|
|
|
|
|
|
|
||
Original Article |
psnyder{at}blue.weeg.uiowa.edu
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, β, and 
). The channel forms the pathway for Na+ absorption in the kidney, and mutations cause disorders of Na+ homeostasis. However, little is known about the mechanisms that control the gating of ENaC. We investigated the gating mechanism by introducing bulky side chains at a position adjacent to the extracellular end of the second membrane spanning segment (549, 520, and 529 in , β, and ENaC, respectively). Equivalent "DEG" mutations in related DEG/ENaC channels in Caenorhabditis elegans cause swelling neurodegeneration, presumably by increasing channel activity. We found that the Na+ current was increased by mutagenesis or chemical modification of this residue and adjacent residues in , β, and ENaC. This resulted from a change in the gating of ENaC; modification of a cysteine at position 520 in βENaC increased the open state probability from 0.12 to 0.96. Accessibility to this side chain from the extracellular side was state-dependent; modification occurred only when the channel was in the open conformation. Single-channel conductance decreased when the side chain contained a positive, but not a negative charge. However, alterations in the side chain did not alter the selectivity of ENaC. This is consistent with a location for the DEG residue in the outer vestibule. The results suggest that channel gating involves a conformational change in the outer vestibule of ENaC. Disruption of this mechanism could be important clinically since one of the mutations that increased Na+ current (N530K) was identified in a patient with renal disease.
Key Words: hypertension • amiloride • sodium channel • epithelia • degenerin
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Garty and Palmer 1997). The channel is a heteromeric complex of , β, and ENaC subunits (Canessa et al. 1994; McDonald et al. 1995). In the kidney collecting duct, ENaC plays a critical role in Na+ homeostasis and blood pressure control; mutations cause inherited forms of hypertension (Liddle's syndrome) and hypotension (pseudohypoaldosteronism type 1; Snyder et al. 1995; Lifton 1996). In the lung and intestine, ENaC controls the quantity and electrolyte composition of the surface liquid (Hummler et al. 1996; Zabner et al. 1998). In cystic fibrosis, defective regulation of ENaC may contribute to the pathogenesis of airway disease (Boucher et al. 1986).
Little is known about the mechanisms that control the gating of ENaC. In contrast to voltage- and ligand-gated ion channels, ENaC conducts current in the absence of an identifiable stimulus (Canessa et al. 1994; Benos et al. 1995; McDonald et al. 1995; Garty and Palmer 1997). However, at the single-channel level, there is wide variability in the gating of the channel (Schild et al. 1995; Snyder et al. 1995). A bimodal Po distribution also has been suggested, with channels existing in either a high or low Po mode (Palmer and Frindt 1996). Sequences in the cytoplasmic NH2 terminus may be involved in the gating of ENaC; mutations in this segment disrupted the ability of the channel to open (Grunder et al. 1997, Grunder et al. 1999), and a loss-of-function mutation associated with pseudohypoaldosteronism type 1 was located in this segment (Grunder et al. 1997). It was also reported that the subunit composition could alter gating: channels comprised of only and β subunits (without ) had a very high Po, whereas channels derived from coexpression of and existed in either a high or low Po state (Fyfe and Canessa 1998). However, in epithelia, all three subunits appear to be required to generate Na+ current (Ishikawa et al. 1998; Snyder 2000).
To investigate the gating mechanisms of ENaC, we examined the effect of mutations in amino acids near the extracellular end of the second membrane-spanning segment. Several findings suggest that this domain might be involved in channel gating. First, in related Caenorhabditis elegans (C. elegans) channels (MEC-4, MEC-10, and DEG-1), dominant gain-of-function mutations in this domain cause neuronal swelling and degeneration, presumably by increasing channel activity (Tavernarakis and Driscoll 1997). These DEG mutations change an alanine to amino acids with a bulky or charged side chain. Second, mutation of a glycine at the equivalent position altered the function of BNC1 (ASIC2), a mammalian member of the DEG/ENaC family expressed in neurons (Price et al. 1996). Mutation of this glycine to bulky or charged amino acids converted BNC1 from a proton-activated channel to a channel that was active at neutral pH (Waldmann et al. 1996; Adams et al. 1998). Finally, in a previous study, we found that covalent modification of cysteines introduced in this domain in ENaC increased Na+ current (Snyder et al. 1999). In each ENaC subunit, a serine is located at the position equivalent to the C. elegans DEG residue (see Fig. 1). Interestingly, mutation of the DEG residue in MEC-4 to serine did not result in neurodegeneration, suggesting that substitution of a serine at this position is a conservative change. In this work, we investigated the mechanism by which DEG mutations in ENaC increase current, and the location of this residue within the channel complex.
|
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, β, and ENaC in pMT3, pcDNA3 (McDonald et al. 1994, McDonald et al. 1995), or pGEM-HE (Volk et al. 2000) were generated as previously described, mutated using the QuickChange kit (Stratagene), and sequenced in the University of Iowa DNA Core. cRNA was transcribed using the Message Machine kit (Ambion).
Expression and Whole-Cell Electrophysiology
For measurement of whole-cell current, cDNAs encoding , β, and ENaC (0.2 ng each; in pMT3 or pcDNA3) were injected into the nucleus of Xenopus oocytes. After incubation in modified Barth's solution at 18°C for 16–24 h, we measured whole-cell Na+ currents by two-electrode voltage clamp with the cells bathed in 116 mM NaCl, 2 mM KCl, 0.4 mM CaCl2, 1 mM MgCl2, and 5 mM Hepes, pH 7.4, with NaOH. Amiloride-sensitive current was determined at –60 mV by adding a maximal dose (100 µM) to the bathing solution. Current–voltage relationships were obtained by stepping from –60 mV to potentials between –120 and +40 mV (20-mV steps) for 300 ms. Permeability ratios were calculated from changes in reversal potential with Na+, Li+, and K+ as the predominant cation in the extracellular bathing solution (Hille 1992).
The response to methanethiosulfonate (MTS) compounds was determined by addition to the bathing solution of 1 mM MTSET ([2-(trimethylammonium)ethyl]methanethiosulfonate bromide), 10 mM MTSES (sodium (2-sulfonatoethyl)MTS), or 1 mM MTSEA-biotincap (N-biotinylcaproylaminoethyl MTS; Toronto Research Chemicals). These compounds have no significant effect on wild-type ENaC currents (Snyder et al. 1999). The percent change in amiloride-sensitive Na+ current was calculated as ((IMTS – Ibasal)/Ibasal) · 100, where IMTS is the amiloride-sensitive current after treatment with an MTS reagent, and Ibasal is the amiloride-sensitive current before treatment.
Single-Channel Currents
, β (wild-type or βS520C), and ENaC were expressed in Xenopus oocytes by cytoplasmic injection of cRNA (2 ng each). 1–3 d after injection, single-channel currents were recorded from devitellinized oocytes by patch-clamp (cell attached configuration). The pipet solution contained 150 mM LiCl, 1 mM CaCl2, 1 mM MgCl2, 5 mM Hepes, pH 7.4, with LiOH. The bath solution contained 150 mM LiCl, 5 mM EDTA, and 5 mM Hepes, pH 7.4, with LiOH. Currents were amplified using an Axopatch 200B amplifier (Axon Instruments) and acquired at 2 kHz using Pulse software (version 8.09; HEKA). Currents were digitally filtered at 100 Hz and analyzed using TAC 3.0 (Bruxton Corporation). Slope conductance was determined between –100 and –40 mV (the conductance was identical in recordings filtered at 100 or 1,000 Hz). Open state probability (Po) was determined at –100 mV in patches containing one to three channels. The majority of patches contained a single channel. Mean open and closed times were determined from patches containing single channels.
To selectively modify channels in the patch, 1 mM MTSET or 10 mM MTSES were included in the patch pipet. In some experiments, we used a lower concentration of MTSET (10 µM), and filled the tip of the pipet with solution lacking MTSET, to allow us to record currents before and after modification of the channel.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and ENaC) in Xenopus oocytes generated 3.2-fold more amiloride-sensitive Na+ current than wild-type ENaC (Fig. 2A and Fig. B). This mutation increased the size of the side chain at the DEG position, and added a positive charge. Mutation of Ser520 to an amino acid with a large neutral side chain (βS520V) or a negatively charged side chain (βS520E) also increased Na+ current (Fig. 2 B). In contrast, a more conservative mutation that did not change the size or charge of the side chain (βS520C) did not increase Na+ current (Fig. 2 B).
|
and ENaC), extracellular MTSET rapidly increased Na+ current 4.3-fold (Fig. 3A and Fig. B). Amiloride completely blocked the stimulated current, indicating that it resulted from the stimulation of ENaC. Also consistent with this interpretation, MTSET had no significant effect on uninjected oocytes (not shown) or oocytes expressing wild-type ENaC (Snyder et al. 1999). MTSET-induced stimulation was irreversible upon removal of MTSET from the bathing solution, but was reversed by the addition of a reducing agent, dithiothreitol (20 mM), to the bathing solution (Fig. 3 A). These results, together with the lack of effect of MTSET on wild-type ENaC, indicate that stimulation resulted from covalent modification of the cysteine introduced at the DEG position.
|
DEG Mutations in and ENaC
The three ENaC subunits share significant sequence similarity, including the serine at the DEG position in all three subunits (Fig. 1). Therefore, we tested the hypothesis that an increase in the size of the side chain at the DEG position in and ENaC would increase Na+ current as it did in βENaC. When we placed a cysteine at the DEG position in (S549C) or ENaC (S529C) (coexpressed with the other two wild-type subunits), Na+ currents were identical to wild-type ENaC (Fig. 4 C). Modification of the cysteine introduced in S549C with MTSET increased Na+ current (Fig. 4 A), although to a lesser extent than modification of βS520C (Fig. 4 B). In contrast, modification of S529C had minimal effect on Na+ current (Fig. 4A and Fig. B; Snyder et al. 1999); either the DEG residue in ENaC was not accessible to modification, or modification did not alter the current. In addition, mutation of S529 to either valine or lysine also failed to increase the current (Fig. 4 C), suggesting that, in ENaC, large side chains at the DEG position do not alter channel function. Conversely, Na+ current decreased when we mutated the DEG residue (Ser549) to valine or lysine in ENaC (Fig. 4 C). This contrasts with the increase in current when S549C was modified by MTSET (Fig. 4 A).
|
or ENaC (positions 550 and 530, respectively; Fig. 4 B). In contrast, modification of a cysteine introduced at the other neighboring position in and βENaC (positions 548 and 519, respectively) did not increase Na+ current. However, in ENaC, modification of this cysteine (position 528) produced a large increase in Na+ current (Fig. 4 B). Thus, stimulation was not specific to the DEG position, but resulted from introduction of a bulky side chain at neighboring positions as well. The data suggest that the DEG region has a similar function in all three ENaC subunits, although there are differences in the function of the specific residues.
A sequence variation in this DEG domain was identified in a patient with diabetic nephropathy, changing Asn530 in ENaC to lysine (N530K; Melander et al. 1998). This change increases the size of the side chain and introduces a positive charge, similar to modification with MTSET. We found that expression of N530K (with wild-type and βENaC) increased Na+ current twofold compared with wild-type ENaC (Fig. 4 D). Thus, a mutation in the DEG domain might alter the function of ENaC in humans.
Effect of Cysteine Modification on Selectivity
ENaC is highly selective for Na+ over K+, and is slightly more permeable to Li+ than Na+ (Benos et al. 1995; Garty and Palmer 1997). This is illustrated by the current–voltage relationships for wild-type ENaC with Na+, Li+, or K+ as the predominant cation in the extracellular bathing solution (Fig. 5 A). To test the hypothesis that the DEG residue contributes to the selectivity properties of ENaC, we determined the cation permeability ratios for mutant and chemically modified channels. Mutation of Ser520 in βENaC to lysine or cysteine did not alter either the Na+/Li+ or K+/Na+ permeability ratios (Fig. 5B and Fig. C, respectively). Modification of a cysteine at this position with either MTSET or MTSES also did not significantly alter selectivity (Fig. 5B and Fig. C). As positive controls, mutation of two adjacent residues in the second membrane-spanning segment (S540C and S542C; Fig. 1, shaded box) changed the selectivity of ENaC (Fig. 5B and Fig. C), similar to previous reports (Kellenberger et al. 1999a,Kellenberger et al. 1999b; Snyder et al. 1999). These results suggest that the DEG residue does not contribute to the selectivity filter of ENaC.
|
and ENaC. To modify the DEG cysteine, MTSET was included in the patch pipet in an independent group of patches. Fig. 6 A shows representative currents from patches containing single channels. Without MTSET, βS520C displayed kinetics similar to previous reports for wild-type ENaC (Fig. 6 A, top; (Palmer and Frindt 1986; Canessa et al. 1994; Snyder et al. 1995; Volk et al. 1995). However, the Po (0.12 ± 0.03; Fig. 6 B) was lower and less variable (Po = 0.005–0.28) than reported for wild-type human ENaC (0.58 ± 0.12; Snyder et al. 1995). MTSET caused a dramatic change in kinetics, converting the βS520C channel from a low to a high Po state (0.96 ± 0.03; Fig. 6A and Fig. B). As illustrated in the single-channel trace (Fig. 6 A), the channel was almost always open, interrupted by only brief closures. Increased Po resulted from both an increase in the mean open time (Fig. 6 D) and a decrease in mean closed time (Fig. 6 E). MTSET had no effect on the gating of wild-type ENaC (not shown). βS520K increased whole-cell Na+ current in the absence of MTSET (Fig. 2 B). This also resulted from an increase in Po (0.96 ± 0.01, n = 4), similar to the modification of βS520C with MTSET
|
). Surprisingly, MTSET decreased the single-channel conductance to 5.8 pS. Thus, an increase in conductance did not contribute to the MTSET-mediated stimulation of ENaC. However, the negatively charged MTSES did not decrease the single-channel conductance (Fig. 6 C), suggesting that the positive charge of MTSET was required. MTSET did not alter the single-channel conductance of wild-type ENaC (not shown).
Modification of βS520C Is State Dependent
To investigate the conformational changes associated with the gating of ENaC, we tested the hypothesis that βS520C was modified selectively in either the open or closed conformation. Modification of the channel by MTSET changes the gating and single-channel conductance, converting ENaC from a low Po, large single-channel conductance (OL) state to a high Po, small single-channel conductance (OS) state. If βS520C was modified in the closed conformation, we predict that the next channel opening would be to the OS/high Po state. This concept is illustrated in Fig. 7 A (top). In contrast, if modification occurred when the channel was open, we predict that the channel would first open into the OL state, followed by a decrease in current to the OS/high Po state at the time of modification (Fig. 7 A, bottom). In the protocol used in Fig. 6, the rate of modification was too fast to allow us to record channel activity at the time of modification. To delay modification, we used a lower concentration of MTSET (10 µM) in the patch pipet, and filled the tip of the pipet with solution lacking MTSET. Using this approach, we were able to observe the transition from the OL/low Po to the OS/high Po state in 10 experiments. An example is shown in Fig. 7 B. The patch contained a single channel that opened only to the OL state during the first 4.6 min of recording (the last 8.5 s are shown, Before Modification). The Po during this time was very low (0.01). The channel then converted (Fig. 7 B, Modification) to the OS/high Po state for the remainder of recording. After modification, the channel had brief infrequent closures (Fig. 7 B, inset c) with a Po close to 1.0, and there was a significant decrease in the single-channel current amplitude (Fig. 7 D), which is consistent with conversion from the OL to the OS state. In Fig. 7 B, inset b shows an expanded time scale to focus on this conversion between states. The channel first opened into the OL state, followed by a decrease in current to the OS state (indicated by the arrowhead). This sequence is consistent with modification of the channel in the open conformation; it was observed in 10/10 experiments (Fig. 7 A). A second example is shown in Fig. 7 C. The three sweeps were taken from the same channel before, during, and after modification with MTSET. This record contains longer closures from the OS state. We did not observe channels open directly into the OS state (Fig. 7 A). Thus, the data suggest that modification of the DEG residue is state-dependent, occurring selectively in the open conformation.
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Several findings suggest that the DEG residue influences the conduction pathway of ENaC. First, a cysteine introduced at this position was accessible to modification with water-soluble thiol-reactive compounds added to the extracellular bathing solution. Second, modification of βS520C with MTSET decreased the single-channel conductance. Third, a positive charge was required; the negatively charged MTSES did not decrease single-channel conductance. This electrostatic effect suggests that the DEG residue is located within the conduction pathway, and is reminiscent of the effect of charged residues in the outer vestibule on the conductance of the nicotinic acetylcholine receptor (Imoto et al. 1988). Also consistent with this model is the previous report that a mutation at the DEG position decreased the ability of a cation (MTSET) to enter the pore and modify a cysteine in ENaC (Eskandari et al. 1999). However, the DEG residue does not appear to be part of the selectivity filter since modification or mutation of this residue did not alter the selectivity of ENaC (Li+ > Na+ >> K+). Thus, the data are consistent with a model in which the DEG residue is located in the outer vestibule of the channel. This also fits with our previous finding that, in ENaC, residues in the DEG domain lie external to the site in the pore of amiloride block (Snyder et al. 1999).
The presence of a bulky side chain at the DEG position produced a dramatic change in the gating of ENaC, converting the channel from a low Po state to a state in which the channel was almost always open. This resulted from a destabilization of the closed state and stabilization of the open state, as reflected by the large decrease in closed time and increase in open time, respectively. The requirement for a bulky side chain suggests that this may be a steric effect; perhaps the bulky side chain interferes with the conformational change required for the channel to close. Such a mechanism was previously proposed to explain the swelling neurodegeneration produced by equivalent mutations in C. elegans (Driscoll and Chalfie 1991). It seems likely that modification of cysteines at the DEG position and surrounding positions in and ENaC increased Na+ current by increasing Po, similar to the modification of βS520C. However, we cannot exclude an increase in the single-channel conductance of these mutant channels.
We used two strategies to alter the side chain at the DEG position: mutagenesis to amino acids with large and/or charged side chains, and modification of an introduced cysteine. Both strategies produced equivalent results, with one exception; modification of S549C increased Na+ current, whereas mutation of this residue to valine or lysine decreased current. The reason for this difference is unclear. It is possible that large side chains at the DEG position in ENaC disrupted the processing of ENaC to the cell surface, resulting in decreased Na+ current. This underscores a significant advantage of the cysteine modification strategy, which allowed us to determine the functional effect of an acute change in the size and/or charge of the side chain. A second possible mechanism involves the number of altered S549 side chains in the channel complex. ENaC contains either two or three subunits (Firsov et al. 1998; Kosari et al. 1998; Snyder et al. 1998; Eskandari et al. 1999). However, we don't know how many of the subunits were modified by MTSET; modification of only one cysteine might be sufficient to increase current. In contrast, when we mutated S549 to valine or lysine, each subunit contained a large side chain. Perhaps one large side chain increases current, but current is decreased when all of the subunits contain large side chains. Finally, the modified cysteine is not identical to lysine or valine; differences in the structure of the side chain could also explain the data. Future work will be required to distinguish between these potential mechanisms. The increase in whole-cell Na+ current with modification of βS520C by MTSET (3.3–4.2 fold; Fig. 3 B and 4 B) was less than predicted (5.4-fold increase) from the increase in Po (8-fold) and decrease in single-channel current (0.67-fold). It is possible that some channels or DEG cysteines may not have been modified by MTSET.
We found that a cysteine introduced at the DEG position was modified only when the channel was in the open conformation. This state-dependent modification suggests that channel gating results from a conformational change that alters the accessibility of the DEG residue to the extracellular bathing solution. Two potential models could explain these results. First, channel gating might result from the opening and closing of a gate in the extracellular domain of ENaC (Fig. 8, top). If the gate was external to the DEG residue, channel closure would block access to this residue. Steric hindrance by the bulky side chain at this position might make it unfavorable for the gate to close. In this model, channel gating does not change the position of the DEG residue in relation to the pore of ENaC. This contrasts with a second potential model in which the DEG residue changes position during the gating conformational change (Fig. 8, bottom). When the channel is open, the DEG residue side chain lines the vestibule where it is accessible to modification. Channel closure moves the DEG residue into a buried inaccessible position. A bulky side chain at the DEG position prevents this conformational change, disrupting channel closing. In this model, the gate lies internal to the DEG residue, either at the selectivity filter or within the intracellular vestibule, similar to K+ channels. The two models shown in Fig. 8 are not mutually exclusive: elements of both models could be true. However, common to both models is the requirement for a conformational change in the outer vestibule in the gating of ENaC.
|
). Thus, the mechanism that underlies the gating of ENaC may be a conserved feature of the DEG/ENaC family.
DEG mutations produce pathology. In C. elegans, DEG mutations in MEC-4, MEC-10, and DEG-1 cause neuronal swelling, lysis, and touch insensitivity. In ENaC, a DEG mutation may also have clinical relevance. Melander and co-workers identified a patient with diabetic nephropathy who had a mutation in the subunit, changing Asn530 to lysine (Melander et al. 1998). Interestingly, we found that this mutation increased Na+ current. Modification of a cysteine at this position with MTSET also increased Na+ current. Thus, its seems possible that mutation of Asn530 might predispose to hypertension, and be a contributing factor to renal disease in this patient. Further work will be required to determine the frequency of this mutation in the population and its role in disease.
|
ACKNOWLEDGMENTS |
|---|
P.M. Snyder was supported by the Roy J. Carver Charitable Trust and by the National Heart, Lung and Blood Institute (grants No. HL-58812 and HL-03575) and National Institute of Diabetes and Digestive Kidney Diseases (grant No. DK-52617) of the National Institutes of Health.
Submitted: 11 May 2000
Revised: 13 October 2000
Accepted: 16 October 2000
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Facebook 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  D. M. Collier and P. M. Snyder Extracellular Chloride Regulates the Epithelial Sodium Channel J. Biol. Chem., October 23, 2009; 284(43): 29320 - 29325. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Haerteis, B. Krueger, C. Korbmacher, and R. Rauh The {delta}-Subunit of the Epithelial Sodium Channel (ENaC) Enhances Channel Activity and Alters Proteolytic ENaC Activation J. Biol. Chem., October 16, 2009; 284(42): 29024 - 29040. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. B. Maarouf, N. Sheng, J. Chen, K. L. Winarski, S. Okumura, M. D. Carattino, C. R. Boyd, T. R. Kleyman, and S. Sheng Novel Determinants of Epithelial Sodium Channel Gating within Extracellular Thumb Domains J. Biol. Chem., March 20, 2009; 284(12): 7756 - 7765. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. M. Collier and P. M. Snyder Extracellular Protons Regulate Human ENaC by Modulating Na+ Self-inhibition J. Biol. Chem., January 9, 2009; 284(2): 792 - 798. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. K. Knight, D. M. Wentzlaff, and P. M. Snyder Intracellular Sodium Regulates Proteolytic Activation of the Epithelial Sodium Channel J. Biol. Chem., October 10, 2008; 283(41): 27477 - 27482. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Lu, F. Echeverri, D. Kalabat, B. Laita, D. S. Dahan, R. D. Smith, H. Xu, L. Staszewski, J. Yamamoto, J. Ling, et al. Small Molecule Activator of the Human Epithelial Sodium Channel J. Biol. Chem., May 2, 2008; 283(18): 11981 - 11994. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. L. Brown, S. M. Fernandez-Illescas, Z. Liao, and M. B. Goodman Gain-of-Function Mutations in the MEC-4 DEG/ENaC Sensory Mechanotransduction Channel Alter Gating and Drug Blockade J. Gen. Physiol., January 29, 2007; 129(2): 161 - 173. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Anantharam, Y. Tian, and L. G. Palmer Open probability of the epithelial sodium channel is regulated by intracellular sodium J. Physiol., July 15, 2006; 574(2): 333 - 347. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. D. Carattino, S. Sheng, J. B. Bruns, J. M. Pilewski, R. P. Hughey, and T. R. Kleyman The Epithelial Na+ Channel Is Inhibited by a Peptide Derived from Proteolytic Processing of Its {alpha} Subunit J. Biol. Chem., July 7, 2006; 281(27): 18901 - 18907. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Pfister, I. Gautschi, A.-N. Takeda, M. van Bemmelen, S. Kellenberger, and L. Schild A Gating Mutation in the Internal Pore of ASIC1a J. Biol. Chem., April 28, 2006; 281(17): 11787 - 11791. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Vukicevic, G. Weder, A. Boillat, A. Boesch, and S. Kellenberger Trypsin Cleaves Acid-sensing Ion Channel 1a in a Domain That Is Critical for Channel Gating J. Biol. Chem., January 13, 2006; 281(2): 714 - 722. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Loffing and L. Schild Functional Domains of the Epithelial Sodium Channel J. Am. Soc. Nephrol., November 1, 2005; 16(11): 3175 - 3181. [Full Text] [PDF]
|
|
|
|
|
|
A. Adebamiro, Y. Cheng, J. P. Johnson, and R. J. Bridges Endogenous Protease Activation of ENaC: Effect of Serine Protease Inhibition on ENaC Single Channel Properties J. Gen. Physiol., September 26, 2005; 126(4): 339 - 352. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. D. Carattino, R. S. Edinger, H. J. Grieser, R. Wise, D. Neumann, U. Schlattner, J. P. Johnson, T. R. Kleyman, and K. R. Hallows Epithelial Sodium Channel Inhibition by AMP-activated Protein Kinase in Oocytes and Polarized Renal Epithelial Cells J. Biol. Chem., May 6, 2005; 280(18): 17608 - 17616. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Kellenberger, I. Gautschi, Y. Pfister, and L. Schild Intracellular Thiol-mediated Modulation of Epithelial Sodium Channel Activity J. Biol. Chem., March 4, 2005; 280(9): 7739 - 7747. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. D. Carattino, S. Sheng, and T. R. Kleyman Mutations in the Pore Region Modify Epithelial Sodium Channel Gating by Shear Stress J. Biol. Chem., February 11, 2005; 280(6): 4393 - 4401. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z.-R. Zhang, G. Cui, X. Liu, B. Song, D. C. Dawson, and N. A. McCarty Determination of the Functional Unit of the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel: ONE POLYPEPTIDE FORMS ONE PORE J. Biol. Chem., January 7, 2005; 280(1): 458 - 468. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Sheng, C. J. Perry, and T. R. Kleyman Extracellular Zn2+ Activates Epithelial Na+ Channels by Eliminating Na+ Self-inhibition J. Biol. Chem., July 23, 2004; 279(30): 31687 - 31696. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. B. Condliffe, H. Zhang, and R. A. Frizzell Syntaxin 1A Regulates ENaC Channel Activity J. Biol. Chem., March 12, 2004; 279(11): 10085 - 10092. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. D. Carattino, S. Sheng, and T. R. Kleyman Epithelial Na+ Channels Are Activated by Laminar Shear Stress J. Biol. Chem., February 6, 2004; 279(6): 4120 - 4126. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. B. Bruns, B. Hu, Y. J. Ahn, S. Sheng, R. P. Hughey, and T. R. Kleyman Multiple epithelial Na+ channel domains participate in subunit assembly Am J Physiol Renal Physiol, October 1, 2003; 285(4): F600 - F609. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Li, S. Sheng, C. J. Perry, and T. R. Kleyman Asymmetric Organization of the Pore Region of the Epithelial Sodium Channel J. Biol. Chem., April 11, 2003; 278(16): 13867 - 13874. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Sheng, C. J. Perry, and T. R. Kleyman External Nickel Inhibits Epithelial Sodium Channel by Binding to Histidine Residues within the Extracellular Domains of alpha and gamma Subunits and Reducing Channel Open Probability J. Biol. Chem., December 13, 2002; 277(51): 50098 - 50111. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Kellenberger, I. Gautschi, and L. Schild An External Site Controls Closing of the Epithelial Na+ Channel ENaC J. Physiol., September 1, 2002; 543(2): 413 - 424. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Kellenberger and L. Schild Epithelial Sodium Channel/Degenerin Family of Ion Channels: A Variety of Functions for a Shared Structure Physiol Rev, July 1, 2002; 82(3): 735 - 767. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. M. Snyder The Epithelial Na+ Channel: Cell Surface Insertion and Retrieval in Na+ Homeostasis and Hypertension Endocr. Rev., April 1, 2002; 23(2): 258 - 275. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. A. Volk, P. M. Snyder, and J. B. Stokes Regulation of Epithelial Sodium Channel Activity through a Region of the Carboxyl Terminus of the alpha -Subunit. EVIDENCE FOR INTRACELLULAR KINASE-MEDIATED REACTIONS J. Biol. Chem., November 16, 2001; 276(47): 43887 - 43893. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Sheng, K. A. McNulty, J. M. Harvey, and T. R. Kleyman Second Transmembrane Domains of ENaC Subunits Contribute to Ion Permeation and Selectivity J. Biol. Chem., November 16, 2001; 276(47): 44091 - 44098. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E.-L. Bassler, T. J. Ngo-Anh, H.-S. Geisler, J. P. Ruppersberg, and S. Grunder Molecular and Functional Characterization of Acid-sensing Ion Channel (ASIC) 1b J. Biol. Chem., August 31, 2001; 276(36): 33782 - 33787. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. C. Askwith, C. J. Benson, M. J. Welsh, and P. M. Snyder DEG/ENaC ion channels involved in sensory transduction are modulated by cold temperature PNAS, May 22, 2001; 98(11): 6459 - 6463. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
