Epithelial Na ؉ channels (ENaCs) are activated by extracellular trypsin or by co-expression with channelactivating proteases, although there is no direct evidence that these proteases activate ENaC by cleaving the channel. We previously demonstrated that the ␣ and ␥ subunits of ENaC are cleaved during maturation near consensus sites for furin cleavage. Using site-specific mutagenesis of channel subunits, ENaC expression in furin-deficient cells, and furin-specific inhibitors, we now report that ENaC cleavage correlates with channel activity. Channel activity in furin-deficient cells was rescued by expression of furin. Our data provide the first example of a vertebrate ion channel that is a substrate for furin and whose activity is dependent on its proteolysis.Epithelial Na ϩ channels are expressed in apical membranes of high resistance Na ϩ -transporting epithelia. These channels have a key role in the regulation of extracellular fluid volume, blood pressure, and airway fluid volume. ENaCs 1 are composed of three structurally related subunits, termed ␣, ␤, and ␥, with a presumed ␣ 2 ␤ 1 ␥ 1 subunit stoichiometry (1, 2), although an alternative stoichiometry has been proposed (3). Each subunit has two membrane-spanning domains that are connected by a large extracellular loop and intracellular NH 2 and COOH termini. Residues preceding and within the second membranespanning domain form the channel pore (4 -6).Previous studies have demonstrated that ENaC activity is regulated by proteases. Extracellular trypsin has been shown to increase channel activity, while extracellular serine protease inhibitors, such as aprotinin and bikunin, have been shown to decrease channel activity (7)(8)(9)(10)(11)(12). Channel activation by proteases likely reflects changes in channel gating (8,12). ENaCs characteristically have long open and closed times, generally on the order of seconds (13,14). However, a population of channels has been described that exhibit only brief (50 ms) openings and have long closed states (13,14). Caldwell et al. (12) recently reported that extracellular trypsin converts these near-silent Na ϩ channels to channels that exhibit the typically long open and closed times. A family of channel-activating serine proteases, referred to as CAPs, have been identified based on their ability to activate ENaC when co-expressed in heterologous systems (7,15,16). These serine proteases include CAP1 (or prostasin), CAP2, CAP3, and a member of a family of transmembrane serine proteases (TMPRSS3) (7,15,16). However, it is not known whether proteolysis of ENaC subunits or cleavage of a distinct regulatory protein is responsible for the activation of Na ϩ channels.We recently reported that maturation of mouse ENaC in both Chinese hamster ovary (CHO) and Madin-Darby canine kidney (MDCK) cells involves proteolytic cleavage of the ␣ and ␥ subunits (17). Expression of individual subunits revealed full-length forms of the ␣, ␤, and ␥ subunits (95, 96, and 93 kDa, respectively) that had immature N-glycans. However, co-expression o...

Three distinct mammalian Na+/Ca2+exchangers have been cloned: NCX1, NCX2, and NCX3. We have undertaken a detailed functional comparison of these three exchangers. Each exchanger was stably expressed at high levels in the plasma membranes of BHK cells. Na+/Ca2+exchange activity was assessed using three different complementary techniques: Na+ gradient-dependent45Ca2+uptake into intact cells, Na+gradient-dependent45Ca2+uptake into membrane vesicles isolated from the transfected cells, and exchange currents measured using giant patches of excised cell membrane. Apparent affinities for the transported ions Na+ and Ca2+ were markedly similar for the three exchangers at both membrane surfaces. Likewise, generally similar responses to changes in pH, chymotrypsin treatment, and application of various inhibitors were obtained. Depletion of cellular ATP inhibited NCX1 and NCX2 but did not affect the activity of NCX3. Exchange activities of NCX1 and NCX3 were modestly increased by agents that activate protein kinases A and C. All exchangers were regulated by intracellular Ca2+. NCX1-induced exchange currents were especially large in excised patches and, like the native myocardial exchanger, were stimulated by ATP. Results may be influenced by our choice of expression system and specific splice variants, but, overall, the three exchangers appear to have very similar properties.

The phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) is accepted to be a direct modulator of ion channel activity. The products of phosphoinositide 3-OH kinase (PI3K), PtdIns(3,4)P 2 and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ), in contrast, are not. We report here activation of the epithelial Na ؉ channel (ENaC) reconstituted in Chinese hamster ovary cells by PI3K. Insulin-like growth factor-I also activated reconstituted ENaC and increased Na ؉ reabsorption across renal A6 epithelial cell monolayers via PI3K. Neither IGF-I nor PI3K affected the levels of ENaC in the plasma membrane. The effects of PI3K and IGF-I on ENaC activity paralleled changes in the plasma membrane levels of the PI3K product phospholipids, PtdIns-(3,4)P 2 /PtdIns(3,4,5)P 3 , as measured by evanescent field fluorescence microscopy. Both PtdIns(3,4)P 2 and PtdIns-(3,4,5)P 3 activated ENaC in excised patches. Activation of ENaC by PI3K and its phospholipid products corresponded to changes in channel open probability. We conclude that PI3K directly modulates ENaC activity via PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3 . This represents a novel transduction pathway whereby growth factors, such as IGF-I, rapidly modulate target proteins independent of signaling elicited by kinases downstream of PI3K.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) are physiologically important second messengers. These molecules bind effector proteins to modulate activity. Several types of ion channels, including the epithelial Na+ channel (ENaC), are phosphoinositide effectors capable of directly interacting with these signaling molecules. Little, however, is known of the regions within ENaC and other ion channels important to phosphoinositide binding and modulation. Moreover, the molecular mechanism of this regulation, in many instances, remains obscure. Here, we investigate modulation of ENaC by PI(3,4,5)P3 and PI(4,5)P2 to begin identifying the molecular determinants of this regulation. We identify intracellular regions near the inner membrane interface just following the second transmembrane domains in β- and γ- but not α-ENaC as necessary for PI(3,4,5)P2 but not PI(4,5)P2 modulation. Charge neutralization of conserved basic amino acids within these regions demonstrated that these polar residues are critical to phosphoinositide regulation. Single channel analysis, moreover, reveals that the regions just following the second transmembrane domains in β- and γ-ENaC are critical to PI(3,4,5)P3 augmentation of ENaC open probability, thus, defining mechanism. Unexpectedly, intracellular domains within the extreme N terminus of β- and γ-ENaC were identified as being critical to down-regulation of ENaC activity and Po in response to depletion of membrane PI(4,5)P2. These regions of the channel played no identifiable role in a PI(3,4,5)P3 response. Again, conserved positive-charged residues within these domains were particularly important, being necessary for exogenous PI(4,5)P2 to increase open probability. We conclude that β and γ subunits bestow phosphoinositide sensitivity to ENaC with distinct regions of the channel being critical to regulation by PI(3,4,5)P3 and PI(4,5)P2. This argues that these phosphoinositides occupy distinct ligand-binding sites within ENaC to modulate open probability.

Aldosterone induces expression and activation of the GTP-dependent signaling switch K-Ras. This small monomeric G protein is both necessary and sufficient for activation of the epithelial Na ؉ channel (ENaC). The mechanism by which K-Ras enhances ENaC activity, however, is uncertain. We demonstrate here that K-Ras activates human ENaC reconstituted in Chinese hamster ovary cells in a GTP-dependent manner. K-Ras influences ENaC activity most likely by affecting open probability. Inhibition of phosphoinositide 3-OH kinase (PI3K) abolished K-Ras actions on ENaC. In contrast, inhibition of other K-Ras effector cascades, including the MAPK and Ral/Rac/Rho cascades, did not affect KRas actions on ENaC. Activation of ENaC by K-Ras, moreover, was sensitive to co-expression of dominant negative p85 PI3K . The G12:C40 effector-specific double mutant of Ras, which preferentially activates PI3K, enhanced ENaC activity in a manner sensitive to inhibition of PI3K. Other effector-specific mutants preferentially activating MAPK and RalGDS signaling had no effect. Constitutively active PI3K activated ENaC independent of K-Ras with the effects of PI3K and K-Ras on ENaC not being additive. We conclude that K-Ras activates ENaC via the PI3K cascade.