Point mutations in the post-M2 region of human α-ENaC regulate cation selectivity

Ji, Hong; Parker, Suzanne; Langloh, Anne Lynn B.; Fuller, Catherine M.; Benos, Dale J.

doi:10.1152/ajpcell.2001.281.1.c64

Cited by 30 publications

(39 citation statements)

References 37 publications

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“…ENaC mutants were constructed as described previously (31). Preparation of oocytes and the two-electrode voltage clamp studies were performed as described in the online supplement (31).…”

Section: Oocyte Expression and Voltage Clamp Studiesmentioning

confidence: 99%

8-(4-Chlorophenylthio)-Guanosine-3′,5′-Cyclic Monophosphate-Na Stimulates Human Alveolar Fluid Clearance by Releasing External Na⁺ Self-Inhibition of Epithelial Na⁺ Channels

Han

Nie²,

et al. 2011

Am J Respir Cell Mol Biol

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Salt absorption via alveolar epithelial Na 1 channels (ENaC) is a critical step for maintaining an airspace free of flooding. Previously, we found that 8-(4-chlorophenylthio)-guanosine-39,59-cyclic monophosphate-Na (CPT-cGMP) activated native and heterologous ENaC. To investigate the potential pharmacological relevance, we applied this compound intratracheally to human lungs and found that ex vivo alveolar fluid clearance was increased significantly. Furthermore, this compound eliminated self-inhibition in human lung H441 cells and in oocytes expressing human abg but not dbg channels. To further elucidate this novel mechanism, we constructed mutants abolishing (b DV348 and g H233R ) or augmenting (a Y458A and g M432G ) self-inhibition. The mutants eliminating self-inhibition lost their responses to CPT-cGMP, whereas those enhancing self-inhibition facilitated the stimulatory effects of this compound. CPT-cGMP was unable to activate a high P o mutant (b S520C ) and plasmin proteolytically cleaved channels. Our data suggest that elimination of self-inhibition of abg ENaC may be a novel mechanism for CPT-cGMP to stimulate salt reabsorption in human lungs.Keywords: lung fluid reabsorption; amiloride-sensitive sodium channel; CPT-cGMP; ENaC self-inhibition Epithelial Na1 channels (ENaC) in polarized absorptive tight epithelium are responsible for the transapical salt influx. Together with basolaterally located Na 1 /K 1 -ATPase, the vectorial solute/fluid transport pathway is crucial for maintaining luminal and body fluid volume (1-3). Five subunits have been cloned: a, b, g, d, and e ENaC. Luminal impermeable reagents and hormones have been confirmed to regulate salt reabsorption in lung, kidney, and colon.ENaC has long been proposed to function as a ligand-gated channel on the basis of observations of other ENaC/DEG family members (1, 4). Acid-sensing Na 1 channels, a branch of the ENaC/DEG super gene family, are gated by external protons. Another branch of channels, termed FaNaC, are manipulated by FMRFamide and related tetrapeptides (5). With respect to ENaC channels, a number of luminal reagents, including parachloromercuribenzoate; benzimidazoly-2-guanidinium; bumetanide; and H 1 , Zn 21 , and Ni 21 ions, have been reported to alter external Na 1 self-inhibition, a temperature-sensitive intrinsic phenomenon of ENaC (1, 4, 6-10). Recently, a small molecule (S6939), cpt-cAMP, halothane, glibenclamide, and extracellular chloride ions (Cl 2 ) have been shown to act as ligands to regulate heterologous ENaC (11-16). Among these, halothane and Cl 2 ions may activate ENaC by eliminating Na 1 self-inhibition (17, 18). We recently found that 8-(4-chlorophenylthio)-guanosine-39,59-cyclic monophosphate-Na (CPT-cGMP) stimulated human abg ENaC in oocytes in a PKG-independent and domain-specific manner (19). The underlying mechanisms are unknown.Several ectodomains have been reported to modify self-inhibition by unknown mechanisms. Given that self-inhibition is governed by the extracellular Na 1 concentrations, the extracellu...

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“…ENaC mutants were constructed as described previously (31). Preparation of oocytes and the two-electrode voltage clamp studies were performed as described in the online supplement (31).…”

Section: Oocyte Expression and Voltage Clamp Studiesmentioning

confidence: 99%

8-(4-Chlorophenylthio)-Guanosine-3′,5′-Cyclic Monophosphate-Na Stimulates Human Alveolar Fluid Clearance by Releasing External Na⁺ Self-Inhibition of Epithelial Na⁺ Channels

Han

Nie²,

et al. 2011

Am J Respir Cell Mol Biol

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“…Mutations of two arginine residues (K504E and K515E) near the carboxyl terminus of the extracellular domain of bovine ␣ENaC alter Na ϩ /K ϩ selectivity, amiloride sensitivity, and gating behavior (43). Mutations of arginine residues (␣R586E-R587E) near the carboxyl-terminal end of human ␣ENaC M2 also resulted in significant changes in K ϩ /Na ϩ and Li ϩ /Na ϩ selectivity (10). A stretch of residues within the amino terminus (preceding the first transmembrane domain) of an acid-sensing ion channel type 2, a member of the ENaC/degenerin family, was identified as a region that has a role in determining K ϩ /Na ϩ selectivity (44).…”

Section: Fig 5 Representative Two-electrode Voltage Clamp Recordingmentioning

confidence: 99%

“…Residues within ␣mENaC M2 where mutations altered Li ϩ /Na ϩ current ratios are underlined. ␣Arg 586 and ␣Arg 587 in ␣hENaC are highlighted to indicate residues where a charge reversal of both side chains increased the K ϩ /Na ϩ current ratio (10). Highlighted residues in Kir 2.1 and Shaker B were proposed to expose their side chains to the conducting pore based on the accessibility to the sulfhydryl reagent MTSET (12,46,47).…”

Section: Fig 5 Representative Two-electrode Voltage Clamp Recordingmentioning

confidence: 99%

“…Langloh et al (9) reported that mutations of the negative-charged amino acids within the M2 domains of human ␣ENaC (E568R, E571R, and D575R) nearly eliminated channel activity, although these mutations did not alter the levels of protein expression at the plasma membrane. Furthermore, mutations of positively charged residues immediately following the ␣-subunit M2 domain altered cation selectivity of human ENaC (10). To explore the functional roles of residues within the M2 domains of ENaC, 18 residues (␣Val 593 -␣Met 610 ) within M2 of the ␣ subunit of mouse ENaC (mENaC) were individually mutated to cysteine.…”

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Second Transmembrane Domains of ENaC Subunits Contribute to Ion Permeation and Selectivity

Sheng¹,

McNulty²,

Harvey³

et al. 2001

Journal of Biological Chemistry

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Epithelial sodium channels (ENaC) are composed of three structurally related subunits (␣, ␤, and ␥). Each subunit has two transmembrane domains termed M1 and M2, and residues conferring cation selectivity have been shown to reside in a pore region immediately preceding the M2 domains of the three subunits. Negatively charged residues are interspersed within the M2 domains, and substitution of individual acidic residues within human ␣-ENaC with arginine essentially eliminated channel activity in oocytes, suggesting that these residues have a role in ion permeation. We examined the roles of M2 residues in contributing to the permeation pore by individually mutating residues within the M2 domain of mouse ␣ENaC to cysteine and systematically characterizing functional properties of mutant channels expressed in Xenopus oocytes by two-electrode voltage clamp. The introduction of cysteine residues at selected sites, including negatively charged residues (␣Glu 595 , ␣Glu 598 , and ␣Asp 602 ) led to a significant reduction of expressed amiloride-sensitive Na ؉ currents. Two mutations (␣E595C and ␣D602C) resulted in K ؉ -permeable channels whereas multiple mutations altered Li ؉ / Na ؉ current ratios. Channels containing ␣D602K or ␣D602A also conducted K ؉ whereas more conservative mutations (␣D602E and ␣D602N) retained wild type selectivity. Cysteine substitution at the site equivalent to ␣Asp 602 within ␤ mENaC (␤D544C) did not alter either Li ؉ /Na ؉ or K ؉ /Na ؉ current ratios, although mutation of the equivalent site within ␥ mENaC (␥D562C) significantly increased the Li ؉ /Na ؉ current ratio. Mutants containing introduced cysteine residues at ␣Glu 595 , ␣Glu 598 , ␣Asp 602 , or ␣Thr 607 did not respond to externally applied sulfhydryl reagent with significant changes in macroscopic currents. Our results suggest that some residues within the M2 domain of ␣ENaC contribute to the channel's conduction pore and that, in addition to the pore region, selected sites within M2 (␣Glu 595 and ␣Asp 602 ) may have a role in conferring ion selectivity.The epithelial sodium channel (ENaC 1 ) mediates Na ϩ transport across high resistance epithelia and has a key role in Na ϩ homeostasis and blood pressure control. This channel is a member of the ENaC/degenerin gene superfamily and is composed of three structurally related subunits, termed ␣, ␤, and ␥ (1). Members of the ENaC/degenerin family are homo-or heterooligomeric proteins whose subunits share a common topology of two membrane spanning domains (M1 and M2) and intracellular amino and carboxyl termini. All three ENaC subunits contribute to the formation of the ion-conducting pore (2-7). Hydropathy analyses of ENaC subunits have identified two large hydrophobic domains consisting of about 45 residues. The second hydrophobic domain contains two regions that are distinct in structure and function. The amino-terminal region is functionally similar to the pore region of many cation channels and is a key element determining pore properties of ENaC, including selectivity, gating, condu...

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“…We have identified a sequence of positively charged amino acids between residues 586 and 591 of human ␣-ENaC (613-624 of the rat ␣-ENaC ortholog) (10) that is identical in all five mammalian ␣-ENaC subunits cloned to date (rat, bovine, human, mouse, and guinea pig) and that is arginine rich (RRFRSRYWSPGR). This region is conserved in ␦-ENaC (RRLRRAWFSWPR) (16) but is not present in either the ␤-or ␥-ENaC subunit or in the ENaC-related Aplysia sodium channel that is gated by FMRF-amide (phenylalanine-methionine-argininephenylalanine) (12).…”

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Intrinsic gating mechanisms of epithelial sodium channels

Fuller

Benos

2002

American Journal of Physiology-Cell Physiology

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The hypothesis that there is a highly conserved, positively charged region distal to the second transmembrane domain in ␣-ENaC (epithelial sodium channel) that acts as a putative receptor site for the negatively charged COOH-terminal ␤-and ␥-ENaC tails was tested in mutagenesis experiments. After expression in Xenopus oocytes, ␣-ENaC constructs in which positively charged arginine residues were converted into negatively charged glutamic acids could not be inhibited by blocking peptides. These observations provide insight into the gating machinery of ENaC.Liddle mutant; amiloride; inside-out patch; voltage clamp; post-M2 region LIDDLE'S SYNDROME is a form of hereditary hypertension produced by mutations within the epithelial sodium channel (ENaC) (1, 17). These mutations result in constitutive channel activation. Both an increase in functional channel number and an increase in singlechannel open probability (P o ) have been reported (5-9, 14, 15). The initial description of Liddle's syndrome identified truncation mutations in the COOH-terminal polypeptide chain of the ␤-ENaC (and subsequently the ␥-ENaC) subunit as being causative for constitutive channel activation (3, 5-9, 14, 15). We proposed the hypothesis that the COOH-terminal chains of ␤ and ␥ could act as intrinsic channel blockers by serving as an inactivation moiety. Our evidence, obtained in both bilayers (7,8) and heterologous expression systems (9), supports this type of mechanism. We tested the hypothesis that the functional gating particle comprised the COOH-terminal tails of both the ␤-and ␥-ENaC subunits associated as a two-strand, antiparallel ␤-sheet. Support for this idea was threefold: 1) the inhibitory effects of adding COOH-terminal ␤-and ␥-ENaC 30-amino acid residue peptides together with ENaC comprising wild-type ␣-and COOH-terminally truncated ␤-and ␥-subunits produced a greater than additive inhibition of the channel; 2) circular dichroism studies showed that the 30-mer ␤ and ␥ peptides formed a ␤-sheet; and 3) when the isoleucines and valines within the 30-mer peptides were replaced by the ␤-sheet, breaking amino acids proline or aspartic acid, the resulting peptides were unable to affect basalactivated ENaC (8). The paradigm that we have developed for ENaC gating is as follows. Because ␣-ENaC itself forms a functional sodium channel (4), there must be an intrinsic gating mechanism in ␣-ENaC alone. We hypothesized that calcium is intimately involved in this process and have presented evidence to this effect (2). The overall gating properties of ␣-ENaC vs. ␣␤␥-ENaC in bilayers do not differ (7). Because the elimination of the cytoplasmic COOH-terminal tails of either or both of the ␤-and ␥-subunits substantially increases single-channel P o (5-9), there must be at least two separate gating processes, one inherent to ␣-ENaC alone and one conferred onto the complex by the ␤-and ␥-subunits.To further elucidate the mechanism underlying the COOH-terminal ␤-and ␥-ENaC tail block of ENaC, we tested the hypothesis that a highly conserved region...

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Point mutations in the post-M2 region of human α-ENaC regulate cation selectivity

Cited by 30 publications

References 37 publications

8-(4-Chlorophenylthio)-Guanosine-3′,5′-Cyclic Monophosphate-Na Stimulates Human Alveolar Fluid Clearance by Releasing External Na⁺ Self-Inhibition of Epithelial Na⁺ Channels

8-(4-Chlorophenylthio)-Guanosine-3′,5′-Cyclic Monophosphate-Na Stimulates Human Alveolar Fluid Clearance by Releasing External Na⁺ Self-Inhibition of Epithelial Na⁺ Channels

Second Transmembrane Domains of ENaC Subunits Contribute to Ion Permeation and Selectivity

Intrinsic gating mechanisms of epithelial sodium channels

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Point mutations in the post-M2 region of human α-ENaC regulate cation selectivity

Cited by 30 publications

References 37 publications

8-(4-Chlorophenylthio)-Guanosine-3′,5′-Cyclic Monophosphate-Na Stimulates Human Alveolar Fluid Clearance by Releasing External Na+ Self-Inhibition of Epithelial Na+ Channels

8-(4-Chlorophenylthio)-Guanosine-3′,5′-Cyclic Monophosphate-Na Stimulates Human Alveolar Fluid Clearance by Releasing External Na+ Self-Inhibition of Epithelial Na+ Channels

Second Transmembrane Domains of ENaC Subunits Contribute to Ion Permeation and Selectivity

Intrinsic gating mechanisms of epithelial sodium channels

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About

8-(4-Chlorophenylthio)-Guanosine-3′,5′-Cyclic Monophosphate-Na Stimulates Human Alveolar Fluid Clearance by Releasing External Na⁺ Self-Inhibition of Epithelial Na⁺ Channels

8-(4-Chlorophenylthio)-Guanosine-3′,5′-Cyclic Monophosphate-Na Stimulates Human Alveolar Fluid Clearance by Releasing External Na⁺ Self-Inhibition of Epithelial Na⁺ Channels