Identification and Functional Characterization of TMEM16A, a Ca2+-activated Cl− Channel Activated by Extracellular Nucleotides, in Biliary Epithelium
2010; Elsevier BV; Volume: 286; Issue: 1 Linguagem: Inglês
10.1074/jbc.m110.164970
ISSN1083-351X
AutoresAmal K. Dutta, Al-karim Khimji, Charles Kresge, Abhijit Bugde, Michael Dougherty, Victoria Esser, Yoshiyuki Ueno, Shannon Glaser, Gianfranco Alpini, Don C. Rockey, Andrew P. Feranchak,
Tópico(s)Drug Transport and Resistance Mechanisms
ResumoCl− 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 [Ca2+]i. The molecular identity of this Ca2+-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 Ca2+-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 Ca2+-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 (Isc). 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. 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 [Ca2+]i. The molecular identity of this Ca2+-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 Ca2+-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 Ca2+-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 (Isc). 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. IntroductionThe formation of bile by the liver depends on the transport functions of two complimentary cell types: hepatocytes and intrahepatic bile duct epithelial cells, known as cholangiocytes. Although cholangiocytes account for only ∼2–5% of the nuclear mass of the liver (1Kumar U. Jordan T.W. Liver. 1986; 6: 369-378Crossref PubMed Scopus (34) Google Scholar), they have a prodigious capacity for secretion (2Preisig R. Cooper H.L. Wheeler H.O. J. Clin. Invest. 1962; 41: 1152-1162Crossref PubMed Scopus (98) Google Scholar, 3Wheeler H.O. Ramos O.L. J. Clin. Invest. 1960; 39: 161-170Crossref PubMed Google Scholar) and may account for up to 40% of bile volume in humans through regulated ion and water transport (4Fitz J.G. Semin. Liver Dis. 2002; 22: 241-249Crossref PubMed Scopus (30) Google Scholar). Opening of Cl− channels in the apical membrane of cholangiocytes represents the driving force for ductular secretion and at least two Cl− conductances contribute (5Fitz J.G. Basavappa S. McGill J. Melhus O. Cohn J.A. J. Clin. Invest. 1993; 91: 319-328Crossref PubMed Scopus (183) Google Scholar). The best characterized pathway involves cAMP-stimulated opening of Cl− channels encoded by the cystic fibrosis transmembrane conductance regulator (CFTR) 2The abbreviations used are: CFTRcystic fibrosis transmembrane conductance regulatorNRCnormal rat cholangiocyte(s)MLCmouse large cholangiocytesMSCmouse small cholangiocytesBDLbile duct-ligated. (5Fitz J.G. Basavappa S. McGill J. Melhus O. Cohn J.A. J. Clin. Invest. 1993; 91: 319-328Crossref PubMed Scopus (183) Google Scholar, 6McGill J.M. Basavappa S. Gettys T.W. Fitz J.G. Am. J. Physiol. 1994; 266: G731-G736Crossref PubMed Google Scholar). Additionally, there is a separate Ca2+-activated Cl− channel (CaCC) that is involved, but the molecular identity of this channel has not been defined (5Fitz J.G. Basavappa S. McGill J. Melhus O. Cohn J.A. J. Clin. Invest. 1993; 91: 319-328Crossref PubMed Scopus (183) Google Scholar, 7Schlenker T. Fitz J.G. Am. J. Physiol. 1996; 271: G304-G310Crossref PubMed Google Scholar).Increasing evidence suggests that the dominant mechanism for regulation of biliary secretion involves (i) autocrine/paracrine release of ATP into bile, (ii) activation by ATP of purinergic receptors, and (iii) mobilization of intracellular calcium and activation of CaCCs. The magnitude of the Cl− secretory response to ATP is greater than that to cAMP in single human biliary epithelial cells and polarized rat cholangiocyte monolayers (8Dutta A.K. Woo K. Doctor R.B. Fitz J.G. Feranchak A.P. Am. J. Physiol. Gastrointest. Liver Physiol. 2008; 295: G1004-G1015Crossref PubMed Scopus (34) Google Scholar). Furthermore, many of the effects of cAMP on bile formation appear to be mediated by ATP release into the duct lumen and subsequent activation of apical P2 receptors (9Minagawa N. Nagata J. Shibao K. Masyuk A.I. Gomes D.A. Rodrigues M.A. Lesage G. Akiba Y. Kaunitz J.D. Ehrlich B.E. Larusso N.F. Nathanson M.H. Gastroenterology. 2007; 133: 1592-1602Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 10Fiorotto R. Spirlì C. Fabris L. Cadamuro M. Okolicsanyi L. Strazzabosco M. Gastroenterology. 2007; 133: 1603-1613Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). These general findings are consistent with the clinical observation that only 10–20% of patients with cystic fibrosis (CF) develop clinically significant liver disease despite absent or abnormal CFTR in biliary epithelium (11Colombo C. Battezzati P.M. Crosignani A. Morabito A. Costantini D. Padoan R. Giunta A. Hepatology. 2002; 36: 1374-1382Crossref PubMed Scopus (285) Google Scholar, 12Feranchak A.P. Sokol R.J. Sem. Liv. Disease. 2001; 21: 471-488Crossref PubMed Scopus (101) Google Scholar). Furthermore, in cells with the CF phenotype, activation of P2 receptors still elicits potent secretory responses (13Paradiso A.M. Ribeiro C.M. Boucher R.C. J. Gen. Physiol. 2001; 117: 53-67Crossref PubMed Scopus (96) Google Scholar, 14Clarke L.L. Harline M.C. Gawenis L.R. Walker N.M. Turner J.T. Weisman G.A. Am. J. Physiol. Gastrointest. Liver Physiol. 2000; 279: G132-G138Crossref PubMed Google Scholar). Together, these findings challenge the conventional model that centers on the concept that cAMP-dependent opening of CFTR-related Cl− channels is the driving force for cholangiocyte secretion. Rather, the operative regulatory pathways appear to take place within the lumen of intrahepatic ducts, where release of ATP into bile is a final common pathway controlling ductular bile formation. Accordingly, molecular definition of the CaCC channel(s) involved has a high priority for future efforts to modulate the volume and composition of bile.Several candidate proteins have been proposed as CaCCs in other epithelia, including CLC and bestrophin family members (15Hartzell C. Putzier I. Arreola J. Annu. Rev. Physiol. 2005; 67: 719-758Crossref PubMed Scopus (482) Google Scholar). However, the biophysical properties of the currents associated with these proteins are not consistent with the ATP-stimulated Cl− currents described in biliary epithelium (8Dutta A.K. Woo K. Doctor R.B. Fitz J.G. Feranchak A.P. Am. J. Physiol. Gastrointest. Liver Physiol. 2008; 295: G1004-G1015Crossref PubMed Scopus (34) Google Scholar). More recently, TMEM16A, a 114-kDa membrane protein with eight putative transmembrane domains, has been identified as a CaCC in other epithelium (16Caputo A. Caci E. Ferrera L. Pedemonte N. Barsanti C. Sondo E. Pfeffer U. Ravazzolo R. Zegarra-Moran O. Galietta L.J. Science. 2008; 322: 590-594Crossref PubMed Scopus (979) Google Scholar, 17Yang Y.D. Cho H. Koo J.Y. Tak M.H. Cho Y. Shim W.S. Park S.P. Lee J. Lee B. Kim B.M. Raouf R. Shin Y.K. Oh U. Nature. 2008; 455: 1210-1215Crossref PubMed Scopus (1002) Google Scholar, 18Schroeder B.C. Cheng T. Jan Y.N. Jan L.Y. Cell. 2008; 134: 1019-1029Abstract Full Text Full Text PDF PubMed Scopus (904) Google Scholar), and the general biophysical properties of the TMEM16A conductance as measured by whole cell patch clamp appear similar to the ATP-stimulated Cl− currents described in biliary cells (8Dutta A.K. Woo K. Doctor R.B. Fitz J.G. Feranchak A.P. Am. J. Physiol. Gastrointest. Liver Physiol. 2008; 295: G1004-G1015Crossref PubMed Scopus (34) Google Scholar). Although TMEM16A is found in liver (19Ferrera L. Caputo A. Ubby I. Bussani E. Zegarra-Moran O. Ravazzolo R. Pagani F. Galietta L.J. J. Biol. Chem. 2009; 284: 33360-33368Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar), its cellular localization and function are unknown.The aim of the present studies was to identify the molecular basis for Ca2+-activated Cl− currents stimulated by extracellular nucleotides in different biliary cell models. We have utilized single cells (human and mouse), polarized biliary monolayers (rat), and novel models of small and large cholangiocytes isolated from distinct areas of the intrahepatic bile ducts (mouse) to critically assess the potential role of TMEM16A in regulating membrane Cl− permeability and biliary secretion. Lastly, we have explored the potential role of IL-4 on TMEM16A channel expression and function, which may have important implications for Cl− secretion during cholestasis.DISCUSSIONIn these studies of human, rat, and mouse biliary epithelial cells, we have identified TMEM16A as the molecular basis for Ca2+-activated Cl− channels stimulated by extracellular nucleotides. Furthermore, we have identified the mechanism by which small cholangiocytes, which do not express CFTR (35Glaser S.S. Gaudio E. Rao A. Pierce L.M. Onori P. Franchitto A. Francis H.L. Dostal D.E. Venter J.K. DeMorrow S. Mancinelli R. Carpino G. Alvaro D. Kopriva S.E. Savage J.M. Alpini G.D. Lab. Invest. 2009; 89: 456-469Crossref PubMed Scopus (99) Google Scholar), contribute to ductular secretion. Lastly, the finding that IL-4 up-regulates biliary TMEM16A expression and nucleotide-stimulated secretion may have important implications for cholestatic liver diseases associated with proinflammatory mediators. Accordingly, these studies represent the first molecular identification of a non-CFTR Cl− channel in biliary epithelium and therefore compliment and extend the original biophysical characterization of Cl− currents in biliary epithelial cells first published almost 20 years ago (5Fitz J.G. Basavappa S. McGill J. Melhus O. Cohn J.A. J. Clin. Invest. 1993; 91: 319-328Crossref PubMed Scopus (183) Google Scholar). As these are the first studies of TMEM16A in biliary epithelium, several points are in order.First, although these studies demonstrate a predominant role of TMEM16A in ATP-stimulated Cl− secretion, we cannot exclude contributions from other Cl− channels or TMEM16 isoforms. TMEM16A/ANO-1 is a 114-kDa membrane protein with 960 amino acids and eight transmembrane domains and is one of the 10 members of TMEM16/ANO family of proteins (16Caputo A. Caci E. Ferrera L. Pedemonte N. Barsanti C. Sondo E. Pfeffer U. Ravazzolo R. Zegarra-Moran O. Galietta L.J. Science. 2008; 322: 590-594Crossref PubMed Scopus (979) Google Scholar, 17Yang Y.D. Cho H. Koo J.Y. Tak M.H. Cho Y. Shim W.S. Park S.P. Lee J. Lee B. Kim B.M. Raouf R. Shin Y.K. Oh U. Nature. 2008; 455: 1210-1215Crossref PubMed Scopus (1002) Google Scholar, 18Schroeder B.C. Cheng T. Jan Y.N. Jan L.Y. Cell. 2008; 134: 1019-1029Abstract Full Text Full Text PDF PubMed Scopus (904) Google Scholar). Only TMEM16A/ANO1 (16Caputo A. Caci E. Ferrera L. Pedemonte N. Barsanti C. Sondo E. Pfeffer U. Ravazzolo R. Zegarra-Moran O. Galietta L.J. Science. 2008; 322: 590-594Crossref PubMed Scopus (979) Google Scholar, 17Yang Y.D. Cho H. Koo J.Y. Tak M.H. Cho Y. Shim W.S. Park S.P. Lee J. Lee B. Kim B.M. Raouf R. Shin Y.K. Oh U. Nature. 2008; 455: 1210-1215Crossref PubMed Scopus (1002) Google Scholar, 18Schroeder B.C. Cheng T. Jan Y.N. Jan L.Y. Cell. 2008; 134: 1019-1029Abstract Full Text Full Text PDF PubMed Scopus (904) Google Scholar) and TMEM16B/ANO2 (42Stephan A.B. Shum E.Y. Hirsh S. Cygnar K.D. Reisert J. Zhao H. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 11776-11781Crossref PubMed Scopus (256) Google Scholar, 43Stöhr H. Heisig J.B. Benz P.M. Schöberl S. Milenkovic V.M. Strauss O. Aartsen W.M. Wijnholds J. Weber B.H. Schulz H.L. J. Neurosci. 2009; 29: 6809-6818Crossref PubMed Scopus (170) Google Scholar) are associated with Ca2+-activated Cl− currents in other epithelia. In the current studies, TMEM16A was identified in all biliary models, whereas TMEM16B was not present in any models. Furthermore, TMEM16A gene silencing decreased protein expression by 75% which corresponded to a 75% decrease in the magnitude of Ca2+-activated Cl− current density as assessed by whole-cell patch clamp. The residual Cl− currents may be secondary to residual TMEM16A protein (due to the less than 100% efficiency of gene silencing), and the correlation between the decrease in TMEM16A protein expression and Cl− currents provides strong evidence that this is the predominant operative channel. However, the possibility that the other TMEM16 isoforms (f, j, and k) identified in biliary epithelium form novel Cl−-permeable heteromultimers cannot be excluded.Second, the studies in polarized NRC monolayers demonstrate that TMEM16A is activated through nucleotides binding P2 receptors on the apical membrane. This is in contrast to CFTR, which is activated by binding of secretin to basolateral receptors (6McGill J.M. Basavappa S. Gettys T.W. Fitz J.G. Am. J. Physiol. 1994; 266: G731-G736Crossref PubMed Google Scholar). Thus, TMEM16A represents a critical member of the purinergic signaling pathway involved in hepatobiliary coupling, by which ATP released into bile by hepatocytes can stimulate P2 receptors on downstream cholangiocytes to increase ductular Cl− transport. Together with the recent findings demonstrating that fluid flow, or shear, at the cholangiocyte apical membrane is a potent stimulus for ATP release (44Woo K. Dutta A.K. Patel V. Kresge C. Feranchak A.P. J. Physiol. 2008; 586: 2779-2798Crossref PubMed Scopus (68) Google Scholar), a model emerges in which increases in bile flow rate or viscosity is transmitted to mechanosensitive ATP release, autocrine/paracrine P2 receptor binding, and TMEM16A-mediated increases in membrane Cl− permeability, ultimately resulting in bile dilution.Third, these studies suggest that TMEM16A represents a potential target for regulation of secretion by cAMP-independent pathways and for pharmacological therapy of cystic fibrosis and other cholestatic liver diseases. In fact, in other secretory cells, the level of expression of Cl− channels other than CFTR is an important determinant of organ level disease in CF (45Clarke L.L. Grubb B.R. Yankaskas J.R. Cotton C.U. McKenzie A. Boucher R.C. Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 479-483Crossref PubMed Scopus (311) Google Scholar). In the CF mouse model for example, increased Ca2+-activated Cl− channel expression in respiratory epithelium is associated with mild pulmonary disease (45Clarke L.L. Grubb B.R. Yankaskas J.R. Cotton C.U. McKenzie A. Boucher R.C. Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 479-483Crossref PubMed Scopus (311) Google Scholar). Additionally, gallbladder epithelium from CF mice exhibits brisk secretory responses in response to UTP (14Clarke L.L. Harline M.C. Gawenis L.R. Walker N.M. Turner J.T. Weisman G.A. Am. J. Physiol. Gastrointest. Liver Physiol. 2000; 279: G132-G138Crossref PubMed Google Scholar), suggesting that nucleotide-stimulated secretion, via Ca2+-activated pathways, represents a viable strategy to increase secretion and bile flow in CF biliary epithelium.Fourth, these studies are the first to identify the molecular basis of secretion in small mouse cholangiocytes isolated from small "upstream" intrahepatic ducts (<15 μm in diameter) (27Ueno Y. Alpini G. Yahagi K. Kanno N. Moritoki Y. Fukushima K. Glaser S. LeSage G. Shimosegawa T. Liver Int. 2003; 23: 449-459Crossref PubMed Scopus (89) Google Scholar). As far as we know, these are the first patch clamp studies to compare the biophysical properties of cholangiocytes isolated from distinct segments of the intrahepatic bile duct. Small cholangiocytes do not express CFTR and do not exhibit cAMP-stimulated Cl− secretion (35Glaser S.S. Gaudio E. Rao A. Pierce L.M. Onori P. Franchitto A. Francis H.L. Dostal D.E. Venter J.K. DeMorrow S. Mancinelli R. Carpino G. Alvaro D. Kopriva S.E. Savage J.M. Alpini G.D. Lab. Invest. 2009; 89: 456-469Crossref PubMed Scopus (99) Google Scholar). It seemed unusual that these small, cholangiocytes forming the smallest ducts, and the first to be exposed to canalicular bile rich in bile acids, did not possess the necessary secretory apparatus to increase fluid secretion and dilute bile. The identification of robust ATP-stimulated Ca2+-activated Cl− efflux via TMEM16A provides an answer.Lastly, cholestasis may have profound effects on cholangiocyte Cl− transport through down-regulation of IP3 receptors (46Shibao K. Hirata K. Robert M.E. Nathanson M.H. Gastroenterology. 2003; 125: 1175-1187Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) and altered Ca2+ and cAMP signaling (46Shibao K. Hirata K. Robert M.E. Nathanson M.H. Gastroenterology. 2003; 125: 1175-1187Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). These effects may be mediated in part by proinflammatory cytokines that are elevated during cholestasis. For example, IL-5 may have direct effects on CFTR expression and cAMP-stimulated Cl− transport (41McGill J.M. Yen M.S. Cummings O.W. Alpini G. LeSage G. Pollok K.E. Miller B. Engle S.K. Stansfield A.P. Am. J. Physiol. Gastrointest. Liver Physiol. 2001; 280: G738-G745Crossref PubMed Google Scholar). In contrast, IL-4 appears to mediate an adaptive response through up-regulation of TMEM16A expression resulting in an increase in transepithelial secretion. Thus, interleukin-mediated effects on channel expression and/or function may be complex within the inflammatory milieu of the cholestatic liver with some interleukins increasing, and others decreasing, cellular transport. Defining the roles of interleukins in channel expression and regulation may have important implications during cholestasis.In summary, these studies have identified the molecular identity of the Ca2+-activated Cl− channel responsible for nucleotide-mediated secretion in biliary epithelium. Modulation of TMEM16A, or other components of the ATP-stimulated secretory apparatus, might provide novel strategies for the management of liver diseases characterized by impaired bile flow. IntroductionThe formation of bile by the liver depends on the transport functions of two complimentary cell types: hepatocytes and intrahepatic bile duct epithelial cells, known as cholangiocytes. Although cholangiocytes account for only ∼2–5% of the nuclear mass of the liver (1Kumar U. Jordan T.W. Liver. 1986; 6: 369-378Crossref PubMed Scopus (34) Google Scholar), they have a prodigious capacity for secretion (2Preisig R. Cooper H.L. Wheeler H.O. J. Clin. Invest. 1962; 41: 1152-1162Crossref PubMed Scopus (98) Google Scholar, 3Wheeler H.O. Ramos O.L. J. Clin. Invest. 1960; 39: 161-170Crossref PubMed Google Scholar) and may account for up to 40% of bile volume in humans through regulated ion and water transport (4Fitz J.G. Semin. Liver Dis. 2002; 22: 241-249Crossref PubMed Scopus (30) Google Scholar). Opening of Cl− channels in the apical membrane of cholangiocytes represents the driving force for ductular secretion and at least two Cl− conductances contribute (5Fitz J.G. Basavappa S. McGill J. Melhus O. Cohn J.A. J. Clin. Invest. 1993; 91: 319-328Crossref PubMed Scopus (183) Google Scholar). The best characterized pathway involves cAMP-stimulated opening of Cl− channels encoded by the cystic fibrosis transmembrane conductance regulator (CFTR) 2The abbreviations used are: CFTRcystic fibrosis transmembrane conductance regulatorNRCnormal rat cholangiocyte(s)MLCmouse large cholangiocytesMSCmouse small cholangiocytesBDLbile duct-ligated. (5Fitz J.G. Basavappa S. McGill J. Melhus O. Cohn J.A. J. Clin. Invest. 1993; 91: 319-328Crossref PubMed Scopus (183) Google Scholar, 6McGill J.M. Basavappa S. Gettys T.W. Fitz J.G. Am. J. Physiol. 1994; 266: G731-G736Crossref PubMed Google Scholar). Additionally, there is a separate Ca2+-activated Cl− channel (CaCC) that is involved, but the molecular identity of this channel has not been defined (5Fitz J.G. Basavappa S. McGill J. Melhus O. Cohn J.A. J. Clin. Invest. 1993; 91: 319-328Crossref PubMed Scopus (183) Google Scholar, 7Schlenker T. Fitz J.G. Am. J. Physiol. 1996; 271: G304-G310Crossref PubMed Google Scholar).Increasing evidence suggests that the dominant mechanism for regulation of biliary secretion involves (i) autocrine/paracrine release of ATP into bile, (ii) activation by ATP of purinergic receptors, and (iii) mobilization of intracellular calcium and activation of CaCCs. The magnitude of the Cl− secretory response to ATP is greater than that to cAMP in single human biliary epithelial cells and polarized rat cholangiocyte monolayers (8Dutta A.K. Woo K. Doctor R.B. Fitz J.G. Feranchak A.P. Am. J. Physiol. Gastrointest. Liver Physiol. 2008; 295: G1004-G1015Crossref PubMed Scopus (34) Google Scholar). Furthermore, many of the effects of cAMP on bile formation appear to be mediated by ATP release into the duct lumen and subsequent activation of apical P2 receptors (9Minagawa N. Nagata J. Shibao K. Masyuk A.I. Gomes D.A. Rodrigues M.A. Lesage G. Akiba Y. Kaunitz J.D. Ehrlich B.E. Larusso N.F. Nathanson M.H. Gastroenterology. 2007; 133: 1592-1602Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 10Fiorotto R. Spirlì C. Fabris L. Cadamuro M. Okolicsanyi L. Strazzabosco M. Gastroenterology. 2007; 133: 1603-1613Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). These general findings are consistent with the clinical observation that only 10–20% of patients with cystic fibrosis (CF) develop clinically significant liver disease despite absent or abnormal CFTR in biliary epithelium (11Colombo C. Battezzati P.M. Crosignani A. Morabito A. Costantini D. Padoan R. Giunta A. Hepatology. 2002; 36: 1374-1382Crossref PubMed Scopus (285) Google Scholar, 12Feranchak A.P. Sokol R.J. Sem. Liv. Disease. 2001; 21: 471-488Crossref PubMed Scopus (101) Google Scholar). Furthermore, in cells with the CF phenotype, activation of P2 receptors still elicits potent secretory responses (13Paradiso A.M. Ribeiro C.M. Boucher R.C. J. Gen. Physiol. 2001; 117: 53-67Crossref PubMed Scopus (96) Google Scholar, 14Clarke L.L. Harline M.C. Gawenis L.R. Walker N.M. Turner J.T. Weisman G.A. Am. J. Physiol. Gastrointest. Liver Physiol. 2000; 279: G132-G138Crossref PubMed Google Scholar). Together, these findings challenge the conventional model that centers on the concept that cAMP-dependent opening of CFTR-related Cl− channels is the driving force for cholangiocyte secretion. Rather, the operative regulatory pathways appear to take place within the lumen of intrahepatic ducts, where release of ATP into bile is a final common pathway controlling ductular bile formation. Accordingly, molecular definition of the CaCC channel(s) involved has a high priority for future efforts to modulate the volume and composition of bile.Several candidate proteins have been proposed as CaCCs in other epithelia, including CLC and bestrophin family members (15Hartzell C. Putzier I. Arreola J. Annu. Rev. Physiol. 2005; 67: 719-758Crossref PubMed Scopus (482) Google Scholar). However, the biophysical properties of the currents associated with these proteins are not consistent with the ATP-stimulated Cl− currents described in biliary epithelium (8Dutta A.K. Woo K. Doctor R.B. Fitz J.G. Feranchak A.P. Am. J. Physiol. Gastrointest. Liver Physiol. 2008; 295: G1004-G1015Crossref PubMed Scopus (34) Google Scholar). More recently, TMEM16A, a 114-kDa membrane protein with eight putative transmembrane domains, has been identified as a CaCC in other epithelium (16Caputo A. Caci E. Ferrera L. Pedemonte N. Barsanti C. Sondo E. Pfeffer U. Ravazzolo R. Zegarra-Moran O. Galietta L.J. Science. 2008; 322: 590-594Crossref PubMed Scopus (979) Google Scholar, 17Yang Y.D. Cho H. Koo J.Y. Tak M.H. Cho Y. Shim W.S. Park S.P. Lee J. Lee B. Kim B.M. Raouf R. Shin Y.K. Oh U. Nature. 2008; 455: 1210-1215Crossref PubMed Scopus (1002) Google Scholar, 18Schroeder B.C. Cheng T. Jan Y.N. Jan L.Y. Cell. 2008; 134: 1019-1029Abstract Full Text Full Text PDF PubMed Scopus (904) Google Scholar), and the general biophysical properties of the TMEM16A conductance as measured by whole cell patch clamp appear similar to the ATP-stimulated Cl− currents described in biliary cells (8Dutta A.K. Woo K. Doctor R.B. Fitz J.G. Feranchak A.P. Am. J. Physiol. Gastrointest. Liver Physiol. 2008; 295: G1004-G1015Crossref PubMed Scopus (34) Google Scholar). Although TMEM16A is found in liver (19Ferrera L. Caputo A. Ubby I. Bussani E. Zegarra-Moran O. Ravazzolo R. Pagani F. Galietta L.J. J. Biol. Chem. 2009; 284: 33360-33368Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar), its cellular localization and function are unknown.The aim of the present studies was to identify the molecular basis for Ca2+-activated Cl− currents stimulated by extracellular nucleotides in different biliary cell models. We have utilized single cells (human and mouse), polarized biliary monolayers (rat), and novel models of small and large cholangiocytes isolated from distinct areas of the intrahepatic bile ducts (mouse) to critically assess the potential role of TMEM16A in regulating membrane Cl− permeability and biliary secretion. Lastly, we have explored the potential role of IL-4 on TMEM16A channel expression and function, which may have important implications for Cl− secretion during cholestasis.
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