Functional Characterization of Pendrin in a Polarized Cell System
2004; Elsevier BV; Volume: 279; Issue: 13 Linguagem: Inglês
10.1074/jbc.m313648200
ISSN1083-351X
AutoresMary P. Gillam, Aniket Sidhaye, Eun Jig Lee, Jonas Rutishauser, Catherine Waeber Stephan, Peter Kopp,
Tópico(s)Nicotinic Acetylcholine Receptors Study
ResumoPendred'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). 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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 was shown to mediate 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 Scholar) and to act as a chloride/formate exchanger (23Scott D.A. Karniski L.P. Am J. Cell. Physiol. 2000; 278: C207-C211Crossref PubMed Google Scholar). 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The perchlorate test was positive with a discharge of 22% of the incorporated iodide 15 min after administration of 0.6 g of KClO4. Magnetic resonance of the inner ear revealed the presence of an enlarged endolymphatic sac (Fig. 1). DNA Sequencing—After obtaining informed consent, blood was collected and DNA was extracted from peripheral leukocytes using standard techniques. Exons 2-21 of the PDS gene were amplified using primers and conditions reported 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 PCR products were purified with Centricon 100 columns (Amicon, Beverely, MA), and both strands were sequenced directly using FS AmpliTaq DNA polymerase with an ABI Prism rhodamine dye primer cycle-sequencing kit following the protocol of the supplier. Sequencing products were analyzed on a 373A Sequencer (Applied Biosystems). Construction of Plasmids and Recombinant Adenoviral Vectors—The human wild type PDS cDNA was generated by reverse transcriptase-PCR using total RNA from normal human thyroid tissue and Pfu polymerase (Stratagene, La Jolla, CA). After PCR amplification with primers containing appropriate linkers, the PDS cDNA was subcloned into the XhoI and BamHI sites of pCMX (31Umesono K. Murakami K.K. Thompson C.C. Evans R.M. Cell. 1991; 65: 1255-1266Abstract Full Text PDF PubMed Scopus (1497) Google Scholar). Construction of the two PDS mutants identified in this patient was performed using the overlap extension methodology with Pfu polymerase (32Horton R. Pease L. McPherson M. Directed Mutagenesis. IRL Press, Oxford1991: 217-247Google Scholar). The cDNAs encoding the wild type and the two mutations were also subcloned in-frame and without stop codon into the vector pEGFPN1 (Clontech, Palo Alto, CA) to create fusion proteins with a carboxyl-terminal green fluorescent protein. The wild type cDNA was also fused to an amino-terminal or carboxyl-terminal His6 epitope. All of the final constructs were verified by direct DNA sequencing using FS AmpliTaq DNA polymerase with an ABI Prism dye primer cycle-sequencing kit following the protocol of the supplier. The human NIS cDNA was kindly provided by Dr. Sissy Jhiang (Columbus, OH). For the creation of a PDS adenovirus, a cassette containing the PDS cDNA driven by the cytomegalovirus promoter/enhancer with a SV40 polyadenylation sequence was subcloned into an adenoviral transfer plasmid based on pcDNA3 (Invitrogen) as described elsewhere (Fig. 2A) (33Lee E.J. Anderson L.M. Thimmapaya B. Jameson J.L. J. Clin. Endocrinol. Metab. 1999; 84: 786-794Crossref PubMed Scopus (75) Google Scholar). This transfer plasmid was used to generate recombinant adenoviruses. Linearized transfer plasmids containing the expression cassette and 393 bp of 5′-adenoviral sequence were ligated with XbaI-digested Ad5 309/356 DNA representing map units 3.6-100. Adenoviral vector 5 (Ad5) 309/356 is a recombinant adenovirus in which the E3 region is deleted. The XbaI digestion removes the E1a region, resulting in a replication-deficient virus. The ligation products were transfected into human embryonic kidney 293 cells in which cellular expression of the E1a protein allows replication of the E1-deleted recombinant viruses. The purification and titration of Ad-PDS was performed by plaque assay. Ad-β-galactosidase, which contains the β-galactosidase cDNA driven by cytomegalovirus promoter, was used as a control. Transduction efficiency of adenoviral vectors in Madin-Darby canine kidney (MDCK) cells was tested using Ad-β-galactosidase. β-Galactosidase expression was detected in 95-100% MDCK cells at 48 h after infection with Ad-β-galactosidase at a multiplicity of infection of 10 plaque-forming units/cell (Fig. 2B). Cell Culture and Iodide Transport in Polarized Cells—MDCK cells (American Tissue Type Collection, Manassas, VA) were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 μg/ml). Cells were transfected with pcDNA3.1 constructs for NIS. Stable clones were isolated using G418 selection. Expression of the transgene was confirmed by reverse transcriptase-PCR. For reverse transcriptase-PCR, the primers for NIS were sense 5′-CTTCTGAACTCGGTCCTCAC-3′ (bp 283-302) and antisense 5′-GTCCAGAATGTATAGCGGCTC-3′ (bp 737-717). Cells expressing NIS were then characterized by documenting perchlorate-sensitive iodide uptake. Because of difficulties in obtaining functional clones stably expressing NIS and pendrin concomitantly (data not shown), an adenoviral system was developed to transduce cells with the PDS cDNA. One of the selected MDCK clones stably expressing NIS was infected with 10 plaque-forming units/cell of recombinant adenoviral PDS vector for functional assays in a bicameral system using Costar 3460 Transwell cell culture chambers (Corning Costar, Cambridge, MA). After formation of a monolayer, electric resistance was measured with a Millicell®-ERS voltohmmeter (Millipore, Bedford, MA) to assure the formation of an intact polarized monolayer. Cells were assayed after reaching a resistance ≥200 ohms/cm2. The solution in the lower chamber consisted of Hanks' buffered saline solution, 10 mm Hepes, pH 7.4, and 10-6m cold NaI and was labeled with Na125I (20 mCi/mmol). Unlabeled uptake solution with 10-6m cold iodide was added to the upper chamber. Radiolabeled iodide was measured in the lower chamber, the intracellular compartment after lysis of the cells in buffered Hanks' buffered saline solution containing 1% Triton X-100, and the upper chamber after 45 min. Iodide Transport in Transiently Transfected Non-polarized Cells—JEG-3 choriocarcinoma, COS, and TSA-201 cells, a clone of human embryonic kidney 293 cells (34Margolskee R.F. McHendry-Rinde B. Horn R. BioTechniques. 1993; 15: 906-911PubMed Google Scholar) were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 μg/ml). Cells were split into 12-well plates the day before transfection and grown to 80% confluency. pCMX plasmids containing the wild type or mutant PDS cDNAs or the NIS cDNA, were transfected (500 ng/well) using the calcium-phosphate method. The empty pCMX vector was included as negative control. In cotransfection experiments of PDS ± NIS constructs, the total amount of added plasmid was kept constant by adding empty vector. Iodide assays were performed 48 h after transfection. Iodide uptake was performed using Hanks' balanced salt solution, 10 mm Hepes, pH 7.4, and 10-5m cold NaI labeled with Na125I (20 mCi/mmol). Intracellular iodide uptake was determined by measuring radiolabeled iodide in the cell lysates, and iodide efflux was determined by measuring iodide release as described previously (35Weiss S.J. Philip N.J. Grollmann E.F. Endocrinology. 1984; 114: 1090-1098Crossref PubMed Scopus (334) Google Scholar). Perchlorate (100 mm) was used as inhibitor as indicated. Immunohistochemistry and Expression of Green Fluorescent Proteins—For immunohistochemistry experiments, MDCK cells were grown as monolayers on Transwell cell culture chambers. After fixation in 4% paraformaldehyde, the polycarbonate membrane was removed and paraffin-embedded in vertical orientation. For the detection of pendrin, slides were incubated with a primary chicken IgY antibody against the carboxyl-terminal epitope CZETELTEEELDVQDEAMRT (Aves, Tigard, OR) or a rabbit anti-NIS antibody kindly provided by Dr. Nancy Carrasco (New York, NY). The secondary antibody was a biotin-streptavidin-conjugated donkey anti-chicken antibody (Jackson ImmunoResearch laboratories) or a mouse anti-rabbit (DAKO) labeled with horseradish peroxidase. Cells were counterstained with Mayer's hematoxylin. For the detection of pendrin fused to an amino-terminal or carboxyl-terminal His6 tag, opossum kidney cells were transfected with the respective cDNAs. The cells were fixed in 1% formaldehyde with or without the addition of 1% saponin incubated with a primary anti-His antibody and subsequently with a fluorescein-labeled secondary goat anti-mouse antibody. Expression of fusion proteins of wild type or mutant pendrin with a carboxyl-terminal green fluorescent protein was determined in opossum kidney cells using a LSM510 META Zeiss confocal microscope. Statistical Analysis—All of the experiments were performed in triplicate in more than six independent experiments. Groups were compared by ANOVA using Statview (Abacus Concepts, Berkeley, CA). Identification of Compound Heterozygous Mutations in the PDS Gene—Sequence analysis revealed that the patient was compound heterozygous for a novel and a recently described PDS gene mutation. One allele was found to harbor insertion 916insG in exon 7 (Fig. 1). This alteration resulted in a frameshift beginning at codon 306 and lead most likely to a premature stop in codon 309, an amino acid in the putative third intracellular loop according to the model proposed by Everett et al. (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). Alternatively, it could have resulted in altered splicing, a possibility that could not be assessed in the absence of thyroidal mRNA. The second allele had a transversion 2027T>A in exon 17 resulting in substitution of leucine at position 676 by glutamine (L676Q), a mutation recently found to be prevalent in Asian patients (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). Of note, the mother of the patient is of Vietnamese origin; however, the parents were not available for genetic testing and the paternal origin of the mutations could therefore not be determined. Pendrin-mediated Apical Iodide Transport in Polarized Cells—A bicameral system using polarized MDCK monolayers was used to study pendrin-mediated iodide transport at the apical membrane. MDCK cells expressing NIS, pendrin, or NIS and pendrin were exposed to a solution containing 10-6m NaI labeled with 20 mCi/mmol 125I in the lower chamber (Fig. 2C). Untransfected MDCK cells served as a negative control. Cells stably transfected with NIS showed a significant uptake in intracellular iodide uptake (Fig. 2D). The release into the apical chamber was higher than in untransfected MDCK cells, most probably because of unspecific transport across the apical membrane following the electrochemical gradient. In contrast, cells expressing NIS and infected with a PDS adenovirus showed significant iodide transport into the apical chamber and, as a consequence of this vectorial transport, a significant drop in intracellular iodide content to levels seen in untransfected MDCK cells. Cells expressing only pendrin did show lower intracellular iodide levels than untransfected MDCK cells but higher levels in the upper chamber. Taken together, the results are consistent with the concept that pendrin mediates vectorial iodide transport at the apical membrane in polarized cells. Pendrin-mediated Iodide Transport in Non-polarized Cells—Transiently transfected JEG-3 cells expressing NIS alone showed a significant perchlorate-sensitive increase in the uptake of iodide compared with cells transfected with empty vector (Fig. 3). In cells transfected only with PDS, iodide uptake was not increased in comparison to control cells at concentrations in the physiologic range of 10-5m NaI. Consistent with our findings in the bicameral system (Fig. 2D), pendrin reduced NIS-mediated intracellular iodide accumulation to control levels in coexpression experiments. In contrast, cotransfection of the two mutants (L676Q + NIS; FS306>309X + NIS) did not result in a decrease in intracellular iodide accumulation in comparison to cells transfected with NIS only (Fig. 4). Similar results were observed in TSA-293 cells (results not shown).Fig. 4Intracellular iodide content of JEG-3 cells expressing wild type and mutant pendrin. In contrast to cells expressing NIS and wild type pendrin, cotransfection of the two mutants (L676Q + NIS; FS306>309X + NIS) did not result in a decrease in intracellular iodide accumulation in comparison to cells transfected with NIS only, indicating that the mutants lost their ability to mediate iodide efflux. Values are the means of triplicates ± S.E.View Large Image Figure Vi
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