Identification and functional characterization of TMEM16A, a Ca2+-activated Cl- channel activated by extracellular nucleotides, in biliary epithelium

Cl(-) channels in the apical membrane of biliary epithelial cells (BECs) provide the driving force for ductular bile formation. Although a cystic fibrosis transmembrane conductance regulator has been identified in BECs and contributes to secretion via secretin binding basolateral receptors and increasing [cAMP](i), an alternate Cl(-) secretory pathway has been identified that is activated via nucleotides (ATP, UTP) binding apical P2 receptors and increasing [Ca(2+)](i). The molecular identity of this Ca(2+)-activated Cl(-) channel is unknown. The present studies in human, mouse, and rat BECs provide evidence that TMEM16A is the operative channel and contributes to Ca(2+)-activated Cl(-) secretion in response to extracellular nucleotides. Furthermore, Cl(-) currents measured from BECs isolated from distinct areas of intrahepatic bile ducts revealed important functional differences. Large BECs, but not small BECs, exhibit cAMP-stimulated Cl(-) currents. However, both large and small BECs express TMEM16A and exhibit Ca(2+)-activated Cl(-) efflux in response to extracellular nucleotides. Incubation of polarized BEC monolayers with IL-4 increased TMEM16A protein expression, membrane localization, and transepithelial secretion (I(sc)). These studies represent the first molecular identification of an alternate, noncystic fibrosis transmembrane conductance regulator, Cl(-) channel in BECs and suggest that TMEM16A may be a potential target to modulate bile formation in the treatment of cholestatic liver disorders.     

A minimal isoform of the TMEM16A protein associated with chloride channel activity

TMEM16A protein, also known as anoctamin-1, has been recently identified as an essential component of Ca(2+)-activated Cl(-) channels. We previously reported the existence of different TMEM16A isoforms generated by alternative splicing. In the present study, we have determined the functional properties of a minimal TMEM16A protein. This isoform, called TMEM16A(0), has a significantly shortened amino-terminus and lacks three alternative segments localized in the intracellular regions of the protein (total length: 840 amino acids). TMEM16A(0) expression is associated with Ca(2+)-activated Cl(-) channel activity as measured by three different functional assays based on the halide-sensitive yellow fluorescent protein, short-circuit current recordings, and patch-clamp technique. However, compared to a longer isoform, TMEM16(abc) (total length: 982 amino acids), TMEM16A(0) completely lacks voltage-dependent activation. Furthermore, TMEM16A(0) and TMEM16A(abc) have similar but not identical responses to extracellular anion replacement, thus suggesting a difference in ion selectivity and conductance. Our results indicate that TMEM16A(0) has the basic domains required for anion transport and Ca(2+)-sensitivity. However, the absence of alternative segments, which are present in more complex isoforms of TMEM16A, modifies the channel gating and ion transport ability.     

TMEM16A(a)/anoctamin-1 shares a homodimeric architecture with CLC chloride channels

TMEM16A/anoctamin-1 has been identified as a protein with the classic properties of a Ca(2+)-activated chloride channel. Here, we used blue native polyacrylamide gel electrophoresis (BN-PAGE) and chemical cross-linking to assess the quaternary structure of the mouse TMEM16A(a) and TMEM16A(ac) splice variants as well as a genetically concatenated TMEM16A(a) homodimer. The constructs carried hexahistidyl (His) tags to allow for their purification using a nondenaturing metal affinity resin. Neither His-tagging nor head-to-tail concatenation of two copies of TMEM16A(a) noticeably affected Ca(2+)-induced measured macroscopic Cl(-) currents compared with the wild-type TMEM16A(a) channel. The digitonin-solubilized, nondenatured TMEM16A(a) protein migrated in the BN-PAGE gel as a homodimer, as judged by comparison with the concatenated TMEM16A(a) homodimer and channel proteins of known oligomeric structures (e.g. the voltage-gated Cl(-) channel CLC-1). Cross-linking with glutaraldehyde corroborated the homodimeric structure of TMEM16A(a). The TMEM16A(a) homodimer detected in Xenopus laevis oocytes and HEK 293 cells dissociated into monomers following denaturation with SDS, and reducing versus nonreducing SDS-PAGE provided no evidence for the presence of intersubunit disulfide bonds. Together, our data demonstrate that the Ca(2+)-activated chloride channel member TMEM16A shares an obligate homodimeric architecture with the hCLC-1 channel.     

Eugenol-induced contractions of saponin-skinned fibers are inhibited by heparin or by a ryanodine receptor blocker

The effects of eugenol on the sarcoplasmic reticulum (SR) and contractile apparatus of chemically skinned skeletal muscle fibers of the frog Rana catesbeiana were investigated. In saponin-skinned fibers, eugenol (5 mmol/L) induced muscle contractions, probably by releasing Ca(2+) from the SR. The Ca(2+)-induced Ca(2+) release blocker ruthenium red (10 micromol/L) inhibited both caffeine- and eugenol-induced muscle contractions. Ryanodine (200 micromol/L), a specific ryanodine receptor/Ca(2+) release channel blocker, promoted complete inhibition of the contractions induced by caffeine, but only partially blocked the contractions induced by eugenol. Heparin (2.5 mg/mL), an inositol 1,4,5-trisphosphate (InsP3) receptor blocker, strongly inhibited the contractions induced by eugenol but had only a small effect on the caffeine-induced contractions. Eugenol neither altered the Ca(2+) sensitivity nor the maximal force in Triton X-100 skinned muscle fibers. These data suggest that muscle contraction induced by eugenol involves at least 2 mechanisms of Ca(2+) release from the SR: one related to the activation of the ryanodine receptors and another through a heparin-sensitive pathway.     

TMEM16A inhibitors reveal TMEM16A as a minor component of calcium-activated chloride channel conductance in airway and intestinal epithelial cells

TMEM16A (ANO1) functions as a calcium-activated chloride channel (CaCC). We developed pharmacological tools to investigate the contribution of TMEM16A to CaCC conductance in human airway and intestinal epithelial cells. A screen of ～110,000 compounds revealed four novel chemical classes of small molecule TMEM16A inhibitors that fully blocked TMEM16A chloride current with an IC(50) < 10 μM, without interfering with calcium signaling. Following structure-activity analysis, the most potent inhibitor, an aminophenylthiazole (T16A(inh)-A01), had an IC(50) of ～1 μM. Two distinct types of inhibitors were identified. Some compounds, such as tannic acid and the arylaminothiophene CaCC(inh)-A01, fully inhibited CaCC current in human bronchial and intestinal cells. Other compounds, including T16A(inh)-A01 and digallic acid, inhibited total CaCC current in these cells poorly, but blocked mainly an initial, agonist-stimulated transient chloride current. TMEM16A RNAi knockdown also inhibited mainly the transient chloride current. In contrast to the airway and intestinal cells, all TMEM16A inhibitors fully blocked CaCC current in salivary gland cells. We conclude that TMEM16A carries nearly all CaCC current in salivary gland epithelium, but is a minor contributor to total CaCC current in airway and intestinal epithelia. The small molecule inhibitors identified here permit pharmacological dissection of TMEM16A/CaCC function and are potential development candidates for drug therapy of hypertension, pain, diarrhea, and excessive mucus production.     

CFTR and TMEM16A are separate but functionally related Cl- channels

Previous reports point out to a functional relationship of the cystic fibrosis transmembrane conductance regulator (CFTR) and Ca(2+) activated Cl(-) channels (CaCC). Recent findings showing that TMEM16A forms the essential part of CaCC, prompted us to examine whether CFTR controls TMEM16A. Inhibition of endogenous CaCC by activation of endogenous CFTR was found in 16HBE human airway epithelial cells, which also express TMEM16A. In contrast, CFBE airway epithelial cells lack of CFTR expression, but express TMEM16A along with other TMEM16-proteins. These cells produce CaCC that is inhibited by overexpression and activation of CFTR. In HEK293 cells coexpressing TMEM16A and CFTR, whole cell currents activated by IMBX and forskolin were significantly reduced when compared with cells expressing CFTR only, while the halide permeability sequence of CFTR was not changed. Expression of TMEM16A, but not of TMEM16F, H or J, produced robust CaCC, which that were inhibited by CaCCinh-A01 and niflumic acid, but not by CFTRinh-172. TMEM16A-currents were attenuated by additional expression of CFTR, and were completely abrogated when additionally expressed CFTR was activated by IBMX and forskolin. On the other hand, CFTR-currents were attenuated by additional expression of TMEM16A. CFTR and TMEM16A were both membrane localized and could be coimmunoprecipitated. Intracellular Ca(2+) signals elicited by receptor-stimulation was not changed during activation of CFTR, while ionophore-induced rise in [Ca(2+)](i) was attenuated after stimulation of CFTR. The data indicate that both CFTR and TMEM16 proteins are separate molecular entities that show functional and molecular interaction.     

Role of the Ca2+ -activated Cl- channels bestrophin and anoctamin in epithelial cells

Two families of proteins, the bestrophins (Best) and the recently cloned TMEM16 proteins (anoctamin, Ano), recapitulate properties of Ca(2+)-activated Cl(-) currents. Best1 is strongly expressed in the retinal pigment epithelium and could have a function as a Ca(2+)-activated Cl(-) channel as well as a regulator of Ca(2+) signaling. It is also present at much lower levels in other cell types including epithelial cells, where it regulates plasma membrane localized Cl(-) channels by controlling intracellular Ca(2+) levels. Best1 interacts with important Ca(2+)-signaling proteins such as STIM1 and can interact directly with other Ca(2+)-activated Cl(-) channels such as TMEM16A. Best1 is detected in the endoplasmic reticulum (ER) where it shapes the dynamic ER structure and regulates cell proliferation, which could be important for renal cystogenesis. Ca(2+)-activated Cl(-) channels of the anoctamin family (TMEM16A) show biophysical and pharmacological properties that are typical for endogenous Ca(2+)-dependent Cl(-) channels. TMEM16 proteins are abundantly expressed and many reports demonstrate their physiological importance in epithelial as well as non-epithelial cells. These channels are also activated by cell swelling and can therefore control cell volume, proliferation and apoptosis. To fully understand the function and regulation of Ca(2+)-activated Cl(-) currents, it is necessary to appreciate that Best1 and TMEM16A are embedded in a protein network and that they probably operate in functional microdomains.     

Synthesis and evaluation of 5,6-disubstituted thiopyrimidine aryl aminothiazoles as inhibitors of the calcium-activated chloride channel TMEM16A/Ano1

Transmembrane protein 16A (TMEM16A), also called Ano1, is a Ca²⁺ activated Cl^? channel expressed widely in mammalian epithelia, as well as in vascular smooth muscle and some tumors and electrically excitable cells. TMEM16A inhibitors have potential utility for treatment of disorders of epithelial fluid and mucus secretion, hypertension, some cancers and other diseases. 4-Aryl-2-amino thiazole T16A_inh-01 was previously identified by high-throughput screening. Here, a library of 47 compounds were prepared that explored the 5,6-disubstituted pyrimidine scaffold found in T16A_inh-01. TMEM16A inhibition activity was measured using fluorescence plate reader and short-circuit current assays. We found that very little structural variation of T16A_inh-01 was tolerated, with most compounds showing no activity at 10?μM. The most potent compound in the series, 9bo, which substitutes 4-methoxyphenyl in T16A_inh-01 with 2-thiophene, had IC₅₀ ～1?μM for inhibition of TMEM16A chloride conductance.     

Identification of the Novel TMEM16A Inhibitor Dehydroandrographolide and Its Anticancer Activity on SW620 Cells

TMEM16A, a calcium-activated chloride channel (CaCC), is highly amplified and expressed in human cancers and is involved in the growth and metastasis of some malignancies. Inhibition of TMEM16A represents a novel pharmaceutical approach for the treatment of cancers and metastases. The purpose of this study is to identify a new TMEM16A inhibitor, investigate the effects of this inhibitor on the proliferation and metastasis of TMEM16A-amplified SW620 cells, and to elucidate the underlying molecular mechanism in vitro. We identified a novel small-molecule TMEM16A inhibitor dehydroandrographolide (DP). By using patch clamp electrophysiology, we showed that DP inhibited TMEM16A chloride currents in Fisher rat thyroid (FRT) cells that were transfected stably with human TMEM16A and in TMEM16A-overexpressed SW620 cells but did not alter cystic fibrosis transmembrane conductance regulator (CFTR) chloride currents. Further functional studies showed that DP suppressed the proliferation of SW620 cells in a dose- and time-dependent manner using MTT assays. Moreover, DP significantly inhibited migration and invasion of SW620 cells as detected by wound-healing and transwell assays. Further mechanistic study demonstrated that knockdown of human TMEM16A decreased the inhibitory effect of DP on the proliferation of SW620 cells and that TMEM16A-dependent cells (SW620 and HCT116) were more sensitive to DP than TMEM16A-independent cells (SW480 and HCT8). In addition, we found that treatment of SW620 cells with DP led to a decrease in TMEM16A protein levels but had no effect on TMEM16A mRNA levels. The current work reveals that DP, a novel TMEM16A inhibitor, exerts its anticancer activity on SW620 cells partly through a TMEM16A-dependent mechanism, which may introduce a new targeting approach for an antitumour therapy in TMEM16A-amplified cancers.     

Inhibition of xanthine oxidase — xanthine — iron mediated lipid peroxidation by eugenol in liposomes

The effect of eugenol on xanthine oxidase (XO) xanthine(X)-Fe⁺³-ADP mediated lipid peroxidation was studied in liver microsomal lipid liposomes. Eugenol inhibited the lipid peroxidation in a dose dependent manner as assessed by formation of thiobarbituric acid reactive substances. When tested for its effect on XO activity per se, (by measuring uric acid formation) eugenol inhibited the enzyme to an extent of 85% at 10 µm concentration and hence formation of O₂ also However, the concentration of eugenol required for XO inhibition was more in presence of metal chelators such as EDTA, EGTA and DETAPAC, but not in presence of deferoxamine, ADP and citrate. The antiperoxidative effect of eugenol was about 35 times more and inhibition of XO was about 5 times higher as compared to the effect of allopurinol. Eugenol did not scavenge O₂ generated by phenazine methosulfate and NAD but inhibited propagation of peroxidation catalyzed by Fe²⁺ EDTA and lipid hydroperoxide containing liposomes. Eugenol inhibits XO-X-Fe⁺³ ADP mediated peroxidation by inhibiting the XO activity per se in addition to quenching various radical species. (Mol Cell Biochem 166: 65-71, 1997)     

The TMEM16 Protein Family: A New Class of Chloride Channels?

Cl⁻ channels play important roles in many physiological processes, including transepithelial ion absorption and secretion, smooth and skeletal muscle contraction, neuronal excitability, sensory perception, and cell volume regulation. The molecular identity of many types of Cl⁻ channels is still unknown. Recently, three research groups have arrived independently at the identification of TMEM16A (also known as anoctamin-1) as a membrane protein strongly related to the activity of Ca²⁺-activated Cl⁻ channels (CaCCs). Site-specific mutagenesis of TMEM16A alters the properties of the channels, thus suggesting that TMEM16A forms, at least in part, the CaCC. TMEM16A is a member of a family that includes nine other membrane proteins. All TMEM16 proteins have a similar structure, with eight putative transmembrane domains and cytosolic amino- and carboxy-termini. TMEM16B expression also evokes the appearance of CaCCs, but with biophysical characteristics (voltage dependence, unitary conductance) different from those associated to TMEM16A. The roles of the other TMEM16 proteins are still unknown. The study of TMEM16 proteins may lead to identification of novel molecular mechanisms underlying ion transport and channel gating by voltage and Ca²⁺.     

The bestrophin- and TMEM16A-associated Ca2+-activated Cl– channels in vascular smooth muscles

The presence of Ca²⁺-activated Cl^– currents (I_Cl(Ca)) in vascular smooth muscle cells (VSMCs) is well established. I_Cl(Ca) are supposedly important for arterial contraction by linking changes in [Ca²⁺]_i and membrane depolarization. Bestrophins and some members of the TMEM16 protein family were recently associated with I_Cl(Ca). Two distinct I_Cl(Ca) are characterized in VSMCs; the cGMP-dependent I_Cl(Ca) dependent upon bestrophin expression and the ‘classical’ Ca²⁺-activated Cl^– current, which is bestrophin-independent. Interestingly, TMEM16A is essential for both the cGMP-dependent and the classical I_Cl(Ca). Furthermore, TMEM16A has a role in arterial contraction while bestrophins do not. TMEM16A’s role in the contractile response cannot be explained however only by a simple suppression of the depolarization by Cl^– channels. It is suggested that TMEM16A expression modulates voltage-gated Ca²⁺ influx in a voltage-independent manner and recent studies also demonstrate a complex role of TMEM16A in modulating other membrane proteins.     

Novel 5-substituted benzyloxy-2-arylbenzofuran-3-carboxylic acids as calcium activated chloride channel inhibitors

Transmembrane protein 16A (TMEM16A) channels are recently discovered membrane proteins that functions as a calcium activated chloride channel (CaCC). CaCCs are major regulators of various physiological processes, such as sensory transduction, epithelial secretion, smooth muscle contraction and oocyte fertilization. Thirty novel 5-substituted benzyloxy-2-arylbenzofuran-3-carboxylic acids (B01-B30) were synthesized and evaluated for their TMEM16A inhibitory activity by using short circuit current measurements in Fischer rat thyroid (FRT) cells expressing human TMEM16A. IC(50) values were calculated using YFP fluorescence plate reader assay. Final compounds, having free carboxylic group displayed significant inhibition. Eight of the novel compounds B02, B13, B21, B23, B25, B27, B28, B29 exhibit excellent CaCCs inhibition with IC(50) value <6 μM, with compound B25 exhibiting the lowest IC(50) value of 2.8 ± 1.3 μM. None of the tested ester analogs of final benzofuran derivatives displayed TMEM16A/CaCCs inhibition.     

The bestrophin- and TMEM16A-associated Ca2+-activated Cl– channels in vascular smooth muscles

The presence of Ca²⁺-activated Cl^– currents (I_Cl(Ca)) in vascular smooth muscle cells (VSMCs) is well established. I_Cl(Ca) are supposedly important for arterial contraction by linking changes in [Ca²⁺]_i and membrane depolarization. Bestrophins and some members of the TMEM16 protein family were recently associated with I_Cl(Ca). Two distinct I_Cl(Ca) are characterized in VSMCs; the cGMP-dependent I_Cl(Ca) dependent upon bestrophin expression and the ‘classical’ Ca²⁺-activated Cl^– current, which is bestrophin-independent. Interestingly, TMEM16A is essential for both the cGMP-dependent and the classical I_Cl(Ca). Furthermore, TMEM16A has a role in arterial contraction while bestrophins do not. TMEM16A’s role in the contractile response cannot be explained however only by a simple suppression of the depolarization by Cl^– channels. It is suggested that TMEM16A expression modulates voltage-gated Ca²⁺ influx in a voltage-independent manner and recent studies also demonstrate a complex role of TMEM16A in modulating other membrane proteins.     

Airway epithelial cells-Functional links between CFTR and anoctamin dependent Cl(-) secretion

Airways consist of a heterogeneous population of cells, comprising ciliated cells, Clara cells and goblet cells. Electrolyte secretion by the airways is necessary to produce the airway surface liquid that allows for mucociliary clearance of the lungs. Secretion is driven by opening of Cl(-) selective ion channels in the apical membrane of airway epithelial cells, through either receptor mediated increase in intracellular cAMP or cytosolic Ca(2+). Traditionally cAMP-dependent and Ca(2+)-dependent secretory pathways are regarded as independent. However, this concept has been challenged recently. With identification of the Ca(2+) activated Cl(-) channel TMEM16A (anoctamin 1) and with detailed knowledge of the cAMP-regulated cystic fibrosis transmembrane conductance regulator (CFTR), it has become possible to look more closely into this relationship.     

Functional Swapping between Transmembrane Proteins TMEM16A and TMEM16F

The transmembrane proteins TMEM16A and -16F each carry eight transmembrane regions with cytoplasmic N and C termini. TMEM16A carries out Ca²⁺-dependent Cl⁻ ion transport, and TMEM16F is responsible for Ca²⁺-dependent phospholipid scrambling. Here we established assay systems for the Ca²⁺-dependent Cl⁻ channel activity using 293T cells and for the phospholipid scramblase activity using TMEM16F^−/− immortalized fetal thymocytes. Chemical cross-linking analysis showed that TMEM16A and -16F form homodimers in both 293T cells and immortalized fetal thymocytes. Successive deletion from the N or C terminus of both proteins and the swapping of regions between TMEM16A and -16F indicated that their cytoplasmic N-terminal (147 amino acids for TMEM16A and 95 for 16F) and C-terminal (88 amino acids for TMEM16A and 68 for 16F) regions were essential for their localization at plasma membranes and protein stability, respectively, and could be exchanged. Analyses of TMEM16A and -16F mutants with point mutations in the pore region (located between the fifth and sixth transmembrane regions) indicated that the pore region is essential for both the Cl⁻ channel activity of TMEM16A and the phospholipid scramblase activity of TMEM16F. Some chemicals such as epigallocatechin-3-gallate and digallic acid inhibited the Cl⁻ channel activity of TMEM16A and the scramblase activity of TMEM16F with an opposite preference. These results indicate that TMEM16A and -16F use a similar mechanism for sorting to plasma membrane and protein stabilization, but their functional domains significantly differ.     

pH and external Ca(2+) regulation of a small conductance Cl(-) channel in kidney distal tubule

A single channel characterization of the Cl(-) channels in distal nephron was undertaken using vesicles prepared from plasma membranes of isolated rabbit distal tubules. The presence in this vesicle preparation of ClC-K type Cl(-) channels was first established by immunodetection using an antibody raised against ClC-K isoforms. A ClC-K1 based functional characterization was next performed by investigating the pH and external Ca(2+) regulation of a small conductance Cl(-) channel which we identified previously by channel incorporation experiments. Acidification of the cis (external) solution from pH 7.4 to 6.5 led to a dose-dependent inhibition of the channel open probability P(O). Similarly, changing the trans pH from 7.4 to 6.8 resulted in a 4-fold decrease of the channel P(O) with no effect on the channel conductance. Channel activity also appeared to be regulated by cis (external) Ca(2+) concentration, with a dose-dependent increase in channel activity as a function of the cis Ca(2+) concentration. It is concluded on the basis of these results that the small conductance Cl(-) channel present in rabbit distal tubules is functionally equivalent to the ClC-K1 channel in the rat. In addition, the present work constitutes the first single channel evidence for a chloride channel regulated by external Ca(2+).     

Direct or Indirect Regulation of Calcium-Activated Chloride Channel by Calcium

Calcium-activated chloride channels (CaCCs) play fundamental roles in numerous physiological processes. Despite their physiological importance, the molecular identity of CaCCs has not been fully investigated until now. Recently, transmembrane 16A (TMEM16A) was demonstrated by three independent research groups to be a strong candidate for the CaCC molecular basis. To further investigate the electrophysiological characteristics, we constructed TMEM16A (abcd) stably transfected HEK293 cell lines and carried out whole-cell and excised inside–out patch-clamp experiments. The TMEM16A channel was Ca²⁺-dependent in both patch configurations. The TMEM16A current could be strongly inhibited by niflumic acid, and when Cl⁻ was substituted by gluconate ions, the current was reduced considerably. In inside–out configuration, TMEM16A channel was time-independent but voltage-dependent, in which the half-maximum activating free Ca²⁺ concentration was 63 nM at 80 mV. While in whole-cell configuration, the current was both time- and voltage-dependent. About the rectification feature, the TMEM16A current also showed distinct characteristics in the two patch configurations. In whole cells, the TMEM16A channel expressed outward rectification at low Ca²⁺ concentration but when the Ca²⁺ concentration was high it became linear. On the contrary, in inside–out configuration, it always expressed outward rectification. Comparing the different characteristics in the two configurations, some underlying mechanisms remain to be identified, which is discussed with respect to direct or indirect activation. There was irreversible rundown in this channel.     

F508del-CFTR increases intracellular Ca(2+) signaling that causes enhanced calcium-dependent Cl(-) conductance in cystic fibrosis

In many cells, increase in intracellular calcium ([Ca(2+)](i)) activates a Ca(2+)-dependent chloride (Cl(-)) conductance (CaCC). CaCC is enhanced in cystic fibrosis (CF) epithelial cells lacking Cl(-) transport by the CF transmembrane conductance regulator (CFTR). Here, we show that in freshly isolated nasal epithelial cells of F508del-homozygous CF patients, expression of TMEM16A and bestrophin 1 was unchanged. However, calcium signaling was strongly enhanced after induction of expression of F508del-CFTR, which is unable to exit the endoplasmic reticulum (ER). Since receptor-mediated [Ca(2+)](i) increase is Cl(-) dependent, we suggested that F508del-CFTR may function as an ER chloride counter-ion channel for Ca(2+). This was confirmed by expression of the double mutant F508del/G551D-CFTR, which remained in the ER but had no effects on [Ca(2+)](i). Moreover, F508del-CFTR could serve as a scavenger for inositol-1,4,5-trisphosphate [IP3] receptor binding protein released with IP(3) (IRBIT). Our data may explain how ER-localized F508del-CFTR controls intracellular Ca(2+) signaling.