Key points are not available for this paper at this time.
The epithelial Na+ channel (ENaC) is a tetramer of two α-, one β-, and one γ-subunit, but little is known about its assembly and processing. Because co-expression of mouse ENaC subunits with three different carboxyl-terminal epitope tags produced an amiloride-sensitive sodium current in oocytes, these tagged subunits were expressed in both Chinese hamster ovary or Madin-Darby canine kidney type 1 epithelial cells for further study. When expressed alone α-(95 kDa), β-(96 kDa), and γ-subunits (93 kDa) each produced a single band on SDS gels by immunoblotting. However, co-expression of αβγENaC subunits revealed a second band for each subunit (65 kDa for α, 110 kDa for β, and 75 kDa for γ) that exhibited N-glycans that had been processed to complex type based on sensitivity to treatment with neuraminidase, resistance to cleavage by endoglycosidase H, and GalNAc-independent labeling with 3HGal in glycosylation-defective Chinese hamster ovary cells (ldlD). The smaller size of the processed α- and γ-subunits is also consistent with proteolytic cleavage. By using α- and γ-subunits with epitope tags at both the amino and carboxyl termini, proteolytic processing of the α- and γ-subunits was confirmed by isolation of an additional epitope-tagged fragment from the amino terminus (30 kDa for α and 18 kDa for γ) consistent with cleavage within the extracellular loop. The fragments remain stably associated with the channel as shown by immunoblotting of co-immunoprecipitates, suggesting that proteolytic cleavage represents maturation rather than degradation of the channel. The epithelial Na+ channel (ENaC) is a tetramer of two α-, one β-, and one γ-subunit, but little is known about its assembly and processing. Because co-expression of mouse ENaC subunits with three different carboxyl-terminal epitope tags produced an amiloride-sensitive sodium current in oocytes, these tagged subunits were expressed in both Chinese hamster ovary or Madin-Darby canine kidney type 1 epithelial cells for further study. When expressed alone α-(95 kDa), β-(96 kDa), and γ-subunits (93 kDa) each produced a single band on SDS gels by immunoblotting. However, co-expression of αβγENaC subunits revealed a second band for each subunit (65 kDa for α, 110 kDa for β, and 75 kDa for γ) that exhibited N-glycans that had been processed to complex type based on sensitivity to treatment with neuraminidase, resistance to cleavage by endoglycosidase H, and GalNAc-independent labeling with 3HGal in glycosylation-defective Chinese hamster ovary cells (ldlD). The smaller size of the processed α- and γ-subunits is also consistent with proteolytic cleavage. By using α- and γ-subunits with epitope tags at both the amino and carboxyl termini, proteolytic processing of the α- and γ-subunits was confirmed by isolation of an additional epitope-tagged fragment from the amino terminus (30 kDa for α and 18 kDa for γ) consistent with cleavage within the extracellular loop. The fragments remain stably associated with the channel as shown by immunoblotting of co-immunoprecipitates, suggesting that proteolytic cleavage represents maturation rather than degradation of the channel. The amiloride-sensitive epithelial Na+ channel (ENaC) 1The abbreviations used are: ENaC, epithelial Na+ channel; CAP, channel activating protease; CHO, Chinese hamster ovary cells; Endo H, endoglycosidase H; ER, endoplasmic reticulum; hENaC, human ENaC; N-glycanase/PNGase F, peptide N-glycanase F; MDCK, Madin-Darby canine kidney cells; mENaC, mouse ENaC; N-glycans, Asn (N)-linked oligosaccharides; rENaC, rat ENaC; xENaC, Xenopus ENaC; HA, hemagglutinin.1The abbreviations used are: ENaC, epithelial Na+ channel; CAP, channel activating protease; CHO, Chinese hamster ovary cells; Endo H, endoglycosidase H; ER, endoplasmic reticulum; hENaC, human ENaC; N-glycanase/PNGase F, peptide N-glycanase F; MDCK, Madin-Darby canine kidney cells; mENaC, mouse ENaC; N-glycans, Asn (N)-linked oligosaccharides; rENaC, rat ENaC; xENaC, Xenopus ENaC; HA, hemagglutinin. is composed of three structurally related subunits, termed α-, β-, and γ-ENaC. The three subunits exhibit limited amino acid sequence identity (30–40%) but are structurally similar with two membrane-spanning domains and cytosolic amino and carboxyl termini. We and others have shown that ENaC expressed in Xenopus oocytes has a subunit stoichiometry of two α-, one β-, and one γ-subunit (1Kosari F. Sheng S. Li J. Mak D.-O.D. Foskett J.K. Kleyman T.R. J. Biol. Chem. 1998; 273: 13469-13474Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 2Firsov D. Gautschi I. Meriallat A.M. Rossier V.C. Schild L. EMBO J. 1998; 17: 344-352Crossref PubMed Scopus (368) Google Scholar), although an alternative subunit ratio has been proposed (3Snyder P.M. Cheng C. Prince L.S. Rogers J.C. Welsh M.J. J. Biol. Chem. 1998; 273: 681-684Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). ENaC subunits assemble in the endoplasmic reticulum (ER) where they also undergo N-linked glycosylation (4Canessa C.M. Meriallat A.M. Rossier B.C. Am. J. Physiol. 1994; 267: C1682-C1690Crossref PubMed Google Scholar, 5Snyder P.M. McDonald F.J. Stokes J.B. Welsh M.J. J. Biol. Chem. 1994; 269: 24379-24383Abstract Full Text PDF PubMed Google Scholar, 6Renard S. Lingueglia E. Voilley N. Lazdunski M. Barbry P. J. Biol. Chem. 1994; 269: 12981-12986Abstract Full Text PDF PubMed Google Scholar, 7Adams C.M. Snyder P.M. Welsh M.J. J. Biol. Chem. 1997; 272: 27295-27300Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). There are 6, 12, and 5 consensus sites (Asn-X-Ser/Thr) for N-linked oligosaccharide (N-glycan) addition present in the extracellular domains of the α-, β- and γ-subunits of mouse ENaC (mENaC), respectively. Based on apparent molecular weights on SDS-PAGE, the majority of these sites are modified during synthesis with an N-glycan core of ∼3 kDa (4Canessa C.M. Meriallat A.M. Rossier B.C. Am. J. Physiol. 1994; 267: C1682-C1690Crossref PubMed Google Scholar, 5Snyder P.M. McDonald F.J. Stokes J.B. Welsh M.J. J. Biol. Chem. 1994; 269: 24379-24383Abstract Full Text PDF PubMed Google Scholar, 7Adams C.M. Snyder P.M. Welsh M.J. J. Biol. Chem. 1997; 272: 27295-27300Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 8Cheng C. Prince L.S. Snyder P.M. Welsh M.J. J. Biol. Chem. 1998; 273: 22693-22700Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 9Weisz O.A. Wang J.-M. Edinger R.S. Johnson J.P. J. Biol. Chem. 2000; 275: 39886-39893Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 10Alvarez de la Rosa D. Li H. Canessa C.M. J. Gen. Physiol. 2002; 119: 427-442Crossref PubMed Scopus (127) Google Scholar, 11May A. Puoti A. Gaeggeler H.P. Horisberger J.D. Rossier B.C. J. Am. Soc. Nephrol. 1997; 8: 1813-1822Crossref PubMed Google Scholar, 12Prince L.S. Welsh M.J. Biochem. J. 1998; 336: 705-710Crossref PubMed Scopus (47) Google Scholar, 13Hanwell D. Ishikawa T. Saleki R. D. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, D. Schild L. Am. J. Physiol. PubMed Google Scholar). on are for with or and for by to from the A. M. PubMed Scopus Google and L. A. Biol. PubMed Scopus Google Scholar). on the and the core from N-glycans on that the complex during to the are modified from a type to a complex type by a of for these two of N-glycans is they from the by in treatment with endoglycosidase N-glycans on to to the complex are for Endo H, and N-glycans on are However, N-glycans are modified during the and these N-glycans remain to treatment with Endo H. ENaC is to the the complex is by treatment of cells with the that cytosolic assembly for the complex O.A. Wang J.-M. Edinger R.S. Johnson J.P. J. Biol. Chem. 2000; 275: 39886-39893Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, H. P. Ishikawa T. Saleki R. Horisberger Schild L. D. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, Canessa C.M. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). However, for processing of N-glycans on ENaC to complex type is D. Ishikawa T. Saleki R. D. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Endo rat ENaC expressed in Madin-Darby canine kidney Prince and Welsh L.S. Welsh M.J. Biochem. J. 1998; 336: 705-710Crossref PubMed Scopus (47) Google that human ENaC α- and in a were to both N-glycanase and Endo and they that N-glycans are from expressed at the of Canessa C.M. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google that three subunits at the of oocytes exhibited and on with the consistent with of complex type N-glycans, but Endo sensitivity of these was de la Rosa de la Rosa D. Li H. Canessa C.M. J. Gen. Physiol. 2002; 119: 427-442Crossref PubMed Scopus (127) Google Endo and of both the α- and of in cells during but the were by the that the Endo of the was also to the Endo of the exhibit a on consistent with de la Rosa D. Li H. Canessa C.M. J. Gen. Physiol. 2002; 119: 427-442Crossref PubMed Scopus (127) Google Scholar, 12Prince L.S. Welsh M.J. Biochem. J. 1998; 336: 705-710Crossref PubMed Scopus (47) Google Scholar). C. M. S. T. C. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google the smaller of two of the on the of rat and S. M. J.B. J. Scopus Google an from a to a smaller of the γ-subunit in from on a However, is that these proteolytic processing of the α- and We of in present for processing of N-glycans on three subunits of αβγENaC to Endo Endo N-glycans on smaller of the α- and and that maturation of ENaC proteolytic processing. and epitope-tagged subunit F. Li J. Kleyman T.R. Am. J. Physiol. PubMed Google were by or in or in and or in tagged α- and with and carboxyl-terminal epitope tags and were were from The epitope was from the of human C.M. J. Google Scholar). P. and T. R. in and of ENaC epitope-tagged subunits expressed in cells were with mouse from mouse from mouse from mouse from or mouse from of ENaC epitope-tagged subunits expressed in cells was with the that to from was used for the in of ENaC subunits from cells was with mouse from mouse from and rat from and second for immunoblotting were from type and glycosylation-defective were as J.B. O.A. Biol. 2000; PubMed Scopus Google Scholar). of subunit was in and cells by with by with at the of the as A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, O.A. Biol. 1994; PubMed Scopus Google Scholar). The was a from of were with as by the and cells were used the type 1 cells were a from M. cells were with using as by the on and ENaC subunits were used were in and in a at with with the from 5 from or from and stably cells were on size for 5 to where The of cells was with to and of with as J.B. O.A. Biol. 2000; PubMed Scopus Google Scholar). in Xenopus type and epitope-tagged ENaC subunit were using the The subunit were oocytes, and were as S. Kleyman T.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The in current in the and of was used to ENaC produced by different subunit from were of sodium and for at from was to the were for at in a at to and were at on a with the mouse and on from to was used for in subunits were with SDS from to by cells 5 to addition of SDS to and as Biol. PubMed Scopus Google Scholar). were by in a and were with in with SDS in and with were in for at to of the for as J.B. O.A. Biol. 2000; PubMed Scopus Google Scholar). weights of were using from on the gels were to size as by the and with in were in with and for at or at by second for 1 at with were with of on the was and using from as by the with from of was from or cells as of ENaC were with Endo from from or N-glycanase/PNGase from as by the and of cells ENaC subunits in were with of from with Biol. PubMed Scopus Google and of subunits as for were to as for and subunits were on the using a the was to was to the with the 3HGal where cells ENaC subunits were with both or to and of as epitope tags were to the carboxyl terminus of the three subunits of mouse ENaC using a epitope was from the sequence of the human and P. and T. R. in co-expression of epitope-tagged ENaC subunits in assembly of amiloride-sensitive sodium of α-, β-, and γ-subunit were and sodium in oocytes by the that of epitope-tagged subunits sodium with amiloride-sensitive in the of similar to have with type ENaC S. Kleyman T.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Sheng S. Kleyman T.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Because these that the epitope-tagged αβγENaC subunits produced amiloride-sensitive sodium they were used to ENaC assembly and processing. The α-, β-, and were expressed in cells to the the epitope tags used to subunits or subunits from The cells were with and ENaC subunits were with were to as shown in When expressed as subunits, was When three subunits were subunit was that channel assembly had a band at kDa was α was expressed alone and with band was on the in using or However, the band was on the αβγENaC was expressed in and were with or second band (65 kDa) was in the three subunits were but was expressed alone in The of the with the but with a similar a band was by but or was expressed The band at in the represents the γ-subunit was to treatment with Endo or N-glycanase that N-glycans from second band at 75 kDa was three subunits were and both the and with the α- and for on from in N-glycan processing. of was for the When expressed the produced a single but expressed as a second band was for The and were also with the the of the was with the The band was on three subunits of ENaC were that processing of on the channel during its to the of processing that within the complex that on are of N-glycans from to complex type and addition of processing was for the αβγENaC was expressed in a glycosylation-defective of cells that the to to L. M. Full Text PDF PubMed Scopus Google Scholar). synthesis of with a core as as the of N-glycans that addition of to However, the with or the of the in cells L. M. Full Text PDF PubMed Scopus Google Scholar). shown in the band was in of cells by and immunoblotting the was expressed alone or as of to the with or in of a for the with the of the The band was the were with Because the of the band was on the of in the that its from processing of N-glycans on the of αβγENaC to complex type and from addition of was confirmed by that addition of 3HGal to the of cells but the produced a band were using a of the in αβγENaC with 3HGal was that 3HGal was to N-glycans and to additional were also in the the on were also three subunits were Because of similar size (65 and 75 kDa) were for both the α- and by immunoblotting αβγENaC additional were to these were processed of the α- and shown in of the α-, β-, or γ-subunit of ENaC from cells αβγENaC and with 3HGal produced the three labeling of the three was on the of consistent with N-glycan processing of these Because is that three ENaC subunits of cells in was of ENaC subunits with shown in of ENaC subunits in SDS produced a single band (65 kDa for the 110 kDa for the and 75 kDa for the to the three that cells were in were to that the for the α-, β-, and subunits with processed from cells αβγENaC or from cells stably αβγENaC were with neuraminidase, endoglycosidase or to the of the N-glycans on each acid from or complex type Endo type N-glycans, N-glycanase both and complex N-glycans from shown in treatment of the from cells the of the band the the on each of the The band was to treatment with Endo and N-glycanase a band at However, the was to treatment with Endo a band at treatment with N-glycanase a band at that the band represents a of the with of its N-glycans processed to the complex the N-glycans on the band are type and of ENaC stably expressed in cells N-glycan and peptide processing of three cells stably with and were on for 5 to treatment of the with of were with and to to were also with sodium to ENaC from the for of the The the band that to are to the of the and and and are to the in the of the processed and for the α- and and The in kDa for ENaC subunits are at the of the of the with the band but the band The band was also to cleavage with Endo a band at N-glycanase treatment produced a band at kDa that of the N-glycans were complex However, the band had type N-glycans was to Endo treatment the band as treatment with of the γ-subunit with a complex treatment produced a in the of the band of the γ-subunit treatment with Endo the band to 75 Because N-glycanase treatment also the band to 75 that the N-glycans on the subunit are However, N-glycanase treatment also revealed at and kDa consistent with the of complex type N-glycans on the of the The epitope-tagged α-, β-, and γ-subunits of ENaC expressed on the of stably cells were also by neuraminidase, Endo H, and N-glycanase were on for 5 to treatment of the with subunits were with with as and to to the of ENaC at the shown in both the and of the both the and of the but the of the γ-subunit were on the the for the γ-subunit by immunoblotting was with the for the α- and is of the of the in the that was expressed in the to of the γ-subunit as the However, the of was in of cells in of with neuraminidase, Endo H, and N-glycanase similar to that in where the of the and the of the exhibit N-glycans that were processed to complex type as shown by treatment with Endo or The smaller size of the processed α- and γ-subunits with the α- and γ-subunits is consistent with proteolytic cleavage of the were to by an epitope at both the amino and carboxyl terminus of the α- and γ-subunits and to the shown in and of or and γ) in or produced the that was by or immunoblotting of the channel with or However, the band was on from cells and a band was on the the band was present was expressed and the band and the band that the fragments stably associated within the channel the and on each in of in and cells and also for proteolytic cleavage of the γ-subunit with α and The γ-subunit was by or immunoblotting of the channel with or However, the band was on from cells α and a band was on The band and the band also that the fragments stably associated within the channel the and on each in The band in from cells in the of α and represents the γ-subunit in the of a band was in but of the γ-subunit to immunoblotting in that fragment from the carboxyl terminus with the channel. The fragment is also present is expressed with or the α- and although a fragment was are consistent with the fragment from a second cleavage of the γ-subunit within the extracellular the the carboxyl terminus in second cleavage the extracellular an epitope to kDa), and by the of the of the carboxyl-terminal epitope on the γ-subunit also the processed of α- and are by the epitope in on of We have shown that N-linked on a of three subunits of mouse ENaC are processed to complex type expressed in or cells and that processing three subunits are processing also to to cleavage of the α- and for N-glycan processing is based on of the subunits on SDS-PAGE, sensitivity to resistance to Endo and of 3HGal N-glycans on labeling with 3HGal is in cells have of the they the that from L. M. Full Text PDF PubMed Scopus Google Scholar). 3HGal to the is the of these 3HGal is used for processing of N-linked is present to on or Because labeling of three subunits of ENaC in cells with 3HGal in the of and of 3HGal in the of is consistent with processing of ENaC N-glycans to complex rather than addition of 3HGal was α, β, or were expressed for in that with three subunits are processed in the complex where for N-glycan processing 3HGal the of the was N-glycan processing the of on SDS the band was the was expressed alone the of and was on immunoblotting three subunits were expressed and de la Rosa de la Rosa D. Li H. Canessa C.M. J. Gen. Physiol. 2002; 119: 427-442Crossref PubMed Scopus (127) Google also two of the in cells at and that were to N-glycanase but the was to Endo However, were to two additional 75 kDa in for the Because additional for the α- and γ-subunits were also 75 kDa by immunoblotting αβγENaC the that N-glycans on three subunits were that with to α-, β-, or revealed the three and that of the channel subunits with SDS to revealed one band for each Based on immunoblotting of with confirmed that the α-, β-, and the γ-subunit exhibit processed of ENaC of α- and is in the that the of that represents the of the subunit in C. M. S. T. C. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google two of the ENaC in rat epithelial type cells at and kDa on SDS-PAGE, but the of α was at the by and immunoblotting with a de la Rosa de la Rosa D. Li H. Canessa C.M. J. Gen. Physiol. 2002; 119: 427-442Crossref PubMed Scopus (127) Google also two of the at and kDa on from cells using and the The also these two of the by of αβγENaC in the of the to the of the and the was to Endo treatment de la Rosa D. Li H. Canessa C.M. J. Gen. Physiol. 2002; 119: 427-442Crossref PubMed Scopus (127) Google Scholar). However, the cells were with a of by in the and the de la Rosa D. Li H. Canessa C.M. J. Gen. Physiol. 2002; 119: 427-442Crossref PubMed Scopus (127) Google that the the with that were to When with to and with acid to of the in the immunoblotting or the on for cleavage of the mouse ENaC by of an with epitope tags at the amino and carboxyl in the extracellular to the membrane-spanning for the of the fragment and the fragment is with the β- and There is also in the that the of represents the of the subunit in S. M. J.B. J. Scopus Google an in the molecular of the γ-subunit of in rat kidney in to on a was a in the and an in a of by immunoblotting with a within the cytosolic carboxyl The that a proteolytic cleavage in the of the extracellular of the γ-subunit by an as for the of the of S. M. J.B. J. 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Chem. 2000; 275: 39886-39893Abstract Full Text Full Text PDF PubMed Scopus (96) Google also a of the on the of cells although C. M. S. T. C. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google the processed of at the of rat epithelial type We of both the processed and of the three subunits in and processed and of the α- and at the that is in the processing of subunit N-glycans in the N-glycans on a single are processed to complex but is to the processing of N-glycans on that with that the processing of the N-glycans on three ENaC subunits is that processing is to cleavage of the α- and γ-subunits within the We have that of N-glycan processing with the or proteolytic processing of the α- or γ-subunit of but is that N-glycan processing is on cleavage of the α- and is from present subunits within a single are processed or subunits within a channel complex are both extracellular as and as and have been to ENaC is ENaC subunits and the of is are to the of the in αβγENaC processing as as the of proteolytic cleavage. The to in ENaC proteolytic processing a to ENaC cleavage the the subunit or the and of the channel. We for
Hughey et al. (Mon,) studied this question.