The cystic fibrosis transmembrane conductance regulator (CFTR), in addition to its well defined Cl ؊ channel properties, regulates other ion channels. CFTR inhibits epithelial Na ؉ channel (ENaC) currents in many epithelial and nonepithelial cells. Because modulation of net NaCl reabsorption has important implications in extracellular fluid volume homeostasis and airway fluid volume and composition, we investigated whether this regulation was reciprocal by examining whether ENaC regulates CFTR. Co-expression of human (h) CFTR and mouse (m) ␣␤␥ENaC in Xenopus oocytes resulted in a significant, 3.7-fold increase in whole-cell hCFTR Cl ؊ conductance compared with oocytes expressing hCFTR alone. The forskolin/3-isobutyl-1-methylxanthine-stimulated whole-cell conductance in hCFTR-mENaC co-injected oocytes was amiloride-insensitive, indicating an inhibition of mENaC following hCFTR activation, and it was blocked by DPC (diphenylamine-2-carboxylic acid) and was DIDS (4,4-diisothiocyanatostilbene-2,2-disulfonic acid)-insensitive. Enhanced hCFTR Cl ؊ conductance was also observed when either the ␣-or ␤-subunit of mENaC was co-expressed with hCFTR, but this was not seen when CFTR was co-expressed with the ␥-subunit of mENaC. Single Cl ؊ channel analyses showed that both CFTR Cl ؊ channel open probability and the number of CFTR Cl ؊ channels detected per patch increased when hCFTR was co-expressed with ␣␤␥mENaC. We conclude that in addition to acting as a regulator of ENaC, CFTR activity is regulated by ENaC.

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 ϳ1.3-2.0-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 epitopetagged ␤-subunit, was significantly reduced in oocytes expressing ␣T663A␤␥ when compared with oocytes expressing ␣T663␤␥. The corresponding polymorphism was generated in the mouse ␣-subunit (m␣A692T) 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 h␣T663A is a functional polymorphism that affects human ENaC surface expression.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 ϩ from the glomerular ultrafiltrate. Volume regulatory hormones, such as aldosterone, have a key role in modifying rates of renal tubular Na ϩ reabsorption through regulation of functional ENaC expression at the apical plasma membrane (1). Epithelial Na ϩ 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 (ϳ30 -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 ϩ 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 m...

The mammalian urinary bladder exhibits transepithelial Na+ absorption that contributes to Na+ gradients established by the kidney. Electrophysiological studies have demonstrated that electrogenic Na+ absorption across the urinary bladder is mediated in part by amiloride-sensitive Na+ channels situated within the apical membrane of the bladder epithelium. We have used a combination of in situ hybridization, Northern blot analysis, and immunocytochemistry to examine whether the recently cloned epithelial Na+ channel (ENaC) is expressed in the rat urinary bladder. In situ hybridization and Northern blot analyses indicate that α-, β-, and γ-rat ENaC (rENaC) are expressed in rat urinary bladder epithelial cells. Quantitation of the levels of α-, β-, and γ-rENaC mRNA expression in rat urinary bladder, relative to β-actin mRNA expression, indicates that, although comparable levels of α- and β-rENaC subunits are expressed in the urinary bladder of rats maintained on standard chow, the level of γ-rENaC mRNA expression is 5- to 10-fold lower than α- or β-rENaC mRNA. Immunocytochemistry, using an antibody directed against α-rENaC, revealed that ENaCs are predominantly localized to the luminal membrane of the bladder epithelium. Together, these data demonstrate that ENaC is expressed in the mammalian urinary bladder and suggest that amiloride-sensitive Na+ transport across the apical membrane of the mammalian urinary bladder epithelium is mediated primarily by ENaC.

The epithelial sodium channel (ENaC) plays a major role in the transepithelial reabsorption of sodium in the renal cortical collecting duct, distal colon, and lung. ENaCs are formed by three structurally related subunits, termed α-, β-, and γENaC. We previously isolated and sequenced cDNAs encoding a portion of mouse α-, β-, and γENaC (α-, β-, and γmENaC). These cDNAs were used to screen an oligo-dT-primed mouse kidney cDNA library. Full-length βmENaC and partial-length α- and γmENaC clones were isolated. Full-length α- and γmENaC cDNAs were subsequently obtained by 5′-rapid amplification of cDNA ends (5′-RACE) PCR. Injection of mouse α-, β-, and γENaC cRNAs into Xenopus oocytes led to expression of amiloride-sensitive ( K i = 103 nM), Na+-selective currents with a single-channel conductance of 4.7 pS. Northern blots revealed that α-, β-, and γmENaC were expressed in lung and kidney. Interestingly, αmENaC was detected in liver, although transcript sizes of 9.8 kb and 3.1 kb differed in size from the 3.2-kb message observed in other tissues. A partial cDNA clone was isolated from mouse liver by 5′-RACE PCR. Its sequence was found to be nearly identical to αmENaC. To begin to identify regions within αmENaC that might be important in assembly of the native heteroligomeric channel, a series of functional experiments were performed using a construct of αmENaC encoding the predicted cytoplasmic NH2 terminus. Coinjection of wild-type α-, β-, and γmENaC with the intracellular NH2 terminus of αmENaC abolished amiloride-sensitive currents in Xenopus oocytes, suggesting that the NH2 terminus of αmENaC is involved in subunit assembly, and when present in a 10-fold excess, plays a dominant negative role in functional ENaC expression.

Epithelial sodium channels (ENaCs) are composed of three structurally related subunits that form a tetrameric channel. The Xenopus laevis oocyte expression system was used to identify regions within the ENaC alpha-subunit that confer a dominant negative phenotype on functional expression of alphabetagamma-ENaC to define domains that have a role in subunit-subunit interactions. Coexpression of full-length mouse alphabetagamma-ENaC with either 1) the alpha-subunit first membrane-spanning domain and short downstream hydrophobic domain (alpha-M1H1); 2) alpha-M1H1 and its downstream hydrophilic extracellular loop (alpha-M1H1-ECL); 3) the membrane-spanning domain of a control type 2 transmembrane protein (glutamyl transpeptidase; gamma-GT) fused to the alpha-ECL (gamma-GT-alpha-ECL); 4) the extracellular domain of a control type 1 transmembrane protein (Tac) fused to the alpha-subunit second membrane-spanning domain and short upstream hydrophobic domain (Tac-alpha-H2M2); or 5) the alpha-subunit cytoplasmic COOH terminus (alpha-Ct) significantly reduced amiloride-sensitive Na+ currents in X. laevis oocytes. Functional expression of Na+ channels was not inhibited when full-length alphabetagamma-ENaC was coexpressed with either 1) the alpha-ECL lacking a signal-anchor sequence, 2) alpha-M1H1 and alpha-Ct expressed as a fusion protein, 3) full-length gamma-GT, or 4) full-length Tac. Furthermore, the expression of ROMK channels was not inhibited when full-length ROMK was coexpressed with either alpha-M1H1-ECL or alpha-Ct. Full-length FLAG-tagged alpha-, beta-, or gamma-ENaC coimmunoprecipitated with myc-tagged alpha-M1H1-ECL, whereas wild-type gamma-GT did not. These data suggest that multiple sites within the alpha-subunit participate in subunit-subunit interactions that are required for proper assembly of the heterooligomeric ENaC complex.