James B. Bruns

Epithelial Na+ channels are fully activated by furin- and prostasin-dependent release of an inhibitory peptide from the gamma-subunit.

Epithelial sodium channels (ENaC) are expressed in the apical membrane of high resistance Na+ transporting epithelia and have a key role in regulating extracellular fluid volume and the volume of airway surface liquids. Maturation and activation of ENaC subunits involves furin-dependent cleavage of the ectodomain at two sites in the α subunit and at a single site within the γ subunit. We now report that the serine protease prostasin further activates ENaC by inducing cleavage of the γ subunit at a site distal to the furin cleavage site. Dual cleavage of the γ subunit is predicted to release a 43-amino acid peptide. Channels with a γ subunit lacking this 43-residue tract have increased activity due to a high open probability. A synthetic peptide corresponding to the fragment cleaved from the γ subunit is a reversible inhibitor of endogenous ENaCs in mouse cortical-collecting duct cells and in primary cultures of human airway epithelial cells. Our results suggest that multiple proteases cleave ENaC γ subunits to fully activate the channel.

Maturation of the Epithelial Na+ Channel Involves Proteolytic Processing of the α- and γ-Subunits

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 [3H]Gal 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 is inhibited by a peptide derived from proteolytic processing of its alpha subunit.

Epithelial sodium channels (ENaCs) mediate Na+ entry across the apical membrane of high resistance epithelia that line the distal nephron, airway and alveoli, and distal colon. These channels are composed of three homologous subunits, termed α, β, and γ, which have intracellular amino and carboxyl termini and two membrane-spanning domains connected by large extracellular loops. Maturation of ENaC subunits involves furin-dependent cleavage of the extracellular loops at two sites within the α subunit and at a single site within the γ subunit. The α subunits must be cleaved twice, immediately following Arg-205 and Arg-231, in order for channels to be fully active. Channels lacking α subunit cleavage are inactive with a very low open probability. In contrast, channels lacking both α subunit cleavage and the tract αAsp-206-Arg-231 are active when expressed in oocytes, suggesting that αAsp-206-Arg-231 functions as an inhibitor that stabilizes the channel in the closed conformation. A synthetic 26-mer peptide (α-26), corresponding to αAsp-206-Arg-231, reversibly inhibits wild-type mouse ENaCs expressed in Xenopus oocytes, as well as endogenous Na+ channels expressed in either a mouse collecting duct cell line or primary cultures of human airway epithelial cells. The IC50 for amiloride block of ENaC was not affected by the presence of α-26, indicating that α-26 does not bind to or interact with the amiloride binding site. Substitution of Arg residues within α-26 with Glu, or substitution of Pro residues with Ala, significantly reduced the efficacy of α-26. The peptide inhibits ENaC by reducing channel open probability. Our results suggest that proteolysis of the α subunit activates ENaC by disassociating an inhibitory domain (αAsp-206-Arg-231) from its effector site within the channel complex.

Recycling of MUC1 Is Dependent on Its Palmitoylation

MUC1 is a mucin-like transmembrane protein expressed on the apical surface of epithelia, where it protects the cell surface. The cytoplasmic domain has numerous sites for phosphorylation and docking of proteins involved in signal transduction. In a previous study, we showed that the cytoplasmic YXXφ motif Y20HPM and the tyrosine-phosphorylated Y60TNP motif are required for MUC1 clathrin-mediated endocytosis through binding AP-2 and Grb2, respectively (Kinlough, C. L., Poland, P. A., Bruns, J. B., Harkleroad, K. L., and Hughey, R. P. (2004) J. Biol. Chem. 279, 53071-53077). Palmitoylation of transmembrane proteins can affect their membrane trafficking, and the MUC1 sequence CQC3RRK at the boundary of the transmembrane and cytoplasmic domains mimics reported site(s) of S-palmitoylation. [3H]Palmitate labeling of Chinese hamster ovary cells expressing MUC1 with mutations in CQC3RRK revealed that MUC1 is dually palmitoylated at the CQC motif independent of RRK. Lack of palmitoylation did not affect the cold detergent solubility profile of a chimera (Tac ectodomain and MUC1 transmembrane and cytoplasmic domains), the rate of chimera delivery to the cell surface, or its half-life. Calculation of rate constants for membrane trafficking of wild-type and mutant Tac-MUC1 indicated that the lack of palmitoylation blocked recycling, but not endocytosis, and caused the chimera to accumulate in a EGFP-Rab11-positive endosomal compartment. Mutations CQC/AQA and Y20N inhibited Tac-MUC1 co-immunoprecipitation with AP-1, although mutant Y20N had reduced rates of both endocytosis and recycling, but a normal subcellular distribution. The double mutant chimera AQA+Y20N had reduced endocytosis and recycling rates and accumulated in EGFP-Rab11-positive endosomes, indicating that palmitoylation is the dominant feature modulating MUC1 recycling from endosomes back to the plasma membrane.

MUC1 membrane trafficking is modulated by multiple interactions.

MUC1 is a mucin-like transmembrane protein found on the apical surface of many epithelia. Because aberrant intracellular localization of MUC1 in tumor cells correlates with an aggressive tumor and a poor prognosis for the patient, experiments were designed to characterize the features that modulate MUC1 membrane trafficking. By following [35S]Met/Cys-labeled MUC1 in glycosylation-defective Chinese hamster ovary cells, we found previously that truncation of O-glycans on MUC1 inhibited its surface expression and stimulated its internalization by clathrin-mediated endocytosis. To identify signals for MUC1 internalization that are independent of its glycosylation state, the ectodomain of MUC1 was replaced with that of Tac, and chimera endocytosis was measured by the same protocol. Endocytosis of the chimera was significantly faster than for MUC1, indicating that features of the highly extended ectodomain inhibit MUC1 internalization. Analysis of truncation mutants and tyrosine mutants showed that Tyr20 and Tyr60 were both required for efficient endocytosis. Mutation of Tyr20 significantly blocked coimmunoprecipitation of the chimera with AP-2, indicating that Y20HPM is recognized as a YXXϕ motif by the μ2 subunit. The tyrosine-phosphorylated Y60TNP was previously identified as an SH2 site for Grb2 binding, and we found that mutation of Tyr60 blocked coimmunoprecipitation of the chimera with Grb2. This is the first indication that Grb2 plays a significant role in the endocytosis of MUC1.

Functional Role of Extracellular Loop Cysteine Residues of the Epithelial Na+ Channel in Na+ Self-inhibition

The epithelial Na+ channel (ENaC) is typically formed by three homologous subunits (α, β, and γ) that possess a characteristic large extracellular loop (ECL) containing 16 conserved cysteine (Cys) residues. We investigated the functional role of these Cys residues in Na+ self-inhibition, an allosteric inhibition of ENaC activity by extracellular Na+. All 16 Cys residues within α and γ ECLs and selected β ECL Cys residues were individually mutated to alanine or serine residues. The Na+ self-inhibition response of wild type and mutant channels expressed in Xenopus oocytes was determined by whole cell voltage clamp. Individual mutation of eight α (Cys-1, -4, -5, -6, -7, -10, -13, or -16), one β (Cys-7), and nine γ (Cys-3, -4, -6, -7, -10, -11, -12, -13, or -16) residues significantly reduced the magnitude of Na+ self-inhibition. Na+ self-inhibition was eliminated by simultaneous mutations of either the last three α ECL Cys residues (Cys-14, -15, and -16) or Cys-7 within both α and γ ECLs. By analyzing the Na+ self-inhibition responses and the effects of a methanethiosulfonate reagent on channel currents in single and double Cys mutants, we identified five Cys pairs within the αECL (αCys-1/αCys-6, αCys-4/αCys-5, αCys-7/αCys-16, αCys-10/αCys-13, and αCys-11/αCys-12) and one pair within the γECL (γCys-7/γCys-16) that likely form intrasubunit disulfide bonds. We conclude that approximately half of the ECL Cys residues in the α and γ ENaC subunits are required to establish the tertiary structure that ensures a proper Na+ self-inhibition response, likely by formation of multiple intrasubunit disulfide bonds.

γ‐Glutamyltranspeptidase: Disulfide Bridges, Propeptide Cleavage, and Activation in the Endoplasmic Reticulum

gamma-Glutamyltranspeptidase (gammaGT) is found primarily on the apical surface of epithelial and endothelial cells, where it degrades reduced and oxidized glutathione (gamma-GluCysGly) by hydrolysis of the unique gamma-glutamyl bond. Glutathione plays a key role in disulfide rearrangement in the endoplasmic reticulum (ER) and acts as a redox buffer. Previous work has shown that overexpression of gammaGT or an inactive splice variant gammaGTDelta7 mediates a redox stress response in the endoplasmic reticulum (ER) characterized by increased levels of BiP and induction of CHOP-10. To determine whether a CX(3)C motif might be the common feature of gammaGT and gammaGTDelta7 that mediates this response, we characterized disulfide bridges in gammaGT that might form between the six highly conserved Cys residues. Using site-directed mutagenesis of gammaGT, expression in Chinese Hamster Ovary (CHO) cells, metabolic labeling, and immunoblotting, our data predict disulfide formation between Cys49 and Cys73 and between Cys191 and Cys195 (the CX(3)C motif). Potential functions for this CX(3)C motif are discussed. In the course of defining the disulfides, we also noted that propeptide cleavage correlated with enzymatic activity. Because recent reports indicate that the homologous Escherichia coli gammaGT is a member of the N-terminal nucleophile (Ntn) hydrolase family, where the amino acid at the new N-terminus functions as the nucleophile for both autocatalytic cleavage and enzymatic activity, the rat gammaGT was similarly characterized. As predicted, mutations at the propeptide cleavage site coincidentally inhibit both heterodimer formation and gammaGT enzymatic activity. Analysis of early cleavage events using cell extraction into SDS indicates that propeptide cleavage occurs while gammaGT is still within the ER. Because activation and cleavage are coincident events, this raises the new question of whether an active glutathionase is present within the ER and what role gammaGT plays in modulating ER glutathione levels that are so critical for proper redox balance and disulfide formation in this compartment.

Epithelial Sodium Channel Exit from the Endoplasmic Reticulum Is Regulated by a Signal within the Carboxyl Cytoplasmic Domain of the α Subunit

Epithelial sodium channels (ENaCs) are assembled in the endoplasmic reticulum (ER) from α, β, and γ subunits, each with two transmembrane domains, a large extracellular loop, and cytoplasmic amino and carboxyl termini. ENaC maturation involves transit through the Golgi complex where Asn-linked glycans are processed to complex type and the channel is activated by furin-dependent cleavage of the α and γ subunits. To identify signals in ENaC for ER retention/retrieval or ER exit/release, chimera were prepared with the interleukin α subunit (Tac) and each of the three cytoplasmic carboxyl termini of mouse ENaC (Tac-Ct) or with γ-glutamyltranspeptidase and each of the three cytoplasmic amino termini (Nt-GGT). By monitoring acquisition of endoglycosidase H resistance after metabolic labeling, we found no evidence of ER retention of any chimera when compared with control Tac or GGT, but we did observe enhanced exit of Tac-αCt when compared with Tac. ER exit of ENaC was assayed after metabolic labeling by following the appearance of cleaved α as cleaved α subunit, but not non-cleaved α, is endoglycosidase H-resistant. Interestingly ER exit of epitope-tagged and truncated α (αΔ624–699-V5) with full-length βγ was similar to wild type α (+βγ), whereas ER exit of ENaC lacking the entire cytoplasmic carboxyl tail of α (αΔ613–699-V5 +βγ) was significantly reduced. Subsequent analysis of ER exit for ENaCs with mutations within the intervening sequence 613HRFRSRYWSPG623 within the context of the full-length α revealed that mutation αRSRYW620 to AAAAA significantly reduced ER exit. These data indicate that ER exit of ENaC is regulated by a signal within the α subunit carboxyl cytoplasmic tail.