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• W2041344034 abstract "The cystic fibrosis transmembrane conductance regulator (CFTR), in addition to its well defined Cl- channel properties, regulates other ion channels. CFTR inhibits murine or rat epithelial Na+ channel (mENaC or rENaC) currents in many epithelial and non-epithelial cells, whereas murine or rat ENaC increases CFTR functional expression. These regulatory interactions are reproduced in Xenopus oocytes where both the open probability and surface expression of wild type CFTR Cl- channels are increased when CFTR is co-expressed with αβγ mENaC, and conversely the activity of mENaC is inhibited after wild type CFTR activation. Using the Xenopus oocyte expression system, differences in functional regulatory interactions were observed when CFTR was co-expressed with either αβγ mENaC or αβγ human ENaC (hENaC). Co-expression of CFTR and αβγ mENaC or hENaC resulted in an ∼3-fold increase in CFTR Cl- current compared with oocytes expressing CFTR alone. Oocytes co-injected with both CFTR and mENaC or hENaC expressed an amiloride-sensitive whole cell current that was decreased compared with that observed with the injection of mENaC or hENaC alone before CFTR activation with forskolin/3-isobutyl-1-methylxanthine. CFTR activation resulted in a further 50% decrease in mENaC-mediated currents, an ∼20% decrease in α-T663-hENaC-mediated currents, and essentially no change in α-A663-hENaC-mediated currents. Changes in ENaC functional expression correlated with ENaC surface expression by oocyte surface biotinylation experiments. Assessment of regulatory interactions between CFTR and chimeric mouse/human ENaCs suggest that the 20 C-terminal amino acid residues of α ENaC confer species specificity regarding ENaC inhibition by activated CFTR. The cystic fibrosis transmembrane conductance regulator (CFTR), in addition to its well defined Cl- channel properties, regulates other ion channels. CFTR inhibits murine or rat epithelial Na+ channel (mENaC or rENaC) currents in many epithelial and non-epithelial cells, whereas murine or rat ENaC increases CFTR functional expression. These regulatory interactions are reproduced in Xenopus oocytes where both the open probability and surface expression of wild type CFTR Cl- channels are increased when CFTR is co-expressed with αβγ mENaC, and conversely the activity of mENaC is inhibited after wild type CFTR activation. Using the Xenopus oocyte expression system, differences in functional regulatory interactions were observed when CFTR was co-expressed with either αβγ mENaC or αβγ human ENaC (hENaC). Co-expression of CFTR and αβγ mENaC or hENaC resulted in an ∼3-fold increase in CFTR Cl- current compared with oocytes expressing CFTR alone. Oocytes co-injected with both CFTR and mENaC or hENaC expressed an amiloride-sensitive whole cell current that was decreased compared with that observed with the injection of mENaC or hENaC alone before CFTR activation with forskolin/3-isobutyl-1-methylxanthine. CFTR activation resulted in a further 50% decrease in mENaC-mediated currents, an ∼20% decrease in α-T663-hENaC-mediated currents, and essentially no change in α-A663-hENaC-mediated currents. Changes in ENaC functional expression correlated with ENaC surface expression by oocyte surface biotinylation experiments. Assessment of regulatory interactions between CFTR and chimeric mouse/human ENaCs suggest that the 20 C-terminal amino acid residues of α ENaC confer species specificity regarding ENaC inhibition by activated CFTR. Cystic fibrosis (CF) 1The abbreviations used are: CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; ΔF508, deletion of phenylalanine 508; ENaC, epithelial sodium channel; hENaC, human ENaC; mENaC, murine ENaC; β-V5, ENaC β subunit containing a C-terminal V5 epitope tag; ns, not significant; IBMX, 3-isobutyl-1-methylxanthine; TEV, two-electrode voltage clamp. results from mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) (1Riordan J.R. Rommens J.M. Kerem B. Alon N. Rozmahel R. Grzelczak Z. Zielenski J. Lok S. Plavsic N. Chou J.L. Drumm M.L. Ianuzzi M.C. Collins F.S. Tsui L.-C. Science. 1989; 245: 1066-1073Crossref PubMed Scopus (5977) Google Scholar). In addition to functioning as a cAMP-activated, ATP-dependent Cl- channel, CFTR influences the transepithelial transport of other solutes, including Na+ via the epithelial sodium channel (ENaC), Cl- via an outwardly rectifying Cl- channel, K+ via Kir1.1, HCO3−, and ATP (2Schwiebert E.M. Egan M.E. Hwang T.H. Fulmer S.B. Allen S.S. Cutting G.R. Guggino W.B. Cell. 1995; 81: 1063-1073Abstract Full Text PDF PubMed Scopus (596) Google Scholar, 3Choi J.Y. Muallem D. Kiselyov K. Lee M.G. Thomas P.J. Muallem S. Nature. 2001; 410: 94-97Crossref PubMed Scopus (342) Google Scholar, 4Konstas A.A. Koch J.P. Tucker S.J. Korbmacher C. J. Biol. Chem. 2002; 277: 25377-25384Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 5Braunstein G.M. Roman R.M. Clancy J.P. Kudlow B.A. Taylor A.L. Shylonsky V.G. Jovov B. Peter K. Jilling T. Ismailov I.I. Benos D.J. Schwiebert L.M. Fitz J.G. Schwiebert E.M. J. Biol. Chem. 2001; 276: 6621-6630Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). Functional interactions between CFTR and rodent ENaC have been observed in epithelial as well as non-epithelial cells (6Briel M. Greger R. Kunzelmann K. J. Physiol. 1998; 508: 825-836Crossref PubMed Scopus (115) Google Scholar, 7Chabot H. Vives M.F. Dagenais A. Grygorczyk C. Berthiaume Y. Grygorczyk R. J. Membr. Biol. 1999; 169: 175-188Crossref PubMed Scopus (46) Google Scholar, 8Ismailov I.I. Awayda M.S. Jovov B. Berdiev B.K. Fuller C.M. Dedman J.R. Kaetzel M. Benos D.J. J. Biol. Chem. 1996; 271: 4725-4732Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 9Ji H.L. Chalfant M.L. Jovov B. Lockhart J.P. Parker S.B. Fuller C.M. Stanton B.A. Benos D.J. J. Biol. Chem. 2000; 275: 27947-27956Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 10Jiang Q. Li J. Dubroff R. Ahn Y.J. Foskett J.K. Engelhardt J. Kleyman T.R. J. Biol. Chem. 2000; 275: 13266-13274Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 11Suaud L. Li J. Jiang Q. Rubenstein R.C. Kleyman T.R. J. Biol. Chem. 2002; 277: 8928-8933Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 12Reddy M.M. Light M.J. Quinton P.M. Nature. 1999; 402: 301-304Crossref PubMed Scopus (202) Google Scholar). The activation of CFTR is generally associated with an inhibition of ENaC (7Chabot H. Vives M.F. Dagenais A. Grygorczyk C. Berthiaume Y. Grygorczyk R. J. Membr. Biol. 1999; 169: 175-188Crossref PubMed Scopus (46) Google Scholar, 8Ismailov I.I. Awayda M.S. Jovov B. Berdiev B.K. Fuller C.M. Dedman J.R. Kaetzel M. Benos D.J. J. Biol. Chem. 1996; 271: 4725-4732Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 10Jiang Q. Li J. Dubroff R. Ahn Y.J. Foskett J.K. Engelhardt J. Kleyman T.R. J. Biol. Chem. 2000; 275: 13266-13274Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 11Suaud L. Li J. Jiang Q. Rubenstein R.C. Kleyman T.R. J. Biol. Chem. 2002; 277: 8928-8933Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 13Hopf A. Schreiber R. Mall M. Greger R. Kunzelmann K. J. Biol. Chem. 1999; 274: 13894-13899Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 14Kunzelmann K. Kiser G.L. Schreiber R. Riordan J.R. FEBS Lett. 1997; 400: 341-344Crossref PubMed Scopus (130) Google Scholar), although activation of CFTR leads to activation of ENaC in the sweat duct (12Reddy M.M. Light M.J. Quinton P.M. Nature. 1999; 402: 301-304Crossref PubMed Scopus (202) Google Scholar) suggesting that the regulatory interactions between these two transporters are complex. The co-expression of CFTR and rodent ENaC in Xenopus oocytes results in regulatory interactions that mimic the airway where there is a decrease in ENaC-mediated Na+ transport in the presence of CFTR (9Ji H.L. Chalfant M.L. Jovov B. Lockhart J.P. Parker S.B. Fuller C.M. Stanton B.A. Benos D.J. J. Biol. Chem. 2000; 275: 27947-27956Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 10Jiang Q. Li J. Dubroff R. Ahn Y.J. Foskett J.K. Engelhardt J. Kleyman T.R. J. Biol. Chem. 2000; 275: 13266-13274Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 11Suaud L. Li J. Jiang Q. Rubenstein R.C. Kleyman T.R. J. Biol. Chem. 2002; 277: 8928-8933Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 14Kunzelmann K. Kiser G.L. Schreiber R. Riordan J.R. FEBS Lett. 1997; 400: 341-344Crossref PubMed Scopus (130) Google Scholar). Furthermore CFTR-mediated Cl- conductance is increased in presence of ENaC in oocytes (4Konstas A.A. Koch J.P. Tucker S.J. Korbmacher C. J. Biol. Chem. 2002; 277: 25377-25384Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 7Chabot H. Vives M.F. Dagenais A. Grygorczyk C. Berthiaume Y. Grygorczyk R. J. Membr. Biol. 1999; 169: 175-188Crossref PubMed Scopus (46) Google Scholar, 8Ismailov I.I. Awayda M.S. Jovov B. Berdiev B.K. Fuller C.M. Dedman J.R. Kaetzel M. Benos D.J. J. Biol. Chem. 1996; 271: 4725-4732Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 9Ji H.L. Chalfant M.L. Jovov B. Lockhart J.P. Parker S.B. Fuller C.M. Stanton B.A. Benos D.J. J. Biol. Chem. 2000; 275: 27947-27956Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 10Jiang Q. Li J. Dubroff R. Ahn Y.J. Foskett J.K. Engelhardt J. Kleyman T.R. J. Biol. Chem. 2000; 275: 13266-13274Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 11Suaud L. Li J. Jiang Q. Rubenstein R.C. Kleyman T.R. J. Biol. Chem. 2002; 277: 8928-8933Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 14Kunzelmann K. Kiser G.L. Schreiber R. Riordan J.R. FEBS Lett. 1997; 400: 341-344Crossref PubMed Scopus (130) Google Scholar). In contrast, the ΔF508-CFTR mutation, the most prevalent mutation found in North American Caucasian patients with CF, does not inhibit the functional expression of rat (15Mall M. Hipper A. Greger R. Kunzelmann K. FEBS Lett. 1996; 381: 47-52Crossref PubMed Scopus (132) Google Scholar) or mouse ENaC (mENaC) (11Suaud L. Li J. Jiang Q. Rubenstein R.C. Kleyman T.R. J. Biol. Chem. 2002; 277: 8928-8933Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar), and mENaC does not enhance the functional expression of ΔF508-CFTR in Xenopus oocytes (11Suaud L. Li J. Jiang Q. Rubenstein R.C. Kleyman T.R. J. Biol. Chem. 2002; 277: 8928-8933Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The isoflavone genistein, which activates the Cl- conductance of both wild type and mutant CFTRs (16al-Nakkash L. Hu S. Li M. Hwang T.C. J. Pharmacol. Exp. Ther. 2001; 296: 464-472PubMed Google Scholar, 17Andersson C. Roomans G.M. Eur. Respir. J. 2000; 15: 937-941Crossref PubMed Scopus (46) Google Scholar, 18Hwang T.C. Wang F. Yang I.C. Reenstra W.W. Am. J. Physiol. 1997; 273: C988-C998Crossref PubMed Google Scholar, 19Illek B. Zhang L. Lewis N.C. Moss R.B. Dong J.Y. Fischer H. Am. J. Physiol. 1999; 277: C833-C839Crossref PubMed Google Scholar), can restore these interactions (11Suaud L. Li J. Jiang Q. Rubenstein R.C. Kleyman T.R. J. Biol. Chem. 2002; 277: 8928-8933Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The mechanism by which this interregulation of CFTR and ENaC occurs in oocytes is unclear and somewhat controversial. Others have suggested that the decrease in ENaC-mediated current after CFTR activation may result from a series resistor error (20Nagel G. Szellas T. Riordan J.R. Friedrich T. Hartung K. EMBO Rep. 2001; 2: 249-254Crossref PubMed Scopus (51) Google Scholar) and have presented data in abstract form that suggest that activation of CFTR in oocytes does not result in a further decrease in hENaC-mediated current (21Nagel G. Szellas T. Grygorczyk R. Barbry P. Pediatr. Pulmonol. 2001; (abstr.): 209Google Scholar). Our previous data (11Suaud L. Li J. Jiang Q. Rubenstein R.C. Kleyman T.R. J. Biol. Chem. 2002; 277: 8928-8933Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) are inconsistent with the hypothesis that the apparent decrease in mENaC-mediated current with CFTR activation is due to a series resistor error. Furthermore such a mechanism cannot account for the increased CFTR functional expression observed with co-expression of rodent ENaC (9Ji H.L. Chalfant M.L. Jovov B. Lockhart J.P. Parker S.B. Fuller C.M. Stanton B.A. Benos D.J. J. Biol. Chem. 2000; 275: 27947-27956Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 10Jiang Q. Li J. Dubroff R. Ahn Y.J. Foskett J.K. Engelhardt J. Kleyman T.R. J. Biol. Chem. 2000; 275: 13266-13274Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 11Suaud L. Li J. Jiang Q. Rubenstein R.C. Kleyman T.R. J. Biol. Chem. 2002; 277: 8928-8933Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 22Suaud L. Carattino M. Kleyman T.R. Rubenstein R.C. J. Biol. Chem. 2002; 277: 50341-50347Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). The present studies were aimed at better understanding the regulatory interactions between CFTR and ENaC and whether there are species-specific differences in these interactions. In this regard, our data are consistent with the decrease in mENaC functional expression that occurs with CFTR activation being due, in part, to decreased mENaC surface expression, while hENaC (with Ala in position α663) functional and surface expression does not decrease following CFTR activation. These observations led us to test two additional hypotheses. First we tested the hypothesis that the C terminus of the ENaC α subunit, which has limited homology between mouse and human, may influence the species-related differences in this interaction. We also tested the hypothesis that a naturally occurring polymorphism in the C terminus of α hENaC, substitution of Ala at residue 663 for Thr (T663A), which we have recently shown to decrease the functional and surface expression of hENaC in oocytes, 2Samaha, F. F., Rubenstein, R. C., Yan, W., Ramkumar, M., Levy, D. I., Ahn, Y. J., Sheng, S., and Kleyman, T. R. (April 6, 2004) J. Biol. Chem. 10.1074/jbc.M401941200 would influence regulatory interactions between CFTR and hENaC. Our data suggest that the C terminus of α hENaC confers species specificity regarding ENaC inhibition by activated CFTR, whereas the αT663A polymorphism has a modest effect on the response of hENaC to activated CFTR. Materials—Forskolin and IBMX were purchased from Sigma. All other reagents were purchased from Fisher. Expression of Human CFTR and Mouse and Human ENaC in Xenopus Oocytes—Human CFTR (wild type and ΔF508) and mouse and human ENaC were expressed in Xenopus oocytes as described previously (10Jiang Q. Li J. Dubroff R. Ahn Y.J. Foskett J.K. Engelhardt J. Kleyman T.R. J. Biol. Chem. 2000; 275: 13266-13274Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 11Suaud L. Li J. Jiang Q. Rubenstein R.C. Kleyman T.R. J. Biol. Chem. 2002; 277: 8928-8933Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Briefly human CFTR and mouse and human α, β, and γ ENaC cRNAs were prepared using a cRNA synthesis kit (mMESSAGE mMACHINE, Ambion Inc., Austin, TX) according to the manufacturer's protocol. cRNA concentrations were determined spectroscopically. Oocytes obtained from adult female Xenopus laevis (NASCO, Fort Atkinson, WI) were enzymatically defolliculated and maintained at 18 °C in modified Barth's saline (88 mm NaCl, 1 mm KCl, 2.4 mm NaHCO3, 0.3 mm Ca(NO3)2, 0.41 mm CaCl2, 0.82 mm MgSO4, 15 mm Hepes, pH 7.6, supplemented with 10 μg/ml sodium penicillin, 10 μg/ml streptomycin sulfate, and 100 μg/ml gentamicin sulfate). Each batch of oocytes obtained from an individual frog was injected (50 nl/oocyte) using a Nanoject II microinjector (Drummond Scientific, Broomall, PA) with α, β, and γ subunits of either mENaC (0.33 ng/subunit) or hENaC (2 ng/subunit), CFTR (wild type or ΔF508, 10 ng), or a combination of ENaC and CFTR cRNAs dissolved in RNase-free water. Electrophysiological Analyses—Whole cell current measurements were performed 24-48 h after injection using the two-electrode voltage clamp method as described previously (10Jiang Q. Li J. Dubroff R. Ahn Y.J. Foskett J.K. Engelhardt J. Kleyman T.R. J. Biol. Chem. 2000; 275: 13266-13274Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 11Suaud L. Li J. Jiang Q. Rubenstein R.C. Kleyman T.R. J. Biol. Chem. 2002; 277: 8928-8933Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Oocytes were placed in a 1-ml chamber containing modified ND96 (96 mm NaCl, 1 mm KCl, 0.2 mm CaCl2, 5.8 mm MgCl2, 10 mm Hepes, pH 7.4) and impaled with micropipettes of 0.5-5-megaohm resistance filled with 3 m KCl. The whole cell currents were measured by voltage clamping the oocytes in 20-mV steps between -140 and +60 mV adjusted for resting transmembrane potential. Whole cell currents (I) were digitized at 200 Hz during the voltage steps, recorded directly onto a hard disk, and analyzed using pClamp 8.1 software (Axon Instruments, Foster City, CA). To reduce error due to series resistance, the voltage clamp (Axon Geneclamp 500B) was configured to clamp the bath potential to 0 mV. In this configuration, we independently monitored the oocyte membrane potential during our clamp protocol and routinely observed membrane potentials that were <5% depolarized from our target holding potentials. The difference in whole cell currents measured in the absence and presence of 10 μm amiloride was used to define the amiloride-sensitive Na+ current that was carried by ENaC. CFTR was activated by perfusion of the oocyte with buffer containing 10 μm forskolin and 500 μm IBMX (forskolin/IBMX) for 25 min (10Jiang Q. Li J. Dubroff R. Ahn Y.J. Foskett J.K. Engelhardt J. Kleyman T.R. J. Biol. Chem. 2000; 275: 13266-13274Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 11Suaud L. Li J. Jiang Q. Rubenstein R.C. Kleyman T.R. J. Biol. Chem. 2002; 277: 8928-8933Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). CFTR Cl- current was defined as the difference between amiloride-insensitive current measured before and after perfusion with forskolin/IBMX. Whole cell currents were recorded at a clamp potential of -100 mV for comparisons. All measurements were performed at room temperature. Assessment of ENaC Surface Expression—A β hENaC-V5 epitope C-terminal fusion protein (hβ-V5) was constructed in pcDNA6/V5-HisA (Invitrogen) as was described for β mENaC-V5 (mβ-V5) (23Hughey R.P. Mueller G.M. Bruns J.B. Kinlough C.L. Poland P.A. Harkleroad K.L. Carattino M.D. Kleyman T.R. J. Biol. Chem. 2003; 278: 37073-37082Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). cRNA for β-V5 was prepared and co-injected into oocytes with αγ ENaC with or without CFTR as described above. After 48 h, oocytes were treated with forskolin/IBMX for 20 min and cell surface proteins of oocytes that were mechanically stripped of their vitellin membranes in hypertonic medium (300 mm sucrose in modified Barth's saline without penicillin, streptomycin, and gentamicin) were labeled with sulfo-N-hydroxysuccinimide-biotin (Pierce) as we have described previously. 2Samaha, F. F., Rubenstein, R. C., Yan, W., Ramkumar, M., Levy, D. I., Ahn, Y. J., Sheng, S., and Kleyman, T. R. (April 6, 2004) J. Biol. Chem. 10.1074/jbc.M401941200 Oocytes (10/group) were subsequently lysed in 0.15 m NaCl, 0.01 m Tris-Cl, pH 8.0, 0.01 m EDTA, 1.0% Nonidet P-40, 0.5% sodium deoxycholate, 1.0 mm phenylmethanesulfonyl fluoride, 0.1 mm Nα-p-tosyl-l-lysine chloromethyl ketone (TLCK), 0.1 mm l-1-tosylamide-2-phenylethyl-chloromethyl ketone, and 2 μg/ml aprotinin for 1 h at 4 °C and centrifuged at 13,000 × g for 15 min at 4 °C, and labeled proteins in the supernatant were precipitated with streptavidin-agarose (Pierce). Immunoblot Analysis—Streptavidin-precipitated proteins or whole oocyte lysates (prepared as above) were incubated in Laemmli sample buffer, resolved by SDS-PAGE, and transferred to nitrocellulose as described previously by our group. 2Samaha, F. F., Rubenstein, R. C., Yan, W., Ramkumar, M., Levy, D. I., Ahn, Y. J., Sheng, S., and Kleyman, T. R. (April 6, 2004) J. Biol. Chem. 10.1074/jbc.M401941200 β-V5 was identified by immunoblot using an anti-V5 monoclonal antibody (Invitrogen), an anti-mouse horseradish peroxidase-conjugated secondary antibody, and ECL (Amersham Biosciences) visualization essentially as described previously by our group (24Rubenstein R.C. Zeitlin P.L. Am. J. Physiol. 2000; 278: C259-C267Crossref PubMed Google Scholar, 25Rubenstein R.C. Egan M.E. Zeitlin P.L. J. Clin. Investig. 1997; 100: 2457-2465Crossref PubMed Scopus (330) Google Scholar). Fluorogram density was quantitated using an AlphaImager 2200 system and version 5.5 software (Alpha Innotech, San Leandro, CA), and the intensity of β-V5 was expressed relative to that of oocytes injected with ENaC alone without forskolin/IBMX stimulation. As in our previous work, 2Samaha, F. F., Rubenstein, R. C., Yan, W., Ramkumar, M., Levy, D. I., Ahn, Y. J., Sheng, S., and Kleyman, T. R. (April 6, 2004) J. Biol. Chem. 10.1074/jbc.M401941200 less than 20% of intracellular β-V5 epitope was biotin-labeled in oocytes expressing only β-V5 and γ ENaC subunits (data not shown). Statistical Analyses—All data are presented as mean ± S.E. Statistical comparisons were performed using the Student's t test. A pairwise t test was used for pre/post-treatment in experiments using an individual oocyte. A two-tailed t test was used when comparing currents obtained from oocytes injected with a cRNA for a single transporter (i.e. ENaC or CFTR versus oocytes co-injected with cRNAs for both ENaC and CFTR). p values ≤0.05 were accepted to indicate statistical significance. Statistical analyses were performed using SigmaStat version 2.03 software. Regulatory Interactions between mENaC and CFTR in Xenopus Oocytes—Several groups have reported that when wild type CFTR and rodent (murine or rat) ENaC were co-expressed in Xenopus oocytes, ENaC-mediated Na+ currents were inhibited in response to CFTR activation in two-electrode voltage clamp (TEV) experiments (7Chabot H. Vives M.F. Dagenais A. Grygorczyk C. Berthiaume Y. Grygorczyk R. J. Membr. Biol. 1999; 169: 175-188Crossref PubMed Scopus (46) Google Scholar, 9Ji H.L. Chalfant M.L. Jovov B. Lockhart J.P. Parker S.B. Fuller C.M. Stanton B.A. Benos D.J. J. Biol. Chem. 2000; 275: 27947-27956Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 10Jiang Q. Li J. Dubroff R. Ahn Y.J. Foskett J.K. Engelhardt J. Kleyman T.R. J. Biol. Chem. 2000; 275: 13266-13274Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 11Suaud L. Li J. Jiang Q. Rubenstein R.C. Kleyman T.R. J. Biol. Chem. 2002; 277: 8928-8933Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Furthermore co-expression of either of these rodent ENaCs enhanced forskolin/IBMX-stimulated CFTR Cl- currents (7Chabot H. Vives M.F. Dagenais A. Grygorczyk C. Berthiaume Y. Grygorczyk R. J. Membr. Biol. 1999; 169: 175-188Crossref PubMed Scopus (46) Google Scholar, 9Ji H.L. Chalfant M.L. Jovov B. Lockhart J.P. Parker S.B. Fuller C.M. Stanton B.A. Benos D.J. J. Biol. Chem. 2000; 275: 27947-27956Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 10Jiang Q. Li J. Dubroff R. Ahn Y.J. Foskett J.K. Engelhardt J. Kleyman T.R. J. Biol. Chem. 2000; 275: 13266-13274Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 11Suaud L. Li J. Jiang Q. Rubenstein R.C. Kleyman T.R. J. Biol. Chem. 2002; 277: 8928-8933Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). In agreement with these previous observations (7Chabot H. Vives M.F. Dagenais A. Grygorczyk C. Berthiaume Y. Grygorczyk R. J. Membr. Biol. 1999; 169: 175-188Crossref PubMed Scopus (46) Google Scholar, 10Jiang Q. Li J. Dubroff R. Ahn Y.J. Foskett J.K. Engelhardt J. Kleyman T.R. J. Biol. Chem. 2000; 275: 13266-13274Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 11Suaud L. Li J. Jiang Q. Rubenstein R.C. Kleyman T.R. J. Biol. Chem. 2002; 277: 8928-8933Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 15Mall M. Hipper A. Greger R. Kunzelmann K. FEBS Lett. 1996; 381: 47-52Crossref PubMed Scopus (132) Google Scholar, 22Suaud L. Carattino M. Kleyman T.R. Rubenstein R.C. J. Biol. Chem. 2002; 277: 50341-50347Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar), oocytes injected with mENaC alone had similar amiloride-sensitive currents before (-3.23 ± 0.75 μA) and after (-3.61 ± 0.85 μA, n = 10, p = ns) treatment with forskolin and IBMX (Fig. 1A). Oocytes co-injected with mENaC and CFTR had reduced amiloride-sensitive current (-1.36 ± 0.20 μA, n = 10) compared with oocytes injected with mENaC alone (-3.23 ± 0.75 μA, p = 0.03), and this amiloride-sensitive current was further reduced in co-injected oocytes upon activation of CFTR with forskolin/IBMX (-0.89 ± 0.20 μA, p = 0.01, Fig. 1A). Also in agreement with our previous data (10Jiang Q. Li J. Dubroff R. Ahn Y.J. Foskett J.K. Engelhardt J. Kleyman T.R. J. Biol. Chem. 2000; 275: 13266-13274Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 11Suaud L. Li J. Jiang Q. Rubenstein R.C. Kleyman T.R. J. Biol. Chem. 2002; 277: 8928-8933Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar), co-injection of mENaC with CFTR increased the functional expression of CFTR (-10.08 ± 1.07 μA, n = 10) compared with oocytes injected with CFTR alone (-2.12 ± 0.42 μA, n = 10, p < 0.001, Fig. 1B). As the mechanism by which these alterations in mENaC functional expression upon co-expression and activation of CFTR occur is not clear, we performed surface biotinylation experiments to assess the amount of mENaC at the oocyte surface. In these experiments, we used mENaC where the β subunit contained a C-terminal V5 epitope tag to increase sensitivity of our immunoblots; control experiments suggested that this β mENaC modification did not influence regulatory interactions between CFTR and mENaC as measured by TEV (data not shown). As shown in Fig. 1C, mENaC(β-V5) expression at the oocyte surface was unaltered by forskolin/IBMX in oocytes injected with mENaC alone. In contrast, mENaC surface expression decreased when mENaC was co-expressed with CFTR and was further decreased by activation of CFTR in oocytes expressing both channels. There was direct correspondence of these changes in surface expression of mENaC(β-V5) and the mENaC-mediated currents by TEV (Fig. 1A), suggesting that alterations in mENaC functional expression are, in part, a result of changes in mENaC expression at the oocyte surface (or “N”). We also assessed whole oocyte expression of mENaC(β-V5) (Fig. 1D). Expression of mENaC(β-V5) was decreased by coinjection of CFTR but was not further decreased upon activation of CFTR with forskolin/IBMX. These data are consistent with CFTR activation causing an acute decrease in mENaC(β-V5) surface and functional expression without altering the total amount of mENaC(β-V5) present in the oocyte. Co-expression of CFTR and hENaC—We next assessed regulatory interactions between CFTR and hENaC as well as the potential influence of the T663A functional polymorphism of α hENaC described recently by our group2 on these interactions. Similar to our data with mENaC (Fig. 1A), forskolin/IBMX did not alter hENaC functional expression in oocytes injected with hENaC alone as is shown in Fig. 2A for α-T663-hENaC (-1.97 ± 0.27 versus -1.94 ± 0.30 μA, n = 21, p = ns) and in Fig. 2B for α-A663-hENaC (-1.85 ± 0.28 versus -1.95 ± 0.31 μA, n = 21, p = ns). Again co-injection of CFTR reduced hENaC functional expression prior to activation of CFTR (α-T663-hENaC: -1.97 ± 0.27 μA, n = 21 versus -0.81 ± 0.09 μA, n = 24, p < 0.001; α-A663-hENaC: -1.85 ± 0.28 μA, n = 21 versus -0.78 ± 0.15 μA, n = 25, p = 0.001). However, activation of CFTR in co-injected oocytes resulted in a further, more modest decrease in α-T663-hENaC functional expression (-0.81 ± 0.09 versus -0.65 ± 0.08 μA, n = 24, p = 0.02, Fig. 2A) compared with mENaC (Fig. 1A), and CFTR activation did not significantly decrease α-A663-hENaC functional expression (-0.78 ± 0.15 versus -0.69 ± 0.14 μA, n = 25, p = ns, Fig. 2B), although the lack of significance of this decrease for α-A663-hENaC may be related to the larger standard errors in these data compared with those of α-T663-hENaC. CFTR functional expression was enhanced by co-injection of α-T663-hENaC (coinjected: -6.32 ± 1.14 μA, n = 24, versus injected with CFTR alone: -2.83 ± 0.50 μA, n = 19, p = 0.009) or α-A663-hENaC (co-injected: -7.78 ± 1.32 μA, n = 25, versus injected with CFTR alone: -3.08 ± 0.40 μA, n = 19, p = 0.004, Fig. 2C) as it was by mENaC (Fig. 1B). We next sought to correlate the functional expression of α-A663-hENaC by TEV with its surface expression by surface biotinylation of oocytes expressing α-A663-hENaC (where the hENaC β subunit contained a C-terminal V5 epitope tag). Again there was direct correlation of α-A663-hENaC(β-V5) surface expression (Fig. 2D) and α-A663-hENaC functional expression by TEV (Fig. 2B). Surface and functional expression of α-A663-hENaC was not altered by treatment with forskolin/IBMX in oocytes injected with α-A663-hENaC alone. Co-injection of CFTR decreased surface and functional expression of α-A663-hENaC (Fig. 2, B and D) as well as whole oocyte expression of hENaC(β-V5) (Fig. 2E). Activation of CFTR with forskolin/IBMX in co-injected oocytes did not further alter surface or whole oocyte expression of hENaC(β-V5). These data suggest that the regulation of mENaC and hENaC by CFTR in oocytes differs and involves changes in mENaC surface expression in response to CFTR activa" @default.
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• W2041344034 title "Cystic Fibrosis Transmembrane Conductance Regulator Differentially Regulates Human and Mouse Epithelial Sodium Channels in Xenopus Oocytes" @default.
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