Interstitial cells of Cajal (ICC) generate electrical slow waves by coordinated openings of ANO1 channels, a Ca 2+ -activated Cl − (CaCC) conductance. Efflux of Cl − during slow waves must be significant, as there is high current density during slow-wave currents and slow waves are of sufficient magnitude to depolarize the syncytium of smooth muscle cells and PDGFRα + cells to which they are electrically coupled. We investigated how the driving force for Cl − current is maintained in ICC. We found robust expression of Slc12a2 (which encodes an Na + -K + -Cl − cotransporter, NKCC1) and immunohistochemical confirmation that NKCC1 is expressed in ICC. With the use of the gramicidin permeabilized-patch technique, which is reported to not disturb [Cl − ] i , the reversal potential for spontaneous transient inward currents ( E STICs ) was −10.5 mV. This value corresponds to the peak of slow waves when they are recorded directly from ICC in situ. Inhibition of NKCC1 with bumetanide shifted E STICs to more negative potentials within a few minutes and reduced pacemaker activity. Bumetanide had no direct effects on ANO1 or Ca V 3.2 channels expressed in HEK293 cells or L-type Ca 2+ currents. Reducing extracellular Cl − to 10 mM shifted E STICs to positive potentials as predicted by the Nernst equation. The relatively rapid shift in E STICs when NKCC1 was blocked suggests that significant changes in the transmembrane Cl − gradient occur during the slow-wave cycle, possibly within microdomains formed between endoplasmic reticulum and the plasma membrane in ICC. Recovery of Cl − via NKCC1 might have additional consequences on shaping the waveforms of slow waves via Na + entry into microdomains.

electrical slow-wave activity drives phasic contractions in the gastrointestinal (GI) tract that are responsible for peristalsis in the stomach and segmental contractions in the small bowel and colon. Electrical slow waves originate in specialized pacemaker cells, known as interstitial cells of Cajal (ICC) (16, 22, 34, 39, 43). A strain of mice with constitutive expression of a reporter molecule, copGFP, in ICC has allowed studies of these cells soon after enzymatic liberation from muscles and has obviated the need for cell culture (30, 47). These studies have begun to piece together the ionic mechanisms responsible for generation and propagation of slow waves (35, 46–48). Studies on intact muscle utilizing electrophysiology and Ca2+ imaging techniques have developed a detailed hypothesis about the propagation of slow waves in ICC networks and how these events interact with electrically coupled smooth muscle cells (SMCs) (2, 7, 23–25, 28, 36).

At the present time, the developing concept of the pacemaker activity GI muscles includes the following (35): 1) ongoing discharge of spontaneous transient inward currents (STICs) is attributable to localized Ca2+ release from intracellular stores via IP 3 receptors (37, 40); 2) STICs cause spontaneous transient depolarizations (STDs) in ICC (9, 15, 19, 20, 40, 48); 3) depolarization activates low-threshold, T-type Ca2+ currents (46); however, some investigators have also suggested that depolarization may enhance IP 3 production (15, 41); 4) Ca2+ entry (or enhanced IP 3 production) coordinates release of Ca2+ from stores, activating a whole-cell slow-wave current (46, 47); 5) cells depolarize to about −10 mV (20); and 6) repolarization resets the slow-wave cycle. STICs and slow-wave currents are due to Cl− efflux via Ca2+-activated Cl− channels (CaCC), and slow waves are absent in mice with genetic deactivation of Ano1 (17, 36, 47). Slow-wave currents in ICC are of large current density, reaching 80 pA/pF (47), which must result in significant efflux of Cl−. Most of the Cl− channels in ICC appear to be localized within microdomains formed by the close apposition between the plasma membrane and the endoplasmic reticulum (49). How the driving force for slow-wave currents (i.e., the difference between the resting membrane potential and E Cl ; E M -E Cl ) is maintained from event to event in these constantly active pacemaker cells has not been clarified.

A previous study using suppression-subtractive hybridization demonstrated that the tunica muscularis of the murine jejunum displays high expression of Slc12a2, which encodes the Na+-K+-Cl− cotransporter (NKCC1) (44). Immunohistochemistry revealed that NKCC1 is expressed by the pacemaker class of ICC in the myenteric region of the small intestine (ICC-MY), and block of NKCC1 with bumetanide reduced or blocked slow waves (18, 44). It was also found that slow waves were of much smaller amplitude in mice with constitutive genetic deactivation of Slc12a2, and bumetanide had no effect on the activity remaining in these mice. Thus studies of whole muscles suggest that NKCC1 is involved, in some manner, in the slow-wave cycle. Realization that ANO1 is a fundamental conductance in slow waves suggests the hypothesis that the role of NKCC1 may be to restore the Cl− gradient after loss of Cl− during slow-wave currents. In the present study, we have measured the reversal potential of STICs (E STICs ), which is likely, based on analysis of reversal potentials (48), to approximate E Cl in freshly isolated and identified ICC from the murine small intestine. We determined how STICs and slow-wave currents are affected by NKCC1 inhibitor bumetanide. Our results confirm that E STICs lies positive to the resting potentials of ICC and that the driving force for STICs must be maintained by NKCC1 to sustain the pacemaker activity of these cells.

MATERIALS AND METHODS Animals. Kit+/copGFP mice (7–12 days old) were used for the electrophysiological experiments on ICC in this study (30). ICC can be identified unequivocally by the expression of copGFP in these mice. Kit+/copGFP mice and smooth muscle myosin heavy chain (smMHC)/Cre/enhanced green fluorescent protein (eGFP) mice (both 5–8 wk old; the latter strain was donated by Dr. Michael Kotlikoff, Cornell University) were used for gene expression studies. Colon SMCs, used for control studies of L-type Ca2+ channels, were obtained from C57BL/6 mice (2–3 mo old; Jackson Laboratory). The mice were anesthetized with isofluorane and killed by cervical dislocation. The institutional Animal Use and Care Committee at the University of Nevada approved all procedures. Preparation of dispersed cells. Small strips of intestinal and colonic muscles were cut and equilibrated in Ca2+-free Hanks solution consisting of the following (in mM): 125.00 NaCl, 5.60 KCl, 15.00 NaHCO 3 , 0.36 Na 2 HPO 4 , 0.40 KH 2 PO 4 , 10.00 glucose, 2.00 sucrose, and 10.00 N-2-hydroxyethylpiperazine-N′-ethanesulfonic acid (HEPES) adjusted to pH 7.2 with Tris. ICC were isolated from small intestinal muscles of Kit+/copGFP mice, using an enzyme solution containing the following: collagenase (Worthington type II, 1.3 mg/ml; Worthington Biochemical), BSA (2 mg/ml; Sigma), trypsin inhibitor (2 mg/ml; Sigma), and ATP (0.27 mg/ml). Cells were plated onto sterile glass coverslips coated with murine collagen (2.5 mg/ml; Falcon/BD) in 35-mm culture dishes. The cells were allowed to settle for 10 min before we added smooth muscle growth medium (Clonetics) supplemented with 2% antibiotic/antimycotic (Gibco) and murine stem cell factor (5 ng/ml; Sigma). The cells were placed into a 95% O 2 -5% CO 2 incubator for 2 h at 37°C (47). Freshly isolated SMCs from small intestine were used in expression studies for comparisons with ICC, and SMCs from colon were used as a source of L-type Ca2+ currents for control studies to evaluate nonspecific effects of bumetanide on this conductance. SMCs were dispersed from small intestinal strips of smMHC/Cre/eGFP mice, as described previously (46). Small pieces of muscle were exposed for 30 ± 2 min at 37°C to a solution containing (per ml) 3.5 mg collagenase (Worthington type II; Worthington Biochemical), 8.0 mg BSA (Sigma), and 8.0 mg trypsin inhibitor (Sigma). SMCs were also dispersed from proximal colons of C57BL/6 mice using the enzyme solution used for small intestinal muscles but with the addition of 1 mg/ml papain and 1 mg/ml dithiothreitol (both from Sigma). Gene expression. Total RNA was isolated from copGFP+ cells, and intestinal SMCs were purified by fluorescence-activated cell sorting (FACS) and from samples of dispersed cells before being sorted with the use of an Illustra RNAspin Mini RNA Isolation Kit (GE Healthcare). qScript cDNA SuperMix (Quanta Biosciences) was used to synthesize first-strand cDNA by the manufacturer's instructions. Quantitative PCR (qPCR) was performed with gene-specific primers (Table 1) using Fast Sybr Green chemistry on the 7900HT Fast Real-Time PCR System (Applied Biosystems). Regression analysis was used to produce standard curves from the mean values of technical triplicate qPCRs of log10 diluted cDNA samples. Unknown amounts of messenger RNA (mRNA) were plotted relative to the standard curve for each set of primers using Microsoft Excel. This gave transcriptional quantification of each gene relative to the endogenous Gapdh standard after log transformation of the corresponding raw data. Evaluation of gene expression in ICC was compared with expression in the unsorted cell population cells from small intestinal muscles of Kit+/copGFP mice, and gene expression in sorted SMCs was compared with the unsorted population of cells from small intestinal muscles of smMHC/Cre/eGFP mice. Table 1. Primers used for qPCR Gene Primer Sequence Accession No. mGapdh-F TGAACGGATTTGGCCGTATTG NM_001289726 mGapdh-R GATGGGCTTCCCGTTGATGA mSlc12a1-F all isoforms CGGCAAGCTGAACATTACCA NM_183354 mSlc12a1-R all isoforms TTAAACTGAGCGCTGGCTTC mSlc12a1-F isoform A AGTCATGCTCTTCATTCGCC NM_183354 mSlc12a1-R isoform A CTCCACCTCCACGAACAAACC mSlc12a1-F isoform B GGGAATTGGTCTCGGTGTGA U20974 mSlc12a1-R isoform B GGCTCCACCTCCTCTGACAT mSlc12a1-F isoform F ATCATTGGCCTGAGCGTAGT NM_001079690 mSlc12a1-R isoform F AAGCCTATTGACCCACCGAA mSlc12a2-F CTTGCGAGAAGGTGCACAATA NM_009194 mSlc12a2-R TCAGGCGGATAACCACAACT mSlc12a3-F CCCTTGTGGACTTTGTGAGC NM_001205311 mSlc12a3-R TTGTTCAGCCACTTGGTGTG mSlc4a1-F ACCTAACCATCCCTGTGACC NM_011403 mSlc4a1-R CCACATAGACCTGACCGGAG mSlc4a2-F CTGGAGGTGGATAGAGAGCG NM_009207 mSlc4a2-R CTCCGCATTCTCAGGGATCT mSlc4a3-F CTTTGAGTTTCACCGGCACA NM_009208 mSlc4a3-R GATAGGGGGTGTCCCTTCTG mSlc4a4-F CAACGACGATTCTGACTGCC NM_053424 mSlc4a4-R TGTGTGGCGTTCAAGGAATG mSlc4a5-F ACACAGACCAGCGGAAAAAC NM_001166067 mSlc4a5-R GCAGACTGGACAAGACGAAC mSlc4a7-F CAGGAGAGTGGGATCCTTCC NM_001033270 mSlc4a7-R CAAAAAGCCGTCCAGTCCTC Immunohistochemistry. For whole-mount immunohistochemistry, the tunica muscularis of small intestine of C57BL/6 mice was fixed in acetone (10 min at 4°C) and then washed with PBS and immersed in PBS with 1% BSA (1 h) to reduce nonspecific binding of antibodies. Muscles were then incubated with anti-c-KIT antibody (ACK2; eBioscience) diluted (1:500) in PBS with 0.5% Triton X-100 for 48 h at 4°C, washed briefly with PBS, and then incubated with anti-NKCC1 antibody (SC-21547; Santa Cruz Biotechnology) diluted (1:100) in PBS with 0.5% Triton X-100 for 48 h at 4°C. After being washed in PBS, the muscles were incubated with secondary antibodies diluted (1:500) in PBS for 1 h at room temperature. The secondary antibodies used for the anti-c-KIT antibody and anti-NKCC1 antibody were Alexa Fluor 594 donkey anti-rat IgG and Alexa Fluor 488 donkey anti-goat IgG (Molecular Probe, Life Technologies), respectively. Controls using no primary antibody confirmed that labeling was specific. For immunohistochemistry, sections of small intestines of C57BL/6 mice were fixed in 4% paraformaldehyde (15 min at 4°C) and were then washed with PBS. Frozen tissue blocks were made with optimum cutting temperature compound (Sakura Finetek) by standard methods, and 10-μm sections were cut with a cryostat (Carl Zeiss). Tissue sections were incubated in 1% BSA (Sigma) in PBS for 1 h at room temperature to block nonspecific antibody binding. After being blocked, tissue sections were incubated with the same primary antibodies overnight at 4°C and then the same secondary antibodies for 1 h at room temperature, as used for whole mounts. After immunolabeling, the samples were examined with a confocal microscope (Olympus FluoView 1000; Olympus), and images were obtained using an ×100 objective (UPlanSApo 100x, NA1.40; Olympus) for whole-mount tissues and an ×60 objective (PlanApo N 60x, NA1.42; Olympus) for sectioned tissues. The micrographs shown are composites of Z-series scans (0.5-μm optical section) through a depth of 6 μm constructed by Olympus Fluoview Ver.2.1c Viewer (FV10-ASW). Electrophysiological experiments. Whole-cell voltage- and current-clamp experiments were performed on ICC identified by the expression of GFP, using the permeabilized patch technique or conventional dialyzed cell techniques. For experiments with permeabilized patches, gramicidin (50 mg/ml, Sigma) was dissolved in ethanol with sonication and diluted with the pipette solution to a final concentration of 100 μg/ml. Membrane currents or potentials were amplified with an Axopatch 200B patch-clamp amplifier (Axon Instruments), digitized with a 16-bit analog-to-digital converter (Digidata 1322A; Axon Instruments), and stored digitally using pCLAMP software (version 9.2; Axon Instruments). Data were sampled at 4 kHz and filtered at 2 kHz using an eight-pole Bessel filter. Changes in holding currents (basal currents) were continuously monitored by miniDigi (Axon Instruments) using Axoscope 9.2 software (Axon Instruments). All data were analyzed using clampfit (pCLAMP version, 9.2; Axon Instruments) and GraphPad Prism (version 3.0; GraphPad Software) software. Pipette tip resistances ranged between 3 and 5 MΩ, and all experiments on ICC were conducted at 30°C using a CL-100 bath heater (Warner Instruments). Solutions and chemicals for patch-clamp experiments. The external bath solution for whole-cell recordings from ICC was a Ca2+-containing physiological salt solution (CaPSS, Solution I; Table 2). The pipette solution used for these experiments was Solution II (Table 2) and Solution III for experiments with reduced external Cl−. In studies of Cl− currents, cells were dialyzed with NMDG-Cl (Solution V) and the external solution to achieve a symmetrical Cl− gradient was Solution VI. The effects of bumetanide (Sigma) and various concentrations of external Cl− were tested using a fast bath perfusion system (AutoMate Scientific). Changes of the bath solution were completed within 1 min. Table 2. The composition of solutions Solutions (mM) I II III IV V VI KCl 5 135 5 0 0 0 NaCl 135 0 0 0 0 0 Na isethionate 0 0 135 0 0 0 CsCl 0 0 0 135 0 0 NMDGCl 0 0 0 0 147.7 150 CaCl 2 2 0 2 0 4.14 2 MgCl 2 1.2 0 1.2 0 0 1 MgATP 0 3 0 3 0 0 NaGTP 0 0.1 0 0.1 0 0 CPD 0 2.5 0 2.5 0 0 Glucose 10 0 10 0 0 0 EGTA 0 0.1 0 0.1 10 0 HEPES 10 10 10 10 10 10 pH 7.4 7.2 7.4 7.2 7.2 7.4 Expression of ANO1 and Ca v 3.2 in HEK293. We expressed Ano1 in HEK293 cells (American Type Culture Collection, ATCC) to test whether the effects of bumetanide noted in this study could be due to direct blocking effects on ANO1 currents. An expressed sequence tag (IMAGE Consortium cDNA clone no. 30547439) homologous to the A isoform of Ano1 from mouse was subcloned into pcDNA3.1 (Invitrogen) using standard molecular biological techniques. For expression in mammalian cells, Ano1 was subcloned in frame into pAcGFP1-N1 vector (Clontech Laboratories), resulting in a plasmid coding for a COOH-terminal GFP-tagged ANO1 fusion protein. To generate the Ano1-AC variant, containing the alternative exon 13, we inserted the 12-nucleotide C segment into Ano1-coding region using the QuickChange XL site-directed mutagenesis kit (Agilent Technologies). The plasmids were sequenced at the Nevada Genomics Centre. HEK293 cells expressing Ano1 were seeded in 12-well plates for transient transfection. The next day, the pAcGFP1-N1 vector, containing the AC variant of mouse Ano1 tagged with eGFP, was transfected into cells using FuGENE 6 transfection reagent (Promega). Cells were used for patch-clamp recordings 24–48 h after transfection. HEK293 cells with stable expression of Ca v 3.2, the dominant T-channel paralog in ICC (4), were donated by Dr. E. Perez-Reyes (University of Virginia). Generation of the cell lines expressing human Ca v 3.2 and the electrophysiological properties of the currents expressed were described previously (12). Briefly, HEK293 cells (A293; ATCC) were transfected with linearized plasmid (pcDNA3, Invitrogen) containing the human heart Ca v 3.2 isoform CACNA1H. Cells were grown in DMEM/F-12 (Thermo Fisher Scientific/Gibco), and the media was supplemented with 10% FBS, 1% penicillin/streptomycin, and 1% sodium pyruvate (all media supplements were from Thermo Fisher Scientific/Gibco). Experiments to test the effects of bumetanide on ANO1, Ca V 3.2, and L-type Ca2+ currents. The dialyzed whole-cell patch-clamp configuration was used to record ANO1 currents from HEK293 cells. These experiments were performed at room temperature using an Axopatch 200B amplifier and pClamp 9.0 software (Axon Instruments). ANO1 currents were recorded in response to step depolarizations from −80 mV to +70 mV using Solution V for the pipette solution and Solution VI as the external solution. Ca v 3.2 currents were recorded from HEK293 cells using dialyzed whole-cell patch-clamp conditions. L-type Ca2+ currents of colonic SMCs, the dominant voltage-dependent inward current in these cells, were studied using amphotericin-permeabilized patches to reduce rundown of the current (21). Amphotericin B (90 mg/ml; Sigma) was dissolved with DMSO with sonication and was diluted in the pipette solution to give a final concentration of 250 μg/ml. The external and pipette solutions used for measurement of Ca2+ currents were Solutions I and Solution IV (Table 2), respectively. Voltage-dependent Ca2+ currents were evoked by depolarizing steps from −80 mV to +60 mV from a holding potential of −80 mV. The voltage dependence of activation was determined from a Bolzmann fit of the data normalized to maximal conductance (G/G max ). Time constants of activation and inactivation were calculated from fits of currents with single or double exponentials. Statistical analysis. Data were tabulated and presented as means ± SE. The n values given represent the number of animals from which cells or tissues were obtained for the specific protocols performed. Differences between data sets were determined with Student's paired t-test and considered significant when P < 0.05.

DISCUSSION In the present study, we confirmed the expression of Slc12a2 transcripts and NKCC1 protein in ICC of the murine small intestine, as suggested previously by comparing expression in intestinal muscles of wild-type and WLacZ/WV and Sl/Sld mice (44). There are two paralogs of Slc12a (Slc12a2 and Slc12a1) that encode NKCC1 and NKCC2, respectively. Slc12a2 is expressed ubiquitously and is involved in volume regulation in many cells and Cl− secretion in epithelial cells (26, 45). Slc12a1 is expressed predominantly in the thick ascending limb of Henle (13). Consistent with these expression patterns, we found that Slc12a2 is the dominant paralog expressed in ICC, and it is far more highly expressed in ICC than in other cells of the tunica muscularis in the small intestine. Immunohistochemistry confirmed that gene expression was matched by protein expression in ICC-MY, and we did not resolve NKCC1-LI in cells other than ICC-MY in the tunica muscularis. These data are consistent with a previous screen of gene expression in ICC-MY and ICC-DMP of the murine small intestine in which Slc12a2 was found to be highly expressed in ICC-MY [i.e., nearly 16-fold higher than in whole muscle (4)]. Although not an exhaustive study of all genes encoding Cl− and bicarbonate (HCO 3 −) transporters, our demonstration of Slc4a2 and Slc4a4 expression in ICC may provide additional insights into Cl− recovery and homeostasis of transmembrane ion gradients. Slc4a2 encodes an electroneutral Cl−/HCO 3 − anion exchanger typically involved in regulation of cell pH (3). ICC have been characterized routinely as having an abundance of mitochondria (11, 31, 32, 38), and the presence and activity of these organelles may be necessary for pacemaker activity and active maintenance of ionic gradients. A major byproduct of mitochondrial respiration and production of ATP is CO 2 , which causes acidification via the reaction: CO 2 + H 2 O ⇄ H+ + HCO 3 −. Acidification has inhibitory effects on electrical rhythmicity in intact GI muscle strips (5). Through the coupled actions of Na+/H+ exchange [Slc9a1 is expressed in ICC (4)], Na+ and Cl− influx can occur in exchange for removal of H+ and HCO 3 − (3). Another mechanism of Cl− influx might occur through the expression of Slc4a4. This is an electrogenic Na+-HCO 3 − symporter that can result in the influx of Na+ and HCO 3 − using the electrochemical gradient for Na+ (6). Transporting HCO 3 − into cells promotes Cl− uptake by subsequent actions of Cl−/HCO 3 − exchange. Thus the proteins encoded by both Slc4a2 and Slc4a4, found to be relatively elevated in ICC, might result in enhancing Na+ and Cl− influx into ICC and could possibly aid in the restoration of the Cl− gradient and the modified slow waves that occur in animals lacking Slc12a2 expression (44). However, under the conditions of our experiments and in the presence of HEPES buffering, mechanisms requiring HCO 3 − would have been excluded, and the bumetanide-sensitive exchange mechanism was important for maintaining E STICs in ICC. It should be noted that, although parallel pathways for Cl− recovery may exist in vivo and in tissues in vitro perfused with physiological buffers, a study performed under these conditions showed that genetic deactivation of Slc12a2 had significant effects on slow waves (44). Thus, if other transporters contribute to maintenance of [Cl−] i , they are not capable of compensating for the loss of NKCC1. Another class of Cl−/HCO 3 − exchangers, encoded by the Slc26a family of genes, showed no appreciable expression in ICC of the small intestine in a previous gene array study (4), and these genes were not further characterized in the present study. Reversal of STICs has been shown to follow equilibrium potential for Cl−, and STICs are inhibited by ANO1 blockers (48). Thus the reversal potential for STICs is likely to approximate E Cl . Previous reports, based on measurements of the activity of [Cl−] i (a[Cl− ] i ) with ion-selective electrodes, set E Cl of SMCs at ∼−25 mV (1), and some investigators have assumed similar levels for E Cl in ICC (14). Such a negative value for E Cl in ICC would be problematic to the idea that current carried by ANO1 channels is responsible for slow waves because intracellular recordings from ICC have shown the cells to depolarize to about −10 mV during slow waves (19, 20). The present study, using gramicidin-perforated patch recording to leave a[Cl−] i undisturbed (8), suggests a level for E Cl in ICC of ∼−9 mV based on the interpolation of the reversal potential of STICs. Gramicidin-perforated patch recording was also used to measure the shift in E STICs when NKCC1 was inhibited or when extracellular Cl− was reduced. We hypothesized that loss of Cl− during pacemaker activity would need to be restored by a transporter that is capable of concentrating Cl− to maintain E Cl at a level that can support inward current carried by Cl−. NKCC transporters are electroneutral and move Na+ and K+ in concert with 2 Cl− ions (26, 33). Thus the stoichiometry of NKCC is 1 Na:1 K:2 Cl. The transporter uses energy stored in the Na+ gradient to concentrate Cl− into ICC, creating an E Cl that is far less negative than the resting membrane potentials [i.e., ∼−60 mV; (17)]. This creates an ∼50-mV driving force for Cl− efflux when ANO1 is activated during STICs and slow-wave currents. NKCC1 is selectively and reversibly blocked by loop diuretics, such as bumetanide (33). In the present study, after ICC were treated with bumetanide, E STICs rapidly shifted toward more negative levels, and STICs and slow-wave currents were reduced in amplitude at normal physiological membrane potentials (i.e., −60 mV). Results from the present study help to explain observations from ion replacement studies on intact GI muscles. Studies in which extracellular Cl− was replaced with less permeant (isethionate) or more permeant (iodide) anions resulted in reduction and/or inhibition of slow waves (10, 14). On the surface, this observation would appear contradictory to the role of a Cl− conductance (ANO1) in generating slow-wave currents because reducing [Cl−] o should increase the gradient for Cl− efflux. As we demonstrated, reducing [Cl−] o to 10 mM caused a substantial positive shift in E Cl . This issue was recognized in the study of El-Sharkawy and Daniel (10), and indeed the initial effect they observed in response to reduced [Cl−] o was an increase in slow-wave amplitude. Without cycle-to-cycle restoration of [Cl−] i , however, the Cl− gradient runs down, shifting E Cl to more negative levels. Such a shift in E Cl decreases the driving force for Cl− current. The permeability of anions through Cl− channels (e.g., iodide) was found to be unimportant in the responses to reduced extracellular Cl− (14). Our data explain this result; if I− could be concentrated into cells in place of Cl−, it would carry charge through ANO1 channels, but I− is a poor substitute for Cl− in NKCC1 (27). Thus substitution of [Cl−] o with [I−] o would have similar consequences as the addition of bumetanide in terms of reducing Cl− recovery into ICC. Despite the important physiological role played by NKCC1 in many cells (26) and in the pacemaker activity of ICC, global knockouts of Slc12a2 survive. A previous report in which the electrical activity was recorded from small intestinal muscles of Slc12a2−/− mice showed that slow waves were reduced in amplitude and frequency and were insensitive to bumetanide treatment (44). These data seem to contrast with the rapid shift in E Cl and cessation of spontaneous activity that we noted after treatment of isolated ICC with bumetanide. It is possible that some form of compensation occurs in global knockouts and/or that another Cl− transporter is capable of providing maintenance of E Cl in the absence of NKCC1. In summary, our experiments have demonstrated the dynamic nature of E STICs (which is likely to be nearly equivalent to E Cl ) in ICC. The large Cl− currents generated during slow waves through activation of ANO1 channels result in loss of Cl− from the microdomains of pacemaker units. Loss of Cl− must be compensated for to sustain pacemaker activity, and NKCC1 appears to have a significant role in maintenance of E Cl . Blocking NKCC1 caused a negative shift in E STICs , collapse of the driving force for efflux of Cl− through ANO1 channels, and inhibition of pacemaker activity.

GRANTS This work was supported by a Program Project Grant from NIDDK , P01 DK41325 .

DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS M.H.Z., T.S.S., S.D.K., and K.M.S. conception and design of research; M.H.Z., T.S.S., M.K., L.E.O., and K.O. performed experiments; M.H.Z., T.S.S., M.K., L.E.O., K.O., and S.D.K. analyzed data; M.H.Z., T.S.S., M.K., L.E.O., K.O., S.D.K., and K.M.S. interpreted results of experiments; M.H.Z., T.S.S., M.K., L.E.O., S.D.K., and K.M.S. prepared figures; M.H.Z., S.D.K., and K.M.S. drafted manuscript; M.H.Z., S.D.K., and K.M.S. edited and revised manuscript; M.H.Z., T.S.S., L.E.O., K.O., S.D.K., and K.M.S. approved final version of manuscript.