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Autophagy is a multiple fusion event, initiating with autophagosome formation and culminating with fusion with endo-lysosomes in a Ca

Autophagy is a multiple fusion event, initiating with autophagosome formation and culminating with fusion with endo-lysosomes in a Ca 2+ -dependent manner. The source of Ca 2+ and the molecular mechanism by which Ca 2+ is provided for this process are not known. The intracellular Ca 2+ permeable channel transient receptor potential mucolipin 3 (TRPML3) localizes in the autophagosome and interacts with the mammalian autophagy-related protein 8 (ATG8) homolog GATE16. Here, we show that lipid-regulated TRPML3 is the Ca 2+ release channel in the phagophore that provides the Ca 2+ necessary for autophagy progress. We generated a TRPML3-GCaMP6 fusion protein as a targeted reporter of TRPML3 compartment localization and channel function. Notably, TRPML3-GCaMP6 localized in the phagophores, the level of which increased in response to nutrient starvation. Importantly, phosphatidylinositol-3-phosphate (PI3P), an essential lipid for autophagosome formation, is a selective regulator of TRPML3. TRPML3 interacted with PI3P, which is a direct activator of TRPML3 current and Ca 2+ release from the phagophore, to promote and increase autophagy. Inhibition of TRPML3 suppressed autophagy even in the presence of excess PI3P, while activation of TRPML3 reversed the autophagy inhibition caused by blocking PI3P. Moreover, disruption of the TRPML3–PI3P interaction abolished both TRPML3 activation by PI3P and the increase in autophagy. Taken together, these results reveal that TRPML3 is a downstream effector of PI3P and a key regulator of autophagy. Activation of TRPML3 by PI3P is the critical step providing Ca 2+ from the phagophore for the fusion process, which is essential for autophagosome biogenesis.

Transient receptor potential (TRP) channels comprise a diverse family of ion channels, the majority of which are calcium permeable and show sophisticated regulatory patterns in response to various environmental cues. Early studies led to the recognition of TRP channels as environmental and chemical sensors. Later studies revealed that TRP channels mediated the regulation of intracellular calcium. Mutations in TRP channel genes result in abnormal regulation of TRP channel function or expression, and interfere with normal spatial and temporal patterns of intracellular local Ca2+ distribution. The resulting dysregulation of multiple downstream effectors, depending on Ca2+ homeostasis, is associated with hallmarks of cancer pathophysiology, including enhanced proliferation, survival and invasion of cancer cells. These findings indicate that TRP channels affect multiple events that control cellular fate and play a key role in cancer progression. This review discusses the accumulating evidence supporting the role of TRP channels in tumorigenesis, with emphasis on prostate cancer. [BMB Reports 2020; 53(3): 125-132].

Despite recent evidence suggesting that adult trematodes require oxygen for the generation of bioenergy and eggshells, information on the molecular mechanism by which the parasites acquire oxygen remains largely elusive. In this study, the structural and expressional features of globin genes identified in Clonorchis sinensis, a carcinogenic trematode parasite that invades the hypoxic biliary tracts of mammalian hosts, were investigated to gain insight into the molecules that enable oxygen metabolism. The number of globin paralogs substantially differed among parasitic platyhelminths, ranging from one to five genes, and the C. sinensis genome encoded at least five globin genes. The expression of these Clonorchis genes, named CsMb (CsMb1—CsMb3), CsNgb, and CsGbX, according to their preferential similarity patterns toward respective globin subfamilies, exponentially increased in the worms coinciding with their sexual maturation, after being downregulated in early juveniles compared to those in metacercariae. The CsMb1 protein was detected throughout the parenchymal region of adult worms as well as in excretory-secretory products, whereas the other proteins were localized exclusively in the sexual organs and intrauterine eggs. Stimuli generated by exogenous oxygen, nitric oxide (NO), and nitrite as well as co-incubation with human cholangiocytes variously affected globin gene expression in live C. sinensis adults. Together with the specific histological distributions, these hypoxia-induced patterns may suggest that oxygen molecules transported by CsMb1 from host environments are provided to cells in the parenchyma and intrauterine eggs/sex organs of the worms for energy metabolism and/or, more importantly, eggshell formation by CsMb1 and CsMb3, respectively. Other globin homologs are likely to perform non-respiratory functions. Based on the responsive expression profile against nitrosative stress, an oxygenated form of secreted CsMb1 is suggested to play a pivotal role in parasite survival by scavenging NO generated by host immune cells via its NO dioxygenase activity., Author summary Trematode parasites that invade mammalian tissues have long been believed to produce bioenergy via anaerobic respiration in their definitive hosts. However, recent studies have revealed that these parasites require considerable amounts of oxygen for the generation of hard eggshells during sexual reproduction as well as energy metabolism. Despite these findings, information on the biological mechanisms and relevant molecules responsible for oxygen uptake in the host environment remains largely elusive. Clonorchis sinensis is a carcinogenic trematode parasite that causes clonorchiasis in humans by infecting the bile ducts. Here, we investigated globin genes/proteins in the liver fluke. The genome of C. sinensis encoded at least five globin paralogs (CsMb1, CsMb2, CsMb3, CsNgb, and CsGbX). Temporal expression of these globin genes coincided with the sexual maturation of C. sinensis. Based on the histological localities and induction profiles upon hypoxia, it could be postulated that the oxygen molecules transported by CsMb1 from host environments are provided to cells in the parenchyma and intrauterine eggs/sex organs of the worms by CsMb1 and CsMb3, respectively, for energy metabolism and eggshell formation. Other globin homologs were likely to perform non-respiratory functions. In addition, the oxygenated form of secreted CsMb1 seemed to participate in the scavenging of nitric oxide generated by host immune cells via its nitric oxide dioxygenase activity to increase the survival of the parasite.

Accumulation of tissue macrophages is a significant characteristic of disease-associated chronic inflammation, and facilitates the progression of disease pathology. However, the functional roles of these bone marrow-derived macrophages (BMDMs) in aging are unclear. Here, we identified agedependent macrophage accumulation in the bone marrow, showing that aging significantly increases the number of M1 macrophages and impairs polarization of BMDMs. We found that age-related dysregulation of BMDMs is associated with abnormal overexpression of the anti-inflammatory interleukin-10. BMDM dysregulation in aging impairs the expression levels of pro-inflammatory cytokines and genes involved in B-cell maturation and activation. Phagocytosis of apoptotic Jurkat cells by BMDMs was reduced because of low expression of phagocytic receptor CD14, indicating that increased apoptotic cells may result from defective phagocytosis of apoptotic cells in the BM of aged mice. Therefore, CD14 may represent a promising target for preventing BMDM dysregulation, and macrophage accumulation may provide diagnostic and therapeutic clues. [BMB Reports 2017; 50(1): 43-48].

Interstitial cells of Cajal (ICCs) function as pacemaker cells in the gastrointestinal (GI) tract and therefore, serve an important role in regulating GI motility. The effects of a species of plum (Prunus mume Siebold & Zucc.) on cultured ICC cluster-induced pacemaker potentials in the mouse small intestine were investigated, and the effects of a methanolic extract of Prunus mume (m-PM) on ICC pacemaker activities were examined using the whole-cell patch-clamp technique. ICC pacemaker membrane potentials were depolarized by m-PM in a concentration dependent manner in current clamp mode. 4-Diphenylacetoxy-N-methyl-piperidine methiodide, which is a muscarinic 3 (M3) receptor antagonist, was able to block m-PM-induced pacemaker potential increases, whereas methoctramine, which is a muscarinic 2 (M2) receptor antagonist, was not. When 1 mM guanosine diphosphate β-5 was present in the pipette solution, m-PM induced slight pacemaker depolarization. Following pretreatment in bath solution of Ca2+-free solution or a Ca2+-ATPase inhibitor in endoplasmic reticulum, the pacemaker currents were inhibited. Furthermore, pretreatment with PD98059, SB203580 or SP600125, which is a c-jun NH2-terminal kinase inhibitor, blocked m-PM-induced ICC potential depolarization. Furthermore, m-PM inhibited transient receptor potential melastatin (TRPM) 7 channels, but did not affect Ca2+-activated Cl- channels. These results suggest that m-PM is able to modulate pacemaker potentials through the muscarinic M3 receptor, via G-protein and external and internal Ca2+, in a mitogen-activated protein kinase and TRPM7-dependent manner. Therefore, m-PM may provide a basis for the development of a novel gastroprokinetic agent.

Dietary supplement and personal care products aiming to provide protection from air pollution have been of great interest for decades. Epidemiology demonstrated that PM10 and PM2.5 particulate matter (PM) are an actual threat to public health worldwide, but the detailed processes of how these particles attack the cells are not fully understood. Here, we report that the measurement of intracellular calcium concentration ([Ca2+]i) using human respiratory or skin cells can illustrate pollutant challenges by triggering Ca2+ influx in these cells. This signal was generated by proteinase-activated receptor-2 (PAR-2), confirmed by competition analyses, and Phellodendron amurense bark extract (PAE), a traditional medicine, was able to control the response and expression of PAR-2. Increase in proinflammatory cytokines and decrease in cell adhesion components could suggest a severe damage status by air pollutants and protection by PAE. Finally, we identified 4-O-feruloylquinic acid (FQA), an active compound of PAE, showing the same effects on Ca2+ influx and PAR-2 regulation. The results presented here should help understand the underlying mechanism of PM insults and the beneficial effect of standardized PAE as dietary supplement or cosmetical ingredient.

Actinomyces meyeri is a Gram positive, strict anaerobic bacterium, which was first described by Meyer in 1911. Primary actinomycotic osteomyelitis is rare and primarily affects the cervicofacial region, including mandible. We present an unusual case of osteomyelitis of a long bone combined with myoabscess due to A. meyeri. A 70-year-old man was admitted for pain and pus discharge of the right elbow. Twenty-five days before admission, he had hit his elbow against a table. MRI of the elbow showed a partial tear of the distal triceps tendon and myositis. He underwent open debridement and partial bone resection for the osteomyelitis of the olecranon. Biopsy showed no sulfur granules, but acute and chronic osteomyelitis. The excised tissue grew A. meyeri and Peptoniphilus asaccharolyticus. Intravenous ceftriaxone was administered and switched to oral amoxicillin. Infection of the extremities of actinomycosis often poses diagnostic difficulties, but it should not be neglected even when the characteristic pathologic findings are not present.

Inositol-1,4,5-triphosphate [IP3] receptors binding protein released with IP3 (IRBIT) was previously reported as an activator of NBCe1-B. Recent studies have characterized IRBIT homologue S-Adenosylhomocysteine hydrolase-like 2 (AHCYL2). AHCYL2 is highly homologous to IRBIT (88%) and heteromerizes with IRBIT. The two important domains in the N-terminus of AHCYL2 are a PEST domain and a coiled-coil domain which are highly comparable to those in IRBIT. Therefore, in this study, we tried to identify the role of those domains in mouse AHCYL2 (Ahcyl2), and we succeeded in identifying PEST domain of Ahcyl2 as a regulation region for NBCe1-B activity. Site directed mutagenesis and coimmunoprecipitation assay showed that NBCe1-B binds to the N-terminal Ahcyl2-PEST domain, and its binding is determined by the phosphorylation of 4 critical serine residues (Ser151, Ser154, Ser157, and Ser160) in Ahcyl2 PEST domain. Also we revealed that 4 critical serine residues in Ahcyl2 PEST domain are indispensable for the activation of NBCe1-B using measurement of intracellular pH experiment. Thus, these results suggested that the NBCe1-B is interacted with 4 critical serine residues in Ahcyl2 PEST domain, which play an important role in intracellular pH regulation through NBCe1-B.

Transient receptor potential canonical (TRPC) family contains a non-selective cation channel, and four TRPC subunits form a functional tetrameric channel. TRPC4/5 channels form not only the homotetrameric channel but also a heterotetrameric channel with TRPC1. We investigated the interaction domain required for TRPC1/4 or TRPC1/5 heteromultimeric channels using FRET and the patch-clamp technique. TRPC1 only localized at the plasma membrane (PM) when it was coexpressed with TRPC4 or TRPC5. The TRPC1/4 or TRPC1/5 heteromultimeric showed the typical outward rectifying I/V curve. When TRPC1 and TRPC4 form a heteromeric channel, the N-terminal coiled-coil domain (CCD) and C-terminal 725-745 region of TRPC1 interact with the N-terminal CCD and C-terminal 700-728 region of TRPC4. However, when TRPC1 and TRPC5 form a heteromeric channel, the N-terminal CCD and C-terminal 673-725 region of TRPC1 interact with the N-terminal CCD and C-terminal 707-735 region of TRPC5. In conclusion, the N-terminal CCD of TRPC channels is essential for the heteromultimeric structure of TRPC channels, whereas specific C-terminal regions are required for unique heteromerization between subgroups of TRPC channels.

The diacylglycerol kinase family, which can attenuate diacylglycerol signaling and activate phosphatidic acid signaling, regulates various signaling transductions in the mammalian cells. Studies on the regulation of diacylglycerol and phosphatidic acid levels by various enzymes, the identification and characterization of various diacylglycerol and phosphatidic acid-regulated proteins, and the overlap of different diacylglycerol and phosphatidic acid metabolic and signaling processes have revealed the complex and non-redundant roles of diacylglycerol kinases in regulating multiple biochemical and biological networks. In this review article, we summarized recent progress in the complex and non-redundant roles of diacylglycerol kinases, which is expected to aid in restoring dysregulated biochemical and biological networks in various pathological conditions at the bed side.

The transient receptor potential canonical (TRPC) 1 channel is widely distributed in mammalian cells and is involved in many physiological processes. TRPC1 is primarily considered a regulatory subunit that forms heterotetrameric channels with either TRPC4 or TRPC5 subunits. Here, we suggest that the regulation of TRPC1/4 and TRPC1/5 heterotetrameric channels by the Gαq-PLCβ pathway is self-limited and dynamically mediated by Gαq and PI(4,5)P2. We provide evidence indicating that Gαq protein directly interacts with either TRPC4 or TRPC5 of the heterotetrameric channels to permit activation. Simultaneously, Gαq-coupled PLCβ activation leads to the breakdown of PI(4,5)P2, which inhibits activity of TRPC1/4 and 1/5 channels.

The transient receptor potential canonical (TRPC) 1 channel is widely distributed in mammalian cells and is involved in many physiological processes. TRPC1 is primarily considered a regulatory subunit that forms heterotetrameric channels with either TRPC4 or TRPC5 subunits. Here, we suggest that the regulation of TRPC1/4 and TRPC1/5 heterotetrameric channels by the Gαq-PLCβ pathway is self-limited and dynamically mediated by Gαq and PI(4,5)P2. We provide evidence indicating that Gαq protein directly interacts with either TRPC4 or TRPC5 of the heterotetrameric channels to permit activation. Simultaneously, Gαq-coupled PLCβ activation leads to the breakdown of PI(4,5)P2, which inhibits activity of TRPC1/4 and 1/5 channels.

Protein phosphatase 1 (PP1) is involved in various signal transduction mechanisms as an extensive regulator. The PP1 catalytic subunit (PP1c) recognizes and binds to PP1-binding consensus residues (FxxR/KxR/K) in NBCe1-B. Consequently, we focused on identifying the function of the PP1-binding consensus residue, 922FMDRLK927, in NBCe1-B. Using site-directed mutagenesis and co-immunoprecipitation assays, we revealed that in cases where the residues were substituted (F922A, R925A, and K927A) or deleted (deletion of amino acids 922-927), NBCe1-B mutants inhibited PP1 binding to NBCe1-B. Additionally, by recording the intracellular pH, we found that PP1-binding consensus residues in NBCe1-B were not only critical for NBCe1-B activity, but also relevant to its surface expression level. Therefore, we reported that NBCe1-B, as a substrate of PP1, contains these residues in the C-terminal region and that the direct interaction between NBCe1-B and PP1 is functionally critical in controlling the regulation of the HCO3- transport. These results suggested that like IRBIT, PP1 was another novel regulator of HCO3- secretion in several types of epithelia.

Ca

Background: Daikenchuto (DKT; TJ-100, TU-100), a traditional herbal medicineis used in modern medicine to treat gastrointestinal (GI) functional disorders. Interstitial cells of Cajal (ICCs) are the pacemaker cells of the GI tract and play important roles in the regulation of GI motility. Objective: The objective of this study was to investigate the effects of DKT on the pacemaker potentials (PPs) of cultured ICCs from murine small intestine. Materials and Methods: Enzymatic digestions were used to dissociate ICCs from mouse small intestine tissues. All experiments on ICCs were performed after 12 h of culture. The whole-cell patch-clamp configuration was used to record ICC PPs (current clamp mode). All experiments were performed at 30-32°C. Results: In current-clamp modeDKT depolarized and concentration-dependently decreased the amplitudes of PPs. Y25130 (a 5-HT3 receptor antagonist) or SB269970 (a 5-HT7 receptor antagonist) did not block DKT-induced PP depolarization, but RS39604 (a 5-HT4 receptor antagonist) did. Methoctramine (a muscarinic M2 receptor antagonist) failed to block DKT-induced PP depolarization, but pretreating 4-diphenylacetoxy-N-methylpiperidine methiodide (a muscarinic M3 receptor antagonist) facilitated blockade of DKT-induced PP depolarization. Pretreatment with an external Ca2+-free solution or thapsigargin abolished PPsand under these conditions, DKT did not induce PP depolarization. Furthermore Ginseng radix and Zingiberis rhizomes depolarized PPs, whereas Zanthoxyli fructus fruit (the third component of DKT) hyperpolarized PPs. Conclusion: These results suggest that DKT depolarizes ICC PPs in an internal or external Ca2+-dependent manner by stimulating 5-HT4 and M3 receptors. Furthermore, the authors suspect that the component in DKT largely responsible for depolarization is probably also a component of Ginseng radix and Zingiberis rhizomes. SUMMARY Daikenchuto (DKT) depolarized and concentration-dependently decreased the amplitudes of pacemaker potentials (PPs)Y25130 (a 5-HT3 receptor antagonist) or SB269970 (a 5-HT7 receptor antagonist) did not block DKT-induced PP depolarization, but RS39604 (a 5-HT4 receptor antagonist) didMethoctramine (a muscarinic M2 receptor antagonist) failed to block DKT-induced PP depolarization, but pretreating 4-DAMP (a muscarinic M3 receptor antagonist) facilitated blockade of DKT-induced PP depolarizationGinseng radix and Zingiberis rhizomes depolarized PPswhereas Zanthoxyli fructus fruit (the third component of DKT) hyperpolarized PPs. Abbreviation used: DKT: Daikenchuto, GI: Gastrointestinal, ICCs: Interstitial cells of Cajal, PPs: Pacemaker Potentials.

Ca2+ influx by plasma membrane Ca2+ channels is the crucial component of the receptor-evoked Ca2+ signal. The two main Ca2+ influx channels of non-excitable cells are the Orai and TRPC families of Ca2+ channels. These channels are activated in response to cell stimulation and Ca2+ release from the endoplasmic reticulum (ER). The protein that conveys the Ca2+ content of the ER to the plasma membrane is the ER Ca2+ sensor STIM1. STIM1 activates the Orai channels and is obligatory for channel opening. TRPC channels can function in two modes, as STIM1-dependent and STIM1-independent. When activated by STIM1, both channel types function at the ER/PM (plasma membrane) junctions. This chapter describes the properties and regulation of the channels by STIM1, with emphasis how and when TRPC channels function as STIM1-dependent and STIM1-independent modes and their unique Ca2+-dependent physiological functions that are not shared with the Orai channels.

Plasma membrane hyperpolarization associated with activation of Ca2+-activated K+ channels plays an important role in sperm capacitation during fertilization. Although Slo3 (slowpoke homologue 3), together with the auxiliary γ2-subunit, LRRC52 (leucine-rich-repeat–containing 52), is known to mediate the pH-sensitive, sperm-specific K+ current KSper in mice, the molecular identity of this channel in human sperm remains controversial. In this study, we tested the classical BKCa activators, NS1619 and LDD175, on human Slo3, heterologously expressed in HEK293 cells together with its functional interacting γ2 subunit, hLRRC52. As previously reported, Slo3 K+ current was unaffected by iberiotoxin or 4-aminopyridine, but was inhibited by ~50% by 20 mM TEA. Extracellular alkalinization potentiated hSlo3 K+ current, and internal alkalinization and Ca2+ elevation induced a leftward shift its activation voltage. NS1619, which acts intracellularly to modulate hSlo1 gating, attenuated hSlo3 K+ currents, whereas LDD175 increased this current and induced membrane potential hyperpolarization. LDD175-induced potentiation was not associated with a change in the half-activation voltage at different intracellular pHs (pH 7.3 and pH 8.0) in the absence of intracellular Ca2+. In contrast, elevation of intracellular Ca2+ dramatically enhanced the LDD175-induced leftward shift in the half-activation potential of hSlo3. Therefore, the mechanism of action does not involve pH-dependent modulation of hSlo3 gating; instead, LDD175 may modulate Ca2+-dependent activation of hSlo3. Thus, LDD175 potentially activates native KSper and may induce membrane hyperpolarization-associated hyperactivation in human sperm.

Conflicting evidence has been obtained regarding whether transient receptor potential cation channels (TRPC) are store-operated channels (SOCs) or receptor-operated channels (ROCs). Moreover, the Ca/Na permeability ratio differs depending on whether the current-voltage (I-V) curve has a doubly rectifying shape or inward rectifying shape. To investigate the calcium permeability of TRPC4 channels, we attached GCaMP6s to TRPC4 and simultaneously measured the current and calcium signals. A TRPC4 specific activator, (-)-englerin A, induced both current and calcium fluorescence with the similar time course. Muscarinic receptor stimulator, carbachol, also induced both current and calcium fluorescence with the similar time course. By forming heteromers with TRPC4, TRPC1 significantly reduced the inward current with outward rectifying I-V curve, which also caused the decrease of calcium fluorescence intensity. These results suggest that GCaMP6s attached to TRPC4 can detect slight calcium changes near TRPC4 channels. Consequently, TRPC4-GCaMP6s can be a useful tool for testing the calcium permeability of TRPC4 channels.

The ubiquitous transient receptor potential canonical (TRPC) channels function as non-selective, Ca(2+)-permeable channels. TRPC channels are activated by stimulation of Gαq-PLC-coupled receptors. Here, we report that TRPC4/TRPC5 can be activated by Gαi. We studied the essential role of Gαi subunits in TRPC4 activation and investigated changes in ion selectivity and pore dilation of the TRPC4 channel elicited by the Gαi2 subunit. Activation of TRPC4 by Gαi2 increased Ca2+ permeability and Ca2+ influx through TRPC4 channels. Co-expression of the muscarinic receptor (M2) and TRPC4 in HEK293 cells induced TRPC4-mediated Ca2+ influx. Moreover, both TRPC4β and the TRPC4β-Gαi2 signaling complex induced inhibition of neurite growth and arborization in cultured hippocampal neurons. Cells treated with KN-93, a CaMKII inhibitor, prevented TRPC4- and TRPC4-Gαi2(Q205L)-mediated inhibition of neurite branching and growth. These findings indicate an essential role of Gαi proteins in TRPC4 activation and extend our knowledge of the functional role of TRPC4 in hippocampal neurons.

Fluid and HCO 3 − secretion is a vital function of secretory epithelia, involving basolateral HCO 3 − entry through the Na + -HCO 3 − cotransporter (NBC) NBCe1-B, and luminal HCO 3 − exit mediated by cystic fibrosis transmembrane conductance regulator (CFTR) and solute carrier family 26 (SLC26) Cl − /HCO 3 − exchangers. HCO 3 − secretion is highly regulated, with the WNK/SPAK kinase pathway setting the resting state and the IRBIT/PP1 pathway setting the stimulated state. However, we know little about the relationships between the WNK/SPAK and IRBIT/PP1 sites in the regulation of the transporters. The first 85 N-terminal amino acids of NBCe1-B function as an autoinhibitory domain. Here we have identified a positively charged module within NBCe1-B(37-65) that is conserved in NBCn1-A and all 20 members of the NBC superfamily except NBCe1-A. This module is required for the interaction and activation of NBCe1-B and NBCn1-A by IRBIT and their regulation by phosphatidylinositol 4,5-bisphosphate (PIP 2 ). Activation of the transporters by IRBIT and PIP 2 is nonadditive but complementary. Phosphorylation of Ser65 mediates regulation of NBCe1-B by SPAK, and phosphorylation of Thr49 is required for regulation by IRBIT and SPAK. Sequence searches using the NBCe1-B regulatory module as a template identified a homologous sequence in the CFTR R domain and Slc26a6 sulfat transporter and antisigma factor antagonist (STAS) domain. Accordingly, the R and STAS domains bind IRBIT, and the R domain is required for activation of CFTR by IRBIT. These findings reveal convergence of regulatory modalities in a conserved domain of the NBC that may be present in other HCO 3 − transporters and thus in the regulation of epithelial fluid and HCO 3 − secretion.

Ca(2+) is a critical factor in the regulation of signal transduction and Ca(2+) homeostasis is altered in different human diseases. The level of Ca(2+) in cells is highly regulated through a diverse class of regulators. Among them is the transient receptor potential vanilloid 6 (TRPV6), which is a Ca(2+) selective channel that absorbs Ca(2+) in the small intestine. TRPV6 is overexpressed in some cancers and exhibits oncogenic potential, but its exact mechanism is still poorly understood. The Numb protein is a cell fate determinant that functions in endocytosis and as a tumor suppressor via the stabilization of p53. Numb protein consisted of four isoforms. Here, we showed a novel function of Numb1, which negatively regulates TRPV6 activity. The expression of Numb1 decreased cytosolic Ca(2+) concentrations in TRPV6-transfected HEK293 cells. When all the isoforms of Numb were depleted using siRNA in a TRPV6 stable cell line, the levels of cytosolic Ca(2+) increased. We observed an interaction between Numb1 and TRPV6 using co-immunoprecipitation. We confirmed this interaction using Fluorescence Resolution Energy Transfer (FRET). We identified the TRPV6 and Numb1 binding site using TRPV6 C-terminal truncation mutants and Numb1 deletion mutants. The binding site in TRPV6 was an aspartic acid at amino acid residue 716, and that binding site in Numb1 was arginine at amino acid residue 434. A Numb1 mutant, lacking TRPV6 binding activity, failed to inhibit TRPV6 activity. Every isoform of Numb knockdown, using an siRNA-based approach in MCF-7 breast cancer cells, not only showed enhanced TRPV6 expression but also both the cytosolic Ca(2+) concentration and cell proliferation were increased. The down-regulated expression of TRPV6 using siRNA increased Numb protein expression; however, the cytosolic influx of Ca(2+) and proliferation of the cell were decreased. To examine downstream signaling during Ca(2+) influx, we performed Western blotting analysis on TRPV6 upregulated cancer cells (MCF-7, PC-3). Taken together, these results demonstrated that Numb1 interacts with TRPV6 through charged residues and inhibits its activity via the regulation of protein expression. Moreover, we provided evidence for a Ca(2+)-regulated cancer cell signaling pathway and that the Ca(2+) channel is a target of cancer cells.

The chloride channel (CLC) family of proteins consists of channels and transporters that share similarities in architecture and play essential roles in physiological functions. Among the CLC family, CLC-1 channels have the representative homodimeric double-barreled structure carrying two gating processes. One is protopore gating that acts on each pore independently by glutamate residue (Eext). The other is common gating that closes both pores simultaneously in association with large conformational changes across each subunit. In skeletal muscle, CLC-1 is associated with maintaining normal sarcolemmal excitability, and a number of myotonic mutants were reported to modify the channel gating of CLC-1. In this study, we characterized highly conserved helix O as a key determinant of structural stability in CLC-1. Supporting this hypothesis, myotonic mutant (G523D) at N-terminal of helix O showed the activation at hyperpolarizing membrane potentials with a reversed voltage dependency. However, introducing glutamate at serine residue (S537) at the C-terminal of the helix O on G523D restored WT-like voltage dependency of the common gate and showed proton insensitive voltage dependency. To further validate this significant site, site-specific mutagenesis experiments was performed on V292 that is highly conserved as glutamate in antiporter and closely located to S537 and showed that this area is essential for channel function. Taken together, the results of our study suggest the importance of helix O as the main contributor for stable structure of evolutionary conserved CLC proteins and its key role in voltage dependency of the CLC-1. Furthermore, the C-terminal of the helix O can offer a clue for possible proton involvement in CLC-1 channel.

Oxidative stress in pancreatic beta cells can inhibit insulin secretion and promote apoptotic cell death. Exendin-4 (EX4), a glucagon-like peptide-1 receptor agonist, can suppress beta cell apoptosis, improve beta cell function and protect against oxidative damage. In this study, we investigated the molecular mechanisms for antioxidative effects of EX4 in pancreatic beta cells. INS-1 cells, a rat insulinoma cell line, were pretreated with EX4 and exposed to palmitate or H2O2. Reactive oxygen species (ROS) production, and glutathione and insulin secretion were measured. The mRNA and protein expression levels of antioxidant genes were examined. The level of nuclear factor erythroid 2-related factor 2 (Nrf2), its binding to antioxidant response element (ARE), and its ubiquination in the presence of EX4 were determined. The Nrf2 signaling pathway was determined using rottlerin (protein kinase [PK]Cδ inhibitor), H89 (PKA inhibitor) and LY294002 (phosphatidylinositide 3-kinase [PI3K] inhibitor). EX4 treatment decreased ROS production, recovered cellular glutathione levels and insulin secretion in the presence of oxidative stress in INS-1 cells. The expression levels of glutamate-cysteine ligase catalytic subunit and heme oxygenase-1 were increased by EX4 treatment. EX4 promoted Nrf2 translocation, ARE binding activity and enhanced stabilization of Nrf2 by inhibition of ubiquitination. Knockdown of Nrf2 abolished the effect of EX4 on increased insulin secretion. Inhibition of PKCδ attenuated Nrf2 translocation and antioxidative gene expression by EX4 treatment. We suggest that EX4 activates and stabilizes Nrf2 through PKCδ activation, contributing to the increase of antioxidant gene expression and consequently improving beta cell function in the presence of oxidative stress.

We examined the effects of mibefradil, a T-type Ca2+ channel inhibitor, on voltage-dependent K+ (Kv) channels in rabbit coronary arterial smooth muscle cells using the whole-cell patch clamp technique. Mibefradil reduced the Kv current amplitude in a dose-dependent manner, with an apparent Kd value of 1.08 μM. Kv current inhibition by mibefradil was highly voltage-dependent over the full activation voltage range (−30 to +10 mV). The decay rate of Kv channel inactivation was accelerated by mibefradil without altering the kinetics of current activation. The rate constants of association and dissociation were 2.23 ± 0.07 μM−1·s−1 and 2.40 ± 0.42 s−1, respectively. Mibefradil had no significant effect on the steady-state activation or inactivation curves. In the presence of mibefradil, the recovery time constant from inactivation was decreased, and the application of train pulses (1 or 2 Hz) increased mibefradil-induced Kv channel inhibition, suggesting that the inhibitory effects of mibefradil were use-dependent. The inhibitory effect of mibefradil on Kv channels was unaffected by extracellular Ca2+-free conditions. Moreover, the absence of ATP inside the pipette did not alter the blocking effect of mibefradil. Therefore, we suggest that mibefradil directly inhibited the Kv current, independently of Ca2+ channel inhibition. Keywords:: mibefradil, voltage-dependent K+ channel, coronary artery

The solute carrier 26 (SLC26) transporters are anion transporters with diverse substrate specificity. Several members are ubiquitous while others show limited tissue distribution. They are expressed in many epithelia and to the extent known, play a central role in anion secretion and absorption. Members of the family are primarily Cl- transporters, although some members transport mainly SO(4)2-, Cl-, HCO(3)- or I-. A defining feature of the family is their functional diversity. Slc26a1 and Slc26a2 function as specific SO(4)2- transporters while Slc26a4 functions as an electroneutral Cl-/I-/HCO(3)- exchanger. Slc26a3 and Slc26a6 function as coupled electrogenic Cl-/HCO(3)- exchangers or as bona fide anion channels. SLC26A7 and SLC26A9 function exclusively as Cl- channels. This short review discusses the functional diversity of the SLC26 transporters.

Transepithelial Cl−and HCO3−transport is critically important for the function of all epithelia and, when altered or ablated, leads to a number of diseases, including cystic fibrosis, congenital chloride diarrhea, deafness, and hypotension ( 78 , 111 , 119 , 126 ). HCO3−is the biological buffer that maintains acid-base balance, thereby preventing metabolic and respiratory acidosis ( 48 ). HCO3−also buffers the pH of the mucosal layers that line all epithelia, protecting them from injury ( 2 ). Being a chaotropic ion, HCO3−is essential for solubilization of ions and macromolecules such as mucins and digestive enzymes in secreted fluids. Most epithelia have a Cl−/HCO3exchange activity in the luminal membrane. The molecular nature of this activity remained a mystery for many years until the discovery of SLC26A3 and the realization that it is a member of a new family of Cl−and HCO3−transporters, the SLC26 family ( 73 , 78 ). This review will highlight structural features, the functional diversity, and several regulatory aspects of the SLC26 transporters.

The epithelial Ca(2+) channel TRPV6 constitutes the apical Ca(2+) entry mechanism in active Ca(2+) transport in kidney and intestine, but little is known about regulation mechanism of TRPV6. We performed yeast two-hybrid screen with TRPV6 C-terminus since TRPV6 has PDZ (Post-synaptic density-95, Drosophila discs-large protein, Zonula occludens protein 1) binding motif at its C terminal end. As a result, we found that 4 PDZ domain-containing protein, PDZK2, interacts with TRPV6 through its fourth PDZ domain. Glutathione S-transferase pull-down assay shows that TRPV6 and PDZK2 directly interact and that TRPV6 C-terminal PDZ binding motif is primarily responsible for this interaction. Mutant Delta4 lacking last 4 amino acid EYQI did not interact with PDZK2. Heterologous overexpression of both TRPV6 and PDZK2 did not show any effect on the activation of TRPV6. On the other hand, the peak current amplitude of mutant Delta4 decreased compared with that of WT TRPV6. When introduced into HEK293 cells expressing TRPV6, PDZ binding motif peptide (EYQI) markedly reduced the peak current amplitude in divalent free (DVF) solution. Knocking down the endogenous PDZK2 of HEK293 cells by RNAi significantly decreased DVF current density. Taken together, we propose that PDZK2 is an essential TRPV6 interacting protein as a physiological modulator of TRPV6.

Interstitial cells of Cajal (ICCs) function as pacemaker cells in the gastrointestinal (GI) tract and therefore, serve an important role in regulating GI motility. The effects of a species of plum (Prunus mume Siebold & Zucc.) on cultured ICC cluster-induced pacemaker potentials in the mouse small intestine were investigated, and the effects of a methanolic extract of Prunus mume (m-PM) on ICC pacemaker activities were examined using the whole-cell patch-clamp technique. ICC pacemaker membrane potentials were depolarized by m-PM in a concentration dependent manner in current clamp mode. 4-Diphenylacetoxy-N-methyl-piperidine methiodide, which is a muscarinic 3 (M3) receptor antagonist, was able to block m-PM-induced pacemaker potential increases, whereas methoctramine, which is a muscarinic 2 (M2) receptor antagonist, was not. When 1 mM guanosine diphosphate β-5 was present in the pipette solution, m-PM induced slight pacemaker depolarization. Following pretreatment in bath solution of Ca2+-free solution or a Ca2+-ATPase inhibitor in endoplasmic reticulum, the pacemaker currents were inhibited. Furthermore, pretreatment with PD98059, SB203580 or SP600125, which is a c-jun NH2-terminal kinase inhibitor, blocked m-PM-induced ICC potential depolarization. Furthermore, m-PM inhibited transient receptor potential melastatin (TRPM) 7 channels, but did not affect Ca2+-activated Cl− channels. These results suggest that m-PM is able to modulate pacemaker potentials through the muscarinic M3 receptor, via G-protein and external and internal Ca2+, in a mitogen-activated protein kinase and TRPM7-dependent manner. Therefore, m-PM may provide a basis for the development of a novel gastroprokinetic agent.

TRPC4 is important regulators of electrical excitability in gastrointestinal myocytes, pancreatic β-cells and neurons. Much is known regarding the assembly and function of these channels including TRPC1 as a homotetramer or a heteromultimer and the roles that their interacting proteins play in controlling these events. Further, they are one of the best-studied targets of G protein-coupled receptors and growth factors in general and Gαi/o and Gαq protein coupled receptor or epidermal growth factor and leptin in particular. However, our understanding of the roles of small G proteins and leptin on TRPC4 channels is still rudimentary. We discuss potential roles for Rasd1 small G protein and leptin in channel activation in addition to their known role in cellular signaling.

Dexmedetomidine is a sedative and analgesic agent that exerts its effects by selectively agonizing α2 adrenoceptor. Histamine is a pathophysiological amine that activates G protein-coupled receptors, to induce Ca(2+) release and subsequent mediate or progress inflammation. Dexmedetomidine has been reported to exert inhibitory effect on inflammation both in vitro and in vivo studies. However, it is unclear that dexmedetomidine modulates histamine-induced signaling and pro-inflammatory cytokine expression. This study was carried out to assess how dexmedetomidine modulates histamine-induced Ca(2+) signaling and regulates the expression of pro-inflammatory cytokine genes encoding interleukin (IL)-6 and -8. To elucidate the regulatory role of dexmedetomidine on histamine signaling, HeLa cells and human salivary gland cells which are endogenously expressed histamine 1 receptor were used. Dexmedetomidine itself did not trigger Ca(2+) peak or increase in the presence or absence of external Ca(2+). When cells were stimulated with histamine after pretreatment with various concentrations of dexmedetomidine, we observed inhibited histamine-induced [Ca(2+)]i signal in both cell types. Histamine stimulated IL-6 mRNA expression not IL-8 mRNA within 2 hrs, however this effect was attenuated by dexmedetomidine. Collectively, these findings suggest that dexmedetomidine modulates histamine-induced Ca(2+) signaling and IL-6 expression and will be useful for understanding the antagonistic properties of dexmedetomidine on histamine-induced signaling beyond its sedative effect.

Background & Aims: Interstitial cells of Cajal are pacemakers in the gastrointestinal tract, regulating rhythmicity by activating nonselective cation channels. In Caenorhabditis elegans, the melastatin-type transient receptor potential (TRPM) channel, especially TRPM7, was suggested as being involved in defecation rhythm. The aim here was to show that the nonselective cation channel in interstitial cells of Cajal in mouse small intestine has properties essentially identical to those of murine TRPM7, heterologously expressed in human embryonic kidney cells. Methods: The patch-clamp technique for whole-cell recording was used in cultured or single interstitial cells of Cajal. TRPM7-specific small interfering RNAs were used for specific inhibition of TRPM7. Results: Electrophysiological and pharmacological properties of the nonselective cation channel in interstitial cells of Cajal were the same as those of TRPM7. Reverse-transcription polymerase chain reaction, Western blotting, and immunohistochemistry all showed abundant and localized expression of TRPM7 messenger RNA and protein in mouse small intestine. Treatment of primary cultured interstitial cells of Cajal with TRPM7-specific small interfering RNA resulted in inhibition of pacemaking activity. Conclusions: TRPM7 is required for intestinal pacemaking. The protein is a likely potential target for pharmacological treatment of motor disorders of the gut.

We investigated which transient receptor potential (TRP) channel is responsible for the nonselective cation channel (NSCC) activated by carbachol (CCh) in murine stomach with RT-PCR and the electrophysiological method. All seven types of TRP mRNA were detected in murine stomach with RT-PCR. When each TRP channel was expressed, the current-voltage relationship of mTRP5 was most similar to that recorded in murine gastric myocytes. mTRP5 showed a conductance order of Cs+> K+> Na+, similar to that in the murine stomach. With 0.2 mM GTPγS in the pipette solution, the current was activated transiently in both NSCC in the murine stomach and the expressed mTRP5. Both NSCC activated by CCh in murine stomach and mTRP5 were inhibited by intracellularly applied anti-Gq/11antibody, PLC inhibitor U-73122, IICR inhibitor 2-aminoethoxydiphenylborate, and nonspecific cation channel blockers La3+and flufenamate. There were two other unique properties. Both the native NSCC and mTRP5 were activated by 1-oleoyl-2-acetyl-sn-glycerol. Without the activation of NSCC by CCh, the NSCC in murine stomach was constitutively active like mTRP5. From the above results, we suggest that mTRP5 might be a candidate for the NSCC activated by ACh or CCh in murine stomach.

Objective beta-Cell dysfunction plays a central role in the pathogenesis of type 2 diabetes (T2D), and the identification of novel approaches to improve beta-cell function is essential to treat this disease. Baicalein, a flavonoid originally isolated from the root of Scutellaria Baicalensis, has been shown to have beneficial effects on beta-cell function. Here, the authors investigated the molecular mechanism responsible for the protective effects of baicalein against palmitate (PA)-induced impaired beta-cell function, and placed focus on the role of heme oxygenase (HO)-1. Methods Rat pancreatic beta-cell line INS-1 cells or mouse pancreatic islets were cultured with PA (500 mu M) to induce lipotoxicity in the presence or absence of baicalein (50 mu M), and the expressions of the ER stress markers, ATF-3, CHOP and GRP78 were detected by Western blotting and/or qPCR. The involvement of HO-1 was evaluated by HO-1 siRNA transfection and using the HO-1 inhibitor ZnPP. Results Baicalein reduced PA-induced ER stress and inflammation and enhanced insulin secretion, and these effects were associated with the induction of HO-1. Furthermore, these protective effects were attenuated by ZnPP and by HO-1 siRNA. Pretreatment of PD98059 (an ERK inhibitor) significantly inhibited the protective effects of baicalein and blocked HO-1 induction. On the other hand, CO production by RuCO (a CO donor) ameliorated PA-induced ER stress, suggesting that CO production followed by HO-1 induction may contribute to the protective effects of baicalein against PA-induced beta-cell dysfunction. Conclusion Baicalein protects pancreatic beta-cells from PA-induced ER stress and inflammation via an ERK-HO-1 dependent pathway. The authors suggest HO-1 induction in pancreatic beta-cells appears to be a promising therapeutic strategy for T2D.

Plasma membrane hyperpolarization associated with activation of Ca2+-activated K+ channels plays an important role in sperm capacitation during fertilization. Although Slo3 (slowpoke homologue 3), together with the auxiliary γ2-subunit, LRRC52 (leucine-rich-repeat-containing 52), is known to mediate the pH-sensitive, sperm-specific K+ current KSper in mice, the molecular identity of this channel in human sperm remains controversial. In this study, we tested the classical BKCa activators, NS1619 and LDD175, on human Slo3, heterologously expressed in HEK293 cells together with its functional interacting γ2 subunit, hLRRC52. As previously reported, Slo3 K+ current was unaffected by iberiotoxin or 4-aminopyridine, but was inhibited by ~50% by 20 mM TEA. Extracellular alkalinization potentiated hSlo3 K+ current, and internal alkalinization and Ca2+ elevation induced a leftward shift its activation voltage. NS1619, which acts intracellularly to modulate hSlo1 gating, attenuated hSlo3 K+ currents, whereas LDD175 increased this current and induced membrane potential hyperpolarization. LDD175-induced potentiation was not associated with a change in the half-activation voltage at different intracellular pHs (pH 7.3 and pH 8.0) in the absence of intracellular Ca2+. In contrast, elevation of intracellular Ca2+ dramatically enhanced the LDD175-induced leftward shift in the half-activation potential of hSlo3. Therefore, the mechanism of action does not involve pH-dependent modulation of hSlo3 gating; instead, LDD175 may modulate Ca2+-dependent activation of hSlo3. Thus, LDD175 potentially activates native KSper and may induce membrane hyperpolarization-associated hyperactivation in human sperm.

Fluid and HCO3−secretion is a vital function of all epithelia and is required for the survival of the tissue. Aberrant fluid and HCO3−secretion is associated with many epithelial diseases, such as cystic fibrosis, pancreatitis, Sjögren's syndrome, and other epithelial inflammatory and autoimmune diseases. Significant progress has been made over the last 20 years in our understanding of epithelial fluid and HCO3−secretion, in particular by secretory glands. Fluid and HCO3−secretion by secretory glands is a two-step process. Acinar cells secrete isotonic fluid in which the major salt is NaCl. Subsequently, the duct modifies the volume and electrolyte composition of the fluid to absorb the Cl−and secrete HCO3−. The relative volume secreted by acinar and duct cells and modification of electrolyte composition of the secreted fluids varies among secretory glands to meet their physiological functions. In the pancreas, acinar cells secrete a small amount of NaCl-rich fluid, while the duct absorbs the Cl−and secretes HCO3−and the bulk of the fluid in the pancreatic juice. Fluid secretion appears to be driven by active HCO3−secretion. In the salivary glands, acinar cells secrete the bulk of the fluid in the saliva that is driven by active Cl−secretion and contains high concentrations of Na+and Cl−. The salivary glands duct absorbs both the Na+and Cl−and secretes K+and HCO3−. In this review, we focus on the molecular mechanism of fluid and HCO3−secretion by the pancreas and salivary glands, to highlight the similarities of the fundamental mechanisms of acinar and duct cell functions, and to point out the differences to meet gland-specific secretions.

Background & Aims Excessive Ca 2+ influx mediates many cytotoxic processes, including those associated with autoimmune inflammatory diseases such as acute pancreatitis and Sjogren syndrome. Transient receptor potential (canonical) channel (TRPC) 3 is a major Ca 2+ influx channel in pancreatic and salivary gland cells. We investigated whether genetic or pharmacologic inhibition of TRPC3 protects pancreas and salivary glands from Ca 2+ -dependent damage. Methods We developed a Ca 2+ -dependent model of cell damage for salivary gland acini. Acute pancreatitis was induced by injection of cerulein into wild-type and Trpc3 −/− mice. Mice were also given the Trpc3-selective inhibitor pyrazole 3 (Pyr3). Results Salivary glands and pancreas of Trpc3 −/− mice were protected from Ca 2+ -mediated cell toxicity. Analysis of Ca 2+ signaling in wild-type and Trpc3 −/− acini showed that Pyr3 is a highly specific inhibitor of Tprc3; it protected salivary glands and pancreas cells from Ca 2+ -mediated toxicity by inhibiting the Trpc3-mediated component of Ca 2+ influx. Conclusions TRPC3-mediated Ca 2+ influx mediates damage to pancreas and salivary glands. Pharmacologic inhibition of TRPC3 with the highly selective TRPC3 inhibitor Pyr3 might be developed for treatment of patients with acute pancreatitis and Sjogren syndrome.

Members of the SLC26 family of anion transporters mediate the transport of diverse molecules ranging from halides to carboxylic acids and can function as coupled transporters or as channels. A unique feature of the two members of the family, Slc26a3 and Slc26a6, is that they can function as both obligate coupled and mediate an uncoupled current, in a channel-like mode, depending on the transported anion. To identify potential features that control the two modes of transport, we performed in silico modeling of Slc26a6, which suggested that the closest potential fold similarity of the Slc26a6 transmembrane domains is to the CLC transporters, despite their minimal sequence identity. Examining the predicted Slc26a6 fold identified a highly conserved glutamate (Glu(-); Slc26a6(E357)) with the predicted spatial orientation similar to that of the CLC-ec1 E148, which determines coupled or uncoupled transport by CLC-ec1. This raised the question of whether the conserved Glu(-) in Slc26a6(E357) and Slc26a3(E367) have a role in the unique transport modes by these transporters. Reversing the Glu(-) charge in Slc26a3 and Slc26a6 resulted in the inhibition of all modes of transport. However, most notably, neutralizing the charge in Slc26a6(E357A) eliminated all forms of coupled transport without affecting the uncoupled current. The Slc26a3(E367A) mutation markedly reduced the coupled transport and converted the stoichiometry of the residual exchange from 2Cl(-)/1HCO(3)(-) to 1Cl(-)/1HCO(3)(-), while completely sparing the current. These findings suggest the possibility that similar structural motif may determine multiple functional modes of these transporters.

IRBIT (IP(3)Rs binding protein released with IP(3)) is a protein originally identified by the Mikoshiba group as an inhibitor of IP(3) receptors function. Subsequently it was found to have multiple functions and regulate the activity of diverse proteins, including regulation of HCO(3)(-) transporters to coordinate epithelial HCO(3)(-) secretion and to determine localization of the Fip1 subunit of the CPSF complex to regulate mRNA processing. This review highlights the remarkably divers functions of IRBIT that are likely only a fraction of all the potential functions of this protein.

Fluid and HCO(3)(-) secretion are fundamental functions of epithelia and determine bodily fluid volume and ionic composition, among other things. Secretion of ductal fluid and HCO(3)(-) in secretory glands is fueled by Na(+)/HCO(3)(-) cotransport mediated by basolateral solute carrier family 4 member 4 (NBCe1-B) and by Cl(-)/HCO(3)(-) exchange mediated by luminal solute carrier family 26, member 6 (Slc26a6) and CFTR. However, the mechanisms governing ductal secretion are not known. Here, we have shown that pancreatic ductal secretion in mice is suppressed by silencing of the NBCe1-B/CFTR activator inositol-1,4,5-trisphosphate (IP(3)) receptor-binding protein released with IP(3) (IRBIT) and by inhibition of protein phosphatase 1 (PP1). In contrast, silencing the with-no-lysine (WNK) kinases and Ste20-related proline/alanine-rich kinase (SPAK) increased secretion. Molecular analysis revealed that the WNK kinases acted as scaffolds to recruit SPAK, which phosphorylated CFTR and NBCe1-B, reducing their cell surface expression. IRBIT opposed the effects of WNKs and SPAK by recruiting PP1 to the complex to dephosphorylate CFTR and NBCe1-B, restoring their cell surface expression, in addition to stimulating their activities. Silencing of SPAK and IRBIT in the same ducts rescued ductal secretion due to silencing of IRBIT alone. These findings stress the pivotal role of IRBIT in epithelial fluid and HCO(3)(-) secretion and provide a molecular mechanism by which IRBIT coordinates these processes. They also have implications for WNK/SPAK kinase-regulated processes involved in systemic fluid homeostasis, hypertension, and cystic fibrosis.