Functional Characterization of Pendrin in a Polarized Cell System

Abstract

Pendred's syndrome is an autosomal recessive disorder characterized by sensorineural deafness, goiter, and impaired iodide organification. It is caused by mutations in the PDS/SLC26A4 gene that encodes pendrin. Functionally, pendrin is a transporter of chloride and iodide in Xenopus oocytes and heterologous mammalian cells and a chloride/base exchanger in β-intercalated cells of the renal cortical collecting duct. The partially impaired thyroidal iodide organification in Pendred's syndrome suggests a possible role of pendrin in iodide transport at the apical membrane of thyroid follicular cells, but experimental evidence for this concept is lacking. The iodide transport properties of pendrin were determined in polarized Madin-Darby canine kidney cells expressing the sodium iodide symporter (NIS), pendrin, or NIS and pendrin using a bicameral system-permitting measurement of iodide content in the basal, intracellular, and apical compartments. Moreover, we determined the functional consequences of two naturally occurring mutations (L676Q and FS306>309X). In polarized Madin-Darby canine kidney cells, NIS mediates uptake at the basolateral membrane. Only minimal amounts of iodide reach the apical compartment in the absence of pendrin. In cells expressing NIS and pendrin, pendrin mediates transport of iodide into the apical chamber. Wild type pendrin also mediates iodide efflux in transiently transfected cells. In contrast, both pendrin mutants lose the ability to promote iodide efflux. These results provide evidence that pendrin mediates apical iodide efflux from polarized mammalian cells loaded with iodide. Consistent with the partial organification defect observed in patients with Pendred's syndrome, naturally occurring mutations of pendrin lead to impaired transport of iodide. Pendred's syndrome is an autosomal recessive disorder characterized by sensorineural deafness, goiter, and impaired iodide organification. It is caused by mutations in the PDS/SLC26A4 gene that encodes pendrin. Functionally, pendrin is a transporter of chloride and iodide in Xenopus oocytes and heterologous mammalian cells and a chloride/base exchanger in β-intercalated cells of the renal cortical collecting duct. The partially impaired thyroidal iodide organification in Pendred's syndrome suggests a possible role of pendrin in iodide transport at the apical membrane of thyroid follicular cells, but experimental evidence for this concept is lacking. The iodide transport properties of pendrin were determined in polarized Madin-Darby canine kidney cells expressing the sodium iodide symporter (NIS), pendrin, or NIS and pendrin using a bicameral system-permitting measurement of iodide content in the basal, intracellular, and apical compartments. Moreover, we determined the functional consequences of two naturally occurring mutations (L676Q and FS306>309X). In polarized Madin-Darby canine kidney cells, NIS mediates uptake at the basolateral membrane. Only minimal amounts of iodide reach the apical compartment in the absence of pendrin. In cells expressing NIS and pendrin, pendrin mediates transport of iodide into the apical chamber. Wild type pendrin also mediates iodide efflux in transiently transfected cells. In contrast, both pendrin mutants lose the ability to promote iodide efflux. These results provide evidence that pendrin mediates apical iodide efflux from polarized mammalian cells loaded with iodide. Consistent with the partial organification defect observed in patients with Pendred's syndrome, naturally occurring mutations of pendrin lead to impaired transport of iodide. Pendred's syndrome, an autosomal recessive disorder characterized by congenital sensorineural deafness, goiter, and impaired iodide organification (1Pendred V. Lancet. 1896; ii: 532Abstract Scopus (267) Google Scholar), is caused by mutations in the PDS 1The abbreviations used are: PDS, Pendred syndrome; SLC26A4, solute carrier 26A4; NIS, sodium iodide symporter; Ad, adenoviral vector; E3, early gene 3; E1, early gene 1; MDCK, Madin-Darby canine kidney; ANOVA, analysis of variance. /SLC26A4 gene (2Everett L.A. Glaser B. Beck J.C. Idol J.R. Buchs A. Heyman M. Adawi F. Hazani E. Nassir E. Baxevanis A.D. Sheffield V.C. Green E.D. Nat. Genet. 1997; 17: 411-422Crossref PubMed Scopus (1011) Google Scholar). It encodes pendrin, a member of the solute carrier family 26A, which contains several anion transporters and the motor protein prestin (3Everett L.A. Green E.D. Hum. Mol. Genet. 1999; 8: 1883-1891Crossref PubMed Scopus (121) Google Scholar, 4Zheng J. Shen W. He D.Z. Long K.B. Madison L.D. Dallos P. Nature. 2000; 405: 149-155Crossref PubMed Scopus (1013) Google Scholar). Mutations in the PDS gene displayed impressive allelic heterogeneity, and >75 mutations have been identified previously (2Everett L.A. Glaser B. Beck J.C. Idol J.R. Buchs A. Heyman M. Adawi F. Hazani E. Nassir E. Baxevanis A.D. Sheffield V.C. Green E.D. Nat. Genet. 1997; 17: 411-422Crossref PubMed Scopus (1011) Google Scholar, 5Fugazzola L. Mannavola D. Cerutti N. Maghnie M. Pagella F. Bianchi P. Weber G. Persani L. Beck-Peccoz P. J. Clin. Endocrinol. Metab. 2000; 85: 2469-2475Crossref PubMed Scopus (81) Google Scholar, 6Gonzalez Trevino O. Karamanoglu Arseven O. Ceballos C. Vives V. Ramirez R. Gomez V.V. Medeiros-Neto G. Kopp P. Eur. J. Endocrinol. 2001; 144: 1-9Crossref PubMed Scopus (49) Google Scholar, 7Kopp P. Curr. Opin. Endocrinol.Diabetes. 1999; 6: 261-269Crossref Scopus (20) Google Scholar, 8Park H.-J. Shaukat S. Liu X.-Z. Hahn S.H. Naz S. Ghosh M. Kim H.-N. Moon S.-K. Abe S. Tukamoto K. Riazuddin S. Kabra M. Erdenetungalag R. Radnaabazar J. Khan S. Pandya A. Usami S.-I. Nance W.E. Wilcox E.R. Riazuddin S. Griffith A.J. J. Med. Genet. 2003; 40: 242-248Crossref PubMed Scopus (270) Google Scholar, 9Tsukamoto K. Suzuki H. Harada D. Namba A. Abe S. Usami S.I. Eur. J. Hum. Genet. 2003; 24: 916-922Crossref Scopus (214) Google Scholar). The majority of PDS mutations are missense mutations, and some of these mutants appear to be retained in the endoplasmic reticulum (10Taylor J.P. Metcalfe R.A. Watson P.F. Weetman A.P. Trembath R.C. J. Clin. Endocrinol. Metabol. 2002; 87: 1778-1784Crossref PubMed Scopus (138) Google Scholar, 11Rotman-Pikielny P. Hirschberg K. Maruvada P. Suzuki K. Royaux I.E. Green E.D. Kohn L.D. Lippincott-Schwartz J. Yen P.M. Hum. Mol. Genet. 2002; 11: 2625-2633Crossref PubMed Scopus (84) Google Scholar). A smaller number of mutations resulted in premature truncations or in alterations of splice donor or acceptor sites. Individuals with Pendred's syndrome from consanguineous families are homozygous for PDS mutations, whereas sporadic cases typically harbor compound heterozygous mutations (12Kopp P. Rev. Endocr. Metabol. Dis. 2000; 1/2: 109-121Crossref Google Scholar). Mutations in the PDS gene are not only found in patients with classic Pendred's syndrome but also in individuals afflicted with familial enlarged vestibular aqueduct (13Li X.C. Everett L.A. Lalwani A.K. Desmukh D. Friedman T.B. Green E.D. Wilcox E.R. Nat. Genet. 1998; 18: 215-217Crossref PubMed Scopus (332) Google Scholar, 14Usami S. Abe S. Weston M.D. Shinkawa H. van Camp G. Kimberling W.J. Hum. Genet. 1999; 104: 188-192Crossref PubMed Scopus (257) Google Scholar). The SLC26A family contains several transporters of sulfate or other anions. The chloride/bicarbonate exchanger SLC26A2/down-regulated in adenoma is expressed in the gastrointestinal tract, and mutations in this gene cause congenital chloride diarrhea (15Höglund P. Haila S. Socha J. Tomaszewski L. Saarialho-Kere U. Karjalainen-Lindsberg M.L. Airola K. Holmberg C. de la Chapelle A. Kere J. Nat. Genet. 1996; 14: 316-319Crossref PubMed Scopus (355) Google Scholar, 16Moseley R.H. Hoglund P. Wu G.D. Silberg D.G. Haila S. de la Chapelle A. Holmberg C. Kere J. Am. J. Physiol. 1999; 276: G185-G192PubMed Google Scholar). SLC26A3/diastrophic dysplasia sulfate transporter is a sulfate transporter predominantly expressed in cartilage, and recessive alterations in this gene are the molecular basis of several forms of chondrodysplasias (17Hastbacka J. de la Chapelle A. Mahtani M. Clines G. Reeve-Daly M. Daly M. Hamilton B. Kusumi K. Trivedi B. Weaver A. Coloma A. Lovett M. Buckler A. Kaitila I. Lander E.S. Cell. 1994; 78: 1073-1087Abstract Full Text PDF PubMed Scopus (625) Google Scholar, 18Superti-Furga A. Haestbacka J. Wilcox W.R. Cohn D.H. van der Harten H.J. Rossi A. Blau N. Rimoin D.L. Steinmann B. Lander E.S. Gitzelmann R. Nat. 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Initial functional studies of pendrin in Xenopus oocytes and Sf9 insect cells, as well as functional studies using thyrocytes from patients with documented Pendred's syndrome, revealed that pendrin is unable to transport sulfate (21Scott D.A. Wang R. Kreman T.M. Sheffield V.C. Karniski L.P. Nat. Genet. 1999; 21: 440-443Crossref PubMed Scopus (516) Google Scholar, 22Kraiem Z. Heinrich R. Sadeh O. Shiloni E. Nassir E. Hazani E. Glaser B. J. Clin. Endocrinol. Metab. 1999; 84: 2574-2576Crossref PubMed Scopus (30) Google Scholar). In Xenopus oocytes, pendrin to uptake of chloride and iodide (21Scott D.A. Wang R. Kreman T.M. Sheffield V.C. Karniski L.P. Nat. Genet. 1999; 21: 440-443Crossref PubMed Scopus (516) Google and to as a exchanger D.A. Karniski L.P. J. Cell. Physiol. 2000; PubMed Google Scholar). in transfected cells and renal from that pendrin is a chloride/base exchanger in β-intercalated cells of the cortical collecting in the kidney M. S. Wang Z. H. Kopp P. Am. J. Physiol. 2001; PubMed Google Scholar, I.E. Karniski L.P. Everett L.A. Suzuki K. Green E.D. U. S. A. 2001; PubMed Scopus Google Scholar). on the of the in patients with Pendred's syndrome and the W. Trembath R. H. J. Med. 2000; Scopus Google Scholar, L.A. K. R. A. S.I. B. Wu Green E.D. Hum. Mol. Genet. 2001; PubMed Scopus Google Scholar), pendrin is also in anion and transport in the but its role in this to be In thyroid follicular cells, pendrin is into the apical membrane I.E. Suzuki K. A. R. Everett L.A. Kohn L.D. Green E.D. 2000; PubMed Scopus Google Scholar, C. V. D. S. B. M. J. Clin. Endocrinol. Metab. 2000; 85: PubMed Scopus Google Scholar). studies in transfected cells that the protein iodide transport in mammalian cells (10Taylor J.P. Metcalfe R.A. Watson P.F. Weetman A.P. Trembath R.C. J. Clin. Endocrinol. Metabol. 2002; 87: 1778-1784Crossref PubMed Scopus (138) Google Scholar, A. S. I. Royaux I.E. Green E.D. Kohn L.D. Suzuki K. J. Clin. Endocrinol. Metab. 2002; 87: PubMed Scopus Google Scholar). These with the impaired iodide organification in patients with Pendred's syndrome, a possible role of pendrin in apical iodide efflux from thyrocytes into the In this we determined the iodide transport properties of type pendrin in polarized and mammalian cells, and the functional consequences of two naturally occurring mutations found in a sporadic with Pendred's in and congenital sensorineural a and are of distinct is and is also a goiter, but a The of the is also but the two of the have in the found to have a at the thyroid enlarged and an of were but several and the of thyroid a and at W. Trembath R. H. J. Med. 2000; Scopus Google Scholar, L.A. K. R. A. S.I. B. Wu Green E.D. Hum. Mol. Genet. 2001; PubMed Scopus Google Scholar, I.E. Suzuki K. A. R. Everett L.A. Kohn L.D. Green E.D. 2000; PubMed Scopus Google Scholar, C. V. D. S. B. M. J. Clin. Endocrinol. Metab. 2000; 85: PubMed Scopus Google Scholar, A. S. I. Royaux I.E. Green E.D. 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Curr. 2001; Full Text Full Text PDF Google Scholar). and were a with and into the with a of A thyroid with documented and a The with a of of the iodide of of of the revealed the of an enlarged and from using of the PDS gene were using and previously (2Everett L.A. Glaser B. Beck J.C. Idol J.R. Buchs A. Heyman M. Adawi F. Hazani E. Nassir E. Baxevanis A.D. Sheffield V.C. Green E.D. Nat. Genet. 1997; 17: 411-422Crossref PubMed Scopus (1011) Google Scholar). The were with and both were using with an the of the were on a of and type PDS by using from thyroid and with the PDS into the and of K. Cell. Full Text PDF PubMed Scopus Google Scholar). of the two PDS mutants identified in this using the with R. L. M. Scholar). The encoding the type and the two mutations were also and into the to with a The type also to an or of the were by using with an the of the The NIS by the of a PDS a the PDS by the with a into an adenoviral on as B. J. Clin. Endocrinol. Metab. 1999; 84: PubMed Scopus Google Scholar). used to the and of were with is a in which the is The the in a The were transfected into kidney cells in which of the protein of the The and of by which contains the by used as a of adenoviral in Madin-Darby canine kidney cells using in cells at with at a of of and in cells were in and were transfected with for were using of the by the for NIS were and expressing NIS were characterized by iodide of in functional expressing NIS and pendrin not an adenoviral to cells with the PDS of the expressing NIS with of adenoviral PDS for functional in a bicameral using of a with a to the of an polarized were a The in the of and and with uptake with iodide to the chamber. iodide in the the compartment of the cells in and the in and cells, a of kidney cells B. R. Google were in and were into the and to the type or PDS or the NIS were transfected using the The as In of PDS NIS the of by were uptake using and with iodide uptake determined by iodide in the and iodide efflux determined by iodide as previously PubMed Scopus Google Scholar). used as as and of Green cells were as on in the membrane and in the of pendrin, were with a the or a by The a or a with were with the of pendrin to an or kidney cells were transfected with the The cells were in with or the of with a and with a of of type or pendrin with a protein determined in kidney cells using a of the were in in were by using of Mutations in the PDS analysis revealed that the compound heterozygous for a and a recently PDS gene found to harbor in resulted in a at and lead to a premature in an in the to the by Everett (2Everett L.A. Glaser B. Beck J.C. Idol J.R. Buchs A. Heyman M. Adawi F. Hazani E. Nassir E. Baxevanis A.D. Sheffield V.C. Green E.D. Nat. Genet. 1997; 17: 411-422Crossref PubMed Scopus (1011) Google Scholar). have resulted in a that not be in the absence of thyroidal The a in in of at by a recently found to be in patients H.-J. Shaukat S. Liu X.-Z. Hahn S.H. Naz S. Ghosh M. Kim H.-N. Moon S.-K. Abe S. Tukamoto K. Riazuddin S. Kabra M. Erdenetungalag R. Radnaabazar J. Khan S. Pandya A. Usami S.-I. Nance W.E. Wilcox E.R. Riazuddin S. Griffith A.J. J. Med. Genet. 2003; 40: 242-248Crossref PubMed Scopus (270) Google Scholar). the of the is of the were not for and the of the mutations not be in bicameral using polarized used to iodide transport at the apical membrane. cells expressing NIS, pendrin, or NIS and pendrin were to a with in the cells as a transfected with NIS a uptake in iodide uptake The into the apical in cells, of transport the apical membrane the In contrast, cells expressing NIS and with a PDS iodide transport into the apical as a of this a in iodide content to in cells. expressing only pendrin iodide cells but in the chamber. the results are with the concept that pendrin mediates iodide transport at the apical membrane in polarized cells. in transfected cells expressing NIS a in the uptake of iodide with cells transfected with In cells transfected only with PDS, iodide uptake not in to cells at in the of Consistent with in the bicameral pendrin iodide to in In contrast, of the two mutants (L676Q not in a in iodide in to cells transfected with NIS only results were observed in cells not iodide content of cells expressing type and pendrin. In to cells expressing NIS and type pendrin, of the two mutants (L676Q not in a in iodide in to cells transfected with NIS that the mutants ability to iodide efflux. are the of In iodide efflux cells transfected with NIS a iodide efflux In contrast, cells with type PDS and NIS a very efflux of iodide. transfected with PDS and NIS an impaired efflux with the cells expressing type pendrin, and the efflux similar to cells expressing only NIS and of Green cells expressing NIS as on at the basolateral membrane In contrast, pendrin in cells expressing pendrin at the apical membrane of cells expressing of type pendrin with an or a revealed that the is located A and and a that the to be (2Everett L.A. Glaser B. Beck J.C. Idol J.R. Buchs A. Heyman M. Adawi F. Hazani E. Nassir E. Baxevanis A.D. Sheffield V.C. Green E.D. Nat. Genet. 1997; 17: 411-422Crossref PubMed Scopus (1011) Google Scholar), is also the and I.E. Suzuki K. A. R. Everett L.A. Kohn L.D. Green E.D. 2000; PubMed Scopus Google Scholar). A protein of pendrin with a protein a with membrane In contrast, the two naturally occurring mutations displayed a distinct suggesting of the in a member of the solute carrier family 26A, been to transport chloride and iodide but not sulfate into Xenopus oocytes (21Scott D.A. Wang R. Kreman T.M. Sheffield V.C. Karniski L.P. Nat. Genet. 1999; 21: 440-443Crossref PubMed Scopus (516) Google Scholar). studies an of chloride and in oocytes D.A. Karniski L.P. J. Cell. Physiol. 2000; PubMed Google and an of chloride with and in transfected kidney cells M. S. Wang Z. H. Kopp P. Am. J. Physiol. 2001; PubMed Google Scholar). The absence of also in renal from with of the gene I.E. Karniski L.P. Everett L.A. Suzuki K. Green E.D. U. S. A. 2001; PubMed Scopus Google Scholar). studies in mammalian cells a role for pendrin in iodide efflux (10Taylor J.P. Metcalfe R.A. Watson P.F. Weetman A.P. Trembath R.C. J. Clin. Endocrinol. Metabol. 2002; 87: 1778-1784Crossref PubMed Scopus (138) Google Scholar, A. S. I. Royaux I.E. Green E.D. Kohn L.D. Suzuki K. J. Clin. Endocrinol. Metab. 2002; 87: PubMed Scopus Google Scholar). uptake at the basolateral membrane into the thyroid follicular on the symporter NIS and the sodium by the J. Physiol. Rev. PubMed Scopus Google Scholar, A. O. O. N. Physiol. Rev. 2000; PubMed Scopus Google Scholar). into the follicular iodide and that were by and an that two recently cloned Wang D. S. F. G. F. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, C. R. A. D. A. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, J.C. H. P. F. C. N. J. Med. 2002; PubMed Scopus Google Scholar). is evidence for the of iodide at the apical membrane P. M. R. Am. J. Physiol. PubMed Google Scholar), not been In thyroid follicular cells, pendrin into the apical membrane I.E. Suzuki K. A. R. Everett L.A. Kohn L.D. Green E.D. 2000; PubMed Scopus Google Scholar, C. V. D. S. B. M. J. Clin. Endocrinol. Metab. 2000; 85: PubMed Scopus Google Scholar). The apical with the impaired iodide organification observed in patients with Pendred's syndrome and the of the ability of pendrin to transport iodide in or a possible role in iodide transport into the thyroid (3Everett L.A. Green E.D. Hum. Mol. Genet. 1999; 8: 1883-1891Crossref PubMed Scopus (121) Google Scholar, J.P. Metcalfe R.A. Watson P.F. Weetman A.P. Trembath R.C. J. Clin. Endocrinol. Metabol. 2002; 87: 1778-1784Crossref PubMed Scopus (138) Google Scholar, I.E. Suzuki K. A. R. Everett L.A. Kohn L.D. Green E.D. 2000; PubMed Scopus Google Scholar, A. S. I. Royaux I.E. Green E.D. Kohn L.D. Suzuki K. J. Clin. Endocrinol. Metab. 2002; 87: PubMed Scopus Google Scholar). a polarized the evidence that pendrin iodide efflux at the apical membrane The of iodide transport in thyroid cells are and and and organification J. Physiol. Rev. PubMed Scopus Google Scholar). Consistent with in thyroid J. Physiol. Rev. PubMed Scopus Google Scholar), cells, in a and expressing both NIS and pendrin, iodide in the of the the and the follicular J. Physiol. Rev. PubMed Scopus Google Scholar), that pendrin is its as an anion as an iodide efflux in cells, which a distinct and in polarized thyrocytes PubMed Scopus Google Scholar, M. U. R. J. Google Scholar, M. U. R. Endocrinol. PubMed Scopus Google Scholar). In the that iodide efflux in apical efflux in the transport of iodide into the follicular M. U. R. J. Google Scholar). The of NIS and pendrin in the heterologous polarized used in this of the and of iodide efflux. the of iodide transport of the two mutations and the organification defect is only partial in this sporadic with Pendred's syndrome, a that is for the majority of patients with this disorder P. Curr. Opin. Endocrinol.Diabetes. 1999; 6: 261-269Crossref Scopus (20) Google Scholar). Moreover, this is as are individuals with Pendred's syndrome with iodide and other forms of thyroid These that iodide reach the follicular of pendrin, as also by the absence of a thyroid in with a of the gene I.E. Karniski L.P. Everett L.A. Suzuki K. Green E.D. U. S. A. 2001; PubMed Scopus Google Scholar). In to the efflux of iodide into the follicular is by the and chloride or a iodide It is that studies with membrane the of two apical iodide P. M. R. Am. J. Physiol. PubMed Google Scholar). of these to a and for iodide whereas the anion a for iodide P. M. R. Am. J. Physiol. PubMed Google Scholar). The apical iodide transport with the of of pendrin mutations, that pendrin to of these the recently identified cloned to as apical iodide transporter in apical iodide efflux in thyroid follicular cells is B. L. B. G. M. J. Clin. Endocrinol. Metab. 2002; 87: PubMed Scopus Google Scholar). PDS gene mutations displayed allelic and and splice mutations (2Everett L.A. Glaser B. Beck J.C. 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Harada D. Namba A. Abe S. Usami S.I. Eur. J. Hum. Genet. 2003; 24: 916-922Crossref Scopus (214) Google Scholar). for the of with these of the in or of that are for anion transport into the membrane. of the two mutations characterized to a and premature of pendrin is not that is retained The is located a of the sulfate transporter and found in the transporters of the a that have a L. Curr. 2001; Full Text Full Text PDF Google Scholar). is to an and also results in of the protein by as previously for other pendrin mutations (10Taylor J.P. Metcalfe R.A. Watson P.F. Weetman A.P. Trembath R.C. J. Clin. Endocrinol. Metabol. 2002; 87: 1778-1784Crossref PubMed Scopus (138) Google Scholar, 11Rotman-Pikielny P. Hirschberg K. Maruvada P. Suzuki K. Royaux I.E. Green E.D. Kohn L.D. Lippincott-Schwartz J. Yen P.M. Hum. Mol. Genet. 2002; 11: 2625-2633Crossref PubMed Scopus (84) Google Scholar), both mutations in protein and at some of the pendrin mutations appear to in an endoplasmic reticulum disorder P. Endocr. Rev. 1998; Google Scholar). in other forms of endoplasmic reticulum a of the mutants retained in is whereas have functional P. Endocr. Rev. 1998; Google Scholar). the missense mutations of pendrin, some into the A and to that the in the endoplasmic functional to reach the membrane. In this evidence for apical iodide efflux from polarized mammalian cells. Moreover, we of of two pendrin mutations that are both retained in the endoplasmic reticulum and of the from studies the of iodide the of pendrin mutants be of in of the transporter and from the of the H. for with the studies and from the for with the