ABCMdb

PMID: 17182731

Suaud L, Yan W, Carattino MD, Robay A, Kleyman TR, Rubenstein RC
Regulatory interactions of N1303K-CFTR and ENaC in Xenopus oocytes: evidence that chloride transport is not necessary for inhibition of ENaC.
Am J Physiol Cell Physiol. 2007 Apr;292(4):C1553-61. Epub 2006 Dec 20., [PubMed]

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No. Mutations Sentence Comment

6 ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys It is published 12 timesAJP - Cell Physiology CALL FOR PAPERS Protein and Vesicle Trafficking, Cytoskeleton Regulatory interactions of N1303K-CFTR and ENaC in Xenopus oocytes: evidence that chloride transport is not necessary for inhibition of ENaC Laurence Suaud,1 Wusheng Yan,1 Marcelo D. Carattino,3 Amal Robay,1 Thomas R. Kleyman,3 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; and 3 Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania Submitted 9 February 2006; accepted in final form 15 December 2006 Suaud L, Yan W, Carattino MD, Robay A, Kleyman TR, Rubenstein RC. Regulatory interactions of N1303K-CFTR and ENaC in Xenopus oocytes: evidence that chloride transport is not necessary for inhibition of ENaC. Login to comment 9 ABCC7 p.Gly551Asp CFTR`s first nucleotide binding fold (NBD-1) may be important in these interactions, as dysfunctional CFTRs containing mutations within NBD-1, such as ⌬F508 and G551D, lack such functional interactions with murine ENaC (mENaC). Login to comment 10 ABCC7 p.Asn1303Lys We hypothesized that a dysfunctional CFTR containing a non-NBD-1 mutation would retain regulatory interactions with mENaC and tested this hypothesis for N1303K-CFTR, where the mutation is located in CFTR`s second nucleotide binding fold (NBD-2). Login to comment 11 ABCC7 p.Asn1303Lys cRNA for ␣beta␥-mENaC and N1303K-CFTR was injected separately or together into Xenopus oocytes. Login to comment 13 ABCC7 p.Asn1303Lys Injection of N1303K (class II trafficking mutation) yielded low levels of CFTR function on activation with forskolin and 3-isobutyl-1-methylxanthine (IBMX). Login to comment 14 ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys In coinjected oocytes, N1303K did not alter mENaC functional expression or surface expression before activation of N1303K. Login to comment 16 ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys However, unlike our observations with ⌬F508, activation of N1303K acutely decreased mENaC functional and surface expression, and N1303K currents were enhanced by coinjection of mENaC. Login to comment 17 ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys Furthermore, genistein only mildly enhanced the functional expression of N1303K-CFTR and did not improve regulation of ENaC by N1303K-CFTR. Login to comment 29 ABCC7 p.Gly551Asp A similar NBD-1/R peptide fragment containing the G551D mutation did not demonstrate forskolin/IBMX-regulated inhibition of ENaC (19). Login to comment 30 ABCC7 p.Gly551Asp CFTRs containing NBD-1 mutations, such as ⌬F508-CFTR and G551D-CFTR, lack regulatory interactions with ␣beta␥-murine ENaC (mENaC) when activated by forskolin-IBMX (31, 32). Login to comment 31 ABCC7 p.Gly551Asp These and other data (4, 29) suggest that form and/or function of NBD-1 is important in the inhibition of ENaC by activated CFTR. Interestingly, regulation of mENaC by ⌬F508 and G551D was restored by further activation or "repair" of these mutants with genistein (31, 32), which may interact with CFTR`s second nucleotide binding fold (NBD-2) (25). Login to comment 32 ABCC7 p.Asn1303Lys N1303K-CFTR, like ⌬F508-CFTR, has defective intracellular trafficking characteristic of a class II CFTR mutation (11). Login to comment 34 ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys We hypothesized that the permissive temperature of the oocyte system would allow trafficking of N1303K-CFTR to the oocyte membrane and that activated N1303K Address for reprint requests and other correspondence: R. C. Rubenstein, Div. of Pulmonary Medicine, Abramson 410C, Children`s Hospital of Philadelphia, 34th St. and Civic Center Blvd., Philadelphia, PA 19104 (e-mail: rrubenst@mail.med.upenn.edu). Login to comment 39 ABCC7 p.Asn1303Lys We observed that N1303K-CFTR has low Cl-conductance but is still able to regulate mENaC on activation. Login to comment 40 ABCC7 p.Gly551Asp ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys We also observed reduced activation of N1303K by genistein and a lack of improved N1303K regulation of mENaC with genistein, which differs significantly from our previous data with ⌬F508 and G551D (31, 32). Login to comment 44 ABCC7 p.Asn1303Lys Expression of human N1303K-CFTR and mouse ENaC in Xenopus oocytes. Login to comment 45 ABCC7 p.Asn1303Lys A cDNA encoding human N1303K-CFTR was constructed by site-directed mutagenesis 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 46 ABCC7 p.Asn1303Lys N1303K-CFTR and mouse ␣beta␥-ENaC were expressed in Xenopus oocytes as previously described (16, 32). Login to comment 47 ABCC7 p.Asn1303Lys Briefly, human N1303K-CFTR and murine ␣-, beta-, and ␥-ENaC cRNAs were prepared with a cRNA synthesis kit (mMESSAGE mMACHINE, Ambion, Austin, TX) according to the manufacturer`s protocol. Login to comment 50 ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys Each batch of oocytes obtained from an individual frog was injected (50 nl/oocyte) with a Nanoject II microinjector (Drummond Scientific, Broomall, PA), with ␣-, beta-, and ␥-subunits of mENaC (0.33 ng/subunit), N1303K-CFTR (10 ng), or a combination of mENaC and N1303K-CFTR cRNAs dissolved in RNase-free water. Login to comment 60 ABCC7 p.Asn1303Lys N1303K-CFTR was activated by perfusion of the oocyte with buffer containing 10 ␮M forskolin and 100 ␮M IBMX for 20 or 25 min (16, 32); here (data not shown) and in our previous work (31-33, 35), maximal activation of WT and mutant CFTR-mediated currents was achieved after 10-15 min of such perfusion. Login to comment 62 ABCC7 p.Asn1303Lys In all experiments, N1303K-CFTR Cl- current 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 66 ABCC7 p.Asn1303Lys Single-channel studies of N1303K-CFTR-mediated Cl-conductance were performed as described by Suaud et al. Login to comment 69 ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys Oocytes expressing N1303K-CFTR alone or both N1303K-CFTR and ␣beta␥-mENaC were placed in a hypertonic solution (bath solution supplemented with 200 mM sucrose, 10 ␮M forskolin, 100 ␮M IBMX, and 50 ␮M genistein) for 5-10 min, and the vitelline membranes were then removed manually. Login to comment 89 ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys 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 N1303K-CFTR vs. oocytes coinjected with cRNAs for both ENaC and N1303K-CFTR). Login to comment 94 ABCC7 p.Asn1303Lys C1554 RESULTS Functional expression of N1303K-CFTR in Xenopus oocytes. Login to comment 95 ABCC7 p.Asn1303Lys We used the Xenopus oocyte expression system and TEV technique to examine the functional expression of N1303K-CFTR. Login to comment 96 ABCC7 p.Asn1303Lys In vitro studies suggest that N1303K-CFTR is a class II CFTR trafficking mutant that does not achieve mature glycosylation and leads to a Cl- permeability defect similar to ⌬F508-CFTR (11). Login to comment 97 ABCC7 p.Asn1303Lys We hypothesized that the permissive temperature of oocytes (18°C) would allow N1303K-CFTR delivery to the oocyte plasma membrane. Login to comment 98 ABCC7 p.Asn1303Lys We measured whole cell currents at varying clamping potentials in oocytes injected with N1303K-CFTR cRNA (Fig. 1) and generated I-voltage (V) curves from data obtained before and after 20 min of incubation with 10 ␮M forskolin-100 ␮M IBMX to activate endogenous protein kinase A (Fig. 1A). Login to comment 100 ABCC7 p.Asn1303Lys These data suggest that expression of N1303K leads to small amounts of forskolin/IBMX-stimulated Cl- current in oocytes. Login to comment 102 ABCC7 p.Gly551Asp The isoflavone genistein increases Cl-transport by activated WT and mutant CFTRs including ⌬F508-CFTR and G551D-CFTR (13, 31, 32). Login to comment 103 ABCC7 p.Asn1303Lys We therefore assessed the effect of genistein on oocytes injected with N1303K-CFTR. Login to comment 104 ABCC7 p.Asn1303Lys Figure 1B demonstrates the I-V relationship of N1303K-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 15 min. Login to comment 106 ABCC7 p.Asn1303Lys These data are consistent with IBMX/forskolin-stimulated N1303K-CFTR-mediated Cl- cur- rent being at best modestly enhanced by genistein in Xenopus oocytes. Login to comment 107 ABCC7 p.Gly551Asp ABCC7 p.Asn1303Lys Interestingly, this stimulation of activated N1303K-CFTR by genistein is minimal compared with our previously observed stimulation of ⌬F508-CFTR and G551D-CFTR by genistein (5.7-fold and 4-fold stimulation by genistein, respectively) (31, 32). Login to comment 108 ABCC7 p.Asn1303Lys To ensure that these small increases in whole oocyte current were not a result of nonspecific activation of endogenous channels, we also assessed I-V relationships of N1303K-injected oocytes in the presence of 5 mM tetraethylamine chloride, 5 mM BaCl2, and 200 ␮M 4,4Ј-diisothiocyanostilbene-2,2Ј-disulfonic acid (DIDS) in the bath solution. Login to comment 110 ABCC7 p.Asn1303Lys Unstimulated N1303K-mediated current at -100 mV was -0.38 Ϯ 0.09 ␮A (n ϭ 8). Login to comment 114 ABCC7 p.Asn1303Lys Expression of N1303K-cystic fibrosis transmembrane conductance regulator (CFTR) in Xenopus oocytes. Login to comment 115 ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys A: N1303K-CFTR was expressed in oocytes and 2-electrode voltage clamp (TEV) performed as described in EXPERIMENTAL PROCEDURES. Whole cell currents were measured by voltage clamping oocytes in 20-mV steps between -140 and ϩ60 mV adjusted for baseline transmembrane potential. Shown are current-voltage (I-V) relationships (n ϭ 25) in ND96 buffer before (F) or after (E) incubation with 10 ␮M forskolin and 100 ␮M 3-isobutyl-1-methylxanthine (IBMX) for 25 min. Error bars contained within symbols are not apparent. B: N1303K-CFTR was expressed in oocytes and TEV performed. Login to comment 118 ABCC7 p.Asn1303Lys C: N1303K-CFTR was expressed in oocytes, and TEV was performed as described above with the exception that the ND96 buffer was supplemented with 5 mM tetraethylamine chloride, 5 mM BaCl2, and 200 ␮M 4,4Ј-diisothiocyanostilbene-2,2Ј-disulfonic acid (DIDS) to block putative endogenous channels. Login to comment 121 ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys C1555REGULATORY INTERACTIONS OF N1303K-CFTR AND ENaC AJP-Cell Physiol • VOL 292 • APRIL 2007 • www.ajpcell.org Coexpression of N1303K-CFTR and ␣beta␥-ENaC. Login to comment 129 ABCC7 p.Asn1303Lys On the basis of these considerations, we hypothesized that activated N1303K-CFTR might retain the ability to decrease mENaC functional and surface expression because it has an intact NBD-1. Login to comment 130 ABCC7 p.Asn1303Lys Furthermore, we felt that if activated N1303K-CFTR decreased mENaC functional and surface expression, it would be inconsistent with the notion that Cl-transport was important in such regulatory interactions. Login to comment 131 ABCC7 p.Asn1303Lys We therefore assessed the interregulation of N1303K-CFTR and ␣beta␥-mENaC in oocytes. Login to comment 132 ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys Figure 2 depicts whole cell currents measured at a holding potential of -100 mV in oocytes injected with N1303K-CFTR (10 ng), ␣beta␥-mENaC (0.33 ng/subunit), or both N1303K-CFTR and ␣beta␥-mENaC. Login to comment 133 ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys Oocytes coinjected with N1303K-CFTR and ␣beta␥-mENaC had 4.1-fold increased forskolin/IBMX-stimulated, amiloride-insensitive current compared with oocytes injected with N1303K-CFTR alone [Fig. 2A, -0.12 Ϯ 0.04 ␮A (N1303K-CFTR) vs. -0.49 Ϯ 0.15 ␮A (N1303K-CFTR/␣beta␥-ENaC); P ϭ 0.02]. Login to comment 134 ABCC7 p.Asn1303Lys Thus coexpression of ␣beta␥-mENaC in oocytes resulted in enhancement of the amiloride-insensitive component of the forskolin/IBMX-stimulated current, which likely represents N1303K-CFTR-mediated Cl- cur- rent. Login to comment 135 ABCC7 p.Asn1303Lys This enhancement of N1303K-CFTR Cl- current by coexpression of ␣beta␥-mENaC in oocytes is similar to the previously described enhancement of WT CFTR current by coexpression of ␣beta␥-mENaC in oocytes (7, 15, 16, 21, 32). Login to comment 137 ABCC7 p.Asn1303Lys Oocytes coinjected with both N1303K-CFTR and ␣beta␥-mENaC expressed amiloride-sensitive whole cell currents (-2.48 Ϯ 0.70 ␮A, n ϭ 26) in the absence of forskolin-IBMX that were similar to the oocytes expressing ␣beta␥-mENaC alone (compare open bars in Fig. 2B). Login to comment 138 ABCC7 p.Asn1303Lys After N1303K-CFTR activation with forskolin-IBMX, the amiloride-sensitive whole cell current was significantly lower (-1.67 Ϯ 0.42; n ϭ 26, p ϭ 0.03), which is similar to our group`s previous observations of WT CFTR behavior in this system (16, 32, 35). Login to comment 139 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp These observations directly contrast with our previous observations with G551D-CFTR, where a similar magnitude of stimulated G551D-mediated current did not result in a decrease in amiloride-sensitive whole oocyte current (31). Login to comment 142 ABCC7 p.Asn1303Lys As shown in Fig. 3A, mENaC(beta-V5) expression at the oocyte surface was unaltered by exposure to forskolin-IBMX for 20 min in oocytes injected with ␣beta␥-mENaC alone and was also unaltered by coinjection of N1303K-CFTR before forskolin-IBMX activation. Login to comment 143 ABCC7 p.Asn1303Lys In contrast, mENaC(beta-V5) surface expression decreased on activation of N1303K-CFTR with forskolin-IBMX for 20 min in coinjected oocytes. Login to comment 145 ABCC7 p.Asn1303Lys These data suggest that activated N1303K-CFTR, even with its minimal Cl-transport activity, retains the ability to regulate the surface and functional expression of ␣beta␥-mENaC in oocytes. Login to comment 147 ABCC7 p.Asn1303Lys Expression of mENaC(beta-V5) was similar in oocytes injected with ␣beta␥-mENaC alone or coinjected with N1303K-CFTR. Login to comment 148 ABCC7 p.Asn1303Lys Activation of N1303K-CFTR did not alter the whole oocyte expression of mENaC(beta-V5). Login to comment 150 ABCC7 p.Asn1303Lys Expression of N1303K-CFTR and murine epithelial Naϩ channel (mENaC) in Xenopus oocytes. Login to comment 151 ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys N1303K-CFTR and ␣beta␥-mENaC were expressed separately or together in oocytes and TEV performed as described in EXPERIMENTAL PROCEDURES. A: stimulated CFTR currents represent changes in whole cell currents that were not inhibited by 10 ␮M amiloride (-100-mV holding potential) in oocytes injected with N1303K-CFTR alone or coinjected with N1303K-CFTR and ␣-, beta-, and ␥-mENaC, after stimulation with 10 ␮M forskolin-100 ␮M IBMX. Login to comment 152 ABCC7 p.Asn1303Lys Means Ϯ SE are illustrated. B: amiloride-sensitive whole cell currents (-100-mV holding potential) were determined in oocytes expressing mENaC or coexpressing mENaC and N1303K-CFTR before (open bars) and after (gray bars) stimulation with 10 ␮M forskolin-100 ␮M IBMX. Login to comment 153 ABCC7 p.Asn1303Lys Data obtained from the same N1303K-CFTR/mENaC-coinjected oocytes are presented in A and B. Means Ϯ SE are illustrated. Login to comment 155 ABCC7 p.Asn1303Lys C1556 activated N1303K-CFTR is similar to WT CFTR in its ability to acutely decrease the amount of mENaC(beta-V) present at the oocyte surface without altering the total amount of mENaC(beta-V5) present in the oocyte. Login to comment 156 ABCC7 p.Asn1303Lys As an additional test of whether extracellular to intracellular Cl-transport was required to observe inhibition of ENaC on N1303K-CFTR activation, we performed a series of ion substitution experiments. Login to comment 157 ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys Oocytes were injected with cRNAs for either N1303K-CFTR (10 ng) or ␣beta␥-mENaC (0.33 ng/subunit) alone or with both N1303K-CFTR and ␣beta␥-mENaC cRNAs. Login to comment 159 ABCC7 p.Asn1303Lys Figure 4A shows an I/V plot for oocytes injected with N1303K-CFTR before and after 25 min of activation with forskolin-IBMX in this Cl- -free bath. Login to comment 160 ABCC7 p.Asn1303Lys These data demonstrate a lack of significant forskolin/IBMX-stimulated current at -100 mV holding potential on activation of N1303K-CFTR. Login to comment 162 ABCC7 p.Asn1303Lys Surface and whole cell expression of ␣-, beta-V5- and ␥-mENaC in oocytes with or without N1303K-CFTR. Login to comment 173 ABCC7 p.Asn1303Lys Function and regulatory interactions of N1303K-CFTR and mENaC in Xenopus oocytes in the absence of extracellular Cl- . Login to comment 175 ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys A: N1303K-CFTR was expressed in oocytes and TEV performed as described in EXPERIMENTAL PROCEDURES. Whole cell currents were measured by voltage clamping oocytes in 20-mV steps between -140 and ϩ60 mV adjusted for baseline transmembrane potential. Shown are I-V relationships before (F) or after (E) incubation with 10 ␮M forskolin and 100 ␮M IBMX for 25 min. Error bars contained within symbols are not apparent. B: amiloride-sensitive whole cell currents (-100-mV holding potential) were determined in oocytes expressing mENaC or coexpressing mENaC and N1303K-CFTR before (open bars) and after (gray bars) stimulation with 10 ␮M forskolin-100 ␮M IBMX for 25 min. Login to comment 177 ABCC7 p.Asn1303Lys C1557REGULATORY INTERACTIONS OF N1303K-CFTR AND ENaC AJP-Cell Physiol • VOL 292 • APRIL 2007 • www.ajpcell.org mV, were -0.30 Ϯ 0.04 and -0.45 Ϯ 0.07 ␮A (mean Ϯ SE; n ϭ 6, P ϭ ns) before and after forskolin-IBMX, respectively. Login to comment 180 ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys Importantly, even in this Cl- -free bath, activation of N1303K-CFTR with forskolin-IBMX led to a significant reduction in the relative amiloride-sensitive whole cell current in oocytes co-injected with N1303K-CFTR and ␣beta␥-mENac (Fig. 4B); the relative amiloride-sensitive current after N1303K activation was 0.79 Ϯ 0.08 (n ϭ 10, P ϭ 0.027). Login to comment 182 ABCC7 p.Asn1303Lys Importantly, even when the medians for these data were compared with nonparametric statistical techniques (Wilcoxon signed rank test), oocytes injected with mENaC alone had no significant change in amiloride-sensitive current in response to forskolin-IBMX, while oocytes coinjected with N1303K-CFTR and ␣beta␥-mENaC again demonstrated significant reduction in amiloride-sensitive current in response to forskolin-IBMX. Login to comment 184 ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys In contrast, oocytes coinjected with N1303K-CFTR and ␣beta␥-mENaC had significantly decreased median amiloride-sensitive currents of -0.32 (-0.50, -0.14) ␮A after forskolin-IBMX compared with -0.39 (-0.54, -0.20) ␮A before N1303K activation (n ϭ 10, P ϭ 0.02). Login to comment 185 ABCC7 p.Asn1303Lys These data are therefore consistent with regulation of mENaC by activated N1303K-CFTR in the absence of inward Cl-transport and suggest that regulation of mENaC by activated CFTR can occur in the absence of Cl-transport. Login to comment 186 ABCC7 p.Asn1303Lys Interregulation of N1303K-CFTR and ␣beta␥-mENaC after forskolin-IBMX-genistein stimulation. Login to comment 187 ABCC7 p.Asn1303Lys Our data suggest that N1303K-CFTR has poor Cl-conductance in response to activation with forskolin-IBMX. Login to comment 188 ABCC7 p.Asn1303Lys This may indicate that N1303K has defects in other functional properties. Login to comment 189 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Asn1303Lys We therefore examined whether the CFTR potentiator genistein, which activates G551D-CFTR, ⌬F508-CFTR, and WT CFTR Cl-conductance and restores functional interactions between ␣beta␥- mENaC and the G551D-CFTR and ⌬F508-CFTR mutants (31, 32), is able to activate N1303K-CFTR and improve its regulation with mENaC. Login to comment 190 ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys As shown in Fig. 5A, the amiloride-insensitive, or N1303K-mediated, component of the forskolin/ IBMX/genistein-stimulated (forskolin-IBMX for 25 min followed by genistein for 15 min) whole cell current in oocytes coexpressing N1303K-CFTR and ␣beta␥-mENaC was 9.3-fold greater than the forskolin/IBMX/genistein-stimulated current measured in oocytes expressing N1303K-CFTR alone [N1303K-CFTR -0.15 Ϯ 0.02 ␮A (n ϭ 19) vs. N1303K-CFTR/mENaC -1.4 Ϯ 0.52 ␮A (n ϭ 19); P ϭ 0.006]. Login to comment 192 ABCC7 p.Gly551Asp In contrast, coinjection of ␣beta␥-mENaC increases forskolin/ IBMX/genistein-stimulated G551D-CFTR-mediated current in oocytes but does not alter G551D`s Po (31). Login to comment 193 ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys We attempted to determine whether this significant increase in forskolin/IBMX/ genistein-stimulated, N1303K-mediated whole oocyte current with coinjection of ␣beta␥-mENaC reflected a change in N1303K-CFTR`s Po. Login to comment 194 ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys Unfortunately, we were unable to observe single-channel CFTR currents in 50 patches obtained from oocytes expressing either N1303K-CFTR alone or N1303K-CFTR with ␣beta␥-mENaC. Login to comment 195 ABCC7 p.Asn1303Lys However, we were able to record mENaC-mediated outward Naϩ single-channel currents in oocytes coexpressing N1303K-CFTR and ␣beta␥-mENaC (data not shown). Login to comment 196 ABCC7 p.Asn1303Lys This suggests that these oocytes were viable and able to express channels, but that the number of N1303K-CFTR channels at the oocyte surface was insufficient to detect single channel events. Login to comment 200 ABCC7 p.Asn1303Lys Influence of genistein on the regulatory interactions between N1303K-CFTR and ␣beta␥-mENaC. Login to comment 201 ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys N1303K-CFTR and ␣beta␥-mENaC were expressed separately or together in oocytes and TEV performed as described in EXPERIMENTAL PROCEDURES. A: changes in whole cell currents (-100-mV holding potential) before (-0.15 Ϯ 0.02 ␮A) and after stimulation with 10 ␮M forskolin-100 ␮M IBMX-50 ␮M genistein that were not inhibited by 10 ␮M amiloride are illustrated. B: amiloride-sensitive whole cell currents (-100-mV holding potential) were determined in oocytes expressing ␣beta␥-mENaC or coexpressing ␣beta␥-mENaC and N1303K-CFTR before (open bars) and after (dark gray bars) stimulation with 10 ␮M forskolin-100 ␮M IBMX-50 ␮M genistein. Login to comment 202 ABCC7 p.Asn1303Lys Data obtained from the same N1303K-CFTR/␣beta␥-mENaC-coinjected oocytes are presented in A and B. Means Ϯ SE are illustrated. Login to comment 205 ABCC7 p.Asn1303Lys In oocytes coexpressing ␣beta␥- mENaC and N1303K-CFTR, the amiloride-sensitive currents also modestly but significantly increased after stimulation with forskolin-IBMX-genistein [-1.9 Ϯ 0.4 ␮A (-forskolin-IBMX- genistein) vs. -3.0 Ϯ 0.5 (ϩforskolin-IBMX-genistein); n ϭ 19, P Ͻ 0.001]. Login to comment 206 ABCC7 p.Gly551Asp These data contrast with our previous observations with WT, ⌬F508-, and G551D-CFTR, where such an increase in ␣beta␥-mENaC-mediated current after forskolin-IBMX-genistein in CFTR/mENaC-coinjected oocytes was not present (31, 32). Login to comment 207 ABCC7 p.Gly551Asp ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys These data are consistent with genistein not further improving regulation of ␣beta␥-mENaC by N1303K-CFTR and therefore being a less effective potentiator of N1303K-CFTR function than it is for ⌬F508- and G551D-CFTR. Login to comment 208 ABCC7 p.Asn1303Lys Synergistic activation of N1303K-CFTR by genistein and ENaC. Login to comment 209 ABCC7 p.Asn1303Lys As discussed above, forskolin/IBMX-stimulated, N1303K-CFTR-mediated currents were increased ϳ1.3-fold with the addition of genistein alone (Fig. 1, A and B). Login to comment 210 ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys Oocytes coinjected with N1303K-CFTR and ␣beta␥-mENaC had approximately fourfold greater amiloride-insensitive, forskolin/IBMX-stimulated N1303K-mediated currents compared with oocytes injected with N1303K-CFTR alone (Fig. 2A). Login to comment 211 ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys Interestingly, oocytes coinjected with N1303K-CFTR and ␣beta␥-mENaC had an ϳ10-fold greater N1303K-mediated current in the presence of genistein compared with oocytes expressing N1303K-CFTR alone and stimulated with forskolin-IBMX (Fig. 5A). Login to comment 212 ABCC7 p.Gly551Asp ABCC7 p.Asn1303Lys ABCC7 p.Ile148Thr These data demonstrate synergistic enhancement of N1303K-CFTR functional expression by genistein and ␣beta␥-mENaC in the presence of forskolin-IBMX, and are similar to our previous data suggesting synergistic enhancement of G551D-CFTR (31) and I148T-CFTR (33) function by genistein and ␣beta␥-mENaC. Login to comment 213 ABCC7 p.Asn1303Lys These data are therefore consistent with genistein and ␣beta␥-mENaC enhancing N1303K-CFTR function in oocytes by different and complementary mechanisms. Login to comment 214 ABCC7 p.Asn1303Lys We attempted to probe the mechanisms underlying this synergy by performing single-channel recordings of N1303K-CFTR, but, as detailed above, these experiments were not successful. Login to comment 215 ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys Furthermore, the quite low NPo for N1303K-CFTR suggested by these experiments, and our group`s previous experience with biochemical detection of surface CFTR expression by surface biotinylation in oocytes, suggests that the surface expression of N1303K-CFTR in oocytes is likely below the limit of experimental detection. Login to comment 242 ABCC7 p.Asn1303Lys The forskolin/ IBMX-stimulated current of N1303K-CFTR is very low (Figs. Login to comment 243 ABCC7 p.Asn1303Lys 1 and 2), yet activation of N1303K leads to decreased ␣beta␥- mENaC functional and surface expression in a fashion similar to WT CFTR (35). Login to comment 244 ABCC7 p.Asn1303Lys Furthermore, the functional expression of mENaC also decreases on activation of N1303K-CFTR when inward Cl-transport is prevented by removing Cl- from the bath solution (Fig. 4). Login to comment 250 ABCC7 p.Asn1303Lys Alternatively, as described here and in our previous C1559REGULATORY INTERACTIONS OF N1303K-CFTR AND ENaC AJP-Cell Physiol • VOL 292 • APRIL 2007 • www.ajpcell.org work (35), activation of CFTR decreases mENaC surface expression. Login to comment 252 ABCC7 p.Asn1303Lys Here, Cl-transport by activated N1303K-CFTR was not required to observed inhibition of mENaC. Login to comment 253 ABCC7 p.Asn1303Lys ABCC7 p.Ile148Thr In contrast, we recently demonstrated that activation of I148T-CFTR, which maintains the ability to transport Cl- at levels similar to WT CFTR (9, 33), did not lead to inhibition of mENaC in oocytes despite supporting significantly higher Cl-transport (33) than N1303K-CFTR did in the present experiments. Login to comment 256 ABCC7 p.Gly551Asp A similar NBD-1/R peptide fragment containing the G551D mutation did not demonstrate forskolin/IBMX-regulated inhibition of ENaC (19). Login to comment 259 ABCC7 p.Gly551Asp We previously showed (31, 32) that CFTRs with NBD-1 mutations (G551D-CFTR and ⌬F508-CFTR) lack the ability to regulate ␣beta␥-mENaC when activated by forskolin-IBMX. Login to comment 260 ABCC7 p.Asn1303Lys Our data here with N1303K are also consistent with a structurally and functionally intact NBD-1 being a critical element in this regulatory interaction. Login to comment 261 ABCC7 p.Asn1303Lys Like ⌬F508-CFTR, N1303K-CFTR is a class II CFTR trafficking mutation that, according to our data here, is able to achieve a low level of functional expression at the permissive temperature of 18°C in oocytes. Login to comment 262 ABCC7 p.Asn1303Lys Yet, unlike ⌬F508-CFTR, N1303K-CFTR has an intact NBD-1 and a mutant NBD-2 and interestingly retains the ability to regulate ␣beta␥-mENaC functional and surface expression in oocytes on activation. Login to comment 263 ABCC7 p.Asn1303Lys These data with N1303K-CFTR suggest that a mutant NBD-2, which lacks intrinsic adenylate kinase activity (26), still allows for regulation of ␣beta␥-mENaC by activated CFTR. Login to comment 266 ABCC7 p.Gly551Asp Genistein activates CFTR (WT, ⌬F508, and G551D) Cl-transport by increasing channel open time and decreasing channel closing, thereby increasing channel Po (1, 34). Login to comment 269 ABCC7 p.Asn1303Lys Genistein only modestly (1.3-fold) enhanced N1303K-CFTR mediated current in these experiments. Login to comment 270 ABCC7 p.Gly551Asp These data contrast with our previous observations that genistein enhanced Cl-transport by WT CFTR and the NBD-1 mutants ⌬F508- and G551D-CFTR by four- to sixfold (31, 32). Login to comment 271 ABCC7 p.Gly551Asp ABCC7 p.Asn1303Lys Furthermore, while genistein improves the regulation of ␣beta␥-mENaC by activated ⌬F508- and G551D-CFTR (31, 32), it did not enhance regulation of ␣beta␥-mENaC by activated N1303K. Login to comment 274 ABCC7 p.Asn1303Lys These data suggest that N1303K-CFTR retains regulatory interactions with ␣beta␥-mENaC in oocytes. Login to comment 275 ABCC7 p.Asn1303Lys Given the low level of N1303K-CFTR functional expression, and our observations that regulation of mENaC occurred in the absence of inward Cl-transport, these data provide additional evidence that CFTR-mediated Cl-transport is not required for such interactions to occur. Login to comment 276 ABCC7 p.Asn1303Lys Rather, these data provide additional support for the notion that NBD-1 is a critical element for these interactions and that the class II CFTR mutants ⌬F508 and N1303K are functionally distinct. Login to comment