Hypotonic Regulation of Mouse Epithelial Sodium Channel in Xenopus laevis Oocytes

2013 ◽  
Vol 246 (12) ◽  
pp. 949-958 ◽  
Author(s):  
Luciano Galizia ◽  
Gabriela I. Marino ◽  
Alejandro Ojea ◽  
Basilio A. Kotsias
Keyword(s):  
Xenopus Laevis ◽  
Sodium Channel ◽  
Epithelial Sodium Channel ◽  
Xenopus Laevis Oocytes ◽  
Hypotonic Regulation

COMMD1 downregulates the epithelial sodium channel through Nedd4–2

2010 ◽  
Vol 298 (6) ◽  
pp. F1445-F1456 ◽  
Author(s):  
Ying Ke ◽  
A. Grant Butt ◽  
Marianne Swart ◽  
Yong Feng Liu ◽  
Fiona J. McDonald
Keyword(s):  
Epithelial Cells ◽  
Cell Surface ◽  
Sodium Channel ◽  
Rat Kidney ◽  
Copper Metabolism ◽  
Epithelial Sodium Channel ◽  
Surface Expression ◽  
Dominant Negative ◽  
Cell Surface Expression ◽  
Xenopus Laevis Oocytes

The epithelial sodium channel (ENaC) is important for the long-term control of Na+ homeostasis and blood pressure. Our previous studies demonstrated that Copper Metabolism Murr1 Domain-containing protein 1 (COMMD1; previously known as Murr1), a protein involved in copper metabolism, inhibited amiloride-sensitive current in Xenopus laevis oocytes expressing ENaC ( J Biol Chem 279: 5429, 2004). In this study, we report that COMMD1 inhibits amiloride-sensitive current in mammalian epithelial cells expressing ENaC, that the COMM domain of COMMD1 is sufficient for this effect, and that knockdown of COMMD1 increases amiloride-sensitive current. COMMD1 is coexpressed with ENaC in rat kidney medulla cells. COMMD1 increased ubiquitin modification of ENaC and decreased its cell surface expression. COMMD1 abolished insulin-stimulated amiloride-sensitive current and attenuated the stimulation of current by activated serum and glucocorticoid-regulated kinase (SGK1). COMMD1 was found to interact with both SGK1 and Akt1/protein kinase B, and knockdown of COMMD1 enhanced the stimulatory effect of both SGK1 and Akt1 on amiloride-sensitive current. COMMD1's effects were reduced in the presence of ENaC proteins containing PY motif mutations, abolished in the presence of a dominant negative form of Nedd4–2, and knockdown of COMMD1 reduced the inhibitory effect of Nedd4–2 on ENaC, but did not enhance current when Nedd4–2 was knocked down. These data suggest that COMMD1 modulates Na+ transport in epithelial cells through regulation of ENaC cell surface expression and this effect is likely mediated via Nedd4–2.

scholarly journals Defining an inhibitory domain in the gamma subunit of the epithelial sodium channel

2010 ◽  
Vol 299 (4) ◽  
pp. F854-F861 ◽  
Author(s):  
Christopher J. Passero ◽  
Marcelo D. Carattino ◽  
Ossama B. Kashlan ◽  
Mike M. Myerburg ◽  
Rebecca P. Hughey ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Epithelial Sodium Channel ◽  
Xenopus Laevis Oocytes ◽  
Airway Epithelia ◽  
Gamma Subunit ◽  
Γ Subunit ◽  
Inhibitory Region ◽  
Inhibitory Domain ◽  
Complete Deletion ◽  
Epithelial Sodium Channel Enac

Proteases activate the epithelial sodium channel (ENaC) by cleaving the large extracellular domains of the α- and γ-subunits and releasing peptides with inhibitory properties. Furin and prostasin activate mouse ENaC by cleaving the γ-subunit at sites flanking a 43 residue inhibitory tract (γE144-K186). To determine whether there is a minimal inhibitory region within this 43 residue tract, we generated serial deletions in the inhibitory tract of the γ-subunit in channels resistant to cleavage by furin and prostasin. We found that partial or complete deletion of a short segment in the γ-subunit, R158-N171, enhanced channel activity. Synthetic peptides overlapping this segment in the γ-subunit further identified a key 11-mer tract, R158-F168 (RFLNLIPLLVF), which inhibited wild-type ENaC expressed in Xenopus laevis oocytes, and endogenous channels in mpkCCD cells and human airway epithelia. Further studies with amino acid-substituted peptides defined residues that are required for inhibition in this key 11-mer tract. The presence of the native γ inhibitory tract in ENaC weakened the intrinsic binding constant of the 11-mer peptide inhibitor, suggesting that the γ inhibitory tract and the 11-mer peptide interact at overlapping sites within the channel.

Differential modulation of a polymorphism in the COOH terminus of the α-subunit of the human epithelial sodium channel by protein kinase Cδ

2006 ◽  
Vol 290 (2) ◽  
pp. F279-F288 ◽  
Author(s):  
Wusheng Yan ◽  
Laurence Suaud ◽  
Thomas R. Kleyman ◽  
Ronald C. Rubenstein
Keyword(s):  
Sodium Channel ◽  
Epithelial Sodium Channel ◽  
Functional Expression ◽  
Surface Expression ◽  
Xenopus Laevis Oocytes ◽  
Α Subunit ◽  
Calphostin C ◽  
Specific Effects ◽  
Oocyte Membrane

The A663T polymorphism of the α-subunit of the human epithelial sodium channel (hENaC) increases the functional and surface expression of αβγ-hENaC in Xenopus laevis oocytes. The context of this residue in the COOH terminus of α-hENaC is important for this effect, as a homologous change in murine ENaC (mENaC), A692T, does not alter functional and surface expression of mENaC. Query of a phosphoprotein database suggested that the α-T663 residue might be phosphorylated by PKCδ. General inhibition of PKC with calphostin C decreased the functional and surface expression of αT663-hENaC and not αA663-hENaC, and was without effect on αA692-mENaC, αT692-mENaC, and a chimeric m(1–678)/h(650–669)αT663, mβγ-ENaC. These data suggest that residues outside of the α-hENaC COOH terminus are important for modulation of αT663-hENaC trafficking by PKC. In contrast, expression of PKCδ decreased the functional and surface expression of αT663-hENaC and the functional expression of m(1–678)/h(650–669)αT663, mβγ-ENaC, and was without effect on αA663-hENaC, αA692-mENaC, or αT692-mENaC. PKCδ did not phosphorylate the COOH terminus of either αT663-hENaC or αA663-hENaC in vitro, suggesting that it acts indirectly to regulate hENaC trafficking. αT663-hENaC was retrieved from the oocyte membrane more slowly than αA663-hENaC, and calphostin C increased the rate of αT663-hENaC removal from the oocyte membrane to a rate similar to that of αA663-hENaC. In contrast, PKCδ did not alter the rate of removal of αT663-hENaC from the oocyte membrane, suggesting that PKCδ altered rates of αT663-hENaC biosynthesis and/or delivery to the plasma membrane. These data are consistent with PKC isoform-specific effects on the intracellular trafficking of αT663- vs. αA663-hENaC.

Intracellular trafficking of a polymorphism in the COOH terminus of the α-subunit of the human epithelial sodium channel is modulated by casein kinase 1

2007 ◽  
Vol 293 (3) ◽  
pp. F868-F876 ◽  
Author(s):  
Wusheng Yan ◽  
Lynn Spruce ◽  
Michael M. Rosenblatt ◽  
Thomas R. Kleyman ◽  
Ronald C. Rubenstein
Keyword(s):  
Sodium Channel ◽  
Intracellular Trafficking ◽  
Epithelial Sodium Channel ◽  
Functional Expression ◽  
Surface Expression ◽  
Casein Kinase ◽  
Xenopus Laevis Oocytes ◽  
Α Subunit ◽  
Casein Kinase 1

The A663T polymorphism of the α-subunit of the human epithelial sodium channel (hENaC) increases the functional and surface expression of αβγ-hENaC in Xenopus laevis oocytes, and the context of this residue in the COOH terminus of α-hENaC is important for this effect. Query of a phosphoprotein database suggested that the α-T663 residue of hENaC might be a substrate for phosphorylation by casein kinase 1 (CK1). We tested the hypotheses that phosphorylation of α-T663-hENaC by CK1 would regulate the increased functional and surface expression of α-T663-hENaC vs. α-A663-hENaC in oocytes. General inhibition of CK1 with IC261 decreased the functional and surface expression of α-T663-hENaC, but not α-A663-hENaC. This decrease in α-T663-hENaC functional expression resulted from reduced delivery of α-T663-hENaC to the oocyte membrane. IC261 also inhibited the functional expression of α-T692-mENaC and a chimeric m(1-678)/h(650-669)α-T663, mβγ ENaC, but not α-A692-mENaC or m(1-678)/h(650-669)α-A663, mβγ ENaC. These data suggest that additional residues outside of the α-hENaC COOH terminus are important for modulation of α-T663-hENaC trafficking by CK1. Overexpression of CK1α did not alter functional expression of α-T663-hENaC. In contrast, modest overexpression of CK1δ enhanced, whereas higher levels of CK1δ overexpression inhibited α-T663-hENaC functional expression. CK1 did not phosphorylate the COOH terminus of either α-T663-hENaC or α-A663-hENaC in vitro. These data suggest that CK1, and perhaps specifically CK1δ, regulates the intracellular trafficking of the α-A663T functional polymorphism of hENaC indirectly by altering the rate of α-T663-hENaC biosynthesis and/or delivery to the plasma membrane.

Membrane topology of the epithelial sodium channel in intact cells

1994 ◽  
Vol 267 (6) ◽  
pp. C1682-C1690 ◽  
Author(s):  
C. M. Canessa ◽  
A. M. Merillat ◽  
B. C. Rossier
Keyword(s):  
Sodium Channel ◽  
Epithelial Sodium Channel ◽  
Functional Expression ◽  
Membrane Topology ◽  
Xenopus Laevis Oocytes ◽  
Intact Cells ◽  
Free Translation ◽  
A Cell ◽  
Hydrophilic Loop

The highly selective amiloride-sensitive epithelial sodium channel is formed of three homologous subunits termed alpha-, beta-, and gamma-rENaC. Each subunit has two putative transmembrane domains (M1 and M2), yielding a protein with a large (approximately 50 kDa) hydrophilic loop (between M1 and M2) and short hydrophilic NH2- and COOH-termini (9 and 10 kDa). All three subunits are glycosylated in a cell-free translation assay, demonstrating that they share in vitro a common pattern of membrane insertion. The membrane topology of the alpha-rENaC subunit in intact cells was studied in Xenopus laevis oocytes. We demonstrate that 1) all six potential N-linked glycosylation sites (N190, N259, N320, N339, N424, and N538) of the large hydrophilic loop are used in intact cells; 2) the glycosylation of alpha-rENaC does not play a significant role in the functional expression of the channel; and 3) the two hydrophobic domains M1 (A109-F131) and M2 (S588-L612) serve in intact cells as start- and stop-transfer signals, respectively. We conclude that alpha-rENaC spans the membrane twice with the short NH2- and COOH-terminal ends on the cytoplasmic side and a large hydrophilic loop in the extracellular space.

Concentration-dependent accumulation of [3H]-deltamethrin in sodium channel Nav1.2/β1 expressing Xenopus laevis oocytes

2007 ◽  
Vol 21 (8) ◽  
pp. 1672-1677 ◽  
Author(s):  
Jennifer A. Watkins ◽  
Connie A. Meacham ◽  
Kevin M. Crofton ◽  
Timothy J. Shafer
Keyword(s):  
Xenopus Laevis ◽  
Sodium Channel ◽  
Xenopus Laevis Oocytes

scholarly journals Plasmin and chymotrypsin have distinct preferences for channel activating cleavage sites in the γ subunit of the human epithelial sodium channel

2012 ◽  
Vol 140 (4) ◽  
pp. 375-389 ◽  
Author(s):  
Silke Haerteis ◽  
Matteus Krappitz ◽  
Alexei Diakov ◽  
Annabel Krappitz ◽  
Robert Rauh ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Cleavage Site ◽  
Double Mutant ◽  
Epithelial Sodium Channel ◽  
Xenopus Laevis Oocytes ◽  
Cleavage Sites ◽  
Channel Activation ◽  
Proteolytic Activation ◽  
Double Mutation ◽  
Γ Subunit

Proteolytic activation of the epithelial sodium channel (ENaC) involves cleavage of its γ subunit in a critical region targeted by several proteases. Our aim was to identify cleavage sites in this region that are functionally important for activation of human ENaC by plasmin and chymotrypsin. Sequence alignment revealed a putative plasmin cleavage site in human γENaC (K189) that corresponds to a plasmin cleavage site (K194) in mouse γENaC. We mutated this site to alanine (K189A) and expressed human wild-type (wt) αβγENaC and αβγK189AENaC in Xenopus laevis oocytes. The γK189A mutation reduced but did not abolish activation of ENaC whole cell currents by plasmin. Mutating a putative prostasin site (γRKRK178AAAA) had no effect on the stimulatory response to plasmin. In contrast, a double mutation (γRKRK178AAAA;K189A) prevented the stimulatory effect of plasmin. We conclude that in addition to the preferential plasmin cleavage site K189, the putative prostasin cleavage site RKRK178 may serve as an alternative site for proteolytic channel activation by plasmin. Interestingly, the double mutation delayed but did not abolish ENaC activation by chymotrypsin. The time-dependent appearance of cleavage products at the cell surface nicely correlated with the stimulatory effect of chymotrypsin on ENaC currents in oocytes expressing wt or double mutant ENaC. Delayed proteolytic activation of the double mutant channel with a stepwise recruitment of so-called near-silent channels was confirmed in single-channel recordings from outside-out patches. Mutating two phenylalanines (FF174) in the vicinity of the prostasin cleavage site prevented proteolytic activation by chymotrypsin. This indicates that chymotrypsin preferentially cleaves at FF174. The close proximity of FF174 to the prostasin site may explain why mutating the prostasin site impedes channel activation by chymotrypsin. In conclusion, this study supports the concept that different proteases have distinct preferences for certain cleavage sites in γENaC, which may be relevant for tissue-specific proteolytic ENaC activation.

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