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J. Biol. Chem., Vol. 279, Issue 23, 23900-23907, June 4, 2004

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Functional Polymorphism in the Carboxyl Terminus of the -Subunit of the Human Epithelial Sodium Channel^*

Frederick F. Samaha¶, Ronald C. Rubenstein¶||**, Wusheng Yan¶**, Mohan Ramkumar, Daniel I. Levy, Yoon J. Ahn, Shaohu Sheng, and Thomas R. Kleyman¶¶

From the Medicine and ^||Pediatrics, University of Pennsylvania, the Philadelphia Veterans Affairs Medical Center, and the ^**Division of Pulmonary Medicine, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104 and the Departments of Medicine and of ^¶¶Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

Received for publication, February 23, 2004 , and in revised form, March 26, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
A common human epithelial sodium channel (ENaC) polymorphism, T663A, is present in the cytoplasmic C terminus of the -subunit, although it is unclear whether this polymorphism segregates with blood pressure. We examined whether this polymorphism was associated with differences in functional Na⁺ channel expression. Whole cell amiloride-sensitive currents in Xenopus oocytes expressing wild type channels (T663) were significantly 1.3–2.0-fold higher than currents measured in oocytes expressing channels with an Ala, Gly or Leu, or Lys at position 663. In contrast, differences in functional human ENaC expression were not observed with oocytes expressing channels having Thr (wild type), Ser, or Asp at this position. The surface expression of channels, measured using an epitope-tagged -subunit, was significantly reduced in oocytes expressing T663A when compared with oocytes expressing T663. The corresponding polymorphism was generated in the mouse -subunit (mA692T) and was not associated with differences in functional -mouse ENaC expression. The polymorphism is present in a region that is not well conserved between human and mouse. We generated a mouse/human chimera by replacement of the distal C terminus of the mouse -subunit with the distal C terminus of the human -subunit. Co-expression of this m(1–678)/h(650–669)T663A chimera with mouse led to a significant reduction in whole cell Na⁺ currents and surface expression when compared with m(1–678)/h(650–669)T663-m. Our results suggest that hT663A is a functional polymorphism that affects human ENaC surface expression.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Epithelial sodium channels (ENaC)¹ are expressed in principal cells in the late distal convoluted tubule and collecting tubule, where they serve as a final site for reabsorption of Na⁺ from the glomerular ultrafiltrate. Volume regulatory hormones, such as aldosterone, have a key role in modifying rates of renal tubular Na⁺ reabsorption through regulation of functional ENaC expression at the apical plasma membrane (1). Epithelial Na⁺ channels are composed of three structurally related subunits, termed -, -, and -ENaC that likely assemble as a 2,1,1 tetramer (2, 3), although an alternative subunit stoichiometry has been proposed (4). The three subunits share limited (30–40%) sequence identity but share a common topology of two membrane-spanning domains and intracellular N and C termini (5–7).

Changes in ENaC functional expression are associated with alterations in blood pressure (8, 9). Na⁺ channel gain-of-function mutations have been identified in patients with Liddle's syndrome, a disorder characterized by volume expansion, hypokalemia, and hypertension (10, 11). ENaC loss-of-function mutations have been identified in patients with type I pseudohypoaldosteronism, a disorder characterized by volume depletion, hypotension, and hyperkalemia (12, 13). Some common human ENaC polymorphisms may segregate with blood pressure (i.e. T594M) (14), suggesting that ENaC polymorphisms that alter functional channel expression may contribute to the development of hypertension in the general population.

A common polymorphism has been described in C terminus of the -subunit of human ENaC (hENaC), T663A (15, 16). The question of whether this polymorphism segregates with blood pressure has been addressed by several groups, and conflicting results have been reported (15, 16). We examined whether this polymorphism was associated with altered Na⁺ channel activity when expressed in Xenopus oocytes. We observed that oocytes expressing wild type T663 channels had significantly higher currents than oocytes expressing T663A. Furthermore, the higher currents observed with T663 channels were associated with higher levels of cell surface expression of channels, suggesting that this polymorphism altered channel trafficking. This polymorphism is present in the distal C terminus of the -subunit, a region that is not well conserved between the human and mouse -subunit. Although the A692T polymorphism in mouse ENaC (mENaC), corresponding to human T663A, was not associated with differences in functional -mENaC expression, replacement of the distal C terminus of the mouse -subunit with the distal C terminus of the human -subunit restored the functional differences that were observed with the human T663A polymorphism.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Preparation of hENaC Mutants—-, -, and hENaC cDNAs were from M. J. Welsh (University of Iowa). Mouse ENaC cDNAs have been described and used by our group previously (17). All mutants and mouse/human chimeras were generated using a PCR-based mutagenesis technique as described previously (18). All sequences were confirmed by automated DNA sequence analyses performed at sequencing facilities at the University of Pennsylvania, the Children's Hospital of Philadelphia, or the University of Pittsburgh.

Expression of ENaC in Xenopus Oocytes—cRNAs for wild type and mutant hENaC and mENaC, wild type hENaC and mENaC, and wild type hENaC and mENaC were synthesized from linearized plasmids containing the appropriate cDNAs using appropriate RNA polymerases (T₃, T₇, or SP6, mMessage mMachine, Ambion Inc., Austin, TX) and stored at –80 °C. cRNA concentration was determined spectroscopically. Stage V-VI oocytes were surgically harvested from female Xenopus laevis (Nasco, Fort Atkinson, WI) and pretreated with 2 mg/ml collagenase (type IV) as described previously (18). Oocytes were injected with 2 ng/subunit of hENaC cRNAs or 0.33 ng/subunit of mENaC cRNAs in 50 nl of H₂O. After injection, oocytes were incubated at 18 °C in modified Barth's saline (MBS) (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO₃, 15mM HEPES, 0.3 mM Ca(NO₃)₂, 0.41 mM CaCl₂, 0.82 mM MgSO₄, pH 7.2) supplemented with 10 µg/ml sodium penicillin, 10 µg/ml streptomycin sulfate, and 100 µg/ml gentamycin sulfate. Animal protocols were approved by the University of Pennsylvania, the University of Pittsburgh, and the Children's Hospital of Philadelphia Institutional Animal Care and Use Committees.

Two-electrode Voltage Clamp—A two-electrode voltage clamp (TEV) was performed 24–48 h after cRNA injection at room temperature using a DigiData 1320 interface and Axon Geneclamp 500B Amplifier (Axon Instruments, Foster City, CA). Data were acquired at 200 Hz, and analyses were performed using pClamp 8.0 or 8.1 software (Axon Instruments) on 833-MHz Pentium III personal computers (Dell Computer, Austin, TX). Pipettes were pulled from borosilicate glass capillaries (World Precision Instruments, Inc., Sarasota, FL) with a micropipette fuller (Sutter Instrument Co., Novato, CA) and had resistances of 0.5–5 megohms when filled with 3 M KCl and inserted into the bath solution. Oocytes were maintained in a recording chamber with 1 ml of bath solution and continuously perfused with bath solution at a flow rate of 4–5 ml/min. The bath solution contained 100 mM sodium gluconate, 2 mM KCl, 1.8 mM CaCl₂, 3 mM BaCl₂, 10 mM tetraethylammonium chloride, and 10 mM HEPES, pH 7.4. A series of voltage steps (1 s) from –140 to +60 mV (adjusted for resting membrane potential) in 20-mV increments were performed, and whole cell currents were recorded 750 ms after the initiation of the –100-mV voltage step for data analysis. ENaC-mediated current was defined as the difference in whole oocyte current at a –100-mV holding potential (adjusted for resting membrane potential) before and after the addition of 10 µM amiloride-HCl (Sigma) to the bath solution.

Cell Surface Expression—Two approaches were used to examine the surface expression of ENaC in oocytes. A chemiluminescence-based assay was performed as described by Zerangue et al. (19) and modified by Yoo et al. (21). Oocytes were injected with 0.33 ng/subunit of m(1–678)/h(650–669)T663-m, m(1–678)/h(650–669)T663-m-FLAG-m, or m(1–678)/h(650–669)T663A-m-FLAG-m ENaC. 42–48 h after injection oocytes were incubated for 10 min at 4 °C in 4% formaldehyde in MBS. Oocytes were subsequently washed three times with MBS containing 10 mg/ml of bovine serum albumin (MBS-BSA), incubated an additional 3 h with MBS-BSA, and then incubated overnight with MBS-BSA supplement with 1 µg/ml mouse monoclonal anti-FLAG antibody (M2, Sigma) at 4 °C. Oocytes were then washed at 4 °C for 1 h in MBS-BSA and incubated with MBS-BSA supplemented with 1 µg/ml horseradish peroxidase-coupled secondary antibody for 1–1.5 h at 4 °C (peroxidase-conjugated AffiniPure F(ab'2) fragment goat anti-mouse IgG, Jackson Immunoresearch Laboratories, West Grove, PA). Cells were extensively washed (12 times over 2 h) at 4 °C and transferred to MBS without BSA. Individual oocytes were placed in 100 µl of SuperSignal Elisa Femto (Pierce) and incubated at room temperature for 1 min. Chemiluminescence was quantitated in a TD-20/20 luminometer (Turner Designs, Sunnyvale, CA).

A second approach to examine surface expression used cell surface biotinylation to identify the surface pool of channels as described previously (22). hENaC with a C-terminal V5 epitope tag (-V5) was generated in pcDNA6 as described previously (23). cRNAs for -V5 hENaC were co-injected into Xenopus oocytes. As a control, oocytes were co-injected only with cRNAs for -V5 and . After 48 h, oocytes were stripped mechanically of their vitelline membranes in hypertonic medium (300 mM sucrose in MBS without penicillin, streptomycin, and gentamycin), and surface proteins were labeled with N-hydroxysulfosuccinimidobiotin (Pierce) as described previously (22). Oocytes (10/group) were subsequently lysed in 0.15 M NaCl, 0.01 M Tris-Cl, pH 8.0, 0.01 M EDTA, 1.0% Nonidet P-40, 0.5% sodium deoxycholate, 1.0 mM phenylmethanesulfonyl fluoride, 0.1 mM N--p-tosyl-L-lysine chloromethyl ketone, 0.1 mM L-1-tosylamide-2-phenylethyl-chloromethyl ketone, and 2 µg/ml aprotinin for 1 h at 4 °C and centrifuged at 13,000 x g for 15 min at 4 °C. Biotinylated proteins were precipitated with streptavidin-agarose (Pierce) and subjected to SDS-PAGE. Biotinylated -subunits were detected on immunoblots probed with an anti-V5 antibody. Densitometry was performed using an AlphaImager 2200 system (AlphaInnotech, San Leandro, CA).

Statistical Analyses—Whole cell amiloride-sensitive current data were expressed relative to that of wild type ENaC. To decrease the influence of batch-to-batch variability in ENaC expression, data were normalized by the mean amiloride-sensitive current for the control condition (usually T663 hENaC) within a batch of oocytes prior to combining data of multiple independent batches for statistical analysis. These data are presented as mean ± S.E. Because a Poisson distribution, rather than a Gaussian distribution, best described these combined data, p values were determined by a two-tailed t test after a square root transformation to better approximate a Gaussian distribution (24). A p value of 0.05 was considered significant. We also independently analyzed the statistical significance of these data without normalization and transformation using the Wilcoxon rank sum/Mann-Whitney U test for non-parametric results and obtained similar p values to the method outlined above (data not shown). Other data that were normally distributed are expressed as mean ± S.E. with p values determined by a two-tailed t test. All data analyses were performed with SigmaStat version 2.03.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
T663A Polymorphism Is Associated with Altered Functional Channel Expression in Xenopus Oocytes—Functional hENaC expression was assessed by TEV in oocytes expressing either T663 hENaC (n = 56) or the T663A polymorphism (T663A)(n = 57). Fig. 1 illustrates current/voltage relationships for oocytes expressing T663 and T663A hENaC before and after the addition of 10 µM amiloride. The linear current/voltage relationships are characteristic of ENaC expressed in oocytes, and the reversal potential of 0 mV is consistent with intracellular Na⁺ loading observed in oocytes expressing ENaC and incubated in MBS (17, 18). Oocytes expressing T663A channels exhibited mean ± S.E. whole cell amiloride-inhibited currents of –2.15 ± 0.29 µA at a –100-mV holding potential that were significantly lower than mean Na⁺ currents measured in oocytes expressing wild type channels (–4.65 ± 0.67 µA, p < 0.001 (Fig. 2 for normalized data)). Given the small difference in whole cell Na⁺ currents we observed with oocytes expressing T663 and T663A, experiments were performed with two different cRNA preparations and four different batches of oocytes to confirm our findings.

View larger version (11K): [in this window] [in a new window]   FIG. 1. Expression of human ENaC in Xenopus oocytes. T663 (A)or T663A (B) -hENaC were expressed in oocytes, and TEV was performed as described under "Experimental Procedures." Whole cell currents were measured by voltage clamping oocytes in 20-mV steps between –140 and +60 mV adjusted for base line transmembrane potential. Shown are current/voltage relationships in the absence (closed circles) or presence (open circles) of 10 µM amiloride. Means ± S.E. are illustrated. Error bars contained within the symbols are not apparent.

View larger version (14K): [in this window] [in a new window]   FIG. 2. The T663A polymorphism reduces hENaC functional expression in oocytes. T663 or T663A hENaC was expressed in oocytes, and TEV was performed. Amiloride-sensitive whole cell currents were determined at a –100-mV holding potential (adjusted for resting membrane potential) and are expressed relative to the mean whole oocyte current for T663hENaC-expressing oocytes. Data are presented as mean ± S.E. with the p value determined by a two-tailed t test as described under "Experimental Procedures."

View larger version (25K): [in this window] [in a new window]   FIG. 3. Effects of 663 mutants on hENaC in oocytes. T663S (A), T663D (B), T663K (C), T663G (D), and T663L (E) were expressed in oocytes, and TEV was performed. The functional expression of these mutant hENaCs was compared with T663 by determination of amiloride-sensitive whole cell currents at a –100-mV holding potential (adjusted for resting membrane potential). These data (mean ± S.E.) are expressed relative to the mean whole cell amiloride-sensitive current measured in oocytes expressing T663hENaC. p values were determined by a two-tailed t test as described under "Experimental Procedures."

View this table: [in this window] [in a new window]   TABLE I Analyses of T663 Mutants Mean whole cell amiloride-sensitive currents in oocytes expressing wild type channels (T663) and oocytes expressing channels with an Ala, Gly, Leu, Lys, Asp, or Ser at position 663 (n = 40–85) are shown. Direct comparisons are indicated in each column.

View larger version (30K): [in this window] [in a new window]   FIG. 4. Residues in the vicinity of hT663A have a role in conferring differences in function ENaC expression. A, alignments of the distal C termini of human and mouse -subunits. Spaces were added to separate a conserved six-residue N-terminal tract (YATLGP) from the remainder of the C terminus and to highlight hT663 and mA692 (in bold). The first and last amino acid residue numbers for each sequence are listed. Sequence alignments were performed with MacVector 7.2 (Accelrys) for the mouse and human -subunits (GenBankTM Accession Numbers AF112185 [GenBank] and L29007 [GenBank] ). B, TEV was performed with oocytes expressing either mouse A692 or A692T. Amiloride-sensitive whole cell currents were determined at the –100-mV holding potential (adjusted for resting membrane potential) and are expressed relative to the mean whole cell amiloride-sensitive current for A692. C, TEV was performed with oocytes expressing either human T663/mouse or human T663A/mouse . Amiloride-sensitive whole cell currents were determined at the –100-mV holding potential and expressed relative to the mean whole cell amiloride-sensitive current for human T663/mouse . D, TEV was performed with oocytes expressing either m(1–678)/h(650–669)T663/mouse or m(1–678)/h(650–669)T663A/mouse . Amiloride-sensitive whole cell currents were determined at the –100-mV holding potential and expressed relative to the mean whole cell amiloride-sensitive current for m(1–678)/h(650–669)T663/mouse . p values were determined with a two-tailed t test as described under "Experimental Procedures."

ENaC Polymorphism Affects Surface Expression of ENaC— The reduced functional expression of T663A channels in oocytes likely reflects changes in the numbers of channels at the plasma membrane or changes in channel open probability. Because these residues are not within the pore of the channel (1, 18), it is unlikely that this polymorphism affects single-channel conductance. Previous studies (25) have also suggested that it is difficult to assess differences in ENaC open probability because the channel has inherently large variability in open probability.

We therefore examined whether differences in functional expression of T663 and T663A hENaC were associated with altered levels of cell surface expression of channels. In these experiments, the -subunit had a C-terminal V5 epitope tag to identify the protein on immunoblots. Cell surface proteins of oocytes expressing T663-V5 or T663A-V5 were labeled by biotinylation and recovered with streptavidin beads. The biotinylated -subunits were subsequently detected by immunoblots. As shown in Fig. 5A, expression of T663A-V5 hENaC resulted in a relative recovery of biotinylated -V5 of 0.64 ± 0.08 compared with that observed when T663-V5 hENaC was expressed (n = 5, p = 0.003). Whole oocyte -V5 expression was similar in the two groups (Fig 5B). These data suggest that the increases in functional expression observed with T663, when compared with T663A, are because of differences in surface expression.

View larger version (18K): [in this window] [in a new window]   FIG. 5. The hT663A polymorphism affects channel surface expression. Human T663 or T663A was expressed in oocytes where the -subunit had a C-terminal V5 epitope tag (-V5). Non-injected oocytes were used as controls. Cell surface biotinylation was performed as described under "Experimental Procedures," and -V5 hENaC recovered by streptavidin-agarose precipitation was quantitated by immunoblot and densitometry. A, representative immunoblot and densitometry of immunoblots probed with an anti-V5 antibody. Densitometry results are expressed as mean ± S.E., n = 5 independent experiments, and normalized to values obtained with oocytes expressing T663-V5. B, representative immunoblot of whole oocyte lysates of T663 and T663A-injected oocytes demonstrating a similar expression of -V5 hENaC. This immunoblot is representative of n = 5 independent experiments.

We also examined whether differences in the functional expression of mouse/human -chimeras (m(1–678)/h(650–669)T663-m and m(1–678)/h(650–669)T663A-m) were associated with altered levels of cell surface expression of channels. Cell surface expression was determined using a -subunit with an extracellular FLAG epitope tag and a chemiluminescence-based assay that does not require stripping of the oocyte vitelline membrane. The mean relative cell surface expression of m(1–678)/h(650–669)T663A-m was 0.59 ± 0.10 (n = 46) compared with that of m(1–678)/h(650–669)T663-m (Fig. 6, 1.00 ± 0.18, n = 47, p = 0.016). Nonspecific labeling was examined in oocytes expressing m(1–678)/h(650–669)T663-m where the -subunit did not contain an epitope tag. Mean nonspecific labeling was 0.18 ± 0.03 (n = 35) of the labeling observed in oocytes expressing epitope-tagged m(1–678)/h(650–669)T663-m. We also performed separate control experiments to assess the labeling of intra-cellular -subunits in this chemiluminescence-based assay. The labeling of oocytes expressing only human -FLAG, which, as discussed above, has markedly reduced surface expression (25, 26), was indistinguishable from that of uninjected oocytes and was significantly less than that of oocytes expressing m(1–678)/h(650–669)T663-m-FLAG (25, 26) (data not shown).

View larger version (20K): [in this window] [in a new window]   FIG. 6. The m(1–678)/h(650–669)T663A polymorphism affects channel surface expression. Surface expression was examined in oocytes co-expressing m(1–678)/h(650–669)T663/mouse -FLAG or m(1–678)/h(650–669)T663A/mouse -FLAG using a chemiluminescence-based assay as described under "Experimental Procedures." Relative surface expression (relative light units, expressed as mean ± S.E.) was derived by normalizing light units/min/oocyte to light units/min/oocyte observed with oocytes expressing m(1–678)/h(650–669)T663/FLAG-tagged mouse /wild type mouse . Oocytes expressing non-epitope-tagged m(1–678)/h(650–669)T663/mouse subunits were used for negative controls and had a mean of 18% (n = 35) of the surface expression of m(1–678)/h(650–669)T663/FLAG-tagged mouse /wild type mouse oocytes (data not shown). Statistical significance was determined using a two-tailed t test as described under "Experimental Procedures." Data are derived from three independent batches of oocytes and two independent preparations of cRNA.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Additional studies were performed to assess whether substitution of other amino acid residues at this site influenced functional ENaC expression. Whole cell amiloride-sensitive currents in oocytes expressing channels with a Thr, Ser, or Asp at position 663 were 1.3–2.0-fold higher than currents measured in oocytes expressing channels with an Ala, Gly or Leu, or Lys at this position (Table I and Figs. 2 and 3). Our observations that channels with Thr, Ser, or Asp at position 663 showed similar levels of functional expression in oocytes suggest that 663 may be a site of phosphorylation by Ser/Thr kinases. At present, we have no direct evidence that this site is phosphorylated in vivo nor whether phosphorylation of this site affects ENaC activity.

T663 is present within a region of the -subunit that is not well conserved between the human and mouse (Fig. 4A). An Ala residue is present at the homologous site in the mouse -subunit (mA692), and we observed that oocytes expressing mouse A692 and A692T had similar levels of amiloride-sensitive currents (Fig. 4B). However, when the distal C terminus of the mouse -subunit was replaced with the distal C terminus of the human -subunit, creating a mouse/human chimera, oocytes co-expressing m(1–678)/h(650–669)T663-m had significantly higher whole cell Na⁺ currents than oocytes co-expressing m(1–678)/h(650–669)T663A-m (Fig. 4D). These results suggest that residues in the vicinity of hT663 have a role in conferring the observed differences in function expression between hT663 and hT663A. Furthermore, the differences in ENaC surface expression that we observed with the hT663A and the m(1–678)/h(650–669)T663A-m polymorphisms (Figs. 5 and 6) were correlated with differences in function expression (Figs. 2 and 4D) and likely reflect differences in the delivery of channels to the plasma membrane and/or differences in the rate of retrieval from the plasma membrane. Our data suggest that the distal C terminus of the human -subunit has a novel site that affects the intracellular trafficking of ENaC that is distinct from the PY motif in the - and -subunits (25, 27, 28).

Increases in ENaC activity in renal collecting ducts may be associated with extracellular fluid volume expansion and hypertension, as has been reported in studies of patients with Liddle's syndrome (8, 10, 29, 30). Whole cell Na⁺ currents measured in oocytes expressing mutations that are found in patients with Liddle's syndrome were significantly higher than currents measured in oocytes expressing wild type ENaC (31, 32). However, correlations between increases in ENaC activity measured in oocytes and increases in blood pressure in humans with specific ENaC mutations or polymorphisms are not a consistent finding. For example, the T594M polymorphism has been found to segregate with blood pressure in selected populations, suggesting that this polymorphism is associated with alterations in ENaC activity (14, 33). Lymphocytes obtained from patients homozygous for T594 exhibited amiloride-sensitive whole cell Na⁺ currents that were enhanced by cAMP (34). Phorbol 12-myristate 13-acetate blocked the activation of Na⁺ currents by cAMP. In contrast, lymphocytes obtained from T594M homozygotes exhibited cAMP-activated whole cell currents that were not blocked by phorbol 12-myristate 13-acetate, suggesting that this polymorphism was associated with altered ENaC activity (34). However, no differences in ENaC activity have been reported with the T594M polymorphism in oocyte expression studies (35).

Although we have observed differences in hENaC activity with the T663A polymorphism with oocyte expression, it is unclear whether the hT663A polymorphism is associated with differences in blood pressure in humans. Ambrosius et al. (15, 36) reported that A663 was more prevalent in African-American subjects than in Caucasian subjects, and its presence (A663) was associated with a modest increase in blood pressure. However, other investigators have not observed differences in blood pressure with the T663A polymorphism (16). Our functional studies in oocytes predict that A663 would be associated with a reduction in blood pressure. Certainly environmental and other genetic factors have an important role in the regulation of blood pressure in humans, and it may be difficult to demonstrate that specific ENaC polymorphisms contribute to essential hypertension in humans despite modest differences in functional activity when assayed in heterologous expression systems (37).

In summary, our results suggest that hT663 has increased activity in oocytes when compared with hT663A. The increase in channel activity observed with hT663 reflects an increase in surface expression, suggesting that this polymorphism modifies the intracellular trafficking of ENaC. Furthermore, our studies using mouse/human -subunit chimeras suggest that residues in the vicinity of hT663 have an important role in conferring differences in functional expression between hT663 and hT663A.

	FOOTNOTES

 
^* This work was supported by a grant from the Veterans Affairs Healthcare Network 4 (to F. F. S.), by Grants DK54354 and DK51391 (to T. R. K.) and DK58046 (to R. C. R.) from the National Institutes of Health, and by a grant from the Cystic Fibrosis Foundation (to Y. J. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ These authors contributed equally to this work.

An Established Investigator of the American Heart Association. To whom correspondence should be addressed: Division of Pulmonary Medicine, Children's Hospital of Philadelphia, 34th St. and Civic Center Blvd., Abramson 410C, Philadelphia, PA 19104. Tel.: 215-590-1281; Fax: 215-590-1283; E-mail: rrubenst{at}mail.med.upenn.edu.

¹ The abbreviations used are: ENaC, epithelial Na⁺ channel; hENaC, human ENaC; mENaC, mouse ENaC; MBS, modified Barth's saline; TEV, two-electrode voltage clamp; BSA, bovine serum albumin; n.s., not significant.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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