Cystic Fibrosis Transmembrane Conductance Regulator Inverts Protein Kinase A-mediated Regulation of Epithelial Sodium Channel Single Channel Kinetics

Abnormal regulation of ion channels by members of the ABC transport protein superfamily has been implicated in hyperinsulinemic hypoglycemia and in excessive Na+absorption by airway epithelia in cystic fibrosis (CF). How ABC proteins regulate ion conductances is unknown, but must generally involve either the number or activity of specific ion channels. Here we report that the cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in CF, reverses the regulation of the activity of single epithelial sodium channels (ENaC) by cAMP. ENaC expressed alone in fibroblasts responded to activation of cAMP-dependent protein kinase with increased open probability (Po) and mean open time, whereas ENaC co-expressed with CFTR exhibited decreasedPo and mean open time under conditions optimal for PKA-mediated protein phosphorylation. Thus, CFTR regulates ENaC at the level of single channel gating, by switching the response of single channel Po to cAMP from an increase to a decrease. Abnormal regulation of ion channels by members of the ABC transport protein superfamily has been implicated in hyperinsulinemic hypoglycemia and in excessive Na+absorption by airway epithelia in cystic fibrosis (CF). How ABC proteins regulate ion conductances is unknown, but must generally involve either the number or activity of specific ion channels. Here we report that the cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in CF, reverses the regulation of the activity of single epithelial sodium channels (ENaC) by cAMP. ENaC expressed alone in fibroblasts responded to activation of cAMP-dependent protein kinase with increased open probability (Po) and mean open time, whereas ENaC co-expressed with CFTR exhibited decreasedPo and mean open time under conditions optimal for PKA-mediated protein phosphorylation. Thus, CFTR regulates ENaC at the level of single channel gating, by switching the response of single channel Po to cAMP from an increase to a decrease. Recent studies (1Burch L. Talbot C. Knowles M.R. Canessa C. Rossier B. Boucher R.C. Am. J. Physiol. 1995; 269: C511-C518Crossref PubMed Google Scholar, 2Hummler E. Barker P. Gatzy J. Beermann F. Verdumo C. Schmidt A. Boucher R. Rossier B.C. Nat. Genet. 1996; 12: 325-328Crossref PubMed Scopus (780) Google Scholar) have identified ENaC as the channel that mediates amiloride sensitive Na+ absorption in mammalian airways. In cystic fibrosis (CF), 1The abbreviations used are:CFcystic fibrosisCFTRcystic fibrosis transmembrane conductance regulatorMOTmean open timeENaCepithelial sodium channel(s)PKAprotein kinase ACScatalytic subunitmPKImyristoylated protein kinase A inhibitorTES2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid. ENaC-mediated Na+ absorption is increased 200–300% in airway epithelia and, abnormally, further stimulated by raising intracellular cAMP (3Boucher R.C. Stutts M.J. Knowles M.R. Cantley L. Gatzy J.T. J. Clin. Invest. 1986; 78: 1245-1252Crossref PubMed Scopus (447) Google Scholar). Because most CF mutations result in little if any functional CFTR in the apical cell membrane of affected epithelia (4Collins F.S. Science. 1992; 256: 774-779Crossref PubMed Scopus (723) Google Scholar), we inferred that normal CFTR must either down-regulate the number of active Na+ channels or decrease the activity of individual Na+ channels. In the present study we have studied the effects of cAMP-dependent protein-phosphorylating conditions on the single channel kinetics of ENaC expressed alone or together with CFTR in NIH 3T3 fibroblasts. cystic fibrosis cystic fibrosis transmembrane conductance regulator mean open time epithelial sodium channel(s) protein kinase A catalytic subunit myristoylated protein kinase A inhibitor 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid. α-, β-, and γ-ENaC subunits were stably expressed in NIH 3T3 cell lines that had been previously transduced with a truncated (inactive) interleukin-2 receptor (ENaC alone cells) or with human CFTR (ENaC + CFTR cells) (5Stutts M.J. Canessa C.M. Olsen J.C. Hamrick M. Cohn J.A. Rossier B.C. Boucher R.C. Science. 1995; 269: 847-850Crossref PubMed Scopus (971) Google Scholar). ENaC-mediated single channel currents were recorded from cell attached and excised membrane patches as described in the figure legends. The single channel conductance (4–5 picosiemens) of ENaC expressed in NIH 3T3 fibroblasts, as well as cation selectivity (Li+ > Na+ > K+), amiloride inhibition (Ki ≈ 0.3 μm) and the slow gating pattern (MOT ≈ 1 s), are similar to what has been reported for the cloned channel expressed in oocytes (6Canessa C.M. Horisberger J. Rossier B.C. Nature. 1993; 361: 467-470Crossref PubMed Scopus (838) Google Scholar, 7Canessa C.M. Schild L. Buell G. Thorens B. Gautschi I. Horisberger J.D. Rossier B.C. Nature. 1994; 367: 463-467Crossref PubMed Scopus (1798) Google Scholar) and for endogenously expressed ENaC in rat cortical collecting tubule (8Pacha J. Frindt G. Antonian L. Silver R.B. Palmer L.G. J. Gen. Physiol. 1993; 102: 25-42Crossref PubMed Scopus (206) Google Scholar) or A6 cells (9Marunaka Y. Eaton D.C. Am. J. Physiol. 1991; 260: C1071-C1084Crossref PubMed Google Scholar) (Fig. 1). These similar results in very different cells suggest that cell specific cytoskeletal or other elements are not critical determinants of the basic biophysical characteristics of ENaC. The basal conductance and amiloride sensitivity of ENaC were not affected by co-expression with CFTR (Fig.1). ENaC present in excised membrane patches exhibited a variable degree of rundown following excision. Rundown was partially reversed (Fig. 2 A, panel i) or prevented (Fig.2 A, panel ii) by exposure of the cytoplasmic surface to PKA catalytic subunit and 2 mm ATP (CS + ATP). Fig.2 A, panel iii, summarizes the results from both paradigms, revealing positive regulation of ENaC activity by PKA. One explanation for a range of basal activity, for rundown following excision, and for variable degree of activation by CS + ATP is that the resting phosphorylation state differs from patch to patch. Moreover, it seemed possible that water-soluble reagents, such as PKA catalytic subunit, might have poor access to hydrophobic compartments within the membrane patch. We tested these possibilities with a specific peptide inhibitor of PKA (mPKI) that had been modified by myristoylation to promote its association with biologic membranes (10Glass D.B. Cheng H.C. Mende-Mueller L. Reed J. Walsh D.A. J. Biol. Chem. 1989; 264: 8802-8810Abstract Full Text PDF PubMed Google Scholar, 11Walsh D.A. Glass D.B. Methods Enzymol. 1991; 201: 304-316Crossref PubMed Scopus (60) Google Scholar). mPKI was effective in (6/6) inside out membrane patches, reversing the effects of exogenous CS + ATP (Fig. 2 A) by inhibiting Po(Fig. 2 A, panel iii) and MOT (not shown) to levels lower than “basal.” This observation suggests that the level of basal phosphorylation in the system influences the gating of ENaC in the absence of external manipulation. The presence of CFTR caused a dramatic change in the regulation of ENaC in excised patches by CS + ATP. Whereas the gating and rundown of ENaC in patches excised from CFTR expressing cells were not obviously abnormal under nonstimulated conditions, exposure to CS + ATP routinely inhibited ENaC activity in two different paradigms (Fig.2 B). First, in 4/5 excised inside out patches, CS + ATP decreased Po (Fig. 2 B, panel i). Second, ENaC in 5/5 patches excised from CFTR expressing cells directly into CS + ATP demonstrated low Po (Fig.2 B, panel ii) and MOT (not shown). mPKI further decreasedPo of ENaC co-expressed with CFTR (Fig.2 B, panels i and iii). Fig. 2 B, panel iii, summarizes the very different pattern of regulation of ENaC by PKA in the presence of CFTR (compare with Fig. 2 A, panel iii). To study PKA and CFTR regulation of ENaC in the absence of excision-induced rundown, we exposed cells to permeant PKA activators (cpt-cAMP + forskolin (cpt-cAMP/FSK)) during cell-attached recording (Fig. 3). In ENaC-only cells cpt-cAMP + forskolin increased ENaC Po (Fig.3 A), whereas in ENaC + CFTR-expressing cells PKA activators routinely decreased Po (Fig. 3 B). This result, coupled with the effects of CS + ATP in excised patches, strongly indicates that the CFTR-mediated regulation of whole cell amiloride-sensitive Na+ current observed previously (5Stutts M.J. Canessa C.M. Olsen J.C. Hamrick M. Cohn J.A. Rossier B.C. Boucher R.C. Science. 1995; 269: 847-850Crossref PubMed Scopus (971) Google Scholar) reflects modulation by CFTR of ENaC single channel gating. The results in Figs. 2 and 3 suggest that negative regulation of ENaC by CFTR reflects an effect on ENaC activity rather than ENaC number. Additional analyses of our data support this conclusion. First, co-expression of CFTR with ENaC did not affect the number of ENaC channels observed per patch (2.17 ± 0.29 (n = 28) without CFTR and 2.29 ± 0.29 (n = 26) with CFTR). Second, the MOT of unambiguous single channel openings in excised, and cell-attached patches under optimal conditions of PKA activation were markedly decreased by the presence of CFTR (Fig. 4). Thus, CFTR negative regulation of ENAC can be explained by decreased activity of individual ENaC channels. Our data reveal a surprisingly strong positive regulation of ENaC alone by PKA. The low Po recorded in the presence of mPKI (Fig. 3) and the high Po and long MOT measured during PKA activation (Fig. 4) indicate that increasing protein phosphorylation increased the time ENaC occupied a stable open conformation. This result differs from the cAMP-dependent increase of the number of endogenous amiloride sensitive Na+ channels seen in A6 epithelial cells (9Marunaka Y. Eaton D.C. Am. J. Physiol. 1991; 260: C1071-C1084Crossref PubMed Google Scholar), which are reported to regulate surface expression of transport elements by membrane insertion and retrieval (12Schafer J.A. Hawk C.T. Kidney Int. 1992; 41: 255-268Abstract Full Text PDF PubMed Scopus (134) Google Scholar), but is similar to cAMP-dependent regulation ofPo of partially purified renal (13Ismailov I.I. McDuffie J.H. Benos D.J. J. Biol. Chem. 1994; 269: 10235-10241Abstract Full Text PDF PubMed Google Scholar) and lung alveolar type II cell Na+ channels (14Senyk O. Ismailov I. Bradford A.L. Baker R.R. Matalon S. Benos D.J. Am. J. Physiol. 1995; 268: C1148-C1156Crossref PubMed Google Scholar). Studies of heterologously expressed ENaC in oocytes (15Mall M. Hipper A. Greger R. Kunzelmann K. FEBS Lett. 1996; 381: 47-52Crossref PubMed Scopus (134) Google Scholar) and of reconstituted ENaC in lipid bilayers (16Ismailov I.I. Awayda M.S. Jovov B. Berdiev B.K. Fuller C.M. Dedman J.R. Kaetzel M.A. Benos D.J. J. Biol. Chem. 1996; 271: 4725-4732Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar) detected no effect of PKA activation on single channel gating. γ-rENaC used in our study contains two consensus PKA phosphorylation sites, but these are not highly conserved across species (6Canessa C.M. Horisberger J. Rossier B.C. Nature. 1993; 361: 467-470Crossref PubMed Scopus (838) Google Scholar, 7Canessa C.M. Schild L. Buell G. Thorens B. Gautschi I. Horisberger J.D. Rossier B.C. Nature. 1994; 367: 463-467Crossref PubMed Scopus (1798) Google Scholar). Thus, PKA regulation of ENaC gating may well involve the phosphorylation and function of an additional protein or proteins, including cytoskeletal components such as actin (17Berdiev B.K. Prat A.G. Cantiello H.F. Ausiello D.A. Fuller C.M. Jovov B. Benos D.J. Ismailov I.I. J. Biol. Chem. 1996; 271: 17704-17710Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Cell-specific expression of these proteins could explain why fibroblasts reproduce the defect in CF airways better than oocytes (15Mall M. Hipper A. Greger R. Kunzelmann K. FEBS Lett. 1996; 381: 47-52Crossref PubMed Scopus (134) Google Scholar). In intact oocytes (15Mall M. Hipper A. Greger R. Kunzelmann K. FEBS Lett. 1996; 381: 47-52Crossref PubMed Scopus (134) Google Scholar), or in ENaC reconstituted in lipid bilayers after expression in oocytes (16Ismailov I.I. Awayda M.S. Jovov B. Berdiev B.K. Fuller C.M. Dedman J.R. Kaetzel M.A. Benos D.J. J. Biol. Chem. 1996; 271: 4725-4732Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar), the presence of CFTR decreased whole cell currents or single channel open probability. Thus, CFTR appears to exert a negative modulatory regulation of ENaC in several distinct cell types, including human airway epithelia, mouse fibroblasts, and amphibian oocytes. The present findings help explain the long standing observation that Na+ absorption across CF airway epithelia is increased and inappropriately further stimulated by cAMP (3Boucher R.C. Stutts M.J. Knowles M.R. Cantley L. Gatzy J.T. J. Clin. Invest. 1986; 78: 1245-1252Crossref PubMed Scopus (447) Google Scholar). In CF airways, the abnormally high rate of basal Na+ absorption reflects the absence of negative regulation of ENaC by CFTR under basal phosphorylating conditions, and increased PKA activity leads only to further absorption. In contrast, CFTR function in normal airways converts the activation of PKA into a stimulus for both inhibition of ENaC-mediated Na+ absorption and stimulation of CFTR-mediated Cl− secretion. Despite previous reports of abnormal regulation of Na+ channel activity in CF (18Chinet T.C. Fullton J.M. Yankaskas J.R. Boucher R.C. Stutts M.J. Am. J. Physiol. 1993; 265: C1050-C1060Crossref PubMed Google Scholar, 19Chinet T.C. Fullton J.M. Yankaskas J.R. Boucher R.C. Stutts M.J. Am. J. Physiol. 1994; 266: C1061-C1068Crossref PubMed Google Scholar, 20Duszyk M. French A.S. Man S.F.P. Biomed. Res. 1991; 12: 17-23Crossref Scopus (9) Google Scholar), this conclusion was in doubt until now, because PKA has been reported to regulate only the number of active amiloride-sensitive Na+ channels in A6 cells (9Marunaka Y. Eaton D.C. Am. J. Physiol. 1991; 260: C1071-C1084Crossref PubMed Google Scholar), and because another genetic disease associated with excessive Na+ reabsorption (Liddle's syndrome) has been attributed solely to increased ENaC number (21Snyder P.M. Price M.P. McDonald F.J. Adams C.M. Volk K.A. Zeiher B.G. Stokes J.B. Welsh M.J. Cell. 1995; 83: 969-978Abstract Full Text PDF PubMed Scopus (401) Google Scholar). More recently, the mutations associated with Liddle's syndrome have been shown to act predominantly by increased ENaCPo and MOT (22Hansson J.H. Schild L. Lu Y. Wilson T.A. Gautschi I. Shimkets R. Nelson-Williams C. Rossier B.C. Lifton R.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11495-11499Crossref PubMed Scopus (368) Google Scholar). This observation, coupled with the present results, make it clear that regulation of ENaC single channel kinetics is broadly implicated in the control of epithelial sodium absorption. A general mechanism of regulation of ion channels by ABC proteins is yet to be identified (23Higgins C.F. Cell. 1995; 82: 693-696Abstract Full Text PDF PubMed Scopus (343) Google Scholar), but it is clear that CFTR regulates ENaC at the level of single channel gating. This observation is an important consideration for understanding the mechanism by which ABC proteins, including not only CFTR but also SUR and MDR (23Higgins C.F. Cell. 1995; 82: 693-696Abstract Full Text PDF PubMed Scopus (343) Google Scholar), can influence other ion channels. Potentially, ABC proteins regulate the activity of other ion channels through transported substrates, as proposed for CFTR-mediated ATP release (24Cantiello H.F. Prat A.G. Reisin I.L. Ercole L.B. Abraham E.H. Amara J.F. Gregory R.J. Ausiello D.A. J. Biol. Chem. 1994; 269: 11224-11232Abstract Full Text PDF PubMed Google Scholar, 25Schwiebert E.M. Egan M.E. Hwang T. Fulmer S.B. Allen S.S. Cutting G.R. Guggino W.B. Cell. 1995; 81: 1063-1073Abstract Full Text PDF PubMed Scopus (597) Google Scholar). Alternatively, ABC proteins may regulate the activity of other ion channels by direct or indirect protein-protein interactions.