2+ transient synchronicity subgroup of icAno1KOs showed, in addition to the reduced synchronization indexes, lower half-width duration, decreased frequency, and increases in CVs for the frequency and amplitude when compared with vehicle-treated controls (P < 0.05 for all using ANOVA followed by Dunn’s multiple-comparison test). The amplitudes were not significantly different between the icAno1KO desynchronized subgroup and vehicle controls (Fig. 3D).
Synchronized profile of Ca2+ transients in ICC-MY network of ICC-specific inducible conditional Ano1KOs.
The majority of FOVs from tamoxifen-treated transgenics, 66 (N = 15) of all 83 (~80%), were classified into the icAno1KO-synchronized subgroup (synchronicity indexes above 0.66) and included activities that were highly synchronized with all ICC-MY firing Ca2+ transients simultaneously (Fig. 2Bb and Supplemental Video S3) or contained a very brief asynchronous interval (Fig. 2Bb and Supplemental Video S4). The icAno1KO synchronicity subgroup showed parameters of Ca2+ transients that did not differ significantly from those of vehicle-treated controls with the exception of the reduced duration. In contrast, more robust effects on the parameters were observed in the icAno1KO-desynchronized subgroup. The frequency was lower while the CVs for frequency, amplitude, and duration were all higher when compared with the icAno1KO synchronicity subgroup (P < 0.05 for all three, ANOVA followed by Dunn’s multiple-comparison test). No differences in the amplitudes were noted among the different treatment groups (Fig. 3). Overall, the high Ca2+ transient synchronicity subgroup shared the properties of the vehicle-treated controls (synchronicity, frequency, and amplitude) but with a reduced duration.
Relationship Between Ca2+ Transient Activity Profile and Ano1 Protein Expression in ICC-MY Network
To examine the relationship between Ca2+ transient profile and the expression of Ano1 and Kit, we utilized a method of marking the FOV in live-fresh tissue just after recording the Ca2+ transient activity in the FOV of interest followed by the IHC assessment. The marking was done using a brief UV laser exposure. Figure 4, A–D, shows representative images for this series of experiments, and Fig. 4, E and F, overall summaries for synchronicity indexes and Ano1 expression levels in these experiments, respectively. Each panel within Fig. 4, A–D, contains Ca2+ transient activity in the imaged FOV, spot location of the FOV originally marked with UV light and visualized after completion of IHC protocol for determination of Ano1 and Kit immunoreactivities. Note that the spot shape was distorted in most cases following tissue fixation and the IHC protocol. Thus for each FOV we could directly link Ca2+ transient profile with level of Ano1 and Kit expressions in ICC-MY. In vehicle controls (n = 14, N = 4), highly synchronized Ca2+ transients (synchronicity index: 0.86 ± 0.04, mean ± SE) were matched to very high colocalization of Ano1 and Kit in ICC-MY network (colocalization score = 9.7 ± 0.1, mean ± SE, 10 = maximum score by visual assessment) (Fig. 4, A and F). In comparison, for all FOVs in the icAno1KO group (i.e., pooled data for icAno1 synchronized and desynchronized in Fig. 4, E and F), the synchronicity median index was 0.92 (25th, 75th percentiles: 0.71, 0.97; n = 28, N = 6 (and the colocalization median score 1.3 (25th, 75th percentiles: 0.5, 2.3; n = 28, N = 6) (P < 0.0001 vs. vehicle controls for Ano1 expression score, Mann-Whitney U-test, Fig. 4F). This observation reveals that Ca2+ transients remained highly synchronized despite a high degree of Ano1 loss in the ICC-MY network. Further subclassification of icANO1 KO data points into synchronized (index: 0.91 ± 0.01, mean ± SE; n = 22, N = 6) and desynchronized profiles (index: 0.26 ± 0.11, mean ± SE; n = 6, N = 2), based on the threshold of 0.66, revealed overall a higher loss of Ano1 in the latter group. The respective colocalization scores were 1.9 ± 0.3 (n = 22, N = 6) and 0.75 ± 0.21 (n = 6, N = 2; P < 0.05, ANOVA and Tukey’s multiple-comparison test, Fig. 4F). Collectively, we provide evidence that Ca2+ transient activity profile of ICC-MY networks relates to the magnitude of Ano1 expression with the following rank order: vehicle control>>>icAno1KO: synchronized > icAno1KO: desynchronized. The ICC-MY network maintained a high degree of Ca2+ transient synchronicity unless Ano1 loss was very severe and widespread and was observed in sites that displayed desynchronized Ca2+ transients.
Electrical Activity in Musculature of Vehicle-Treated Controls
Electrical activity in jejunum full-thickness musculature preparations devoid of mucosa was recorded in 87 cells (N = 10) and 91 cells (N = 9) from vehicle controls and icAno1KO mice, respectively. Control musculature (n = 87, N = 10) showed regular electrical slow waves arising from the resting membrane potential of −58.6 ± 0.89 mV (mean ± SE) and amplitude of 10.9 (7.0, 21.3) mV [median (25th, 75th percentiles)], rate of rise of upstroke 0.050 (0.016, 0.167) mV/ms [median (25th, 75th percentiles)], frequency of 32.0 (21.9, 34.8) cpm [median (25th, 75th percentiles)], duration at half-maximum amplitude of 928.4 ± 25.6 ms (mean ± SE), and inter-slow-wave interval 554.8 (440.7, 825.8) ms [median (25th, 75th percentiles)] (Figs. 5A and 6).
Electrical Activity in Musculature of Inducible Conditional Ano1KOs
Smooth muscle cells in jejunum musculature (n = 91, N = 9), obtained from icANO1 KOs, showed variable patterns of electrical activities including recordings without slow waves (26% of the time, n = 24, N = 5) and with slow waves (74%, n = 67, N = 7) (Figs. 5B and 6). When pooled together, the resting membrane potential of all icAno1KOs showed 2.2 mV (median) depolarization vs. vehicle controls (P < 0.01, Mann-Whitney U-test, Fig. 6A). When compared with each other, the resting membrane potential in recordings without slow waves (i.e., “icAno1KO lacking SW” in Fig. 6A) was depolarized by 8.5 mV (median) relative to both vehicle controls and by 7.9 mV (median) relative to those icAno1KOs with slow waves (P < 0.001, ANOVA and ANOVA Dunn’s multiple-comparison posttest, Fig. 6A). There was no significant difference in the resting membrane potential between vehicle controls and icAno1KOs with slow waves.
Abnormal slow waves in musculature of ICC-specific inducible conditional Ano1KOs.
Comparison of the electrical slow-wave properties in icAno1KOs (n = 67, N = 7) with those of vehicle controls (Mann-Whitney U-test) revealed that the former displayed a 54% reduction in the slow-wave duration (at half-amplitude) median (P < 0.001), 72% increase in the median for inter-slow-wave interval (P < 0.001) and 10% higher median for frequency (P < 0.001). The amplitude and rate of rise of the upstroke were not statistically different (P > 0.05) between the two (Fig. 6, B–E).
Irregular amplitude slow-wave pattern in musculature of icAno1KOs.
A more detailed examination of slow waves measured in icAno1KO jejunum musculature revealed additional diversity as shown in the representative traces in Fig. 5B, b–d). At least two different profiles were found. The first profile (43 cells, 7 mice) showed a regular slow-wave pattern of relatively constant regular amplitude oscillations (Fig. 5Bb), referred to herein as icAno1KO-regular slow-wave (SW) subgroup. The second type of slow-wave profile (24 cells, 4 mice) contained variable amplitude slow waves including separate periods of high amplitudes and irregular lower amplitudes and hence defined as icAno1KO-irregular SW subgroup. The high amplitude phase events were comparable to those observed for the first profile occurring at regular single amplitude events. The lower amplitude containing phase consisted of multiple events superimposed onto an apparent single slow-wave event or distinct fast events of small amplitudes. This oscillatory profile was consistent with integration of multiple independent pacemaker units. When the independent pacemaker units were in-phase, constant generation of large amplitude slow waves with longer duration intervals occurred. When they were out-of-phase, separate events could be distinguished. The integration of independent pacemaker units is further supported by the unusually high frequency (104 cpm) slow-wave oscillation pattern shown in Fig. 5Bd that shows regular out-of-phase recording of distinct oscillators occurring at the same frequency. Comparison of the slow-wave properties for icAno1 regular and irregular SW subgroups revealed no significant differences in the resting membrane potential and the frequency, and the inter-slow-wave interval (Fig. 6). Significant differences were noted for the amplitude [constant amplitude slow-wave pattern: 15.8 (11.5, 20.3) mV, median (25th, 75th percentiles), n = 43, N = 7; irregular amplitude slow-wave pattern: 8.4 (6.8, 13.5) mV, median (25th, 75th percentiles), n = 24, N = 4; P < 0.01, Mann-Whitney U-test], rate of rise of upstroke [constant amplitude slow-wave pattern: 0.112 (0.022, 0.146) mV/ms, median (25th, 75th percentiles), n = 43, N = 7; irregular amplitude slow-wave pattern: 0.032 (0.023, 0.046) mV/ms, median (25th, 75th percentiles), n = 24, N = 4; P < 0.001, Mann-Whitney U-test], and the slow-wave duration at half-amplitude [constant amplitude slow-wave pattern: 462.2 (413.9, 508.2) ms, median (25th, 75th percentiles), n = 43, N = 7; irregular amplitude slow-wave pattern: 348.8 (254.4, 433.0) ms, median (25th, 75th percentiles); n = 24, N = 4, P < 0.01, Mann-Whitney U-test]. The greater reduction in the slow-wave duration for the irregular amplitude subgroup is consistent with a greater loss of Ano1 in ICC-MY network and Ano1 channel activity underlying the plateau phase.
High Degree of Ano1 and Kit Coexpression in ICC, Highly Synchronized Ca2+ Transients, and Regular Slow Waves in Jejenum of Tamoxifen-Treated Kit+/+;Ano1Fl/Fl Mice
To address the possibility that either the single functional Kit allele in KitERT/+;Ano1Fl/Fl mice or tamoxifen-treatment per se could have effects on the expression of Ano1 and on Ca2+ transients and electrical slow waves, Kit+/+;Ano1Fl/Fl mice (Ano1 floxed only without CreERT2) were treated with tamoxifen for the same duration and dose as KitCreERT2/+;Ano1Fl/Fl transgenic mice described above. Figure 7A illustrates that Kit and Ano1 immunoreactivities in ICC-MY and ICC-DMP networks were comparable to those of vehicle-treated KitCreERT2/+;Ano1Fl/Fl transgenic mice. In addition, Ca2+ transients in ICC-MY (Fig. 7B) and electrical slow waves (Fig. 7C) in musculature were also normal. Table 1 details the properties of Ca2+ transients and electrical slow waves obtained for Kit+/+;Ano1Fl/Fl mice treated with tamoxifen.
ICC show a high level of Ano1 expression along with Kit receptor tyrosine kinase (13, 24, 51). Even though expression of Ano1 in ICC was first established in 2009 (13, 18, 24), its precise functions still remain incompletely understood. This study was undertaken to advance the knowledge of Ano1 in ICC of adult small intestine using a cell-specific inducible conditional Ano1 knockout model. The novel findings reveal maintenance of a high degree of Ca2+ transient synchronicity in ICC-MY network and musculature slow-wave rhythmicity despite strong loss of Ano1 in ICC, contribution of Ano1 to the Ca2+ transient duration in ICC-MY and slow-wave duration (plateau) in musculature, and requirement of extensive widespread loss of Ano1 throughout the ICC-MY network for desynchronization of Ca2+ transients and loss of the slow-wave activity accompanied by depolarization. The above insights for the roles of Ano1 in ICC were made possible by utilization of the inducible conditional Ano1 mouse model displaying partial and incomplete loss of Ano1 throughout the ICC-MY network, unlike the global constitutive model featuring a complete loss of Ano1 in the ICC-MY network and elsewhere described previously (13, 24, 51). Our study is the first to our knowledge to use Cre-loxP technology to specifically target knockdown of an ion channel in ICC.
Until our report, the understanding of the role of Ano1 in ICC and the gastrointestinal tract relied primarily on the global constitutive Ano1 mouse model (13, 24, 51). These genetic knockout mice lacked slow waves in the gastrointestinal musculature and displayed associated depolarization of ~5–10 mV (24, 51). Furthermore, the recorded Ca2+ transients in the ICC-MY network were not synchronized or coordinated, unlike wild-type controls (51). Ano1, thus, was concluded to play a role in slow-wave activity, regulation of the membrane potential, and synchronization of Ca2+ transients across the network. Additionally Ano1KOs displayed reduced contractility of isolated intestinal segments and of whole tissue measured with spatiotemporal mapping (51). The observation that global constitutive Ano1KOs still displayed Ca2+ transients, albeit desynchronized, was somewhat unexpected at the time. In the present study, we also found preservation of robust Ca2+ transients in ICC that were identified as Ano1 negative (see Fig. 4, C and D), albeit with a decrease in Ca2+ transient duration. This is consistent with a mechanism whereby the activity of Ano1 channels is not required for Ca2+ transient generation but contributes to longer ICC depolarization, allowing more sustained Ca2+ entry and a higher overall Ca2+ load in the intracellular stores. When Ca2+ transients in ICC-MY and slow waves in the musculature were simultaneously measured in the intestine, they showed 1:1 temporal correlation (40) consistent with prior observations showing both activities have similar time course when recorded separately (17, 31, 51). The basis for the disconnect between the lack of slow waves in the musculature and the presence of Ca2+ transients in ICC-MY in constitutive Ano1KOs still remains to be elucidated. Potentially, ICC-MY generate Ca2+ transients independent of voltage entirely driven by a metabotropic mechanism. Alternatively, ICC-MY generate voltage oscillations that correlate with Ca2+ transients that do not effectively spread to the musculature, resulting in the lack of slow waves and depolarization. Simultaneous measurements of electrical activity and Ca2+ within same ICC-MY in the network would obviously provide valuable insights; however, technical difficulties still remain a hurdle. At present, the success rate of recording from ICC in situ is extremely low and at best < 1% using a specialized preparation (26). Another challenge inherent to the global constitutive Ano1KOs is their severe phenotype; they die shortly after birth within hours/days and live up to 21 days on a rare occasion (24, 44). Thus this genetic model is unsuitable to study the role of Ano1 in adults. We, hence, employed an inducible conditional genetic model to specifically knockout Ano1 expression in ICC. We chose the inducible conditional model rather than just conditional since our aim was to study the role of Ano1 in adults with ICC networks normally developed. Furthermore, the use of the conditional cell-specific model alone in the absence of induction provides a potential for the development of compensatory mechanisms complicating data interpretations.
The inducible ICC-specific conditional Ano1 knockout model featured the loss of Ano1 expression occurring in a nonuniform mosaic/patchy pattern with no apparent disruption to the Kit-positive ICC networks. Overall, small intestines of icAno1KOs showed higher loss of Ano1 protein in ICC networks than Ano1 mRNA in whole musculature (Figs. 1 and 4F). We interpret these differences to contribution from ICC where recombination did not occur at one or both alleles, potentially causing Ano1 mRNA upregulation and probable minor impact from non-ICC cells including Ano1-positive cells in the serosa (our unpublished observation). The incomplete loss of Ano1 in icAno1KOs, in contrast to the complete absence in constitutive knockouts, allowed us to opportunistically investigate the function of Ano1 in ICC-MY under the partial loss condition revealing primarily shortening of Ca2+ transient duration. Furthermore, loci in the network with moderate or severe loss of Ano1 could be identified and linked to the profile of Ca2+ transient activity. We show that in ICC-MY networks of icAno1 knockouts, Ca2+ transients’ profiles specifically synchronization, amplitude, frequency, and their coefficients of variability remained similar to those of vehicle controls even in cases of severe but not widespread loss of Ano1 (Fig. 4). This finding indicates a very high degree of functional reserve for Ano1 in regulation of Ca2+ transient synchronicity in ICC-MY networks (Fig. 8). How mechanistically very few Ano1(+) ICC are able to influence the vast majority of Ano1(−) ICC to maintain synchronicity of the network remains to be determined. Perhaps the robust intrinsic electrical activity slow wave in ICC-MY reinforced by the presence of Ano1 propagates to neighboring ICC-MY lacking Ano1 without them exhibiting pronounced dysfunction in electrical activity and thus maintaining largely normal Ca2+ transients. The coupling between Ano1(+) and Ano1(−) ICC most likely occurs via gap junctions known to exist among adjacent ICC (55) and passage of voltage and potentially intracellular messengers such as Ca2+ or inositol 1,4,5-trisphosphate (IP3) (58). The evidence for the role of gap junctions is supported by dye injections directly into ICC illustrating robust spreading of the marker from single ICC-MY to multiple adjacent ICC-MY in the network and by the desynchronization of Ca2+ transients by gap junction blockers (5, 14, 26, 40). High space constants for voltage decay in the gut musculature with intact ICC networks in the range of ~0.6–2.6 mm (20, 21, 48) relative to FOVs imaged in this study (up to 634 μm × 634 μm) imply strong voltage coupling in musculature and especially for neighboring ICC that could drive synchronicity of Ca2+ transients in the network. Since both Ca2+ and IP3 have been shown to be critical in the regulation and modulation of Ca2+ transients in the ICC-MY of the small intestine (31, 40) and they also can pass via gap junctions (25), they too likely play a role in ICC-ICC communication. It is probable that Ano1 channels provide depolarization contributing to active propagation of voltage or Ca2+ via T-type Ca2+ channels or other Ca2+ entry pathways for efficient ICC-to-ICC communication as proposed recently (49) and to more sustained Ca2+ entry and a higher overall Ca2+ load in the intracellular stores. It can be further speculated that, at some threshold distance away, the signal generated from the Ano1(+) ICC-MY and passing through a series of Ano1(−) ICC will dissipate allowing intrinsic activity of Ano1(−) ICC-MY to set their Ca2+ transient activity, pacing, and result in desynchronization of Ca2+ transients among adjacent ICC-MY. Thus, Ca2+ transient activity in ICC-MY network depends on the level of Ano1 protein expression. Under partial loss of Ano1 in ICC-MY, the primary effect is on Ca2+ transient duration and the rich expression of Ano1 ensures retention of Ca2+ transients’ synchronicity.
Microelectrode recordings in musculature of small intestines from icAno1KOs, showing patchiness in the expression of Ano1 in the ICC-MY network, displayed diverse electrical activities including slow waves with amplitudes either regular or irregular, reduced plateau duration, increased inter-slow-wave interval, higher frequency, and lack of slow-wave activity accompanied by depolarization compared with vehicle controls (Figs. 5 and 6). We propose that the type of electrical activity depends on the level of expression of Ano1 in the ICC-MY network (see Fig. 8). Submaximum, but not widespread deficit of Ano1, leads to slow waves with reduced plateau phase with and without regular amplitudes. In contrast extensive loss of Ano1, just as in global constitutive Ano1KOs (24, 51), underlies the loss of slow-wave activity and associated depolarization. Our results implicating a role of Ano1 in slow-wave plateau are consistent with prior observations based on the effects of altering extracellular Cl− concentrations in guinea pig antrum (57), rabbit and mouse small intestine (11, 26), and dog colon (4), and of pharmacological inhibitors of Ano1 including DIDS, niflumic acid, benzbromazone, dichlorphene, and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) in the small intestine (23, 24). In some cases but not all, the reduced slow-wave duration was accompanied by effects on amplitude, frequency, and/or resting membrane potential. Our results are currently not consistent with the primary role of Ano1 in the generation of the upstroke (rate of rise) or peak amplitude as there were no differences between controls and icAno1KOs for these parameters. We, however, cannot exclude that in the presence of even higher loss of Ano1 in ICC-MY than we could obtain under the experimental conditions of our study, these parameters may be affected. When recorded specifically in ICC-MY of the mouse small intestine, a reduction of extracellular Cl− concentration and DIDS, a nonspecific Ano1 channel inhibitor, primarily reduced the slow-wave duration and only subsequent application of Ni2+, a nonselective T-type Ca2+ channel inhibitor, completely abolished the pacemaker slow wave in ICC-MY (26). The latter observation supports a role of T-type Ca2+ channel in the slow-wave upstroke. The nonselective nature of currently available Ano1 inhibitors and Cl− concentration substitution prevents drawing definite conclusions (6, 51). At present, the genetic approach of knocking out Ano1 especially in the cell(s) of interest, as done in this study for ICC, constitutes a robust way to study the function of Ano1.
The pacing mechanism of slow waves is thought to depend on intracellular Ca2+ handling and cycling involving endoplasmic reticulum Ca2+ stores, IP3-induced Ca2+ release, and sodium, potassium, and chloride cotransporter 1 (NKCC1) (33, 62, 63). The discrepancy between the increase in the slow-wave frequency in the musculature and the reduced or unchanged frequency of Ca2+ transients in ICC-MY in icAno1KO small intestines was likely due to the nature of the measured activities. Ca2+ transient measurements were made directly in ICC-MY networks while microelectrode recordings reflect an integrated activity of a syncytium of smooth muscle cells and ICC in the musculature. The irregular amplitude slow-wave activity pattern was similar to that reported for newborn BALB/C (61) and CD1 mice (30). The irregular amplitude slow-wave profile of conditional Ano1 knockouts also shares features of the slow-wave activity observed in the presence of gap junction blockers heptanol and 8-β glycyrrhetinic acid in intestinal and stomach musculature (21, 58). Ca2+ transients also become desynchronized among adjacent ICC in the presence of the gap junction blockade (40). It is, thus, likely that the functional abnormalities in icAno1KOs were due to suboptimal coupling among ICC or clusters of ICC forming distinct pacemaker units or independent oscillators that were not synchronized due to lack of Ano1 in ICC and perhaps directly depending on Ano1 presence in the processes of ICC-MY. Inefficient coupling among ICC clusters has been attributed to abnormal slow-wave pacing in the gastric antrum and body musculature of diabetic mice (10). Thus, Ano1 not only ensures synchronization or coordination of Ca2+ transients in ICC-MY network (this study and Ref. 51) but also appears to efficiently integrate the pacemaker activity generated from distinct ICC oscillators to be in-phase.
Our results support that Ano1 channels expressed in ICC contribute to the resting membrane potential of small intestinal smooth muscle cells in situ. Both small intestines of Ano1 constitutive knockout mice (24, 51) and a subset of the cells lacking slow waves in icAno1KOs (Figs. 5Ba and 6A) showed depolarization when compared with controls. Interestingly, gastrointestinal musculature lacking intact ICC networks (e.g., W and Sl mutants) (22, 35, 39, 60) also displayed depolarization of the resting membrane potential, and the ICC-MY network sets the membrane potential gradient across the muscle wall thickness (15). Since in small intestines of icAno1 and global constitutive Ano1KOs ICC networks appear intact, as judged by normal Kit immunoreactivity, the absence of Ano1 in ICC links Ano1 directly to membrane potential regulation in the musculature. The mechanism directly linking Ano1 in ICC to the smooth muscle membrane potential remains to be established. It may be that ICC express Ano1 channels that are active under the resting conditions (i.e., in between slow waves), and this voltage propagates to the smooth muscle syncytium. Indeed, a unique exon-0 containing Ano1 channel, imparting a higher sensitivity to Ca2+ at basal Ca2+ concentrations, was found in human gastrointestinal tract and characterized when recombinantly expressed in HEK-293 (53) could play a role. Alternatively, the slow kinetics of Ano1 channels activated during the plateau phase of the slow wave and corresponding rise in global Ca2+ during the Ca2+ transient event could allow them to be active under the resting conditions. Other ion channels, directly setting the resting membrane potential in ICC or intestinal smooth muscle, could respond to membrane potential changes mediated by Ano1.
The qRT-PCR and IHC observations (Fig. 1) illustrate that the loss of Ano1 in ICC networks was not complete but rather displayed mosaicism and contained ICC with functional floxed and excised Ano1 alleles next to each other in various degrees of patchiness. Mosaicism is a not an uncommon feature in studies employing Cre-loxP system for targeting a gene of interest (16, 65). The Kit promoter driven ERT-Cre expression system has been previously used by others to study ICC after crossing into mice containing several different floxed genes (3, 27, 28), but not yet an ion channel, and shown to drive the Cre expression in >90% of ICC in the small intestine following a 3- to 5-day treatment with tamoxifen. A similar high level of Cre-loxP recombination to tamoxifen was inferred in ICC-DMP of mice driving expression of the Ca2+ reporter GCaMP3 (3). Thus it is unlikely that inefficient Cre inducibility was the reason for the mosaic loss of Ano1 in ICC observed in this study. The genetic context of the loxP sites could also affect the access of the Cre recombinase to the loxP sites and may account for the lower than expected rate of recombination (59). The availability of our icAno1KO model displaying partial and not uniform loss of Ano1 in ICC-MY network opportunistically allowed the study of the association between the degree of Ano1 channel expression in the ICC-MY network and profile of Ca2+ transients’ activity. In addition to the role of Ano1 in regulating pacemaker activity of ICC-MY, Ano1 is likely involved in regulating their or ICC stem cell growth, maintenance, or survival. The observations of reduced number of proliferating ICC in the presence of T16A(inh)-A01, an Ano1 inhibitor, and lower degree of proliferation for ICC in Ano1 global constitutive KOs in culture support this function (37, 52). Global constitutive Ano1 knockout mice had no apparent loss of Kit-positive ICC, and we suggested that preservation of the ICC network by population from ICC stem cells could compensate for reduced proliferation in those mice (51).
In summary, Ano1 expressed in ICC-MY plays a role in the generation and regulation of Ca2+ transients in ICC-MY networks, and slow-wave activity in the musculature, especially its plateau phase, dependent on the level of Ano1 expression. The Ano1 ICC-MY-dependent functions (synchronization of Ca2+ transients, generation of slow waves, and contribution to the resting membrane potential) remain resilient to submaximum loss of Ano1 expression in the network and require widespread and extensive loss of the protein for robust dysfunction. Under partial loss, the primary effect of Ano1 is on the duration of ICC-MY Ca2+ transients and musculature slow waves. These insights, summarized in Fig. 8, were made possible by utilization of the inducible conditional Ano1 knockout model featuring incomplete loss of Ano1 and patchy variable expression of this ion channel, unlike the global constitutive KOs studied previously (13, 24, 51).
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK57061.
No conflicts of interest, financial or otherwise, are declared by the author(s).
J.M., S.A.S., S.T.E., and C.C. performed experiments; J.M., S.J.G., S.A.S., P.D., S.T.E., C.C., U.O., D.S., S.K., T.O., and G.F. analyzed data; J.M., S.J.G., P.D., S.T.E., C.C., U.O., D.S., S.K., T.O., and G.F. interpreted results of experiments; J.M., S.J.G., and G.F. prepared figures; J.M., S.J.G., S.A.S., P.D., S.T.E., C.C., U.O., D.S., S.K., T.O., and G.F. drafted manuscript; J.M., S.J.G., S.A.S., P.D., S.T.E., U.O., D.S., S.K., T.O., and G.F. edited and revised manuscript; J.M., S.J.G., S.A.S., P.D., S.T.E., C.C., U.O., D.S., S.K., T.O., and G.F. approved final version of manuscript.
We thank Kristy Zodrow, Dr. Amelia Mazzone, and Gary Stoltz for excellent help, and Dr. Joseph H. Szurszewski for valuable comments during manuscript preparation.
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