Downregulation of Epithelial Sodium Channel (ENaC) by CFTR Co-expressed in Xenopus Oocytes is Independent of Cl − Conductance

Proton-gated Na؉ channels (ASIC) are new members of the epithelial sodium channel/degenerin gene family. ASIC3 mRNA has been detected in the homogenate of pulmonary tissues. However, whether ASIC3 is expressed in the apical membranes of lung epithelial cells and whether it regulates cystic fibrosis transmembrane conductance regulator (CFTR) function are not known at the present time. Using reverse transcription-PCR, we found that the ASIC3 mRNA was expressed in the human airway mucosal gland (Calu-3) and human airway epithelial (16HBE14o) cells. Indirect immunofluorescence microscopy revealed that ASIC3 was co-segregated with CFTR in the apical membranes of Calu-3 cells. Proton-gated, amiloride-sensitive short circuit Na ؉ currents were recorded across Calu-3 monolayers mounted in an Ussing chamber. In whole-cell patch clamp studies, activation of CFTR channels with cAMP reduced proton-gated Na ؉ current in Calu-3 cells from ؊154 ؎ 28 to ؊33 ؎ 16 pA (n ‫؍‬ 5, p < 0.05) at ؊100 mV. On the other hand, cAMP-activated CFTR activity was significantly inhibited following constitutive activation of putative ASIC3 at pH 6.0. Immunoassays showed that both ASIC3 and CFTR proteins were expressed and co-immunoprecipitated mutually in Calu-3 cells. Similar results were obtained in human embryonic kidney 293T cells following transient co-transfection of ASIC3 and CFTR. Our results indicate that putative CFTR and ASIC3 channels functionally interact with each other, possibly via an intermolecular association. Because acidic luminal fluid in the cystic fibrosis airway and lung tends to stimulate ASIC3 channel expression and activity, the interaction of ASIC3 and CFTR may contribute to defective salt and fluid transepithelial transport in the cystic fibrotic pulmonary system.

supporting

confidence: 90%

Interregulation of Proton-gated Na+ Channel 3 and Cystic Fibrosis Transmembrane Conductance Regulator

Shrestha

et al. 2006

“…Functional interactions between CFTR and rodent ENaC have been observed in epithelial as well as non-epithelial cells (6 -12). The activation of CFTR is generally associated with an inhibition of ENaC (7,8,10,11,13,14), although activation of CFTR leads to activation of ENaC in the sweat duct (12) suggesting that the regulatory interactions between these two transporters are complex. The co-expression of CFTR and rodent ENaC in Xenopus oocytes results in regulatory interactions that mimic the airway where there is a decrease in ENaCmediated Na ϩ transport in the presence of CFTR (9 -11, 14).…”

Section: Cystic Fibrosis (Cf)mentioning

confidence: 99%

Cystic Fibrosis Transmembrane Conductance Regulator Differentially Regulates Human and Mouse Epithelial Sodium Channels in Xenopus Oocytes

Yan

Samaha

Mohan

et al. 2004

The cystic fibrosis transmembrane conductance regulator (CFTR), in addition to its well defined Cl ؊ channel properties, regulates other ion channels. CFTR inhibits murine or rat epithelial Na ؉ channel (mENaC or rENaC) currents in many epithelial and non-epithelial cells, whereas murine or rat ENaC increases CFTR functional expression. These regulatory interactions are reproduced in Xenopus oocytes where both the open probability and surface expression of wild type CFTR Cl ؊ channels are increased when CFTR is co-expressed with ␣␤␥ mENaC, and conversely the activity of mENaC is inhibited after wild type CFTR activation. Using the Xenopus oocyte expression system, differences in functional regulatory interactions were observed when CFTR was co-expressed with either ␣␤␥ mENaC or ␣␤␥ human ENaC (hENaC). Co-expression of CFTR and ␣␤␥ mENaC or hENaC resulted in an ϳ3-fold increase in CFTR Cl ؊ current compared with oocytes expressing CFTR alone. Oocytes co-injected with both CFTR and mENaC or hENaC expressed an amiloride-sensitive whole cell current that was decreased compared with that observed with the injection of mENaC or hENaC alone before CFTR activation with forskolin/3-isobutyl-1-methylxanthine. CFTR activation resulted in a further 50% decrease in mENaC-mediated currents, an ϳ20% decrease in ␣-T663-hENaC-mediated currents, and essentially no change in ␣-A663-hENaC-mediated currents. Changes in ENaC functional expression correlated with ENaC surface expression by oocyte surface biotinylation experiments. Assessment of regulatory interactions between CFTR and chimeric mouse/human ENaCs suggest that the 20 C-terminal amino acid residues of ␣ ENaC confer species specificity regarding ENaC inhibition by activated CFTR.

“…Previous patch clamp studies in native Na ϩ -absorptive epithelia have shown that the feedback inhibition involves changes in intracellular factors, including membrane potential, cytosolic pH, Ca 2ϩ , and Na ϩ concentrations, and cell metabolism (1). Furthermore, in recent studies using both molecular biological and electrophysiological techniques, cytosolic pH, Na ϩ , and/or Ca 2ϩ concentration have been also shown to affect the activity of the cloned rENaC when expressed in different heterologous expression systems (15)(16)(17)(18)(19)(20), although these cytosolic factors may not act on the ENaC protein directly (18).Focusing on the relationship between cell metabolism and Na ϩ channel activity, Palmer et al (21) and Garty et al (22) observed that inhibition of cell metabolism decreased the apical membrane Na ϩ permeability in the toad urinary bladder, although it is not known whether metabolites such as ATP were responsible for this effect. Frindt et al (23) also suggested a role for metabolic changes in mediating negative feedback regulation of amiloride-sensitive Na ϩ channels in rat cortical collecting tubules; they found that increasing Na ϩ entry into cells by the addition of cAMP led to a decrease in channel activity in cell-attached patches, where the channels were apparently pro-…”

mentioning

confidence: 99%

mentioning

confidence: 99%

Regulation of the Epithelial Na+ Channel by Cytosolic ATP

Ishikawa

Jiang

Stutts

et al. 2003

The epithelial Na ؉ channel (ENaC), composed of three subunits (␣␤␥), is expressed in various Na ؉ -absorbing epithelia and plays a critical role in salt and water balance and in the regulation of blood pressure. By using patch clamp techniques, we have examined the effect of cytosolic ATP on the activity of the rat ␣␤␥ENaC (rENaC) stably expressed in NIH-3T3 cells and in MadinDarby canine kidney epithelial cells. The inward wholecell current attributable to rENaC activity ran down when these cells were dialyzed with an ATP-free pipette solution in the conventional whole-cell voltage-clamping technique. This run down was prevented by 2 mM ATP (but not by AMP or ADP) in the pipette solution or by the poorly or non-hydrolyzable analogues of ATP (adenosine 5 -O-(thiotriphosphate) and adenosine 5 -(␤,␥-imino)triphosphate) in both cell lines, suggesting that protection from run down was mediated through non-hydrolytic nucleotide binding. Accordingly, we demonstrate binding of ATP (but not AMP) to ␣rENaC expressed in Madin-Darby canine kidney cells, which was inhibited upon mutation of the two putative nucleotide-binding motifs of ␣rENaC. Single channel analyses indicated that the run down of currents observed in the whole-cell recording was attributable to run down of channel activity, defined as NP o (the product of the number of channels and open probability). We propose that this novel ATP regulation of ENaC may be, at least in part, involved in the fine-tuning of ENaC activity under physiologic and pathophysiologic conditions.The amiloride-sensitive epithelial Na ϩ channel (ENaC) 1 regulates a variety of physiological functions in different Na ϩ -transporting epithelia, such as those in the distal nephron, distal colon, lung epithelia, and duct cells of exocrine glands (1). ENaC is composed of three partially homologous subunits, ␣, ␤, and ␥ (2, 3). The physiological importance of ENaC has been underscored by the recent identification of either loss of function mutations in the channel leading to pseudohypoaldosteronism (PHA1) (4 -6) or gain of function mutations causing pseudoaldosteronism (Liddle's syndrome), a hereditary form of arterial hypertension (7-9). Moreover, abnormally high ENaC activity has been associated with cystic fibrosis due to loss of the normal inhibitory effect of CFTR on ENaC (10 -13), and gene targeting of ␣ENaC in mice led to newborn death from pulmonary edema (14).The activity of ENaC is tightly regulated not only by various hormones such as aldosterone and vasopressin but also by intracellular factors via mechanisms that are not yet fully understood (1). A mechanism by which the activity of ENaC is regulated in native Na ϩ -transporting epithelia is called the "feedback inhibition," which is defined as channel down-regulation due to the transport of Na ϩ across the apical membrane and its accumulation intracellularly (1). This mechanism would allow cells to adjust optimal Na ϩ movement across the apical membrane, thereby protecting themselves from Na ϩ load and the energy consumption re...