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• W2019401224 abstract "The pancreatic duct expresses cystic fibrosis transmembrane conductance regulator (CFTR) and HCO 3− secretory and salvage mechanisms in the luminal membrane. Although CFTR plays a prominent role in HCO 3− secretion, the role of CFTR in HCO 3− salvage is not known. In the present work, we used molecular, biochemical, and functional approaches to study the regulatory interaction between CFTR and the HCO 3− salvage mechanism Na+/H+ exchanger isoform 3 (NHE3) in heterologous expression systems and in the native pancreatic duct. We found that CFTR regulates NHE3 activity by both acute and chronic mechanisms. In the pancreatic duct, CFTR increases expression of NHE3 in the luminal membrane. Thus, luminal expression of NHE3 was reduced by 53% in ducts of homozygote ΔF508 mice. Accordingly, luminal Na+-dependent and HOE694- sensitive recovery from an acid load was reduced by 60% in ducts of ΔF508 mice. CFTR and NHE3 were co-immunoprecipitated from PS120 cells expressing both proteins and the pancreatic duct of wild type mice but not from PS120 cells lacking CFTR or the pancreas of ΔF508 mice. The interaction between CFTR and NHE3 required the COOH-terminal PDZ binding motif of CFTR, and mutant CFTR proteins lacking the C terminus were not co-immunoprecipitated with NHE3. Furthermore, when expressed in PS120 cells, wild type CFTR, but not CFTR mutants lacking the C-terminal PDZ binding motif, augmented cAMP-dependent inhibition of NHE3 activity by 31%. These findings reveal that CFTR controls overall HCO 3− homeostasis by regulating both pancreatic ductal HCO 3− secretory and salvage mechanisms.AF307992 AF307993 The pancreatic duct expresses cystic fibrosis transmembrane conductance regulator (CFTR) and HCO 3− secretory and salvage mechanisms in the luminal membrane. Although CFTR plays a prominent role in HCO 3− secretion, the role of CFTR in HCO 3− salvage is not known. In the present work, we used molecular, biochemical, and functional approaches to study the regulatory interaction between CFTR and the HCO 3− salvage mechanism Na+/H+ exchanger isoform 3 (NHE3) in heterologous expression systems and in the native pancreatic duct. We found that CFTR regulates NHE3 activity by both acute and chronic mechanisms. In the pancreatic duct, CFTR increases expression of NHE3 in the luminal membrane. Thus, luminal expression of NHE3 was reduced by 53% in ducts of homozygote ΔF508 mice. Accordingly, luminal Na+-dependent and HOE694- sensitive recovery from an acid load was reduced by 60% in ducts of ΔF508 mice. CFTR and NHE3 were co-immunoprecipitated from PS120 cells expressing both proteins and the pancreatic duct of wild type mice but not from PS120 cells lacking CFTR or the pancreas of ΔF508 mice. The interaction between CFTR and NHE3 required the COOH-terminal PDZ binding motif of CFTR, and mutant CFTR proteins lacking the C terminus were not co-immunoprecipitated with NHE3. Furthermore, when expressed in PS120 cells, wild type CFTR, but not CFTR mutants lacking the C-terminal PDZ binding motif, augmented cAMP-dependent inhibition of NHE3 activity by 31%. These findings reveal that CFTR controls overall HCO 3− homeostasis by regulating both pancreatic ductal HCO 3− secretory and salvage mechanisms. AF307992 AF307993 cystic fibrosis cystic fibrosis transmembrane conductance regulator Na+/H+ exchanger ERM-binding phosphoprotein 50 2′7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester polymerase chain reaction reverse transcription-PCR wild type ΔF508 luminal membrane vasoactive intestinal polypeptide Fluid secretion and the control of the ionic composition of biological fluids are essential for the function of many secretory epithelia, including the respiratory, digestive, and reproductive systems. HCO 3− is an important constituent of secreted fluids by virtue of its function as the biological pH buffer and its effect on the solubility of proteins and ions in biological fluids. Accordingly, the mechanisms underlying HCO 3− homeostasis at rest and during stimulation have attracted much attention in recent years. The importance of HCO 3− homeostasis is evident from the marked reduction in Cl−- and HCO 3− -dependent fluid secretion in the pancreatic juice of cystic fibrosis (CF)1 patients (1Johansen P.G. Anderson C.M. Hadorn B. Lancet. 1968; 1: 455-460Abstract Google Scholar). CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR functions as a Cl−channel, and many mutations causing CF reduce or abolish Cl− channel activity (2Pilewski J.M. Frizzell R.A. Physiol. Rev. 1999; 79: S215-S255Crossref PubMed Scopus (379) Google Scholar). However, several CFTR mutations retain normal or even elevated Cl− channel activity although they are associated with mild or severe forms of CF (3Vankeerberghen A. Wei L. Jaspers M. Cassiman J.J. Nilius B. Cuppens H. Hum. Mol. Genet. 1998; 7: 1761-1769Crossref PubMed Scopus (52) Google Scholar). This suggests that CFTR may have other functions, besides conducting Cl−, that are essential for normal fluid and electrolyte transport. Indeed, CFTR regulates the activity of the epithelial Na+ channel ENaC (4Stutts M.J. Canessa C.M. Olsen J.C. Hamrick M. Cohn J.A. Rossier B.C. Boucher R.C. Science. 1995; 269: 847-850Crossref PubMed Scopus (948) Google Scholar), and CFTR supports Cl−/HCO 3− exchange activity in heterologous expression systems (5Lee M.G. Wigley W.C. Zeng W. Noel L.E. Marino C.R. Thomas P.J. Muallem S. J. Biol. Chem. 1999; 274: 3414-3421Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar) and in the mouse pancreatic duct (6Lee M.G. Choi J.Y. Luo X. Strickland E. Thomas P.J. Muallem S. J. Biol. Chem. 1999; 274: 14670-14677Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Importantly, aberrant HCO 3− transport in cells expressing CFTR mutants that retain Cl− channel activity correlates with the pancreatic status of the CF phenotype (7Choi J.Y. Muallem D. Kiselyov K. Lee M.G. Thomas P.J. Muallem S. Nature. 2001; 410: 94-97Crossref PubMed Scopus (331) Google Scholar). Another HCO 3− -transporting mechanism in tissues that express CFTR is HCO 3− -absorbing processes that may function as HCO 3− salvage mechanisms in the resting state (8Lee M.G. Ahn W. Choi J.Y. Luo X. Seo J.T. Schultheis P.J. Shull G.E. Kim K.H. Muallem S. J. Clin. Invest. 2000; 105: 1651-1658Crossref PubMed Scopus (56) Google Scholar). In the pancreatic duct (8Lee M.G. Ahn W. Choi J.Y. Luo X. Seo J.T. Schultheis P.J. Shull G.E. Kim K.H. Muallem S. J. Clin. Invest. 2000; 105: 1651-1658Crossref PubMed Scopus (56) Google Scholar) and the kidney proximal tubule (9Choi J.Y. Shah M. Lee M.G. Schultheis P.J. Shull G.E. Muallem S. Baum M. J. Clin. Invest. 2000; 105: 1141-1146Crossref PubMed Scopus (106) Google Scholar), 50% of HCO 3− absorption is mediated by the Na+/H+ exchanger isoform 3 (NHE3) and 50% by a novel, yet unidentified, Na+-dependent mechanism. Hence, HCO 3− homeostasis in secretory epithelia is accomplished by both HCO 3− -absorbing (resting state) and HCO 3− -secretory (stimulated state) mechanisms. An intriguing feature of HCO 3− homeostasis is the possibility that the activity of multiple proteins involved in this process is regulated by CFTR through interaction with the scaffolding protein ERM (ezrin, radixin,moesin)-binding phosphoprotein 50 (EBP50) (also known as NHE-regulatory factor 1, or NHERF1). EPB50 has two PDZ domains and a C-terminal ERM binding domain. Separate reports showed that the first PDZ domain of EBP50 preferentially binds the C terminus of CFTR (10Short D.B. Trotter K.W. Reczek D. Kreda S.M. Bretscher A. Boucher R.C. Stutts M.J. Milgram S.L. J. Biol. Chem. 1998; 273: 19797-19801Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar) and that NHE3 interacts with EBP50 via a nontraditional association that requires the second PDZ domain and the C terminus of EBP50 (11Weinman E.J. Steplock D. Tate K. Hall R.A. Spurney R.F. Shenolikar S. J. Clin. Invest. 1998; 101: 2199-2206Crossref PubMed Scopus (88) Google Scholar). Likewise, CFTR and NHE3 both associate with the closely related protein E3KARP (12Sun F. Hug M.J. Lewarchik C.M. Yun C. Bradbury N.A. Frizzell R.A. J. Biol. Chem. 2000; 275: 29539-29546Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). NHE3 activity is acutely inhibited by cAMP-dependent phosphorylation, and EBP50 or E3KARP is required to mediate this inhibition, perhaps by binding to ezrin, which may function as a protein kinase A anchoring protein (13Yun C.H.C. Oh S. Zizak M. Steplock D. Tsao S. Tse C.M. Weinman E.J. Donowitz M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3010-3015Crossref PubMed Scopus (400) Google Scholar). Since both CFTR and NHE3 can associate with EBP50 and cAMP regulates both activities, we considered the possibility that CFTR modulates the activity of NHE3 by association with EBP50 or other related proteins (13Yun C.H.C. Oh S. Zizak M. Steplock D. Tsao S. Tse C.M. Weinman E.J. Donowitz M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3010-3015Crossref PubMed Scopus (400) Google Scholar, 14Lamprecht G. Weinman E.J. Yun C.H.C. J. Biol. Chem. 1998; 273: 29972-29978Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). In this manner, CFTR can regulate not only HCO 3− secretion but also HCO 3− salvage. In the present work, we used biochemical and functional approaches to study the interaction of CFTR and NHE3 with EBP50 and the functional significance of this interaction in heterologous expression systems and the native pancreatic duct. In pancreatic duct and PS120 cells expressing NHE3 and EBP50, expression of CFTR augmented the cAMP-dependent inhibition of NHE3 activity. Most notably, CFTR in the pancreatic duct affected not only the activity but also the expression of NHE3 protein in the luminal membrane. These findings reveal a new mechanism by which CFTR regulates ion transport at the luminal membrane and highlight the multiple functions of CFTR in regulating the overall HCO 3− homeostasis in the pancreatic duct and possibly in other CFTR-expressing epithelia. BCECF-AM was purchased from Molecular Probes, Inc. (Eugene, OR). Polyclonal antiserum directed against human EBP50 was generated in rabbits as described previously (15Mohler P.J. Kreda S.M. Boucher R.C. Sudol M. Stutts M.J. Milgram S.L. J. Cell Biol. 1999; 147: 879-890Crossref PubMed Scopus (165) Google Scholar). Rabbit antisera 1566 and 1568 specific for NHE3 were generated as described previously (16Amemiya M. Loffing J. Lotscher M. Kaissling B. Alpern R.J. Moe O.W. Kidney Int. 1995; 48: 1206-1215Abstract Full Text PDF PubMed Scopus (342) Google Scholar). Monoclonal antibodies against an R domain and a C terminus of CFTR were purchased from R & D Systems (Minneapolis, MN). The solution A, used for microdissection and perfusion, contained (in mm) 140 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 HEPES (pH 7.4 with NaOH), and 10 glucose. Na+-free solutions were prepared by replacing Na+ withN-methyl-d-glucamine+ from solution A. An NHE-deficient cell line PS120, originally developed by Pouyssegur et al. (17Pouyssegur J. Sardet C. Franchi A. L'Allemain G. Paris S. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 4833-4837Crossref PubMed Scopus (435) Google Scholar), and the pCMV-NHE3 mammalian expression vector (18Park K. Olschowska J.A. Richardson L.A. Bookstein C. Chang E.B. Melvin J.E. Am. J. Physiol. 1999; 39: G470-G478Google Scholar) were provided by Drs. K. Park and J. E. Melvin (University of Rochester). Mammalian expressing pCMV-CFTR (pCMVNot6.2) vector was a gift from Dr. J. Rommens at the Hospital for Sick Children (Toronto, Canada). Mammalian expressing Ad-CFTR virus was purchased from the Institute of Human Gene Therapy (Philadelphia, PA), and pcDNA3.1-EBP50 was generated by subcloning the full-length EBP50 cDNA from pET-EBP50 (15Mohler P.J. Kreda S.M. Boucher R.C. Sudol M. Stutts M.J. Milgram S.L. J. Cell Biol. 1999; 147: 879-890Crossref PubMed Scopus (165) Google Scholar) to pcDNA3.1 (Invitrogen, Groningen, The Netherlands). PS120 cells were maintained in DMEM-HG (Life Technologies, Inc.) supplemented with 10% fetal bovine serum and penicillin (50 units/ml)/streptomycin (50 μg/ml). The pCMV-NHE3 construct was stably transfected into a PS120 cell line using LipofectAMINE Plus Reagent (Life Technologies). NHE3 stable transfectants (PS120/NHE3) were selected by resistance to the antibiotic Geneticin (G418; Life Technologies, Inc.) and by an H+-killing method (19Wakabayashi S. Fafournoux P. Sardet C. Pouyssegur J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2424-2428Crossref PubMed Scopus (236) Google Scholar). Oligonucleotide-directed mutagenesis using the GeneEditor mutagenesis kit (Promega, Madison, WI) was performed in the CFTR expression vector pCMVNot6.2 to delete the C-terminal 4 (ΔDTRL) or 26 (S1455X) amino acids. Briefly, mutants were selected based upon the incorporation of a second-site mutation in β-lactamase, which alters its substrate specificity, allowing resistance of transformed bacteria to cefotaxime and cerftriaxone in addition to ampicillin. Incorporation of the mutation was verified by DNA sequencing. The mutagenesis primers were as follows: ΔDTRL, 5′-GGA GAC AGA AGA AGA GGT G T A AGA TAC AAG GCT TTA GAG AG-3′; S1455X, 5′-GCT CTT TCC CCA CCG GAA CTGAAG CAA GTG CAA GTC TAA GCC-3′. mRNA transcripts of mouse EBP50, E3KARP, and PDZK1 (20Kocher O. Comella N. Tognazzi K. Brown L.F. Lab. Invest. 1998; 78: 117-125PubMed Google Scholar) were analyzed in pancreatic and PS120 cells using RT-PCR. Among several primer sets designed based on the mouse sequences, at least one set for each protein detected the correct band in samples from hamster as well as mouse tissues. The primer sequences were as follows: 1) mEBP50, sense (5′-CTA AGC CAG GCC AGT TCA TCC GAG CAG T-3′) and antisense (5′-TGG GGT CAG AGG AGG AGG AGG AGG TAG A-3′), size of PCR product 447 base pairs; 2) mE3KARP, sense (5′-GAG GCC CGG CTG CTG GTA GTC G-3′) and antisense (5′-CAT CTG TGG TGC CCG CTT GTT GA-3′), size of PCR product 312 base pairs; 3) mPDZK1, sense (5′-GAC AAG GCT GGG CTG GAG AAT GAG GAC-3′) and antisense (5′-CGA AGA GTG CGA GGC TGT GCT GAG AGT-3′), size of PCR product 297 base pairs. RNA was extracted from the tissue using Trizol solution (Life Technologies, Inc.) and reverse transcribed using random hexamer primers and an RNase H− reverse transcriptase (Life Technologies). The cDNA was amplified using specific primers and a Taq polymerase (Promega), and the products were separated on a 1.5% agarose gel containing 0.1 μg/ml ethidium bromide. The identities of all amplified products were verified by nucleotide sequencing. Precleared pancreatic or PS120 lysates (∼2 mg of protein) were mixed with the appropriate antibodies and incubated overnight at 4 °C in lysis buffer. Immune complexes were collected by binding to protein G- (monoclonal antibody against R domain of CFTR) or protein A- (all other antibodies) Sepharose and washing four times with lysis buffer prior to electrophoresis. The immunoprecipitates or lysates (20 μg of protein) were suspended in SDS sample buffer and separated by SDS-polyacrylamide gel electrophoresis. The separated proteins were transferred to polyvinylidene difluoride membranes, and the membranes were blocked by a 1-h incubation at room temperature in 5% nonfat dry milk in a solution containing 20 mm Tris-HCl, pH 7.5, 150 mm NaCl, and 0.05% Tween 20. The proteins were detected by 1-h incubations with the appropriate primary and secondary antibodies. The pancreata from several wild type (WT) and homozygous ΔF508 (ΔF) mice were embedded in OCT (Miles, Elkhart, IN), frozen in liquid N2, and cut into 4-μm sections. Immunostaining of frozen sections was performed as previously reported (8Lee M.G. Ahn W. Choi J.Y. Luo X. Seo J.T. Schultheis P.J. Shull G.E. Kim K.H. Muallem S. J. Clin. Invest. 2000; 105: 1651-1658Crossref PubMed Scopus (56) Google Scholar). Briefly, the sections were fixed and permeabilized by incubation with cold methanol for 10 min at −20 °C. After removal of the methanol, slices were washed twice with phosphate-buffered saline, and the tissue area was encircled using a hydrophobic marker (Pap Pen;Zymed Laboratories Inc., South San Francisco, CA). Nonspecific binding sites were blocked by incubation for 1 h at room temperature with 0.1 ml of phosphate-buffered saline containing 5% goat serum, 1% bovine serum albumin, and 0.1% gelatin (blocking medium). After blocking, the sections were stained by incubation with the appropriate primary antiserum followed by fluorophore-tagged secondary antibodies. For double labeling experiments, these primary and secondary incubations were repeated with antibodies against the second protein of interest. Then the sections were incubated in a bisbenzimide (10 μg/ml in phosphate-buffered saline) solution to stain DNA and sealed with a coverslip using a Mowiol-based (Calbiochem) mounting medium. Images were collected with a Leica TCS-NT confocal microscope. When desired, NHE3 expression in the LM of the pancreatic duct was compared between samples from wild type and ΔF mice. In this case, all of the images were taken at the same laser intensity, and the same recording conditions and staining intensities of specified regions were analyzed using an imaging software (MCID version 3.0; Brook University, St. Catharines, Ontario, Canada). Briefly, the pixel counts of a 1-μm square region in the nucleus (FNuc; background) and a midportion of the LM (FLM) of the same cell were measured. The ratio of (FLM − FNuc)/FNuc from multiple cells taken from at least three separate sections and from separate animals were averaged and compared between each group. A cystic fibrosis mouse model in which the ΔF508 mutation was introduced in the mouse CFTR gene targeting in embryonic stem cells (21Zeiher B.G. Eichwald E. Zabner J. Smith J.J. Puga A.P. McCray Jr., P.B. Capecchi M.R. Welsh M.J. Thomas K.R. J. Clin. Invest. 1995; 96: 2051-2064Crossref PubMed Scopus (248) Google Scholar) was obtained from Dr. K. R. Thomas (University of Utah). The mice were maintained on a standard diet, and genotyping was carried out during days 7–14 postpartum, as described (22Zeng W. Lee M.G. Yan M. Diaz J. Benjamin I. Marino C.R. Kopito R. Freedman S. Cotton C. Muallem S. Thomas P.J. Am. J. Physiol. 1997; 273: C442-C455Crossref PubMed Google Scholar). The procedure for preparation and perfusion of the main pancreatic duct was identical to that described previously (8Lee M.G. Ahn W. Choi J.Y. Luo X. Seo J.T. Schultheis P.J. Shull G.E. Kim K.H. Muallem S. J. Clin. Invest. 2000; 105: 1651-1658Crossref PubMed Scopus (56) Google Scholar). Briefly, the mice were anesthetized, the abdomen was opened, and the duct lumen was cannulated using a modified 31-gauge needle. After ligating the proximal end of the common duct, the pancreas was removed into a dish containing ice-cold solution A supplemented with 0.02% soybean trypsin inhibitor and 0.1% bovine serum albumin. The main duct was cleared of acini and connective tissue, and the proximal end of the main duct was cut in order to facilitate retrograde luminal perfusion. After transferring to the perfusion chamber and during pHi measurement, the ducts were continuously perfused with separate bath and luminal solutions. For measurement of pHi in PS120/NHE3 cells, the glass coverslips with cells attached to them were washed once with solution A and assembled to form the bottom of a perfusion chamber. The cells were loaded with BCECF by a 10-min incubation at room temperature in solution A containing 2.5 μm BCECF-AM. After dye loading, the cells were perfused with appropriate solutions, and pHi was measured by photon counting using a fluorescence measuring system (Delta Ram; PTI Inc., South Brunswick, NJ). In the case of CFTR transfection with pCMVNot6.2 or its C-terminal deleted mutants, a green fluorescent protein-expressing plasmid (Life Technologies) was co-transfected with the CFTR constructs, and pHi measurements were performed with cells expressing high levels of green fluorescent protein as previously reported (5Lee M.G. Wigley W.C. Zeng W. Noel L.E. Marino C.R. Thomas P.J. Muallem S. J. Biol. Chem. 1999; 274: 3414-3421Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). For pHi measurement in the microdissected ducts, the cannulated ducts were transferred to a perfusion chamber placed on the stage of an inverted microscope. The bath was perfused at a flow rate of 6 ml/min, and the lumen was perfused at a flow rate of 150 μl/min using solution A that was maintained at 37 °C. BCECF was loaded by including 2.5 μm BCECF-AM in the luminal perfusate for 5 min, and dye loading was monitored. After completion of dye loading, the lumen was washed by perfusing solution A, and pHi measurements were performed according to the specified protocols. The fluorescence ratios of 490/440 nm were calibrated intracellularly by perfusing the cells or the ducts with solutions containing 145 mm KCl, 10 mm HEPES, and 5 μm nigericin with pH adjusted to 6.2–7.8. Na+/H+ exchange activity was measured using a standard protocol (23Lee M.G. Schultheis P.J. Yan M. Shull G.E. Bookstein C. Chang E. Tse M. Donowitz M. Park K. Muallem S. J. Physiol. ( Lond. ). 1998; 513: 341-357Crossref PubMed Scopus (63) Google Scholar). The cells were acidified by an NH 4+ pulse and subsequent perfusion with a Na+-free solution. The maximal Na+-dependent pHi recovery was measured in cells acidified to pHi 6.4–6.5. Buffer capacity was calculated by measuring ΔpHi in response to 5–20 mm NH4Cl pulses. In each experiment, the buffer capacity (βi) showed a negative linear relationship with pHi between 6.4 and 7.3. The βi of PS120 cells (30.2 ± 2.1 mm/pH unit at pHi 7.0) was lower than that of the pancreatic duct cells (48.6 ± 6.2). However, forskolin treatment or any gene modulation did not significantly change βi. Therefore, all the results of NHE activity are expressed as ΔpH/min, and this value was directly analyzed without compensating for βi. The results are presented as mean ± S.E., and, when appropriate, statistical analysis was determined using Student'st test or analysis of variance. p < 0.05 was considered statistically significant. To set the stage for analyzing the interaction between CFTR and NHE3 in model systems and native cells, we first determined which of the adapter proteins known to interact with CFTR are expressed in these cells (10Short D.B. Trotter K.W. Reczek D. Kreda S.M. Bretscher A. Boucher R.C. Stutts M.J. Milgram S.L. J. Biol. Chem. 1998; 273: 19797-19801Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar, 12Sun F. Hug M.J. Lewarchik C.M. Yun C. Bradbury N.A. Frizzell R.A. J. Biol. Chem. 2000; 275: 29539-29546Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). These include EBP50 and E3KARP, both of which contain two PDZ domains and associate with ezrin via the C terminus. We also examined the expression of the related mRNA encoding PDZK1 (also called CAP70), a protein that contains four PDZ domains (20Kocher O. Comella N. Tognazzi K. Brown L.F. Lab. Invest. 1998; 78: 117-125PubMed Google Scholar). Although PDZK1 is known to associate with CFTR (24Wang S. Yue H. Derin R.B. Guggino W.B. Li M. Cell. 2000; 103: 169-179Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar), it is not known whether it also associates with NHE3 or ezrin via an ERM binding domain. The RT-PCR analysis shown in Fig.1 A reveals expression of mRNA for EBP50, E3KARP (13Yun C.H.C. Oh S. Zizak M. Steplock D. Tsao S. Tse C.M. Weinman E.J. Donowitz M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3010-3015Crossref PubMed Scopus (400) Google Scholar), and PDZK1 (20Kocher O. Comella N. Tognazzi K. Brown L.F. Lab. Invest. 1998; 78: 117-125PubMed Google Scholar) in hamster lung and the mouse pancreas. On the other hand, only mRNA for EBP50 was detected in PS120 cells. Expression of EBP50 in PS120 cells was unexpected, since a previous study reported that this cell line does not express EBP50 (13Yun C.H.C. Oh S. Zizak M. Steplock D. Tsao S. Tse C.M. Weinman E.J. Donowitz M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3010-3015Crossref PubMed Scopus (400) Google Scholar). To substantiate the RT-PCR findings, expression of EBP50 in the hamster fibroblast-derived PS120 clone was first verified by sequencing the amplified RT-PCR product. As shown in Fig. 1 B, EBP50 in PS120 cells showed the highest similarity to Chinese hamster EBP50 with 99% homology based on nucleotide sequences (GenBankTMaccession numbers AF307992 and AF307993). The 1% sequence difference is probably due to the use of mutagenic agents during the selection of the NHE-deficient cells (17Pouyssegur J. Sardet C. Franchi A. L'Allemain G. Paris S. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 4833-4837Crossref PubMed Scopus (435) Google Scholar) or due to substrain differences. Expression of EBP50 protein in our PS120 cells was examined by Western blotting using a polyclonal antiserum generated against full-length human EBP50. Fig. 1 C shows expression of EBP50 protein in both PS120 cells and the mouse pancreas. Previous works reported that EBP50 can associate with CFTR or NHE3 (10Short D.B. Trotter K.W. Reczek D. Kreda S.M. Bretscher A. Boucher R.C. Stutts M.J. Milgram S.L. J. Biol. Chem. 1998; 273: 19797-19801Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar,11Weinman E.J. Steplock D. Tate K. Hall R.A. Spurney R.F. Shenolikar S. J. Clin. Invest. 1998; 101: 2199-2206Crossref PubMed Scopus (88) Google Scholar). We first confirmed these findings in our PS120 cells that were transfected with either CFTR or NHE3 (not shown). Subsequently, we asked whether CFTR and NHE3 exist in the same complex when expressed in PS120 cells and in the in vivo situation. For these experiments, NHE3 was stably expressed in PS120 cells to generate the PS120/NHE3 clone, and a portion of PS120/NHE3 cells was infected with Ad-CFTR. The blot in Fig. 2 Ashows that expression of CFTR had no measurable effect on expression of NHE3 protein in PS120/NHE3 cells. Lysates were prepared from control and CFTR-expressing PS120/NHE3 cells, and an antibody recognizing the R domain of CFTR was used to immunoprecipitate CFTR from PS120/NHE3 cells and PS120/NHE3 cells co-expressing CFTR. NHE3 was found in the CFTR immunoprecipitates from the cells infected with Ad-CFTR (Fig.2 C), demonstrating that exogenously expressed CFTR and NHE3 may associate in a stable complex in PS120 cells. To determine whether CFTR and NHE3 also associate in native cells, we performed similar experiments using mouse pancreata from WT and ΔF mice. For these experiments, the amount of lysate used was adjusted to contain the same amount of NHE3 (Fig. 2 B). NHE3 was found in CFTR immunoprecipitates from the pancreas of WT mouse (Fig.2 D). In contrast, only a very small amount of NHE3 was found in CFTR immunoprecipitates from the pancreas of ΔF mouse. The small amount of NHE3 found to associate with ΔF508 CFTR can be accounted for by the small amount of CFTR that is found in the luminal membrane of these mice (22Zeng W. Lee M.G. Yan M. Diaz J. Benjamin I. Marino C.R. Kopito R. Freedman S. Cotton C. Muallem S. Thomas P.J. Am. J. Physiol. 1997; 273: C442-C455Crossref PubMed Google Scholar). The findings in Fig. 2 provide the first evidence that NHE3 and CFTR exist in the same multiprotein complex, both in model systems and in native cells. To study the effect of CFTR on NHE3 activity, we first determined the optimal conditions to study this interaction. In previous work, the cAMP-dependent and EBP50-mediated inhibition of NHE3 activity was studied in serum-deprived, Go/G1-arrested PS120 cells that were transfected with EBP50 (13Yun C.H.C. Oh S. Zizak M. Steplock D. Tsao S. Tse C.M. Weinman E.J. Donowitz M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3010-3015Crossref PubMed Scopus (400) Google Scholar). Fig. 3 shows the results of similar experiments in our PS120/NHE3 cells. Serum deprivation for 18 h increased NHE3 activity by ∼41%. This is attributable to an increase in NHE3 expression under these conditions. 2O. W. Moe, unpublished observation. Exogenous overexpression of EBP50 in PS120/NHE3 cells reduced NHE3 activity in unstimulated cells by ∼35%. Preliminary studies showed that the optimal experimental condition to consistently observe regulation of NHE3 by CFTR is to maintain the PS120/NHE3 cells in 10% serum and to rely on the EBP50 already expressed in the cells. Having established optimal conditions for measuring NHE3 activity in PS120 fibroblasts, we next examined the effect of increased cAMP on NHE3 activity of control PS120/NHE3 cells and cells transfected with CFTR (Fig. 4). Treatment of PS120/NHE3 cells with forskolin dose-dependently inhibited NHE3 activity. At 0.1 and 10 μm forskolin, NHE3 activity of PS120/NHE3 cells was 91.6 ± 6.1 and 70.8 ± 5.1% of control, respectively. Importantly, expression of CFTR had no effect on basal NHE3 activity (rates of pHi recovery: 0.86 ± 0.09 ΔpH/min (n = 6) in control and 0.94 ± 0.10 ΔpH/min (n = 9) in CTFR-expressing PS120/NHE3 cells). Incubation of CFTR-expressing PS120/NHE3 cells with forskolin also inhibited NHE3 activity in a dose-dependent manner. However, the inhibition was nearly maximal at 0.1 μmforskolin; at 0.1 and 10 μm forskolin, NHE3 activity in CFTR-expressing cells was 60.3 ± 5.1 and 53.4 ± 6.2% of control, respectively. Thus, we conclude that activation of CFTR augments cAMP-mediated inhibition of NHE3. Fig. 4 shows that CFTR markedly augments the inhibition of NHE3 by cAMP. CFTR binds to EBP50 through its C-terminal DTRL sequence, a signature PDZ domain-binding motif (10Short D.B. Trotter K.W. Reczek D. Kreda S.M. Bretscher A. Boucher R.C. Stutts M.J. Milgram S.L. J. Biol. Chem. 1998; 273: 19797-19801Abstract Full Text Full Text PDF PubMed Scopus" @default.
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• W2019401224 title "Regulatory Interaction between the Cystic Fibrosis Transmembrane Conductance Regulator and HCO <mml:math xmlns:mml=http://www.w3.org/1998/Math/MathML altimg=si2.gif><mml:msubsup><mml:mrow /><mml:mrow><mml:mn>3</mml:mn></mml:mrow><mml:mrow><mml:mo>−</mml:mo></mml:mrow></mml:msubsup></mml:math> Salvage Mechanisms in Model Systems and the Mouse Pancreatic Duct" @default.
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