ABCMdb

PMID: 16822950

Suaud L, Yan W, Rubenstein RC
Abnormal regulatory interactions of I148T-CFTR and the epithelial Na+ channel in Xenopus oocytes.
Am J Physiol Cell Physiol. 2007 Jan;292(1):C603-11. Epub 2006 Jul 5., [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCC7 p.Asn1303Lys ABCC7 p.Ile148Thr First published 5 July 2006;Am J Physiol Cell Physiol Laurence Suaud, Wusheng Yan and Ronald C. Rubenstein oocytesXenopuschannel in+the epithelial Na Abnormal regulatory interactions of I148T-CFTR and You might find this additional info useful... 35 articles, 19 of which can be accessed free at:This article cites http://ajpcell.physiology.org/content/292/1/C603.full.html#ref-list-1 2 other HighWire hosted articlesThis article has been cited by [PDF][Full Text][Abstract] , April 1, 2007; 292 (4): C1553-C1561.Am J Physiol Cell Physiol Ronald C. Rubenstein Laurence Suaud, Wusheng Yan, Marcelo D. Carattino, Amal Robay, Thomas R. Kleyman and chloride transport is not necessary for inhibition of ENaC oocytes: evidence thatXenopusRegulatory interactions of N1303K-CFTR and ENaC in [PDF][Full Text][Abstract] , January , 2011; 300 (1): L88-L101.Am J Physiol Lung Cell Mol Physiol Yael Grumbach Ronald C. Rubenstein, Shannon R. Lockwood, Ellen Lide, Rebecca Bauer, Laurence Suaud and airway epithelial cells F508-CFTR in∆Regulation of endogenous ENaC functional expression by CFTR and including high resolution figures, can be found at:Updated information and services http://ajpcell.physiology.org/content/292/1/C603.full.html can be found at:AJP - Cell PhysiologyaboutAdditional material and information http://www.the-aps.org/publications/ajpcell This infomation is current as of August 8, 2011. Login to comment 7 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr It is published 12 timesAJP - Cell Physiology CALL FOR PAPERS Protein and Vesicle Trafficking, Cytoskeleton Abnormal regulatory interactions of I148T-CFTR and the epithelial Naϩ channel in Xenopus oocytes Laurence Suaud,1 Wusheng Yan,1 and Ronald C. Rubenstein1,2 1 Division of Pulmonary Medicine, Children`s Hospital of Philadelphia, and 2 Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania Submitted 23 February 2006; accepted in final form 30 June 2006 Suaud L, Yan W, Rubenstein RC. Abnormal regulatory interactions of I148T-CFTR and the epithelial Naϩ channel in Xenopus oocytes. Login to comment 12 ABCC7 p.Ile148Thr We tested this hypothesis for I148T-CFTR, where the mutation is located in CFTR`s first intracellular loop. Login to comment 13 ABCC7 p.Ile148Thr I148T-CFTR has been associated with a severe CF phenotype, perhaps because of defects in its regulation of bicarbonate transport, but it transports chloride similarly to wild-type CFTR in model systems (Choi JY, Muallem D, Kiselyov K, Lee MG, Thomas PJ, Muallem S. Login to comment 15 ABCC7 p.Ile148Thr cRNAs encoding ␣beta␥-mENaC and I148T-CFTR were injected separately or together into Xenopus oocytes. Login to comment 18 ABCC7 p.Ile148Thr Injection of I148T-CFTR cRNA alone yielded high levels of CFTR functional expression. Login to comment 19 ABCC7 p.Ile148Thr In coinjected oocytes, mENaC functional and surface expression was not altered by activation of I148T-CFTR with forskolin/ IBMX. Login to comment 20 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr Furthermore, the CFTR potentiator genistein both enhanced functional expression of I148T-CFTR and restored regulation of mENaC surface expression by activated I148T-CFTR. Login to comment 21 ABCC7 p.Ile148Thr These data suggest that the ability to transport chloride is not a critical determinant of regulation of mENaC by activated CFTR in Xenopus oocytes and provide further evidence that I148T-CFTR is dysfunctional despite maintaining the ability to transport chloride. Login to comment 31 ABCC7 p.Gly551Asp These data are consistent with our observations that mutant CFTRs in which the mutation is within NBD-1, such as ⌬F508-CFTR and G551D-CFTR, lack characteristic regulatory interactions with ␣beta␥-mENaC when activated by forskolin/3-isobutyl-1-methylxanthine (IBMX) (33, 34). Login to comment 33 ABCC7 p.Ile148Thr We aimed to test these alternate hypotheses by examining the regulatory interactions of murine ENaC (mENaC) with I148T-CFTR. Login to comment 34 ABCC7 p.Ile148Thr I148T is a rare mutation of CFTR where the mutation is localized in CFTR`s first cytoplasmic loop (CL-1). Login to comment 36 ABCC7 p.Ile148Thr sickkids.on.ca/cftr), I148T can be associated with phenotypically severe, pancreatic-insufficient CF. Login to comment 37 ABCC7 p.Ile148Thr However, this association has been questioned by others, because I148T-containing alleles from patients with CF can harbor a second mutation in cis, 3199del6, which causes deletion of I1023 and V1024, that may itself be sufficient to cause CFTR dysfunction (10). Login to comment 38 ABCC7 p.Ile148Thr Interestingly, in heterologous cells in the absence of this second mutation in cis, I148T-CFTR appears to maintain the ability to transport chloride at levels similar to wild-type CFTR (WT-CFTR) but appears deficient in its ability to regulate HCO3 - transport (9); this defective regulation of HCO3 - transport is hypothesized to underlie the pancreatic dysfunction associated with this mutation in CF. Login to comment 39 ABCC7 p.Ile148Thr Our data suggest that cAMP-activated I148T-CFTR has robust capability to conduct Address for reprint requests and other correspondence: R. C. Rubenstein, Division of Pulmonary Medicine, Children`s Hospital of Philadelphia, Abramson 410C, 34th St. and Civic Center Blvd., Philadelphia, PA 19104 (e-mail: rrubenst@mail.med.upenn.edu). Login to comment 49 ABCC7 p.Ile148Thr Expression of human I148T-CFTR and mouse ENaC in Xenopus oocytes. Login to comment 50 ABCC7 p.Ile148Thr Human I148T-CFTR was constructed by site-directed mutagenesis of WT-CFTR cDNA by using a PCR-based mutagenesis technique, and its sequence was confirmed by automated analysis in The Children`s Hospital of Philadelphia Nucleic Acid and Protein Core. Login to comment 51 ABCC7 p.Ile148Thr I148T-CFTR and ␣beta␥-mENaC were expressed in Xenopus oocytes as previously described (16, 33, 34, 37). Login to comment 52 ABCC7 p.Ile148Thr I148T-CFTR and mouse ␣-, beta-, and ␥-ENaC cRNAs were prepared using the mMESSAGE mMACHINE cRNA synthesis kit (Ambion, Austin, TX) according to the manufacturer`s protocol, and cRNA concentrations were determined spectroscopically. Login to comment 55 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr Each batch of oocytes obtained from an individual frog was injected using a Nanoject II microinjector (Drummond Scientific, Broomall, PA) with either ␣-, beta-, and ␥-subunits of mENaC (0.33 ng/subunit), I148T-CFTR (10 ng), or a combination of ␣beta␥-mENaC and I148T-CFTR cRNAs dissolved in RNase-free water (50 nl/ oocyte). Login to comment 65 ABCC7 p.Ile148Thr I148T-CFTR was activated by perfusion of the oocyte with modified ND96 buffer containing 10 ␮M forskolin and 100 ␮M IBMX for 25 min (16, 33, 34, 37). Login to comment 67 ABCC7 p.Ile148Thr In all experiments, chloride current carried by I148T-CFTR was defined as the difference between amiloride-insensitive current measured before and after perfusion with forskolin/IBMX (or before and after perfusion with forskolin/IBMX/genistein). Login to comment 73 ABCC7 p.Ile148Thr cRNAs for ␣beta-V5␥-mENaC were either injected into Xenopus oocytes alone or coinjected with cRNA encoding I148T-CFTR. Login to comment 83 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr A two-tailed t-test was used when comparing currents obtained from oocytes injected with a cRNA for a single transporter (i.e., ␣beta␥-mENaC or I148T-CFTR) versus oocytes coinjected with cRNAs for both ␣beta␥- mENaC and I148T-CFTR. Login to comment 87 ABCC7 p.Ile148Thr RESULTS Functional expression of I148T-CFTR in Xenopus oocytes. Login to comment 88 ABCC7 p.Ile148Thr We used the Xenopus oocyte expression system and TEV technique to examine the functional expression of I148T-CFTR. Login to comment 89 ABCC7 p.Ile148Thr Prior data suggest that I148T-CFTR exhibits intracellular maturation (as assessed by molecular weight shifts due to glycolytic processing) and functional chloride transport similar to that of WT-CFTR but is deficient in its ability to regulate HCO3 - transport (4, 9, 32). Login to comment 90 ABCC7 p.Ile148Thr We measured whole cell currents at varying clamping potentials in oocytes injected with 10 ␮g of I148T-CFTR cRNA and generated current/voltage (I-V) curves from data obtained before and after 20 min of incubation with 10 ␮M forskolin and 100 ␮M IBMX to activate endogenous protein kinase A (Fig. 1A). Login to comment 91 ABCC7 p.Ile148Thr I148T-CFTR had a CFTR-characteristic linear I-V relationship, and whole cell currents, measured at a holding potential of -100 mV, increased 28-fold from -0.12 Ϯ 0.02 to -3.42 Ϯ 1.18 ␮A (mean Ϯ SE, n ϭ 19, P Յ 0.001) in response to forskolin/IBMX. Login to comment 93 ABCC7 p.Ile148Thr These data suggest that expression of I148T leads to robust forskolin/IBMX-stimulated chloride currents in oocytes that are similar in magnitude to those of WT-CFTR. Login to comment 94 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Ile148Thr C604 REGULATORY INTERACTIONS OF I148T-CFTR AND ENaC AJP-Cell Physiol • VOL 292 • JANUARY 2007 • www.ajpcell.org onAugust,2011ajpcell.physiology.orgDownloadedfrom The isoflavone and CFTR potentiator genistein increases chloride transport by activated WT and mutant CFTRs, including ⌬F508-CFTR and G551D-CFTR (13, 33, 34). Login to comment 95 ABCC7 p.Ile148Thr We assessed whether genistein would also enhance the function of I148T-CFTR. Login to comment 96 ABCC7 p.Ile148Thr Figure 1B demonstrates the I-V relationship of I148T- expressing oocytes before and after incubation with 10 ␮M forskolin/100 ␮l IBMX for 25 min followed by incubation with 10 ␮M forskolin/100 ␮M IBMX/50 ␮M genistein for an additional 20 min. Login to comment 98 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Ile148Thr This enhancement of activated I148T-CFTR by genistein is similar in magnitude to the effect of genistein on WT-CFTR (ϳ2-fold; Ref. 34) but slightly smaller in magnitude compared with our previously observed enhancement of ⌬F508-CFTR-and G551D-CFTR-mediated currents by genistein (5.7and 4-fold, respectively) (33, 34). Login to comment 99 ABCC7 p.Ile148Thr The forskolin/IBMX-stimulated currents mediated by I148T-CFTR were markedly reduced in Fig. 1B vs. Fig. 1A. Login to comment 102 ABCC7 p.Ile148Thr Regulatory interactions of I148T-CFTR and ␣beta␥-ENaC. Login to comment 108 ABCC7 p.Gly551Asp Our data regarding the regulatory interactions of ⌬F508- and G551D-CFTR also are consistent with the data of others suggesting that an intact NBD-1 is required for activated CFTR to inhibit mENaC-mediated conductance (3, 33, 34). Login to comment 109 ABCC7 p.Ile148Thr We therefore assessed the interregulation of I148T-CFTR and mENaC in oocytes. Login to comment 110 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr Figure 2 depicts whole cell currents measured at a holding potential of -100 mV in oocytes injected with I148T-CFTR (10 ng) or ␣beta␥-mENaC (0.33 ng/subunit) or both I148T-CFTR and ␣beta␥-mENaC cRNAs. Login to comment 111 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr Oocytes coinjected with I148T-CFTR and ␣beta␥-mENaC had no change in forskolin/IBMX-stimulated, amiloride-insensitive (I148T-CFTR-mediated) current compared with oocytes injected with I148T-CFTR alone [ICl ϭ -3.20 Ϯ 1.20 (I148T-CFTR, n ϭ 19) vs. -2.86 Ϯ 0.78 ␮A (I148T-CFTR/␣beta␥- mENaC, n ϭ 17), P ϭ not significant (NS); Fig. 2A]. Login to comment 112 ABCC7 p.Ile148Thr Thus coexpression of ␣beta␥-mENaC in oocytes does not alter the functional expression of I148T-CFTR. Login to comment 115 ABCC7 p.Ile148Thr Oocytes coinjected with both I148T-CFTR and ␣beta␥-mENaC expressed amiloride-sensitive whole cell currents before forskolin/IBMX stimulation (-0.32 Ϯ 0.10 ␮A, n ϭ 17) that were significantly lower than the amiloride-sensitive whole cell currents recorded under the same conditions in oocytes injected with ␣beta␥-mENaC alone (-2.06 Ϯ 0.62 ␮A, n ϭ 18, P ϭ 0.01). Login to comment 116 ABCC7 p.Gly551Asp These observations are similar to our group`s data regarding CFTRs with appropriate intracellular trafficking, such as WT-CFTR (34, 37) and G551D-CFTR (33). Login to comment 117 ABCC7 p.Ile148Thr However, unlike WT-CFTR, activation of I148T-CFTR with forskolin/IBMX did not lead to a further Fig. 1. Login to comment 118 ABCC7 p.Ile148Thr Expression of I148T-CFTR in Xenopus oocytes. Login to comment 119 ABCC7 p.Ile148Thr A: I148T-CFTR was expressed in oocytes and two-electrode voltage clamp (TEV) was performed as described in EXPERIMENTAL PROCEDURES. Login to comment 122 ABCC7 p.Ile148Thr B: I148T-CFTR was expressed in oocytes and TEV was performed. Login to comment 123 ABCC7 p.Ile148Thr I-V relationships before stimulation of I148T-CFTR (F), after stimulation with 10 ␮M forskolin and 100 ␮M IBMX (E), and after stimulation with 10 ␮M forskolin, 100 ␮M IBMX, and 50 ␮M genistein () are shown. Login to comment 126 ABCC7 p.Ile148Thr C605REGULATORY INTERACTIONS OF I148T-CFTR AND ENaC AJP-Cell Physiol • VOL 292 • JANUARY 2007 • www.ajpcell.org reduction in ␣beta␥-mENaC-mediated amiloride-sensitive whole cell currents in coinjected oocytes (-0.32 Ϯ 0.10 ␮A before forskolin/IBMX vs. -0.40 Ϯ 0.13 ␮A after forskolin/IBMX, n ϭ 17, P ϭ NS; Fig. 2B). Login to comment 127 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp These data are similar to our previous data for the CFTR mutants ⌬F508 and G551D that have defective regulatory interactions with mENaC; activation of ⌬F508 and G551D with forskolin/IBMX also does not lead to acute decreases in mENaC functional expression (33, 34). Login to comment 128 ABCC7 p.Ile148Thr These data are therefore consistent with activated I148T-CFTR having defective regulatory interactions with ␣beta␥-mENaC in Xenopus oocytes despite maintaining a robust ability to transport chloride. Login to comment 130 ABCC7 p.Ile148Thr Expression of I148T-CFTR and murine epithelial sodium channel ␣beta␥-subunits (␣beta␥-mENaC) in Xenopus oocytes. Login to comment 131 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr I148T-CFTR (10 ng of cRNA) and ␣beta␥-mENaC (0.33 ng cRNA/subunit) were expressed separately or together in oocytes, and TEV was performed as described in EXPERIMENTAL PROCEDURES. A: changes in whole cell currents that were not inhibited by 10 ␮M amiloride (-100 mV holding potential) in oocytes injected with I148T-CFTR alone or in oocytes coinjected with I148T-CFTR and ␣beta␥-mENaC after stimulation with 10 ␮M forskolin/100 ␮M IBMX are shown. Login to comment 132 ABCC7 p.Ile148Thr B: amiloride-sensitive whole cell currents (-100 mV holding potential) were determined in oocytes expressing ␣beta␥-mENaC or coexpressing ␣beta␥-mENaC and I148T-CFTR before (open bars) and after (shaded bars) stimulation with 10 ␮M forskolin/100 ␮M IBMX. Login to comment 133 ABCC7 p.Ile148Thr Data obtained from the same I148T-CFTR/␣beta␥- mENaC coinjected oocytes are shown in A and B. Values are means Ϯ SE. Login to comment 134 ABCC7 p.Ile148Thr Fig. 3. Influence of I148T-CFTR on surface and whole oocyte expression of mENaC. Login to comment 135 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr Influence of I148T-CFTR on surface and whole oocyte expression of mENaC. Login to comment 136 ABCC7 p.Ile148Thr ␣beta␥-mENaC (0.33 ng cRNA/subunit) where the beta-subunit contained a COOH-terminal V5 epitope was expressed in oocytes without or with I148T-CFTR (10 ng of cRNA). Login to comment 149 ABCC7 p.Ile148Thr As shown in Fig. 3A, mENaC(beta-V5) expression at the oocyte surface was unaltered by treatment with forskolin/IBMX in oocytes injected with ␣beta␥-mENaC alone and decreased after coinjection of I148T-CFTR in the absence of forskolin/IBMX activation. Login to comment 150 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr As shown in Fig. 3A, mENaC(beta-V5) expression at the oocyte surface was unaltered by treatment with forskolin/IBMX in oocytes injected with ␣beta␥-mENaC alone and decreased after coinjection of I148T-CFTR in the absence of forskolin/IBMX activation. Login to comment 151 ABCC7 p.Ile148Thr Furthermore, mENaC surface expression did not change upon activation of I148T-CFTR with forskolin/IBMX in coinjected oocytes. Login to comment 153 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr The whole oocyte content of mENaC(beta-V5) was not altered by acute treatment with forskolin/IBMX in oocytes injected with ␣beta␥-mENaC alone, was decreased by coinjection with I148T-CFTR cRNA, and was not further decreased upon activation of I148T-CFTR by forskolin/IBMX. Login to comment 154 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr The whole oocyte content of mENaC(beta-V5) was not altered by acute treatment with forskolin/IBMX in oocytes injected with ␣beta␥-mENaC alone, was decreased by coinjection with I148T-CFTR cRNA, and was not further decreased upon activation of I148T-CFTR by forskolin/IBMX. Login to comment 156 ABCC7 p.Ile148Thr These data are therefore consistent with I148T-CFTR, in the absence of activation, maintaining the ability to decrease the whole oocyte expression of mENaC. Login to comment 157 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr These data are therefore consistent with I148T-CFTR, in the absence of activation, maintaining the ability to decrease the whole oocyte expression of mENaC. Login to comment 158 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr This decrease in whole oocyte expression in coinjected oocytes parallels the decrease in surface expression and may result from I148T-CFTR decreasing the synthesis of mENaC and its trafficking to the oocyte membrane or increasing the rate at which mENaC is degraded either before or after reaching the plasma membrane. Login to comment 159 ABCC7 p.Gly551Asp ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr Interregulation of I148T-CFTR and ␣beta␥ mENaC after IBMX/forskolin/genistein stimulation. Login to comment 160 ABCC7 p.Gly551Asp ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr We examined whether the CFTR potentiator genistein, which improves functional interactions between ␣beta␥-mENaC and the G551D-CFTR and ⌬F508-CFTR mutants (33, 34), is similarly able to restore I148T-CFTR`s regulatory interactions with mENaC. Login to comment 161 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr As shown in Fig. 4A, the amiloride-insensitive, or I148T-CFTR-mediated, component of the forskolin/IBMX/genistein-stimulated whole cell current in oocytes coexpressing I148T-CFTR and ␣beta␥-mENaC was 1.7-fold greater than the forskolin/IBMX/ gensitein-stimulated current measured in oocytes expressing I148T-CFTR alone (I148T-CFTR: -3.06 Ϯ 0.81 ␮A, n ϭ 21 vs. I148T-CFTR/␣beta␥-mENaC: -5.29 Ϯ 0.81 ␮A, n ϭ 23, P ϭ 0.005). Login to comment 162 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr As described above, I148T-CFTR-mediated currents were increased approximately threefold with the addition of genistein alone (Fig. 1B), and coinjected oocytes had no greater amiloride-insensitive forskolin/IBMX-stimulated currents than oocytes injected with I148T-CFTR alone (Fig. 2A). Login to comment 163 ABCC7 p.Gly551Asp ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr In contrast, I148T-CFTR/mENaC coinjected oocytes had ϳ5.5-fold greater current with addition of genistein (-5.29 Ϯ 0.81 ␮A, n ϭ 23) than in forskolin/IBMX-stimulated oocytes injected with I148T-CFTR alone (-0.97 Ϯ; 0.31 ␮A, n ϭ 19). Login to comment 164 ABCC7 p.Gly551Asp ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr These data suggest synergistic enhancement of I148T-CFTR functional expression by genistein and ␣beta␥-mENaC in the presence forskolin/IBMX, and are qualitatively similar to our previous observations of synergistic activation of G551D-CFTR by genistein and ␣beta␥-mENaC (33). Login to comment 165 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr These data are therefore consistent with genistein improving the regulation of I148T-CFTR by ␣beta␥-mENaC in oocytes. Login to comment 166 ABCC7 p.Ile148Thr I148T-CFTR (10 ng of cRNA) and ␣beta␥-mENaC (0.33 ng cRNA/subunit) were expressed separately or together in oocytes, and TEV was performed as described in EXPERIMENTAL PROCEDURES. A: changes in whole cell currents (-100 mV holding potential) after stimulation with 10 ␮M forskolin/100 ␮M IBMX/50 ␮M genistein that were not inhibited by 10 ␮M amiloride. Login to comment 167 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr Influence of genistein on the regulatory interactions between I148T-CFTR and ␣beta␥-mENaC. Login to comment 168 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr I148T-CFTR (10 ng of cRNA) and ␣beta␥-mENaC (0.33 ng cRNA/subunit) were expressed separately or together in oocytes, and TEV was performed as described in EXPERIMENTAL PROCEDURES. A: changes in whole cell currents (-100 mV holding potential) after stimulation with 10 ␮M forskolin/100 ␮M IBMX/50 ␮M genistein that were not inhibited by 10 ␮M amiloride. Login to comment 169 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr B: amiloride-sensitive whole cell currents (-100 mV holding potential) were determined in oocytes expressing ␣beta␥-mENaC or coexpressing ␣beta␥-mENaC and I148T-CFTR before (open bars) and after (shaded bars) stimulation with 10 ␮M forskolin/100 ␮M IBMX/50 ␮M genistein. Login to comment 170 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr Data obtained from the same I148T-CFTR/␣beta425;-mENaC coinjected oocytes are presented in A and B. Values are means Ϯ SE. Login to comment 171 ABCC7 p.Gly551Asp ABCC7 p.Ile148Thr C607REGULATORY INTERACTIONS OF I148T-CFTR AND ENaC AJP-Cell Physiol • VOL 292 • JANUARY 2007 • www.ajpcell.org -4.44 Ϯ 1.12 ␮A (ϩforskolin/IBMX/genistein) n ϭ 17, P ϭ 0.048] in oocytes injected with ␣beta␥-mENaC alone (Fig. 4B). Login to comment 172 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr In oocytes coexpressing ␣beta␥-mENaC and I148T-CFTR, the amiloride-sensitive currents did not change following stimulation with forskolin/IBMX/gensitein [-0.82 Ϯ 0.17 ␮A (-forskolin/IBMX/gensitein) vs. -0.84 Ϯ 0.18 (ϩforskolin/IBMX/ genistein), n ϭ 23, P ϭ NS]. Login to comment 173 ABCC7 p.Gly551Asp ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr These data are similar to our previous observations with WT-, ⌬F508-, and G551D-CFTR, where this forskolin/IBMX/genistein stimulation in mENaC-mediated current observed in oocytes injected with ␣beta␥- mENaC alone was not present in CFTR/␣beta␥-mENaC coinjected oocytes; such data are consistent with improved regulation of ␣beta␥-mENaC by the forskolin/IBMX-activated mutant CFTRs in the presence of genistein (33, 34). Login to comment 174 ABCC7 p.Ile148Thr We performed surface biotinylation and whole oocyte expression experiments to probe the mechanism by which genistein acts on mENaC and influences mENaC regulation by activated I148T-CFTR (Fig. 5). Login to comment 175 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr Data shown in Fig. 5A suggest that mENaC(beta-V5) expression at the oocyte surface was unaltered by treatment with forskolin/IBMX/genistein in oocytes injected with ␣beta␥-mENaC alone, was decreased with coinjection of I148T-CFTR in the absence of forskolin/IBMX/ genistein activation, and was further decreased upon activation of I148T-CFTR with forskolin/IBMX/genistein in coinjected oocytes. Login to comment 176 ABCC7 p.Ile148Thr Furthermore, in coinjected oocytes, mENaC(beta-V5) surface expression acutely decreased upon activation of I148T-CFTR with forskolin/IBMX/genistein. Login to comment 178 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr Furthermore, in coinjected oocytes, mENaC(beta-V5) surface expression acutely decreased upon activation of I148T-CFTR with forskolin/IBMX/genistein. Login to comment 180 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr These data are therefore consistent with genistein restoring the regulation of mENaC surface expression by activated I148T-CFTR. Login to comment 181 ABCC7 p.Ile148Thr These data are consistent with genistein not acutely altering whole oocyte expression of mENaC in oocytes injected with either ␣beta␥- mENaC alone or upon activation of I148T-CFTR in coinjected oocytes. Login to comment 182 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr Similar to Fig. 3B, the whole oocyte content of mENaC(beta-V5) was not altered by acute treatment with forskolin/IBMX/genistein in oocytes injected with ␣beta␥- mENaC alone, was decreased by coinjection with I148T-CFTR cRNA, and was not further decreased upon activation of I148T-CFTR by forskolin/IBMX/genistein. Login to comment 183 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr These data are consistent with genistein not acutely altering whole oocyte expression of mENaC in oocytes injected with either ␣beta␥- mENaC alone or upon activation of I148T-CFTR in coinjected oocytes. Login to comment 186 ABCC7 p.Ile148Thr ␣beta␥-mENaC (0.33 ng cRNA/subunit) where the beta-subunit contained a COOH-terminal V5 epitope was expressed in oocytes without or with I148T-CFTR (10 ng of cRNA). Login to comment 197 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr In the present study, we assessed the functional interactions of I148T-CFTR and mENaC to gain further insight into the mechanism underlying these interactions, as well as to examine in more detail potential functional defects in I148T-CFTR. Login to comment 198 ABCC7 p.Gly551Asp These findings are discussed in the context of our group`s previous observations regarding the interactions of mENaC with WT-, ⌬F508-, and G551D-CFTR, which are summarized in Table 1. Login to comment 199 ABCC7 p.Ile148Thr I148T-CFTR/mENaC functional interactions. Login to comment 200 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr In the present study, we assessed the functional interactions of I148T-CFTR and mENaC to gain further insight into the mechanism underlying these interactions, as well as to examine in more detail potential functional defects in I148T-CFTR. Login to comment 201 ABCC7 p.Gly551Asp ABCC7 p.Ile148Thr These findings are discussed in the context of our group`s previous observations regarding the interactions of mENaC with WT-, ⌬F508-, and G551D-CFTR, which are summarized in Table 1. Login to comment 202 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr I148T-CFTR/mENaC functional interactions. Login to comment 203 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr Previous observations that I148T-CFTR maintains robust chloride transport function but may be clinically associated with a CF phenotype, perhaps because of an inability to regulate bicarbonate transport (9), prompted us to test the hypothesis that I148T-CFTR may also have defective interactions with ENaC. Login to comment 204 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr Of note, the I148T-CFTR construct used in this work, and in the work of Choi et al. Login to comment 205 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr (9), did not harbor a second mutation in cis, 3199del6, which causes deletion of I1023 and V1024, as has been described in patients with CF and an I148T allele. Login to comment 206 ABCC7 p.Ile148Thr However, despite this robust chloride transport, we found that I148T-CFTR had functional and regulatory interactions with mENaC that differed significantly from those of WT-CFTR previously observed by our group (34, 37). Login to comment 207 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr Thus any functional defects in I148T-CFTR noted in this or the previous studies regarding bicarbonate transport (9) are solely due to the I148T mutation and are not potentially confounded by a second mutation in cis. Login to comment 208 ABCC7 p.Ile148Thr Our data confirm the observations of others (9) that I148T-CFTR maintains the ability to transport chloride at levels similar to WT-CFTR. Login to comment 209 ABCC7 p.Ile148Thr However, despite this robust chloride transport, we found that I148T-CFTR had functional and regulatory interactions with mENaC that differed significantly from those of WT-CFTR previously observed by our group (34, 37). Login to comment 210 ABCC7 p.Ile148Thr These data provide additional molecular support for the hypothesis that I148T-CFTR may be associated with a clinically apparent CF phenotype due to aberrant functional interactions with the transporters of other ions that result in defective regulation of transport of these other ions, such as Naϩ and bicarbonate, in epithelia. Login to comment 213 ABCC7 p.Ile148Thr Furthermore, our present data suggest that forskolin/IBMX-stimulated I148T-CFTR does not acutely alter mENaC functional and surface expression despite the presence of robust ability to transport chloride. Login to comment 216 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr Furthermore, our present data suggest that forskolin/IBMX-stimulated I148T-CFTR does not acutely alter mENaC functional and surface expression despite the presence of robust ability to transport chloride. Login to comment 217 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr However, it seems somewhat unlikely to us that intracellular Cl- would increase significantly more with activation of WT- vs. I148T-CFTR given the similar levels of whole oocyte CFTR-mediated current measured by TEV in the present study for I148T-CFTR and in our previous studies for WT-CFTR (34, 37). Login to comment 219 ABCC7 p.Ile148Thr We have not directly measured changes in intracellular Cl- because of expression of WT- vs. I148T-CFTR in these experiments. Login to comment 220 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr However, it seems somewhat unlikely to us that intracellular Cl- would increase significantly more with activation of WT- vs. I148T-CFTR given the similar levels of whole oocyte CFTR-mediated current measured by TEV in the present study for I148T-CFTR and in our previous studies for WT-CFTR (34, 37). Login to comment 222 ABCC7 p.Gly551Asp ABCC7 p.Ile148Thr Wild type N/A Same Decrease* Decrease* Increased 34, 37 ⌬F508 NBD-1 Decreased No change No change No change 34 G551D NBD-1 Same Decrease No change Increased 33 I148T Intracellular loop 1 Same Decrease* No change* No change Present study WT, wild type; mENaC, murine epithelial sodium channel; NBD-1, nucleotide binding domain-1. Login to comment 224 ABCC7 p.Ile148Thr C609REGULATORY INTERACTIONS OF I148T-CFTR AND ENaC AJP-Cell Physiol • VOL 292 • JANUARY 2007 • www.ajpcell.org regulation, these data also implicate NBD-1 as a critical element in such regulation. Login to comment 225 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Ile148Thr Wild type N/A Same Decrease* Decrease* Increased 34, 37 32c;F508 NBD-1 Decreased No change No change No change 34 G551D NBD-1 Same Decrease No change Increased 33 I148T Intracellular loop 1 Same Decrease* No change* No change Present study WT, wild type; mENaC, murine epithelial sodium channel; NBD-1, nucleotide binding domain-1. Login to comment 226 ABCC7 p.Ile148Thr At first glance, I148T-CFTR should have a structurally and functionally intact NBD-1, because this mutation is located within CFTR`s first cytoplasmic loop. Login to comment 227 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr C609REGULATORY INTERACTIONS OF I148T-CFTR AND ENaC AJP-Cell Physiol • VOL 292 • JANUARY 2007 • www.ajpcell.org regulation, these data also implicate NBD-1 as a critical element in such regulation. Login to comment 228 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Ile148Thr That a similar NBD-1/R peptide fragment containing the G551D mutation (20) and the CFTR mutants G551D (33) and ⌬F508 (34) do not manifest forskolin/ IBMX-regulated inhibition of ENaC also suggests that a structurally and functionally intact NBD-1 is required for this regulatory interaction. Login to comment 229 ABCC7 p.Ile148Thr At first glance, I148T-CFTR should have a structurally and functionally intact NBD-1, because this mutation is located within CFTR`s first cytoplasmic loop. Login to comment 230 ABCC7 p.Ile148Thr One would therefore have predicted that I148T-CFTR should demonstrate forskolin/ IBMX-dependent inhibition of mENaC functional and surface expression. Login to comment 231 ABCC7 p.Ile148Thr Since I148T-CFTR does not acutely decrease mENaC functional activity and surface expression upon stimulation, these data suggest either that domains of CFTR other than NBD-1 may be important in the regulatory interactions of CFTR and mENaC or that changes in CFTR`s first cytoplasmic loop may influence NBD-1 form and/or function. Login to comment 232 ABCC7 p.Gly551Asp Genistein potentiates CFTR (WT, ⌬F508, and G551D) chloride transport by increasing channel open probability (1, 36). Login to comment 234 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr In the present study, we have demonstrated that genistein is able to potentiate chloride transport by I148T-CFTR, to enable mENaC to potentiate chloride transport by I148T-CFTR in a synergistic fashion, and to restore the ability of activated I148T-CFTR to acutely decrease the surface expression of mENaC in coinjected oocytes. Login to comment 235 ABCC7 p.Gly551Asp ABCC7 p.Ile148Thr Genistein potentiates CFTR (WT, ⌬F508, and G551D) chloride transport by increasing channel open probability (1, 36). Login to comment 236 ABCC7 p.Gly551Asp These effects are similar to genistein`s actions to potentiate ⌬F508- and G551D-CFTR-mediated chloride transport and improve the regulatory interactions of these mutants with mENaC (33, 34). Login to comment 237 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr In the present study, we have demonstrated that genistein is able to potentiate chloride transport by I148T-CFTR, to enable mENaC to potentiate chloride transport by I148T-CFTR in a synergistic fashion, and to restore the ability of activated I148T-CFTR to acutely decrease the surface expression of mENaC in coinjected oocytes. Login to comment 238 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr These latter two observations suggest that genistein improves the regulatory interactions of I148T-CFTR with mENaC. Login to comment 239 ABCC7 p.Gly551Asp ABCC7 p.Ile148Thr These effects are similar to genistein`s actions to potentiate ⌬F508- and G551D-CFTR-mediated chloride transport and improve the regulatory interactions of these mutants with mENaC (33, 34). Login to comment 240 ABCC7 p.Ile148Thr In conclusion, we have demonstrated that I148T-CFTR has abnormal regulatory interactions with mENaC in Xenopus oocytes despite maintaining robust levels of chloride transport. Login to comment 241 ABCC7 p.Ile148Thr Our data suggest that genistein slightly increases mENaC functional expression without altering surface expression in the absence of CFTR but is also able to "repair" the regulatory interactions between I148T-CFTR and mENaC. Login to comment 242 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr If there is a common mechanism by which activated CFTR regulates HCO3 - transport and ENaC trafficking/surface expression, we can speculate that genistein may also repair regulation of HCO3 - transport by I148T-CFTR. Login to comment 243 ABCC7 p.Ile148Thr In conclusion, we have demonstrated that I148T-CFTR has abnormal regulatory interactions with mENaC in Xenopus oocytes despite maintaining robust levels of chloride transport. Login to comment 245 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr These data provide further molecular and physiological evidence that the I148T mutation is associated with significant CFTR regulatory dysfunction and are consistent with I148T being a CF disease-causing mutation. Login to comment