Regulation of the Epithelial Na+ Channel by Cytosolic ATP

Abstract

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...

Molecular cloning and functional characterization of the Aplysia FMRFamide-gated Na+ channel

“…The amplified cDNAs were gel-purified, and cloned into a SmaI site of pBluescript II SK(+) (Stratagene). We sequenced both strands of two clones (#11, 17), and their sequences were confirmed to be identical to AkFaNaC. The EcoRI-XbaI fragments of the clones (#11, 17) were blunted, and cloned into the EcoRV site of pSD64TR, a modified vector originating from pSP64 (Promega, USA), to make pSD64TR-AkFaNaC.…”

Characteristics and Pharmacological Regulation of Epithelial Na+ Channel (ENaC) and Epithelial Na+ Transport

“…In this report (102), we have indicated that run-down of ENaC activity (NP o ) to 50% of its initial activity (NP o ) occurs 5 min after making excised outside-out patches with no (0 mM) cytosolic ATP, while cytosolic ATP of 2 mM almost completely maintains ENaC activity (NP o ) at its initial value with little run-down of ENaC activity in a hydrolysisindependent mechanism at least within 5 min after making excised outside-out patches. This report (102) also provides further evidence that ATP binds to aENaC and determines ATP binding site of aENaC. Further, ATP is required for non-genomic action of aldosterone on ENaC activity (NP o ) (103).…”

“…In addition to this information (48), we have reported that ATP itself directly acts on ENaC by measuring the activity of ENaC (NP o ) existing in excised outside-out patches (102). In this report (102), we have indicated that run-down of ENaC activity (NP o ) to 50% of its initial activity (NP o ) occurs 5 min after making excised outside-out patches with no (0 mM) cytosolic ATP, while cytosolic ATP of 2 mM almost completely maintains ENaC activity (NP o ) at its initial value with little run-down of ENaC activity in a hydrolysisindependent mechanism at least within 5 min after making excised outside-out patches.…”

Cytosolic potassium controls CFTR deactivation in human sweat duct

“…Figure 5 shows that CFTR-gCl was still normally deactivated after K ϩ substitution with Na ϩ , even after removing Ca 2ϩ . (9,10,12,15,36,38). Even though such coordination is essential to maintaining the functional integrity of the cell in the face of changing rates of transepithelial salt transport, very little is known about the molecular mechanisms involved in this vital process.…”

“…Several physiological strategies have been reported to be involved in this process, such as 1) metabolic coupling in which availability of ATP is linked to cystic fibrosis transmembrane conductance regulator (CFTR; see Refs. 1 and 20) and epithelial Na ϩ channel (ENaC) activity in the apical membrane (15) and K ϩ recycling in the basolateral membrane (36), 2) cross talk between Na ϩ channels in the apical membrane and K ϩ channels in the basolateral membrane (9,12), and 3) permissive control of Ca 2ϩ -dependent activation of multiple Na ϩ transport pathways by intracellular Cl Ϫ (29). Recent evidence also suggests that cytosolic changes in Na ϩ and Cl Ϫ concentrations could control apical salt transport by regulating ENaC channels (8,10).…”