ABCMdb

PMID: 12386156

Suaud L, Carattino M, Kleyman TR, Rubenstein RC
Genistein improves regulatory interactions between G551D-cystic fibrosis transmembrane conductance regulator and the epithelial sodium channel in Xenopus oocytes.
J Biol Chem. 2002 Dec 27;277(52):50341-7. Epub 2002 Oct 16., 2002-12-27 [PubMed]

Sentences

No. Mutations Sentence Comment

0 ABCC7 p.Gly551Asp Genistein Improves Regulatory Interactions between G551D-Cystic Fibrosis Transmembrane Conductance Regulator and the Epithelial Sodium Channel in Xenopus Oocytes* Received for publication, September 19, 2002, and in revised form, October 11, 2002 Published, JBC Papers in Press, October 16, 2002, DOI 10.1074/jbc.M209641200 Laurence Suaud‡§, Marcelo Carattino§¶, Thomas R. Kleyman¶, and Ronald C. Rubenstein‡ʈ** From the ‡Division of Pulmonary Medicine, Children`s Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, ʈDepartment of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, and ¶Department of Medicine, Renal-Electrolyte Division, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 The cystic fibrosis transmembrane conductance regulator (CFTR) in addition to its well defined Cl-channel properties regulates other ion channels. Login to comment 3 ABCC7 p.Gly551Asp Using the Xenopus oocyte expression system, different functional regulatory interactions were observed between G551D-CFTR and ␣beta␥-mENaC. Login to comment 4 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp The co-expression of G551D-CFTR and ␣beta␥- mENaC resulted in a 5-fold increase in G551D-CFTR Cl- current compared with oocytes expressing G551D-CFTR alone. Login to comment 5 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Oocytes co-injected with both G551D-CFTR and ENaC expressed an amiloride-sensitive whole cell current that was similar to that observed before and after G551D-CFTR activation with forskolin/isobutylmethylxanthine. Login to comment 6 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Treatment with genistein both enhanced the functional expression of G551D-CFTR and improved regulatory interactions between G551D-CFTR and ENaC. Login to comment 7 ABCC7 p.Gly551Asp These data suggest that genistein may be useful in patients with cystic fibrosis and the G551D-CFTR mutation. Login to comment 16 ABCC7 p.Gly551Asp The G551D mutation in CFTR results in a channel that is appropriately localized to the apical membrane but does not normally conduct Clin response to cAMP stimulation (20). Login to comment 17 ABCC7 p.Gly551Asp Genistein can improve G551D-CFTRs cAMP-dependent chloride transport (19). Login to comment 18 ABCC7 p.Gly551Asp Others have shown that G551D-CFTR does not inhibit sodium transport by ENaC in Xenopus oocytes (6). Login to comment 19 ABCC7 p.Gly551Asp We hypothesized that genistein would restore the functional interactions between G551D-CFTR and mENaC and examined this interaction in the Xenopus oocyte expression system. Login to comment 20 ABCC7 p.Gly551Asp ENaC was able to enhance the functional expression of G551D-CFTR in the absence of genistein, in contrast to our previous results examining interactions between ⌬F508-CFTR and ENaC (11). Login to comment 21 ABCC7 p.Gly551Asp In agreement with previous data, we observed that G551D-CFTR did not inhibit the functional expression of ENaC after activation with IBMX/forskolin (6). Login to comment 22 ABCC7 p.Gly551Asp As we observed with ⌬F508-CFTR, genistein restored functional interactions between G551D-CFTR and ENaC. Login to comment 25 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Expression of Human G551D-CFTR and Mouse ENaC in Xenopus Oocytes-Human G551D-CFTR and mouse ENaC were expressed in Xenopus oocytes as described previously (10, 11). Login to comment 26 ABCC7 p.Gly551Asp Human G551D-CFTR and mouse ␣-, beta-, and ␥-ENaC cRNAs were prepared using a cRNA synthesis kit (mMESSAGE mMachine, Ambion Inc, Austin, TX) according to the manufacturer`s protocol. Login to comment 40 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Each batch of oocytes obtained from an individual frog was injected (50 nl/oocyte) using a Nanoject II microinjector (Drummond Scientific, Broomall, PA) with ␣, beta, and ␥ subunits of mENaC (0.33 ng/subunit), G551D-CFTR (10 ng), or a combination of mENaC and G551D-CFTR cRNAs dissolved in RNase-free water. Login to comment 49 ABCC7 p.Gly551Asp G551D-CFTR was activated by perfusion of the oocyte with buffer containing 10 ␮M forskolin and 100 ␮M IBMX for 25 min (10, 11). Login to comment 51 ABCC7 p.Gly551Asp In all experiments, G551D-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 59 ABCC7 p.Gly551Asp Expression of G551D-CFTR in Xenopus oocytes. Login to comment 60 ABCC7 p.Gly551Asp G551D-CFTR was expressed in oocytes, and TEV was performed as described under "Experimental Procedures." Login to comment 64 ABCC7 p.Gly551Asp C, G551D-CFTR was expressed in oocytes, and TEV was performed as described with the exception that the ND96 buffer was supplemented with 5 mM tetraethylamine chloride, 5 mM BaCl2, and 200 ␮M DIDS (4,4Ј-diisothiocyanatostilbene-2,2Ј-disulfonic acid). Login to comment 76 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp 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 G551D-CFTR versus oocytes co-injected with a cRNAs for both ENaC and G551D-CFTR). Login to comment 78 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp RESULTS Functional Expression of G551D-CFTR in Xenopus Oocytes-We used the Xenopus oocyte expression system and two-electrode voltage clamp technique to examine the functional expression of G551D-CFTR. Login to comment 79 ABCC7 p.Gly551Asp We measured whole cell currents at varying clamping potentials in oocytes expressing G551D-CFTR (Fig. 1) and generated I/V curves from data obtained prior to 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 81 ABCC7 p.Gly551Asp These data are consistent with previous observations that forskolin/IBMX-stimulated chloride current in G551D-CFTR-expressing oocytes is very low (14). Login to comment 82 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp As the isoflavone genistein is able to stimulate G551D-CFTR chloride conductance (19), whole cell currents in G551D-CFTR-expressing oocytes were assessed before and after additional stimulation with 50 ␮M genistein (in addition to IBMX and forskolin). Login to comment 83 ABCC7 p.Gly551Asp Fig. 1B demonstrates the I/V relationship prior to and after activation of G551D-CFTR by 25 min of incubation with 10 ␮M forskolin and 100 ␮M IBMX followed by 15 min of incubation with 10 ␮M forskolin, 100 ␮M IBMX, and 50 ␮M genistein. Login to comment 85 ABCC7 p.Gly551Asp These data are consistent with IBMX/forskolin-stimulated G551D-CFTR-mediated chloride current being enhanced by genistein in Xenopus oocytes. Login to comment 88 ABCC7 p.Gly551Asp Forskolin, IBMX, and genistein did not alter whole cell currents in oocytes that had not been injected with G551D-CFTR (n ϭ 3). Login to comment 89 ABCC7 p.Gly551Asp We also assessed the I/V relationship of G551D-CFTR-injected oocytes in the presence of inhibitors of putative endogenous channels (Fig. 1C). Login to comment 92 ABCC7 p.Gly551Asp Co-expression of G551D-CFTR and ␣beta␥-ENaC-Several groups have reported (7, 9-11) that when WT CFTR and ENaC were co-expressed in Xenopus oocytes, ENaC-mediated Naϩ currents were inhibited in response to CFTR activation. Login to comment 94 ABCC7 p.Gly551Asp Therefore, we assessed the interregulation of G551D-CFTR and mENaC. Login to comment 95 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Whole cell currents measured at a holding potential of -100 mV in oocytes injected with G551D-CFTR (10 ng) or ␣beta␥-ENaC (0.33 ng/subunit) or both G551D-CFTR and ␣beta␥-ENaC are shown Fig. 2. Login to comment 97 ABCC7 p.Gly551Asp Expression of G551D-CFTR and ENaC in Xenopus oocytes. Login to comment 98 ABCC7 p.Gly551Asp G551D-CFTR and ␣beta␥-ENaC were expressed separately or together in oocytes, and TEV was performed as described under "Experimental Procedures." Login to comment 100 ABCC7 p.Gly551Asp B, amiloride-sensitive whole cell currents (-100-mV holding potential) were determined in oocytes expressing ENaC or co-expressing ENaC and G551D-CFTR prior to (open bars) and following (gray bars) stimulation with 10 ␮M forskolin and 100 ␮M IBMX. Login to comment 101 ABCC7 p.Gly551Asp Data obtained from the same G551D-CFTR/ ENaC co-injected oocytes are presented in panels A and B. Means Ϯ S.E. are illustrated. Login to comment 104 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Oocytes co-injected with G551D-CFTR and ␣beta␥-ENaC had 5-fold increased forskolin/IBMX-stimulated, amiloride-insensitive current compared with oocytes injected with G551D-CFTR alone (Fig. 2A, ICl ϭ -0.1 Ϯ 0.0 ␮A (G551D-CFTR) versus -0.5 Ϯ 0.1 ␮A (G551D-CFTR/␣beta␥- ENaC), p ϭ 0.007). Login to comment 105 ABCC7 p.Gly551Asp Thus, a co-expression of ENaC in oocytes resulted in enhanced forskolin/IBMX-stimulated G551D-CFTR Cl- current similar to its previously described enhancement of WT CFTR current (7, 9-11, 21). Login to comment 106 ABCC7 p.Gly551Asp Oocytes co-injected with both G551D-CFTR and ENaC expressed amiloride-sensitive whole cell currents (-2.8 Ϯ 0.6 ␮A, n ϭ 25) in the absence of forskolin/IBMX that were significantly lower than the amiloride-sensitive whole cell currents recorded in the absence of forskolin/IBMX in oocytes injected with ENaC alone (-5.3 Ϯ 1.0 ␮A, n ϭ 22). Login to comment 107 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp However, similar levels of amiloride-sensitive whole cell currents were observed in oocytes co-injected with G551D-CFTR and ENaC prior to and after G551D-CFTR activation with forskolin/IBMX (-2.8 Ϯ 0.6 ␮A versus -2.5 Ϯ 0.6 ␮A, n ϭ 25, p ϭ n.s.) (Fig. 2B), which is similar to ⌬F508-CFTR behavior and differs from WT CFTR behavior in this system (10, 11). Login to comment 108 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp G551D-CFTR/ENaC Interregulation with IBMX/Forskolin Does Not Require Sodium Transport-When Naϩ in the ND96 bath buffer was replaced by the impermeant cation NMDGϩ , oocytes co-injected with G551D-CFTR and ␣beta␥-ENaC had a 6-fold increased forskolin/IBMX-stimulated whole cell current compared with oocytes injected with G551D-CFTR alone (ICl ϭ -5.1 Ϯ 1.4-fold stimulation (ND96), n ϭ 25, versus -6.3 Ϯ 1.7-fold stimulation (NMDG-CL96), n ϭ 18, p ϭ n.s.) (Fig. 3). Login to comment 109 ABCC7 p.Gly551Asp These data suggest that ENaC enhancement of forskolin/ IBMX-mediated activation of G551D-CFTR is not dependent on extracellular Naϩ , Naϩ influx, or inward Naϩ conductance. Login to comment 110 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Interregulation of G551D-CFTR and ␣beta␥-mENaC after IBMX/Forskolin/Genistein Stimulation-G551D-CFTR exhibits a poor Cl-conductance in response to cAMP. Login to comment 111 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp As this might reflect the presence of alterations in other functional properties of this mutant, we examined whether genistein, which activates G551D-CFTR Cl-conductance, would restore functional interactions between G551D-CFTR and ENaC. Login to comment 112 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp As shown in Fig. 4A, the amiloride-insensitive component of the forskolin/ IBMX/genistein-stimulated whole cell current in oocytes coexpressing G551D-CFTR and ␣beta␥-ENaC was 4-fold greater than the forskolin/IBMX/genistein-stimulated current meas- FIG. 4. Login to comment 113 ABCC7 p.Gly551Asp Genistein restores regulation interactions between G551D-CFTR and ␣beta␥-ENaC. Login to comment 114 ABCC7 p.Gly551Asp G551D-CFTR and ␣beta␥-ENaC were expressed separately or together in oocytes, and TEV was performed as described under "Experimental Procedures." Login to comment 116 ABCC7 p.Gly551Asp B, amiloride-sensitive whole cell currents (-100-mV holding potential) were determined in oocytes expressing ENaC or co-expressing ENaC and G551D-CFTR prior to (open bars) and following (dark gray bars) stimulation with 10 ␮M forskolin, 100 ␮M IBMX, and 50 ␮M genistein. Login to comment 117 ABCC7 p.Gly551Asp Data obtained from the same G551D-CFTR/ENaC-co-injected oocytes are presented in panels A and B. Means Ϯ S.E. are illustrated. FIG. 3. Login to comment 118 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp G551D-CFTR/ENaC regulatory interactions restored by genistein do not require Na؉ transport. Whole cell currents (-100-mV holding potential) were determined in oocytes expressing G551D-CFTR alone (open bars) or co-expressing G551D-CFTR and ␣beta␥- ENaC (closed bars). Login to comment 119 ABCC7 p.Gly551Asp ND96, changes in whole cell currents that were not inhibited by 10 ␮M amiloride (presumed to be G551D-CFTR-mediated) were determined following stimulation with 10 ␮M forskolin and 100 ␮M IBMX in standard ND96 (NaCl) buffer. NMDG-Cl96, changes in whole cell currents were determined following stimulation with 10 ␮M forskolin and 100 ␮M IBMX in oocytes bathed in a Naϩ -free solution (Naϩ was replaced with NMDG). Login to comment 120 ABCC7 p.Gly551Asp Data (means Ϯ S.E.) are expressed relative to the mean change in whole cell current of oocytes injected with G551D-CFTR alone. Login to comment 121 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ured in oocytes expressing G551D-CFTR alone (-0.3 Ϯ 0.0 ␮A (G551D-CFTR, n ϭ 21) versus -1.2 Ϯ 0.4 ␮A (G551D-CFTR/ ENaC, n ϭ 20), p ϭ 0.03). Login to comment 123 ABCC7 p.Gly551Asp Amiloride-sensitive whole cell currents recorded before and after stimulation by forskolin/ IBMX/genistein in oocytes expressing ENaC and G551D-CFTR were significantly lower than the amiloride-sensitive currents obtained both before and after stimulation by forskolin/IBMX/ genistein in oocytes expressing ENaC alone (Fig. 4B). Login to comment 124 ABCC7 p.Gly551Asp Although amiloride-sensitive whole cell currents significantly increased in oocytes expressing ENaC alone following stimulation by forskolin/IBMX/genistein (Fig. 4B), amiloride-sensitive currents recorded in oocytes co-expressing ENaC and G551D-CFTR remained stable following stimulation with forskolin/ IBMX/genistein (-2.1 Ϯ 0.7 ␮A (-forskolin/IBMX/genistein) versus -2.0 Ϯ 0.3 (ϩforskolin/IBMX/genistein), n ϭ 20, p ϭ n.s.). Login to comment 125 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp These data suggest that the activation of G551D-CFTR by forskolin/IBMX in the presence of genistein partially restored the inhibition of ENaC activity by activated G551D-CFTR and are similar to our observations for ⌬F508-CFTR (11). Login to comment 126 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp We again assessed whether the enhanced forskolin/IBMX/ genistein-stimulated amiloride-insensitive whole cell currents recorded in oocytes co-injected with G551D-CFTR and ENaC compared with oocytes injected with G551D-CFTR alone depended on ENaC-mediated sodium transport. Login to comment 127 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Oocytes co-injected with G551D-CFTR and ␣beta␥-ENaC had a 3.75-fold increased forskolin/IBMX/genistein-stimulated G551D-CFTR Cl- current compared with oocytes injected with G551D-CFTR alone both in ND96 (NaCl) buffer and in NMDG-Cl buffer (Fig. 5). Login to comment 128 ABCC7 p.Gly551Asp These data suggest that ENaC enhancement of forskolin/ IBMX/genistein-mediated activation of G551D-CFTR is not dependent on extracellular Naϩ , Naϩ influx, or inward Naϩ conductance. Login to comment 129 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Synergistic Activation of G551D-CFTR by Genistein and ENaC-In Fig. 6, we have replotted the whole oocyte forskolin/ IBMX-stimulated (Fig. 3, NMDG-Cl- ) or forskolin/IBMX/genistein-stimulated (Fig. 5, NMDG-Cl- ) currents and expressed these relative to that of oocytes injected with G551D-CFTR alone and stimulated with IBMX/forskolin. Login to comment 131 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp These data demonstrate synergistic activation of G551D-CFTR function by genistein and ENaC in the presence of forskolin/IBMX and suggest that genistein and ENaC enhance G551D-CFTR function in oocytes by different and complementary mechanisms. Login to comment 132 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Influence of ␣beta␥-mENaC on G551D-CFTR Single Channel Activity in Oocytes-We next sought to assess the mechanism by which ␣beta␥-mENaC increased G551D-CFTR-mediated current in co-injected oocytes. Login to comment 133 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Oocytes were injected with G551D-CFTR alone or co-injected with ENaC, and G551D-CFTR was stimulated with forskolin/IBMX/genistein prior to single channel recordings of chloride transport (Fig. 7). Login to comment 134 ABCC7 p.Gly551Asp At a pipette potential of -80 mV in the cell-attached mode, single channel currents in G551D-CFTR-injected oocytes (1.08 Ϯ 0.13 pA, n ϭ 6) FIG. 6. Login to comment 135 ABCC7 p.Gly551Asp Synergistic activation of G551D-CFTR by genistein and ENaC. Login to comment 136 ABCC7 p.Gly551Asp Whole oocyte IBMX/forskolin-stimulated (Fig. 3, NMDG-Cl- ) or IBMX/forskolin/genistein-stimulated (Fig. 5, NMDG-Cl- ) currents are replotted and expressed relative to that of oocytes injected with G551D-CFTR alone and stimulated with IBMX/forskolin. Login to comment 138 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp G551D-CFTR/ENaC regulatory interactions restored by genistein do not require Na؉ transport. Whole cell currents (-100-mV holding potential) were determined in oocytes expressing G551D-CFTR alone (open bars) or co-expressing G551D-CFTR and ␣beta␥- ENaC (closed bars). Login to comment 139 ABCC7 p.Gly551Asp ND96, changes in whole cell currents that were not inhibited by 10 ␮M amiloride (presumed to be G551D-CFTR-mediated) were determined following stimulation with 10 ␮M forskolin, 100 ␮M IBMX, and 50 ␮M genistein in standard ND96 (NaCl) buffer. NMDG-Cl96, changes in whole cell currents were determined following stimulation with 10 ␮M forskolin, 100 ␮M IBMX, and 50 ␮M genistein in oocytes bathed in a Naϩ -free solution (Naϩ was replaced with NMDG). Login to comment 140 ABCC7 p.Gly551Asp Data (means Ϯ S.E.) are expressed relative to the mean change in whole cell current of oocytes injected with G551D-CFTR alone. Login to comment 141 ABCC7 p.Gly551Asp were similar in magnitude to the currents recorded in oocytes co-expressing G551D-CFTR and ENaC (0.92 Ϯ 0.14 pA, n ϭ 6, p ϭ n.s.). Login to comment 142 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp The Po for G551D-CFTR in oocytes co-injected with ENaC (0.24 Ϯ 0.6, mean Ϯ S.E., n ϭ 6) was not significantly different from that determined in oocytes injected with G551D-CFTR alone (0.29 Ϯ 0.2, n ϭ 6, p ϭ n.s.). Login to comment 143 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp We did not observe a difference in number of G551D-CFTR channels/patch from oocytes injected with G551D-CFTR alone (n ϭ 2, 3, 1, 2, 1, and 4 for n ϭ 6 patches) or co-injected with G551D-CFTR and ENaC (n ϭ 3, 1, 1, 4, 2, and 2 for n ϭ 6 patches). Login to comment 144 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Given the difficulties in estimating small changes in channel density from single channel analyses and the lack of a difference in G551D-CFTR open probability following stimulation with forskolin/IBMX/ genistein in oocytes expressing G551D-CFTR alone or co-expressing G551D-CFTR and ENaC, our results are consistent with the increase in forskolin/IBMX/genistein-stimulated G551D-CFTR currents in the presence of ENaC, reflecting an increase in the number of G551D-CFTR channels at the plasma membrane. Login to comment 145 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp As genistein increases the Po for G551D-CFTR (22), these data are consistent with genistein and ENaC enhancing G551D-CFTR function in oocytes by different and complementary mechanisms. Login to comment 150 ABCC7 p.Gly551Asp The first nucleotide binding fold (nucleotide binding domain (NBD-1)) of CFTR in which G551D mutation is located seems important for this interaction. Login to comment 152 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp A similar NBD1/R peptide fragment containing the G551D mutation did not demonstrate forskolin/IBMX-regulated inhibition of ENaC (14). Login to comment 153 ABCC7 p.Gly551Asp G551D-CFTR when co-expressed with ␣beta␥-ENaC and activated by IBMX/forskolin was unable to inhibit ENaC in our experiments. Login to comment 158 ABCC7 p.Gly551Asp Genistein activates CFTR (WT, ⌬F508, and G551D) (16, 22) by increasing channel open time and decreasing channel closing, thereby increasing channel Po. Login to comment 161 ABCC7 p.Gly551Asp Co-expression of ␣beta␥-ENaC does not alter the open probability of forskolin/IBMX/genistein-stimulated G551D-CFTR. Login to comment 163 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp The upper tracings show 60 s of a continuous recording performed in the cell-attached mode performed as described under "Experimental Procedures" in an oocyte expressing G551D-CFTR alone or an oocyte co-expressing G551D-CFTR and ␣beta␥-ENaC. Login to comment 165 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp B, G551D-CFTR open probability (mean Ϯ S.E.) determined in forskolin/IBMX/genistein-treated oocytes expressing either G551D-CFTR alone or co-expressing G551D-CFTR and ENaC. Login to comment 166 ABCC7 p.Gly551Asp being able to activate both ⌬F508- and G551D-CFTR as these two mutants have intact NBD-2s and further suggests that in some way an interaction of NBD-2 influences NBD-1 form and/or function. Login to comment 168 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp In oocyte co-injected with G551D-CFTR and ␣beta␥-mENaC, the enhancement of G551D-CFTR Cl- current most probably correlates with an increase in the N or the number of Cl-channels present at the oocyte surface as the measured open probability of forskolin/IBMX/genistein-stimulated G551D-CFTR was the same in the presence or absence of ENaC. Login to comment 169 ABCC7 p.Gly551Asp We also observed that amiloride-inhibited whole oocyte currents decreased with co-expression of G551D-CFTR (in these studies) or with WT CFTR in our previous studies (11). Login to comment 171 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp One potential explanation for these observations is that co-expression of ENaC increases G551D-CFTR N, whereas co-expression of G551D-CFTR could decrease either ENaC N or open probability. Login to comment 172 ABCC7 p.Gly551Asp Previous studies in a number of systems have suggested that co-expression of WT CFTR decreases ENaC open probability (21, 27); however, such an effect on open probability was not noted for G551D-CFTR in lipid bilayers (8). Login to comment 173 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Our data do not directly address this issue, but if G551D-CFTR does not decrease ENaC open probability in oocytes (similar to its behavior in lipid bilayers), we can speculate that G551D-CFTR inhibits ENaC functional expression via an N effect. Login to comment 177 ABCC7 p.Gly551Asp The co-expression of an appropriately folded and trafficked CFTR (G551D-CFTR as shown in Figs. Login to comment 180 ABCC7 p.Gly551Asp Another potential site of interaction/competition between CFTR and ENaC is at the plasma membrane where cAMP-activated WT CFTR but not G551D-CFTR and ⌬F508-CFTR inhibited ENaC function. Login to comment 181 ABCC7 p.Gly551Asp One possibility is that both G551D-CFTR and ⌬F508-CFTR undergo more rapid removal from the plasma membrane when compared with WT CFTR as has been suggested for ⌬F508-CFTR (25). Login to comment 182 ABCC7 p.Gly551Asp Another possibility is that G551D-CFTR and ⌬F508-CFTR, perhaps because of their aberrant NBD-1s, are not able to interact or interact differently with either ENaC or proteins important in transducing cAMP-activated CFTR signal to inhibit ENaC function. Login to comment