- Email: [email protected]
Airway epithelial cells—Functional links between CFTR and anoctamin dependent Cl− secretion
The International Journal of Biochemistry & Cell Biology 44 (2012) 1897–1900
Contents lists available at SciVerse ScienceDirect
The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel
Cells in focus
Airway epithelial cells—Functional links between CFTR and anoctamin dependent Cl− secretion Karl Kunzelmann∗ , Yuemin Tian, Joana Raquel Martins, Diana Faria, Patthara Kongsuphol, Jiraporn Ousingsawat, Luisa Wolf, Rainer Schreiber Institut für Physiologie, Universität Regensburg, Universitätsstraße 31, D-93053 Regensburg, Germany
a r t i c l e
i n f o
Article history: Received 3 February 2012 Received in revised form 2 June 2012 Accepted 11 June 2012 Available online 16 June 2012 Keywords: Ca2+ activated Cl− currents CaCC TMEM16A Ano1 Anoctamin CFTR Crosstalk
a b s t r a c t 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 Ca2+ . Traditionally cAMP-dependent and Ca2+ -dependent secretory pathways are regarded as independent. However, this concept has been challenged recently. With identification of the Ca2+ 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. © 2012 Elsevier Ltd. All rights reserved.
Cell facts • Ciliated airway epithelial cells and Clara cells form part of the airway surface epithelium and secrete Cl− , similar to submucosal glands. • Secretion is possible though activation of the cAMP and Ca2+ dependent Cl− channels CFTR and TMEM16A, respectively. • In contrast to the traditional view, Ca2+ and Cl− dependent Cl− secretion do not occur independent of each other but are functionally interrelated at different levels.
anions to the luminal side of the airway epithelium to form a watery electrolyte layer, necessary for the transport of mucous out of the lungs (mucociliary clearance). Proper airway surface liquid (also called periciliary liquid) is due to a balance between electrolyte absorption, enabled essentially by Na+ absorption through amiloride-sensitive epithelial Na+ channels, and Cl− secretion by two apparently independent types of Cl− channels, namely cAMPregulated cystic fibrosis transmembrane conductance regulator (CFTR) and Ca2+ activated Cl− channels (TMEM16A; anoctamin 1) (Hollenhorst et al., 2011; Ousingsawat et al., 2011; Yang et al., 2008). In the present review we will focus on Cl− secretion and summarize current evidence for a co-regulation of both Cl− channels.
1. Introduction
2. Cell origin and plasticity
Human airways consist of more proximal conducting airways and distal respiratory airways, essentially formed by alveoli. The conducting airways (nose, mouth, trachea, bronchi, and bronchioli) transport, clean, warm, and humidify the air. Humidifying inhaled air is part of the airway defence and is fundamental to proper lung function. It is provided by epithelial cells that actively secrete
The contribution of individual cell types (ciliated cells, Clara cells, serous cells, goblet cells, brush cells, neuroendocrine cells, basal cells) to ion secretion depends on the location within the airway epithelium. Most types of airway epithelial cells, like ciliated cells, Clara cells, and goblet cells, are able to secrete ions. Thus CFTR and TMEM16A are expressed quite broadly along the airways, although some hot spots for expression may exist (Kreda et al., 2005). It is commonly accepted that CFTR is indispensable for cAMP-dependent Cl− secretion, however it remains currently an unresolved issue to what degree TMEM16A contributes to Ca2+
∗ Corresponding author. Tel.: +49 0941 943 4302; fax: +49 0941 943 4315. E-mail address: [email protected] (K. Kunzelmann). 1357-2725/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biocel.2012.06.011
1898
K. Kunzelmann et al. / The International Journal of Biochemistry & Cell Biology 44 (2012) 1897–1900
Fig. 1. Cellular model for cAMP and Ca2+ dependent Cl− secretion and role of TMEM16 proteins in airways. (A) RT-PCR-analysis for expression of all ten TMEM16-proteins in native human airways. (B) In human airways both surface epithelium and submucosal glands contribute to Cl− secretion. (C) Cellular model for cAMP and Ca2+ dependent Cl− secretion showing apical cAMP-regulated CFTR and Ca2+ -activated TMEM16A Cl− channels. Cl− is taken up by the basolateral Na+ /2Cl− /K+ cotransporter NKCC1. Na+ is pumped out by the basolateral Na+ /K+ -ATPase, while basolateral K+ channels (KCNQ1/KCNE3 and KCNN4) recycle K+ to the basolateral (serosal) side and hyperpolarize the membrane voltage (Vm) to supply the necessary driving force for luminal Cl− exit.
dependent Cl− secretion in human airways, and whether the other nine members of the TMEM16 family are also of relevance for Cl− secretion (Schreiber et al., 2010). As shown in Fig. 1A, several members of the TMEM16 family are expressed throughout native human airways. Moreover, our own work suggests that all 10 TMEM16 (A–K) proteins are able to form Cl− channels (data not shown). According to siRNA-knockdown in airway cell lines, and data from TMEM16A-knockout mice, TMEM16A is the predominant component of Ca2+ activated Cl− currents in airways (Ousingsawat et al., 2009).
interactom, CFTR may either inhibit ion transport (e.g. by ENaC or NHE3) or activate transport (e.g. by SLC26A6 or SLC26A9). Although the molecular mechanisms for the interaction with other transport proteins has not been described in detail in each case, numerous factors and second messenger pathways have been identified through which CFTR may control the activity
3. Functions The cellular models for both cAMP and Ca2+ dependent Cl− secretion are shown in Fig. 1B and C. Cl− is taken up by the basolateral Na+ /2Cl− /K+ cotransporter NKCC1. Na+ is pumped out by the basolateral Na+ /K+ -ATPase and basolateral K+ channels (KCNQ1/KCNE3 and KCNN4) recycle K+ to the basolateral (serosal) side and hyperpolarize the membrane voltage (Vm) to supply the necessary driving force for luminal Cl− exit (Greger et al., 1990). In general, cAMP/CFTR dependent Cl− secretion is much more sustained than Ca2+ /TMEM16A-dependent secretion, due to the transient nature of the intracellular Ca2+ signal and therefore only transient activation of TMEM16A. Moreover cAMP and protein kinase A are required for sufficient activation of basolateral Cl− uptake (NKCC1) and hyperpolarization of Vm (KCNQ1/KCNE3). Intracellular cAMP and Ca2+ signals are elicited through stimulation of luminal purinergic (P2Y2,4,6 and A2B) receptors, or basolateral hormonal stimulation (prostaglandin, muscarinic, and other receptors). It is, however, not completely clear to what extent basolaterally elicited signals “talk” to apical transport proteins and vice versa. Obviously apical and basolateral signals are compartmentalized to a large extent, and are confined to apical and basolateral membranes (Paradiso et al., 2001). CFTR is a very well studied ion channel. Meanwhile numerous functional interactions with other membrane ion transport proteins have been demonstrated (Fig. 2A). Within such a functional
Fig. 2. The functional CFTR interactom. (A) CFTR interacts with and controls the activity of numerous membrane transport proteins such as Cl− channels (CaCC/TMEM16A, ORCC/TMEM16F, SLC26A9, ClC-3, ICl-swell ), exchangers (SLC26A5, SLC26A6, NHE3), K+ channels (ROMK1, ROMK2, SK4, KCNQ1), as well as cation channels (ENaC, TRPV) and aquaporins. (B) CFTR has been shown to be regulated by a number of cytosolic messengers and to control the activity of several second messenger cascades. For many of them a direct physical interaction has been demonstrated.
K. Kunzelmann et al. / The International Journal of Biochemistry & Cell Biology 44 (2012) 1897–1900
1899
Fig. 3. Crosstalk between cAMP and Ca2+ dependent secretion. Crosstalk between cAMP- and Ca2+ -dependent Cl− secretion may occur at several levels. (i) Purinergic receptors such as P2Y2 /P2Y6 increase both intracellular cAMP and Ca2+ . (ii) Intracellular Ca2+ affects the activity of enzymes that control intracellular cAMP (adenylate cyclase, AC; phosphodiesterase, PD). (iii) Intracellular cAMP affects proteins that control intracellular Ca2+ levels (SERCA). (iv) Intracellular Cl− concentration affects Ca2+ transport over the ER-membrane (via the Cl− channel Best1) and Ca2+ influx (via ORAI/TRP). (v) CFTR and TMEM16A/F may interact directly or through scaffold proteins (NHERF1 and other PDZ-domain proteins). (vi) CFTR translocates Gq-coupled receptors to the plasma membrane that allow for Ca2+ increase and activation of TMEM16A. (vii) Novel messengers like IRBIT link IP3/Ca2+ signaling to CFTR.
and properties of other ion transport proteins, including Ca2+ activated Cl− channels or the outwardly rectifying Cl− channel ORCC (Guggino and Stanton, 2006) (Fig. 2B). We found recently that another member of the TMEM16-family, TMEM16F (Ano6) is the major component of ORCC, which is typically activated when cells move into apoptosis (Martins et al., 2011). Crosstalk between cAMP and Ca2+ dependent secretion has long been suggested and may occur at different levels (Fig. 3): It is known, for example, that purinergic receptors such as P2Y2 or P2Y6 couple to both intracellular messengers, cAMP and Ca2+ (Faria et al., 2009; Schreiber and Kunzelmann, 2005). It is also known that intracellular Ca2+ affects the activity of enzymes that control intracellular cAMP levels such as adenylate cyclase and phosphodiesterase (Namkung et al., 2010). Inversely cAMP affects proteins that control intracellular Ca2+ levels, such as the endoplasmic reticulum Ca2+ -ATPase SERCA or IP3 receptors (Bruce et al., 2003). Intracellular Cl− concentrations affect Ca2+ transport over the ERmembrane (via the Cl− channel Best1) and Ca2+ influx (via ORAI and TRP channels) (Barro Soria et al., 2009). CFTR and TMEM16A/F may interact directly or through scaffold proteins such as NHERF1 or other PDZ-domain proteins. Recently we demonstrated that CFTR translocates Gq-coupled receptors to the plasma membrane of Xenopus oocytes and allows for Ca2+ increase and activation of TMEM16A (Kongsuphol et al., 2011). Finally, other novel messengers like the IP3-receptor binding protein IRBIT may link IP3/Ca2+ signaling to CFTR (Yang et al., 2009). 4. Associated pathologies Although there is still a controversial discussion as to what degree hyperabsorption of Na+ by the airway epithelium contributes to lung disease in cystic fibrosis (CF), it is well accepted that missing Cl− secretion by CFTR is the central defect in CF. Notably enhanced Ca2+ activated Cl− currents have been detected in the airways of CFTR-knockout mice and in numerous studies with primary or permanent human airway epithelial cell lines (Grubb et al., 1994; Martins et al., in press; Ribeiro et al., 2005). More important, significant differences were also found in native non-CF and CF airways ex vivo (Mall et al., 2003). Why is Ca2+ activated
Cl− secretion different in human CF-airways? We found no differences in expression of the Ca2+ activated Cl− channels Best1 or TMEM16A, which could explain the differences (Martins et al., in press). However, differences in Ca2+ signaling were detected between CF- and non-CF airway epithelial cells. These differences are probably due to the so-called ER-stress caused by accumulation of mutant F508del-CFTR in the endoplasmic reticulum and are also induced by infection of the airways (Martins et al., in press; Ribeiro et al., 2005). Although CaCC appears enhanced in CF, it is nevertheless not able to compensate for defective CFTR. This is explained by an only small contribution of CaCC to airway Cl− secretion and by the fact that activation of CaCC is largely transient, in contrast to the permanent CFTR-dependent Cl− secretion. In fact the nontransient component of Ca2+ dependent Cl− secretion elicited through stimulation of purinergic P2Y-receptors is probably due to activation of CFTR rather than activation of TMEM16A. This has been demonstrated in Xenopus oocytes expressing both CFTR and P2Y2 -receptors, and in human airway epithelial cells (Faria et al., 2009; Namkung et al., 2010). The situation in murine airways is remarkably different to human airways, as most Cl− secretion relies on TMEM16A rather than CFTR. Nevertheless, in human submucosal glands, Cl− secretion relies on both CFTR and TMEM16A. cAMP-activated Ca2+ signaling is required for CFTR-mediated fluid secretion by serous cells in porcine and human airways (Lee and Foskett, 2010). Ca2+ activated Cl− secretion by TMEM16A and its regulation through both apical purinergic signaling as well as basolateral cholinergic stimulation, probably controls the airway surface liquid and therefore has a role in mucociliary clearance. Thus, in mouse tracheas from TMEM16A knockout mice we found a reduced particle transport, suggesting impaired mucociliary lung clearance in these animals (Ousingsawat et al., 2009). In fact, these animals may develop a lung phenotype that is similar to that of CF patients (Rock et al., 2009). Novel pharmacological compounds that directly activate TMEM16A leading to a more sustained Cl− secretion, would therefore be most welcome drugs, not only for the treatment of cystic fibrosis lung disease but also to target Sjörgren syndrome and other malfunctions of other exocrine glands (Namkung et al., 2011).
1900
K. Kunzelmann et al. / The International Journal of Biochemistry & Cell Biology 44 (2012) 1897–1900
Acknowledgments The work was supported by Mukoviszidose e.V. (Projekt-Nr. S02/10) and DFG SFB699/A7. References Barro Soria R, AlDehni F, Almaca J, Witzgall R, Schreiber R, Kunzelmann K. ER localized bestrophin1 acts as a counter-ion channel to activate Ca2+ dependent ion channels TMEM16A and SK4. Pflugers Archiv 2009;459:485–97. Bruce JI, Straub SV, Yule DI. Crosstalk between cAMP and Ca2+ signaling in nonexcitable cells. Cell Calcium 2003;34:431–44. Faria D, Schreiber R, Kunzelmann K. CFTR is activated through stimulation of purinergic P2Y2 receptors. Pflugers Archiv 2009;457:1373–80. Greger R, Kunzelmann K, Gerlach L. Mechanisms of chloride transport in secretory epithelia. Annals of the New York Academy of Sciences 1990;574:403–15. Grubb BR, Vick RN, Boucher RC. Hyperabsorption of Na+ and raised Ca2+ mediated Cl− secretion in nasal epithelia of CF mice. The American Journal of Physiology 1994;266:C1478–83. Guggino WB, Stanton BA. New insights into cystic fibrosis: molecular switches that regulate CFTR. Nature Reviews: Molecular Cell Biology 2006;7:426–36. Hollenhorst MI, Richter K, Fronius M. Ion transport by pulmonary epithelia. Journal of Biomedicine and Biotechnology 2011;2011:174306. Kongsuphol P, Schreiber R, Kraidith K, Kunzelmann K. CFTR induces extracellular acid sensing in Xenopus oocytes which activates endogenous Ca(2+)-activated Cl(−) conductance. Pflugers Archiv 2011;462:479–87. Kreda SM, Mall M, Mengos A, Rochelle LG, Yankaskas J, Riordan JR, et al. CFTR in human airways. Molecular Biology of the Cell 2005;16:2154–67. Lee RJ, Foskett JK. cAMP-activated Ca2+ signaling is required for CFTR-mediated serous cell fluid secretion in porcine and human airways. The Journal of Clinical Investigation 2010;120:3137–48. Mall M, Gonska T, Thomas J, Schreiber R, Seydewitz HH, Kuehr J, et al. Modulation of Ca2+ activated Cl− secretion by basolateral K+ channels in human normal and cystic fibrosis airway epithelia. Pediatric Research 2003;53:608–18. Martins JR, Faria D, Kongsuphol P, Reisch B, Schreiber R, Kunzelmann K. Anoctamin 6 is an essential component of the outwardly rectifying chloride channel. Proceedings of the National Academy of Sciences of the United States of America 2011.
Martins JR, Kongsuphol P, Sammels E, Daimène S, AlDehni F, Clarke L, et al. F508del-CFTR increases intracellular Ca2+ signaling that causes enhanced calcium-dependent Cl− conductance in cystic fibrosis. Biochimica et Biophysica Acta, in press. Namkung W, Finkbeiner WE, Verkman AS. CFTR-adenylyl cyclase I association is responsible for UTP activation of CFTR in well-differentiated primary human bronchial cell cultures. Molecular Biology of the Cell 2010;21: 2639–48. Namkung W, Yao Z, Finkbeiner WE, Verkman AS. Small-molecule activators of TMEM16A, a calcium-activated chloride channel, stimulate epithelial chloride secretion and intestinal contraction. The FASEB Journal 2011. Ousingsawat J, Martins JR, Schreiber R, Rock JR, Harfe BD, Kunzelmann K. Loss of TMEM16A causes a defect in epithelial Ca2+ dependent chloride transport. The Journal of Biological Chemistry 2009;284:28698–703. Ousingsawat J, Kongsuphol P, Schreiber R, Kunzelmann K. CFTR and TMEM16A are separate but functionally related Cl channels. Cellular Physiology and Biochemistry 2011;28:715–24. Paradiso AM, Ribeiro CM, Boucher RC. Polarized signaling via purinoceptors in normal and cystic fibrosis airway epithelia. The Journal of General Physiology 2001;117:53–68. Ribeiro CM, Paradiso AM, Carew MA, Shears SB, Boucher RC. Cystic fibrosis airway epithelial Ca2+ i signaling. The mechanism for the larger agonist-mediated Ca2+ i signals in human cystic fibrosis airway epithelia. The Journal of Biological Chemistry 2005. Rock JR, O’Neal WK, Gabriel SE, Randell SH, Harfe BD, Boucher RC, et al. Transmembrane protein 16A (TMEM16A) is a Ca2+ regulated Cl− – secretory channel in mouse airways. The Journal of Biological Chemistry 2009;284:14875–80. Schreiber R, Kunzelmann K. Purinergic P2Y6 receptors induce Ca2+ and CFTR dependent Cl− secretion in mouse trachea. Cellular Physiology and Biochemistry 2005;16:99–108. Schreiber R, Uliyakina I, Kongsuphol P, Warth R, Mirza M, Martins JR, et al. Function of epithelial anoctamins. The Journal of Biological Chemistry 2010;285: 7838–45. Yang YD, Cho H, Koo JY, Tak MH, Cho Y, Shim WS, et al. TMEM16A confers receptor-activated calcium-dependent chloride conductance. Nature 2008;455: 1210–5. Yang D, Shcheynikov N, Zeng W, Ohana E, So I, Ando H, et al. IRBIT coordinates epithelial fluid and HCO3 -secretion by stimulating the transporters pNBC1 and CFTR in the murine pancreatic duct. The Journal of Clinical Investigation 2009;119:193–202.
Contents lists available at SciVerse ScienceDirect
The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel
Cells in focus
Airway epithelial cells—Functional links between CFTR and anoctamin dependent Cl− secretion Karl Kunzelmann∗ , Yuemin Tian, Joana Raquel Martins, Diana Faria, Patthara Kongsuphol, Jiraporn Ousingsawat, Luisa Wolf, Rainer Schreiber Institut für Physiologie, Universität Regensburg, Universitätsstraße 31, D-93053 Regensburg, Germany
a r t i c l e
i n f o
Article history: Received 3 February 2012 Received in revised form 2 June 2012 Accepted 11 June 2012 Available online 16 June 2012 Keywords: Ca2+ activated Cl− currents CaCC TMEM16A Ano1 Anoctamin CFTR Crosstalk
a b s t r a c t 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 Ca2+ . Traditionally cAMP-dependent and Ca2+ -dependent secretory pathways are regarded as independent. However, this concept has been challenged recently. With identification of the Ca2+ 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. © 2012 Elsevier Ltd. All rights reserved.
Cell facts • Ciliated airway epithelial cells and Clara cells form part of the airway surface epithelium and secrete Cl− , similar to submucosal glands. • Secretion is possible though activation of the cAMP and Ca2+ dependent Cl− channels CFTR and TMEM16A, respectively. • In contrast to the traditional view, Ca2+ and Cl− dependent Cl− secretion do not occur independent of each other but are functionally interrelated at different levels.
anions to the luminal side of the airway epithelium to form a watery electrolyte layer, necessary for the transport of mucous out of the lungs (mucociliary clearance). Proper airway surface liquid (also called periciliary liquid) is due to a balance between electrolyte absorption, enabled essentially by Na+ absorption through amiloride-sensitive epithelial Na+ channels, and Cl− secretion by two apparently independent types of Cl− channels, namely cAMPregulated cystic fibrosis transmembrane conductance regulator (CFTR) and Ca2+ activated Cl− channels (TMEM16A; anoctamin 1) (Hollenhorst et al., 2011; Ousingsawat et al., 2011; Yang et al., 2008). In the present review we will focus on Cl− secretion and summarize current evidence for a co-regulation of both Cl− channels.
1. Introduction
2. Cell origin and plasticity
Human airways consist of more proximal conducting airways and distal respiratory airways, essentially formed by alveoli. The conducting airways (nose, mouth, trachea, bronchi, and bronchioli) transport, clean, warm, and humidify the air. Humidifying inhaled air is part of the airway defence and is fundamental to proper lung function. It is provided by epithelial cells that actively secrete
The contribution of individual cell types (ciliated cells, Clara cells, serous cells, goblet cells, brush cells, neuroendocrine cells, basal cells) to ion secretion depends on the location within the airway epithelium. Most types of airway epithelial cells, like ciliated cells, Clara cells, and goblet cells, are able to secrete ions. Thus CFTR and TMEM16A are expressed quite broadly along the airways, although some hot spots for expression may exist (Kreda et al., 2005). It is commonly accepted that CFTR is indispensable for cAMP-dependent Cl− secretion, however it remains currently an unresolved issue to what degree TMEM16A contributes to Ca2+
∗ Corresponding author. Tel.: +49 0941 943 4302; fax: +49 0941 943 4315. E-mail address: [email protected] (K. Kunzelmann). 1357-2725/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biocel.2012.06.011
1898
K. Kunzelmann et al. / The International Journal of Biochemistry & Cell Biology 44 (2012) 1897–1900
Fig. 1. Cellular model for cAMP and Ca2+ dependent Cl− secretion and role of TMEM16 proteins in airways. (A) RT-PCR-analysis for expression of all ten TMEM16-proteins in native human airways. (B) In human airways both surface epithelium and submucosal glands contribute to Cl− secretion. (C) Cellular model for cAMP and Ca2+ dependent Cl− secretion showing apical cAMP-regulated CFTR and Ca2+ -activated TMEM16A Cl− channels. Cl− is taken up by the basolateral Na+ /2Cl− /K+ cotransporter NKCC1. Na+ is pumped out by the basolateral Na+ /K+ -ATPase, while basolateral K+ channels (KCNQ1/KCNE3 and KCNN4) recycle K+ to the basolateral (serosal) side and hyperpolarize the membrane voltage (Vm) to supply the necessary driving force for luminal Cl− exit.
dependent Cl− secretion in human airways, and whether the other nine members of the TMEM16 family are also of relevance for Cl− secretion (Schreiber et al., 2010). As shown in Fig. 1A, several members of the TMEM16 family are expressed throughout native human airways. Moreover, our own work suggests that all 10 TMEM16 (A–K) proteins are able to form Cl− channels (data not shown). According to siRNA-knockdown in airway cell lines, and data from TMEM16A-knockout mice, TMEM16A is the predominant component of Ca2+ activated Cl− currents in airways (Ousingsawat et al., 2009).
interactom, CFTR may either inhibit ion transport (e.g. by ENaC or NHE3) or activate transport (e.g. by SLC26A6 or SLC26A9). Although the molecular mechanisms for the interaction with other transport proteins has not been described in detail in each case, numerous factors and second messenger pathways have been identified through which CFTR may control the activity
3. Functions The cellular models for both cAMP and Ca2+ dependent Cl− secretion are shown in Fig. 1B and C. Cl− is taken up by the basolateral Na+ /2Cl− /K+ cotransporter NKCC1. Na+ is pumped out by the basolateral Na+ /K+ -ATPase and basolateral K+ channels (KCNQ1/KCNE3 and KCNN4) recycle K+ to the basolateral (serosal) side and hyperpolarize the membrane voltage (Vm) to supply the necessary driving force for luminal Cl− exit (Greger et al., 1990). In general, cAMP/CFTR dependent Cl− secretion is much more sustained than Ca2+ /TMEM16A-dependent secretion, due to the transient nature of the intracellular Ca2+ signal and therefore only transient activation of TMEM16A. Moreover cAMP and protein kinase A are required for sufficient activation of basolateral Cl− uptake (NKCC1) and hyperpolarization of Vm (KCNQ1/KCNE3). Intracellular cAMP and Ca2+ signals are elicited through stimulation of luminal purinergic (P2Y2,4,6 and A2B) receptors, or basolateral hormonal stimulation (prostaglandin, muscarinic, and other receptors). It is, however, not completely clear to what extent basolaterally elicited signals “talk” to apical transport proteins and vice versa. Obviously apical and basolateral signals are compartmentalized to a large extent, and are confined to apical and basolateral membranes (Paradiso et al., 2001). CFTR is a very well studied ion channel. Meanwhile numerous functional interactions with other membrane ion transport proteins have been demonstrated (Fig. 2A). Within such a functional
Fig. 2. The functional CFTR interactom. (A) CFTR interacts with and controls the activity of numerous membrane transport proteins such as Cl− channels (CaCC/TMEM16A, ORCC/TMEM16F, SLC26A9, ClC-3, ICl-swell ), exchangers (SLC26A5, SLC26A6, NHE3), K+ channels (ROMK1, ROMK2, SK4, KCNQ1), as well as cation channels (ENaC, TRPV) and aquaporins. (B) CFTR has been shown to be regulated by a number of cytosolic messengers and to control the activity of several second messenger cascades. For many of them a direct physical interaction has been demonstrated.
K. Kunzelmann et al. / The International Journal of Biochemistry & Cell Biology 44 (2012) 1897–1900
1899
Fig. 3. Crosstalk between cAMP and Ca2+ dependent secretion. Crosstalk between cAMP- and Ca2+ -dependent Cl− secretion may occur at several levels. (i) Purinergic receptors such as P2Y2 /P2Y6 increase both intracellular cAMP and Ca2+ . (ii) Intracellular Ca2+ affects the activity of enzymes that control intracellular cAMP (adenylate cyclase, AC; phosphodiesterase, PD). (iii) Intracellular cAMP affects proteins that control intracellular Ca2+ levels (SERCA). (iv) Intracellular Cl− concentration affects Ca2+ transport over the ER-membrane (via the Cl− channel Best1) and Ca2+ influx (via ORAI/TRP). (v) CFTR and TMEM16A/F may interact directly or through scaffold proteins (NHERF1 and other PDZ-domain proteins). (vi) CFTR translocates Gq-coupled receptors to the plasma membrane that allow for Ca2+ increase and activation of TMEM16A. (vii) Novel messengers like IRBIT link IP3/Ca2+ signaling to CFTR.
and properties of other ion transport proteins, including Ca2+ activated Cl− channels or the outwardly rectifying Cl− channel ORCC (Guggino and Stanton, 2006) (Fig. 2B). We found recently that another member of the TMEM16-family, TMEM16F (Ano6) is the major component of ORCC, which is typically activated when cells move into apoptosis (Martins et al., 2011). Crosstalk between cAMP and Ca2+ dependent secretion has long been suggested and may occur at different levels (Fig. 3): It is known, for example, that purinergic receptors such as P2Y2 or P2Y6 couple to both intracellular messengers, cAMP and Ca2+ (Faria et al., 2009; Schreiber and Kunzelmann, 2005). It is also known that intracellular Ca2+ affects the activity of enzymes that control intracellular cAMP levels such as adenylate cyclase and phosphodiesterase (Namkung et al., 2010). Inversely cAMP affects proteins that control intracellular Ca2+ levels, such as the endoplasmic reticulum Ca2+ -ATPase SERCA or IP3 receptors (Bruce et al., 2003). Intracellular Cl− concentrations affect Ca2+ transport over the ERmembrane (via the Cl− channel Best1) and Ca2+ influx (via ORAI and TRP channels) (Barro Soria et al., 2009). CFTR and TMEM16A/F may interact directly or through scaffold proteins such as NHERF1 or other PDZ-domain proteins. Recently we demonstrated that CFTR translocates Gq-coupled receptors to the plasma membrane of Xenopus oocytes and allows for Ca2+ increase and activation of TMEM16A (Kongsuphol et al., 2011). Finally, other novel messengers like the IP3-receptor binding protein IRBIT may link IP3/Ca2+ signaling to CFTR (Yang et al., 2009). 4. Associated pathologies Although there is still a controversial discussion as to what degree hyperabsorption of Na+ by the airway epithelium contributes to lung disease in cystic fibrosis (CF), it is well accepted that missing Cl− secretion by CFTR is the central defect in CF. Notably enhanced Ca2+ activated Cl− currents have been detected in the airways of CFTR-knockout mice and in numerous studies with primary or permanent human airway epithelial cell lines (Grubb et al., 1994; Martins et al., in press; Ribeiro et al., 2005). More important, significant differences were also found in native non-CF and CF airways ex vivo (Mall et al., 2003). Why is Ca2+ activated
Cl− secretion different in human CF-airways? We found no differences in expression of the Ca2+ activated Cl− channels Best1 or TMEM16A, which could explain the differences (Martins et al., in press). However, differences in Ca2+ signaling were detected between CF- and non-CF airway epithelial cells. These differences are probably due to the so-called ER-stress caused by accumulation of mutant F508del-CFTR in the endoplasmic reticulum and are also induced by infection of the airways (Martins et al., in press; Ribeiro et al., 2005). Although CaCC appears enhanced in CF, it is nevertheless not able to compensate for defective CFTR. This is explained by an only small contribution of CaCC to airway Cl− secretion and by the fact that activation of CaCC is largely transient, in contrast to the permanent CFTR-dependent Cl− secretion. In fact the nontransient component of Ca2+ dependent Cl− secretion elicited through stimulation of purinergic P2Y-receptors is probably due to activation of CFTR rather than activation of TMEM16A. This has been demonstrated in Xenopus oocytes expressing both CFTR and P2Y2 -receptors, and in human airway epithelial cells (Faria et al., 2009; Namkung et al., 2010). The situation in murine airways is remarkably different to human airways, as most Cl− secretion relies on TMEM16A rather than CFTR. Nevertheless, in human submucosal glands, Cl− secretion relies on both CFTR and TMEM16A. cAMP-activated Ca2+ signaling is required for CFTR-mediated fluid secretion by serous cells in porcine and human airways (Lee and Foskett, 2010). Ca2+ activated Cl− secretion by TMEM16A and its regulation through both apical purinergic signaling as well as basolateral cholinergic stimulation, probably controls the airway surface liquid and therefore has a role in mucociliary clearance. Thus, in mouse tracheas from TMEM16A knockout mice we found a reduced particle transport, suggesting impaired mucociliary lung clearance in these animals (Ousingsawat et al., 2009). In fact, these animals may develop a lung phenotype that is similar to that of CF patients (Rock et al., 2009). Novel pharmacological compounds that directly activate TMEM16A leading to a more sustained Cl− secretion, would therefore be most welcome drugs, not only for the treatment of cystic fibrosis lung disease but also to target Sjörgren syndrome and other malfunctions of other exocrine glands (Namkung et al., 2011).
1900
K. Kunzelmann et al. / The International Journal of Biochemistry & Cell Biology 44 (2012) 1897–1900
Acknowledgments The work was supported by Mukoviszidose e.V. (Projekt-Nr. S02/10) and DFG SFB699/A7. References Barro Soria R, AlDehni F, Almaca J, Witzgall R, Schreiber R, Kunzelmann K. ER localized bestrophin1 acts as a counter-ion channel to activate Ca2+ dependent ion channels TMEM16A and SK4. Pflugers Archiv 2009;459:485–97. Bruce JI, Straub SV, Yule DI. Crosstalk between cAMP and Ca2+ signaling in nonexcitable cells. Cell Calcium 2003;34:431–44. Faria D, Schreiber R, Kunzelmann K. CFTR is activated through stimulation of purinergic P2Y2 receptors. Pflugers Archiv 2009;457:1373–80. Greger R, Kunzelmann K, Gerlach L. Mechanisms of chloride transport in secretory epithelia. Annals of the New York Academy of Sciences 1990;574:403–15. Grubb BR, Vick RN, Boucher RC. Hyperabsorption of Na+ and raised Ca2+ mediated Cl− secretion in nasal epithelia of CF mice. The American Journal of Physiology 1994;266:C1478–83. Guggino WB, Stanton BA. New insights into cystic fibrosis: molecular switches that regulate CFTR. Nature Reviews: Molecular Cell Biology 2006;7:426–36. Hollenhorst MI, Richter K, Fronius M. Ion transport by pulmonary epithelia. Journal of Biomedicine and Biotechnology 2011;2011:174306. Kongsuphol P, Schreiber R, Kraidith K, Kunzelmann K. CFTR induces extracellular acid sensing in Xenopus oocytes which activates endogenous Ca(2+)-activated Cl(−) conductance. Pflugers Archiv 2011;462:479–87. Kreda SM, Mall M, Mengos A, Rochelle LG, Yankaskas J, Riordan JR, et al. CFTR in human airways. Molecular Biology of the Cell 2005;16:2154–67. Lee RJ, Foskett JK. cAMP-activated Ca2+ signaling is required for CFTR-mediated serous cell fluid secretion in porcine and human airways. The Journal of Clinical Investigation 2010;120:3137–48. Mall M, Gonska T, Thomas J, Schreiber R, Seydewitz HH, Kuehr J, et al. Modulation of Ca2+ activated Cl− secretion by basolateral K+ channels in human normal and cystic fibrosis airway epithelia. Pediatric Research 2003;53:608–18. Martins JR, Faria D, Kongsuphol P, Reisch B, Schreiber R, Kunzelmann K. Anoctamin 6 is an essential component of the outwardly rectifying chloride channel. Proceedings of the National Academy of Sciences of the United States of America 2011.
Martins JR, Kongsuphol P, Sammels E, Daimène S, AlDehni F, Clarke L, et al. F508del-CFTR increases intracellular Ca2+ signaling that causes enhanced calcium-dependent Cl− conductance in cystic fibrosis. Biochimica et Biophysica Acta, in press. Namkung W, Finkbeiner WE, Verkman AS. CFTR-adenylyl cyclase I association is responsible for UTP activation of CFTR in well-differentiated primary human bronchial cell cultures. Molecular Biology of the Cell 2010;21: 2639–48. Namkung W, Yao Z, Finkbeiner WE, Verkman AS. Small-molecule activators of TMEM16A, a calcium-activated chloride channel, stimulate epithelial chloride secretion and intestinal contraction. The FASEB Journal 2011. Ousingsawat J, Martins JR, Schreiber R, Rock JR, Harfe BD, Kunzelmann K. Loss of TMEM16A causes a defect in epithelial Ca2+ dependent chloride transport. The Journal of Biological Chemistry 2009;284:28698–703. Ousingsawat J, Kongsuphol P, Schreiber R, Kunzelmann K. CFTR and TMEM16A are separate but functionally related Cl channels. Cellular Physiology and Biochemistry 2011;28:715–24. Paradiso AM, Ribeiro CM, Boucher RC. Polarized signaling via purinoceptors in normal and cystic fibrosis airway epithelia. The Journal of General Physiology 2001;117:53–68. Ribeiro CM, Paradiso AM, Carew MA, Shears SB, Boucher RC. Cystic fibrosis airway epithelial Ca2+ i signaling. The mechanism for the larger agonist-mediated Ca2+ i signals in human cystic fibrosis airway epithelia. The Journal of Biological Chemistry 2005. Rock JR, O’Neal WK, Gabriel SE, Randell SH, Harfe BD, Boucher RC, et al. Transmembrane protein 16A (TMEM16A) is a Ca2+ regulated Cl− – secretory channel in mouse airways. The Journal of Biological Chemistry 2009;284:14875–80. Schreiber R, Kunzelmann K. Purinergic P2Y6 receptors induce Ca2+ and CFTR dependent Cl− secretion in mouse trachea. Cellular Physiology and Biochemistry 2005;16:99–108. Schreiber R, Uliyakina I, Kongsuphol P, Warth R, Mirza M, Martins JR, et al. Function of epithelial anoctamins. The Journal of Biological Chemistry 2010;285: 7838–45. Yang YD, Cho H, Koo JY, Tak MH, Cho Y, Shim WS, et al. TMEM16A confers receptor-activated calcium-dependent chloride conductance. Nature 2008;455: 1210–5. Yang D, Shcheynikov N, Zeng W, Ohana E, So I, Ando H, et al. IRBIT coordinates epithelial fluid and HCO3 -secretion by stimulating the transporters pNBC1 and CFTR in the murine pancreatic duct. The Journal of Clinical Investigation 2009;119:193–202.