Regulation of the epithelial Na+ channel by the protein kinase CK2

CK2 is a ubiquitous, pleiotropic, and constitutively active Ser/Thr protein kinase that controls protein expression, cell signaling, and ion channel activity. Phosphorylation sites for CK2 are located in the C terminus of both beta- and gamma-subunits of the epithelial Na(+) channel (ENaC). We examined the role of CK2 on the regulation of both endogenous ENaC in native murine epithelia and in Xenopus oocytes expressing rENaC. In Ussing chamber experiments with mouse airways, colon, and cultured M1-collecting duct cells, amiloride-sensitive Na(+) transport was inhibited dose-dependently by the selective CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB). In oocytes, ENaC currents were also inhibited by TBB and by the structurally unrelated inhibitors heparin and poly(E:Y). Expression of a trimeric channel lacking both CK2 sites (alphabeta(S631A)gamma(T599A)) produced a largely attenuated amiloride-sensitive whole cell conductance and rendered the mutant channel insensitive to CK2. In Xenopus oocytes, CK2 was translocated to the cell membrane upon expression of wt-ENaC but not of alphabeta(S631A)gamma(T599A)-ENaC. Phosphorylation by CK2 is essential for ENaC activation, and to a lesser degree, it also controls membrane expression of alphabetagamma-ENaC. Channels lacking the Nedd4-2 binding motif in beta-ENaC (R561X, Y618A) no longer required the CK2 site for channel activity and siRNA-knockdown of Nedd4-2 eliminated the effects of TBB. This implies a role for CK2 in inhibiting the Nedd4-2 pathway. We propose that the C terminus of beta-ENaC is targeted by this essential, conserved pleiotropic kinase that directs its constitutive activity toward many cellular protein complexes.     

The cytosolic termini of the beta- and gamma-ENaC subunits are involved in the functional interactions between cystic fibrosis transmembrane conductance regulator and epithelial sodium channel

Epithelial sodium channel (ENaC) and cystic fibrosis transmembrane conductance regulator (CFTR) are co-localized in the apical membrane of many epithelia. These channels are essential for electrolyte and water secretion and/or reabsorption. In cystic fibrosis airway epithelia, a hyperactivated epithelial Na(+) conductance operates in parallel with defective Cl(-) secretion. Several groups have shown that CFTR down-regulates ENaC activity, but the mechanisms and the regulation of CFTR by ENaC are unknown. To test the hypothesis that ENaC and CFTR regulate each other, and to identify the region(s) of ENaC involved in the interaction between CFTR and ENaC, rENaC and its mutants were co-expressed with CFTR in Xenopus oocytes. Whole cell macroscopic sodium currents revealed that wild type (wt) alphabetagamma-rENaC-induced Na(+) current was inhibited by co-expression of CFTR, and further inhibited when CFTR was activated with a cAMP-raising mixture (CKT). Conversely, alphabetagamma-rENaC stimulated CFTR-mediated Cl(-) currents up to approximately 6-fold. Deletion mutations in the intracellular tails of the three rENaC subunits suggested that the carboxyl terminus of the beta subunit was required both for the down-regulation of ENaC by activated CFTR and the up-regulation of CFTR by ENaC. However, both the carboxyl terminus of the beta subunit and the amino terminus of the gamma subunit were essential for the down-regulation of rENaC by unstimulated CFTR. Interestingly, down-regulation of rENaC by activated CFTR was Cl(-)-dependent, while stimulation of CFTR by rENaC was not dependent on either cytoplasmic Na(+) or a depolarized membrane potential. In summary, there appear to be at least two different sites in ENaC involved in the intermolecular interaction between CFTR and ENaC.     

Activation of large conductance sodium channels upon expression of amiloride-sensitive sodium channel in Sf9 insect cells.

The amiloride-sensitive epithelial sodium channels (ENaC) mediate Na(+) reabsorption in epithelial tissues including distal nephron, colon, lung, and secretory glands and plays a critical role in pathophysiology of hypertension and cystic fibrosis. The ENaC is a multimeric protein composed of alpha-ENaC, beta-ENaC, and gamma-ENaC subunits. To study the biochemical properties of the channel, the subunit cDNAs of rat colon ENaC (rENaC) were subcloned into baculoviruses, and the corresponding proteins were expressed in Sf9 insect cells. The functional characteristics of the expressed rENaC were studied in planar lipid bilayers. The results show that expression of alpha-rENaC and alphabetagamma-rENaC in Sf9 insect cells results in the generation of cation-selective large conductance channels. Although the large conductance channels observed in the alpha-rENaC-containing membranes were unaffected by amiloride, the large conductance channels found in alphabetagamma-rENaC complex-containing membranes exhibited voltage-dependent flickering in the presence of micromolar amiloride. Possible implications of these observations are discussed.     

Cystic fibrosis transmembrane conductance regulator differentially regulates human and mouse epithelial sodium channels in Xenopus oocytes

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 alphabetagamma 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 alphabetagamma mENaC or alphabetagamma human ENaC (hENaC). Co-expression of CFTR and alphabetagamma mENaC or hENaC resulted in an approximately 3-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 approximately 20% decrease in alpha-T663-hENaC-mediated currents, and essentially no change in alpha-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 alpha ENaC confer species specificity regarding ENaC inhibition by activated CFTR.     

Preferential assembly of epithelial sodium channel (ENaC) subunits in Xenopus oocytes: role of furin-mediated endogenous proteolysis

The epithelial sodium channel (ENaC) is preferentially assembled into heteromeric alphabetagamma complexes. The alpha and gamma (not beta) subunits undergo proteolytic cleavage by endogenous furin-like activity correlating with increased ENaC function. We identified full-length subunits and their fragments at the cell surface, as well as in the intracellular pool, for all homo- and heteromeric combinations (alpha, beta, gamma, alphabeta, alphagamma, betagamma, and alphabetagamma). We assayed corresponding channel function as amiloride-sensitive sodium transport (I(Na)). We varied furin-mediated proteolysis by mutating the P1 site in alpha and/or gamma subunit furin consensus cleavage sites (alpha(mut) and gamma(mut)). Our findings were as follows. (i) The beta subunit alone is not transported to the cell surface nor cleaved upon assembly with the alpha and/or gamma subunits. (ii) The alpha subunit alone (or in combination with beta and/or gamma) is efficiently transported to the cell surface; a surface-expressed 65-kDa alpha ENaC fragment is undetected in alpha(mut)betagamma, and I(Na) is decreased by 60%. (iii) The gamma subunit alone does not appear at the cell surface; gamma co-expressed with alpha reaches the surface but is not detectably cleaved; and gamma in alphabetagamma complexes appears mainly as a 76-kDa species in the surface pool. Although basal I(Na) of alphabetagamma(mut) was similar to alphabetagamma, gamma(mut) was not detectably cleaved at the cell surface. Thus, furin-mediated cleavage is not essential for participation of alpha and gamma in alphabetagamma heteromers. Basal I(Na) is reduced by preventing furin-mediated cleavage of the alpha, but not gamma, subunits. Residual current in the absence of furin-mediated proteolysis may be due to non-furin endogenous proteases.     

Regulation of epithelial Na(+) channels by actin in planar lipid bilayers and in the Xenopus oocyte expression system

The hypothesis that actin interactions account for the signature biophysical properties of cloned epithelial Na(+) channels (ENaC) (conductance, ion selectivity, and long mean open and closed times) was tested using planar lipid bilayer reconstitution and patch clamp techniques. We found the following. 1) In bilayers, actin produced a more than 2-fold decrease in single channel conductance, a 5-fold increase in Na(+) versus K(+) permselectivity, and a substantial increase in mean open and closed times of wild-type alphabetagamma-rENaC but had no effect on a mutant form of rENaC in which the majority of the C terminus of the alpha subunit was deleted (alpha(R613X)betagamma-rENaC). 2) When alpha(R613X)betagamma-rENaC was heterologously expressed in oocytes and single channels examined by patch clamp, 12.5-pS channels of relatively low cation permeability were recorded. These characteristics were identical to those recorded in bilayers for either alpha(R613X)betagamma-rENaC or wild-type alphabetagamma-rENaC in the absence of actin. Moreover, we show that rENaC subunits tightly associate, forming either homo- or heteromeric complexes when prepared by in vitro translation or when expressed in oocytes. Finally, we show that alpha-rENaC is properly assembled but retained in the endoplasmic reticulum compartment. We conclude that actin subserves an important regulatory function for ENaC and that planar bilayers are an appropriate system in which to study the biophysical and regulatory properties of these cloned channels.     

Cell-specific expression of epithelial sodium channel alpha, beta, and gamma subunits in aldosterone-responsive epithelia from the rat: localization by in situ hybridization and immunocytochemistry

A highly selective, amiloride-sensitive, epithelial sodium channel from rat colon (rENaC), composed of three homologous subunits termed alpha, beta, and gamma rENaC, has been cloned by functional expression and was proposed to mediate electrogenic sodium reabsorption in aldosterone- responsive epithelia. To determine whether rENaC could account for sodium absorption in vivo, we studied the cellular localization of the sodium channel messenger RNA subunits by in situ hybridization and their cellular and subcellular distribution by immunocytochemistry in the kidney, colon, salivary, and sweat glands of the rat. In the kidney, we show that the three subunit mRNAs are specifically co- expressed in the renal distal convoluted tubules (DCT), connecting tubules (CNT), cortical collecting ducts (CCD), and outer medullary collecting ducts (OMCD), but not in the inner medullary collecting ducts (IMCD). We demonstrate co-localization of alpha, beta, and gamma subunit proteins in the apical membrane of a majority of cells of CCD and OMCD. Our data indicate that alpha, beta, and gamma subunit mRNAs and proteins are co-expressed in the distal nephron (excepting IMCD), a localization that correlates with the previously described physiological expression of amiloride-sensitive electrogenic sodium transport. Our data, however, suggest that another sodium transport protein mediates electrogenic amiloride-sensitive sodium reabsorption in IMCD. We also localized rENaC to the surface epithelial cells of the distal colon and to the secretory ducts of the salivary gland and sweat gland, providing further evidence consistent with the hypothesis that the highly selective, amiloride-sensitive sodium channel is physiologically expressed in aldosterone-responsive cells.     

Stimulation of the epithelial sodium channel (ENaC) by cAMP involves putative ERK phosphorylation sites in the C termini of the channel's beta- and gamma-subunit

The mechanisms involved in the regulation of the epithelial sodium channel (ENaC) via the cAMP pathway are not yet completely understood. The aim of the present study was to investigate cAMP-mediated ENaC regulation in Xenopus laevis oocytes heterologously expressing the three subunits (alphabetagamma) of rat ENaC and to determine the ENaC regions important for mediating the stimulatory effect of cAMP. In oocytes treated for about 24 h with 1 mm 3-isobutyl-1-methylxanthine (IBMX) and 1 microm forskolin (FSK) so as to increase intracellular cAMP, the amiloride-sensitive whole cell current (DeltaI(Ami)) was on average 10-fold larger than DeltaI(Ami) in matched control oocytes. This effect on DeltaI(Ami) was paralleled by an increase in ENaC surface expression caused by a reduced rate of ENaC retrieval. In addition, IBMX/FSK also enhanced ENaC open probability from about 0.2 to 0.5. The stimulatory effect of IBMX/FSK was dependent on the presence of intact PY motifs in the C termini of the channel. Mutagenesis of putative protein kinase A and CK-2 consensus motifs in the cytosolic domains of the channel did not reveal critical sites involved in mediating the stimulatory effect of IBMX/FSK. In contrast, site-directed mutagenesis of two putative ERK-consensus motifs (T613A in betaENaC and T623A in gammaENaC) largely reduced the stimulatory effect of IBMX/FSK. Phosphorylation of these ERK sites has previously been reported to enhance the interaction of ENaC and Nedd4 (Shi, H., Asher, C., Chigaev, A., Yung, Y., Reuveny, E., Seger, R., and Garty, H. (2002) J. Biol. Chem. 277, 13539-13547). Using co-expression experiments we demonstrated that mutating the two ERK sites attenuates the inhibitory effect of Nedd4-2 on ENaC currents. We conclude that an increase in intracellular cAMP favors the dephosphorylation of the two ERK sites, which reduces channel retrieval and increases P(O) by modulating ENaC/Nedd4 interaction. This defines a novel regulatory pathway likely to be relevant for cAMP-induced stimulation of ENaC in vivo.     

Regulation of rENaC mRNA by dietary NaCl and steroids: organ, tissue, and steroid heterogeneity

Rats on a low-NaCl diet have a highNa⁺ channel activity in colon andkidney. To address the mechanism of this increased activity, wemeasured mRNA levels of three Na⁺channel subunits in epithelial tissue (rENaC) from rats having been fedeither a low (0.13%)- or high (8%)-NaCl diet for 2-3 wk. Thesize of the mRNA for each of the rENaC subunits as determined byNorthern blot was unaffected by diet. RNase protection assay showedheterogeneity of response by organs and subunit. In lung, there was noeffect of diet on any of the three subunits. In descending colon, thelow-NaCl diet increased - and -rENaC mRNA, with no effect on-rENaC mRNA. In the kidney, the response to dietary NaCl wasdependent on the region. In cortex and outer medulla, diet had noeffect on any of the subunits. Rats fed the low-NaCl diet had greater-rENaC in inner medulla but not - or -rENaC mRNA. We nextasked whether acute administration of pure glucocorticoid (GC) ormineralocorticoid (MC) hormones to adrenalectomized rats reproduced theeffects of a low-NaCl diet. Six hours after administration of GC or MC,a somewhat different heterogeneity occurred. In lung, -rENaC mRNAwas increased but only in response to GC. In colon, either GC or MCincreased - or -rENaC, and there was no effect on -rENaC. Inkidney, either GC or MC increased -rENaC, without an effect on -or -rENaC. In contrast to the response to a low-NaCl diet, all threeregions were similarly affected by acute steroids. These resultsdemonstrate a striking heterogeneity in response to physiologicalstimuli that regulate ENaC function. The mRNA levels of each of therENaC subunits can be determined by the type of steroid and by factorsunique to the organ and even to the specific region of the kidney.

     

Progesterone down-regulates the open probability of the amiloride-sensitive epithelial sodium channel via a Nedd4-2-dependent mechanism

Activation of the mitogen-activated protein (MAP) kinase cascade by progesterone in Xenopus oocytes leads to a marked down-regulation of activity of the amiloride-sensitive epithelial sodium channel (ENaC). Here we have studied the signaling pathways involved in progesterone effect on ENaC activity. We demonstrate that: (i) the truncation of the C termini of the alphabetagammaENaC subunits results in the loss of the progesterone effect on ENaC; (ii) the effect of progesterone was also suppressed by mutating conserved tyrosine residues in the Pro-X-X-Tyr (PY) motif of the C termini of the beta and gamma ENaC subunits (beta(Y618A) and gamma(Y628A)); (iii) the down-regulation of ENaC activity by progesterone was also suppressed by co-expression ENaC subunits with a catalytically inactive mutant of Nedd4-2, a ubiquitin ligase that has been previously demonstrated to decrease ENaC cell-surface expression via a ubiquitin-dependent internalization/degradation mechanism; (iv) the effect of progesterone was significantly reduced by suppression of consensus sites (beta(T613A) and gamma(T623A)) for ENaC phosphorylation by the extracellular-regulated kinase (ERK), a MAP kinase previously shown to facilitate the binding of Nedd4 ubiquitin ligases to ENaC; (v) the quantification of cell-surface-expressed ENaC subunits revealed that progesterone decreases ENaC open probability (whole cell P(o), wcP(o)) and not its cell-surface expression. Collectively, these results demonstrate that the binding of active Nedd4-2 to ENaC is a crucial step in the mechanism of ENaC inhibition by progesterone. Upon activation of ERK, the effect of Nedd4-2 on ENaC open probability can become more important than its effect on ENaC cell-surface expression.     

Epithelial sodium channel inhibition by AMP-activated protein kinase in oocytes and polarized renal epithelial cells

The epithelial Na(+) channel (ENaC) regulates epithelial salt and water reabsorption, processes that require significant expenditure of cellular energy. To test whether the ubiquitous metabolic sensor AMP-activated kinase (AMPK) regulates ENaC, we examined the effects of AMPK activation on amiloride-sensitive currents in Xenopus oocytes and polarized mouse collecting duct mpkCCD(c14) cells. Microinjection of oocytes expressing mouse ENaC (mENaC) with either active AMPK protein or an AMPK activator inhibited mENaC currents relative to controls as measured by two-electrode voltage-clamp studies. Similarly, pharmacological AMPK activation or overexpression of an activating AMPK mutant in mpkCCD(c14) cells inhibited amiloride-sensitive short circuit currents. Expression of a degenerin mutant beta-mENaC subunit (S518K) along with wild type alpha and gamma increased the channel open probability (P(o)) to approximately 1. However, AMPK activation inhibited currents similarly with expression of either degenerin mutant or wild type mENaC. Single channel recordings under these conditions demonstrated that neither P(o) nor channel conductance was affected by AMPK activation. Moreover, expression of a Liddle's syndrome-type beta-mENaC mutant (Y618A) greatly enhanced ENaC whole cell currents relative to wild type ENaC controls and prevented AMPK-dependent inhibition. These findings indicate that AMPK-dependent ENaC inhibition is mediated through a decrease in the number of active channels at the plasma membrane (N), presumably through enhanced Nedd4-2-dependent ENaC endocytosis. The AMPK-ENaC interaction appears to be indirect; AMPK did not bind ENaC in cells, as assessed by in vivo pull-down assays, nor did it phosphorylate ENaC in vitro. In summary, these results suggest a novel mechanism for coupling ENaC activity and renal Na(+) handling to cellular metabolic status through AMPK, which may help prevent cellular Na(+) loading under hypoxic or ischemic conditions.     

No evidence for inhibition of ENaC through CFTR-mediated release of ATP

Both purinergic stimulation and activation of cystic fibrosis transmembrane conductance regulator (CFTR) increases Cl(-) secretion and inhibit amiloride-sensitive Na(+) transport. CFTR has been suggested to conduct adenosine 5'-triphosphate (ATP) or to control ATP release to the luminal side of epithelial tissues. Therefore, a possible mechanism on how CFTR controls the activity of epithelial Na(+) channels (ENaC) could be by release of ATP or uridine 5'-triphosphate (UTP), which would then bind to P2Y receptors and inhibit ENaC. We examined this question in native tissues from airways and colon and in Xenopus oocytes. Inhibition of amiloride-sensitive transport by both CFTR and extracellular nucleotides was observed in colon and trachea. However, nucleotides did not inhibit ENaC in Xenopus oocytes, even after coexpression of P2Y(2) receptors. Using different tools such as hexokinase, the P2Y inhibitor suramin or the Cl(-) channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), we did not detect any role of a putative ATP secretion in activation of Cl(-) transport or inhibition of amiloride sensitive short circuit currents by CFTR. In addition, N(2),2'-O-dibutyrylguanosine 3',5'-cyclic monophosphate (cGMP) and protein kinase G (PKG)-dependent phosphorylation or the nucleoside diphosphate kinase (NDPK) do not seem to play a role for the inhibition of ENaC by CFTR, which, however, requires the presence of extracellular Cl(-).     

Protein kinase regulation of a cloned epithelial Na+ channel

We examined the regulation of a cloned epithelial Na+ channel (alpha beta gamma-rENaC) by protein kinase A (PKA) and protein kinase C (PKC). Experiments were performed in Xenopus oocytes and in planar lipid bilayers. At a holding potential of -100 mV, amiloride-sensitive current averaged -1,279 +/- 111 nA (n = 7) in alpha beta gamma-rENaC- expressing oocytes. Currents in water-injected oocytes were essentially unresponsive to 10 microM amiloride. A 1-h stimulation of PKC with 100 nM of PMA inhibited whole-cell currents in Xenopus oocytes to 17.1 +/- 1.8, and 22.1 +/- 2.6% of control (n = 7), at holding potentials of - 100 and +40 mV, respectively. Direct injection of purified PKC resulted in similar inhibition to that observed with PMA. Additionally, the inactive phorbol ester, phorbol-12-myristate-13-acetate, 4-O-methyl, was without effect on alpha beta gamma-rENaC currents. Pretreatment with the microtubule inhibitor colchicine (100 microM) did not modify the inhibitory effect of PMA; however, pretreatment with 20 microM cytochalasin B decreased the inhibitory action of PMA to < 20% of that previously observed. In vitro-synthesized alpha beta gamma-rENaC formed an amiloride-sensitive Na(+)-selective channel when incorporated into planar lipid bilayers. Addition of PKC, diacyl-glycerol, and Mg-ATP to the side opposite that which amiloride blocked, decreased the channel''s open probability (Po) from 0.44 +/- 0.06 to 0.13 +/- 0.03 (n = 9). To study the effects of PKA on alpha beta gamma-rENaC expressed in Xenopus oocytes, cAMP levels were elevated with 10 microM forskolin and 1 mM isobutyl-methyl-xanthine. This cAMP-elevating cocktail did not cause any stimulation of alpha beta gamma-rENaC currents in either the inward or outward directions. This lack of activation was also observed in oocytes preinhibited with PMA and in oocytes pretreated with cytochalasin B and PMA. Neither alpha-rENaC nor alpha beta gamma-rENaC incorporated into planar lipid bilayers could be activated with PKA and Mg-ATP added to either side of the membrane, as Po remained at 0.63 +/- 0.06 (n = 7) and 0.45 +/- 0.05 (n = 9), respectively. We conclude that: alpha beta gamma-rENaC is inhibited by PKC, and that alpha beta gamma- rENaC is not activated by PKA.     

Rat ENaC expressed in Xenopus laevis oocytes is activated by cAMP and blocked by Ni(2+)

We used oocytes of the South African clawed toad Xenopus laevis to express the three subunits of the epithelial Na(+) channel from rat distal colon (rENaC). We combined conventional dual-microelectrode voltage-clamp with continuous capacitance (C(m)) measurements and noise analysis to evaluate the effects of cAMP and Ni(2+) on rENaC. Control oocytes or rENaC-expressing oocytes exhibited no spontaneous fluctuations in current. However, in rENaC-expressing oocytes amiloride induced a marked plateau-shaped rise of the power density spectra. Recordings using four different concentrations of amiloride revealed that the blocker-channel interactions were of the first order. A cocktail of the membrane permeant cAMP analogue chlorophenylthio-cAMP and IBMX (cAMP cocktail) increased amiloride-sensitive current (I(ami)) and conductance (G(ami)). Furthermore, C(m) was also increased following cAMP application, indicating an increase in plasma membrane surface area. Noise analysis showed that cAMP increased the number of active channels in the oocyte membrane while single-channel current decreased. From these data we conclude that cAMP triggered exocytotic delivery of preformed rENaCs to the plasma membrane. Ni(2+) (2.5 mM) inhibited about 60% of the rENaC current and conductance while C(m) remained unaffected. Noise analysis revealed that this inhibition could be attributed to a decrease in the apparent channel density, while single-channel current did not change significantly. These observations argue for direct effects of Ni(2+) on channel activity rather than induction of endocytotic removal of active channels from the plasma membrane.     

Determination of epithelial Na+ channel subunit stoichiometry from single-channel conductances

The epithelial Na(+) channel (ENaC) is a multimeric membrane protein consisting of three subunits, alpha, beta, and gamma. The total number of subunits per functional channel complex has been described variously to follow either a tetrameric arrangement of 2alpha:1beta:1gamma or a higher-ordered stoichiometry of 3alpha:3beta:3gamma. Therefore, while it is clear that all three ENaC subunits are required for full channel activity, the number of the subunits required remains controversial. We used a new approach, based on single-channel measurements in Xenopus oocytes to address this issue. Individual mutations that alter single-channel conductance were made in pore-lining residues of ENaC alpha, beta, or gamma subunits. Recordings from patches in oocytes expressing a single species, wild type or mutant, of alpha, beta, and gamma showed a well-defined current transition amplitude with a single Gaussian distribution. When cRNAs for all three wild-type subunits were mixed with an equimolar amount of a mutant alpha-subunit (either S589D or S592T), amplitudes corresponding to pure wild-type or mutant conductances could be observed in the same patch, along with a third intermediate amplitude most likely arising from channels with at least one wild-type and at least 1 mutant alpha-subunit. However, intermediate or hybrid conductances were not observed with coexpression of wild-type and mutant betaG529A or gammaG534E subunits. Our results support a tetrameric arrangement of ENaC subunits where 2alpha, 1beta, and 1gamma come together around central pore.     

Subunit arrangement of gamma-aminobutyric acid type A receptors

The GABA(A) receptors are ligand-gated chloride channels. The subunit stoichiometry of the receptors is controversial; four, five, or six subunits per receptor molecule have been proposed for alphabeta receptors, whereas alphabetagamma receptors are assumed to be pentamers. In this study, alpha-beta and beta-alpha tandem cDNAs from the alpha1 and beta2 subunits of the GABA(A) receptor were constructed. We determined the minimal length of the linker that is required between the two subunits for functional channel expression for each of the tandem constructs. 10- and 23-amino acid residues are required for alpha-beta and beta-alpha, respectively. The tandem constructs either alone or in combination with each other failed to express functional channels in Xenopus oocytes. Therefore, we can exclude tetrameric or hexameric alphabeta GABA(A) receptors. We can also exclude proteolysis of the tandem constructs. In addition, the tandem constructs were combined with single alpha, beta, or gamma subunits to allow formation of pentameric arrangements. In contrast to the combination with alpha subunits, the combination with either beta or gamma subunits led to expression of functional channels. Therefore, a pentameric arrangement containing two alpha1 and three beta2 subunits is proposed for the receptor composed of alpha and beta subunits. Our findings also favor an arrangement betaalphagammabetaalpha for the receptor composed of alpha, beta, and gamma subunits.     

Female gender hormones regulate mRNA levels and function of the rat lung epithelial Na channel

The epithelial Nachannel (ENaC) plays a critical role in the active reabsorption ofalveolar fluid at the time of birth or during pulmonary edema. Althoughrat (r) ENaC is regulated by glucocorticoids during fetal development,there are no data regarding the influence of gender hormones on ENaCexpression or function. We report higher levels of mRNAs encoding the-rENaC subunit or the cystic fibrosis transmembrane conductanceregulator (CFTR) in the lungs of nonpregnant adult female relative toadult male Wistar rats. Combined, but not separate, administration ofprogesterone and 17-estradiol increased mRNA levels encoding-rENaC, -rENaC, and CFTR within 24 h. We also found adose-dependent increase in rENaC functional activity (as assessed bythe amiloride-sensitive short-circuit current across primary monolayercultures of alveolar epithelial cells mounted in Ussing chambers) aftera 5-day incubation of cells in medium containing progesterone and17-estradiol. These findings suggest a gender-dependent influence onthe lung's ability to recover from pulmonary edema and on the degreeof airway fluid hydration in cystic fibrosis.

     

WW domains of Nedd4 bind to the proline-rich PY motifs in the epithelial Na+ channel deleted in Liddle's syndrome.

The amiloride-sensitive epithelial sodium channel (ENaC) plays a major role in sodium transport in kidney and other epithelia, and in regulating blood pressure. The channel is composed of three subunits (alphabetagamma) each containing two proline-rich sequences (P1 and P2) at its C-terminus. The P2 regions in human beta and gammaENaC, identical to the rat betagammarENaC, were recently shown to be deleted in patients with Liddle's syndrome (a hereditary form of hypertension), leading to hyperactivation of the channel. Using a yeast two-hybrid screen, we have now identified the rat homologue of Nedd4 (rNedd4) as the binding partner for the P2 regions of beta and gammarENaC. rNedd4 contains a Ca2+ lipid binding (CaLB or C2) domain, three WW domains and a ubiquitin ligase (Hect) domain. Our yeast two-hybrid and in vitro binding studies revealed that the rNedd4-WW domains mediate this association by binding to the P2 regions, which include the PY motifs (XPPXY) of either betarENaC (PPPNY) or gammarENaC (PPPRY). SH3 domains were unable to bind these sequences. Moreover, mutations to Ala of Pro616 or Tyr618 within the betarENaC P2 sequence (to PPANY or PPPNA, respectively), recently described in Liddle's patients, led to abrogation of rNedd4-WW binding. Nedd4-WW domains also bound to the proline-rich C-terminus (containing the sequence PPPAY) of alpharENaC, and endogenous Nedd4 co-immunoprecipitated with alpharENaC expressed in MDCK cells. These results demonstrate that the WW domains of rNedd4 bind to the PY motifs deleted from beta or gammaENaC in Liddle's syndrome patients, and suggest that Nedd4 may be a regulator (suppressor) of the epithelial Na+ channel.     

The catalytic and GAF domains of the rod cGMP phosphodiesterase (PDE6) heterodimer are regulated by distinct regions of its inhibitory gamma subunit

The central effector of visual transduction in retinal rod photoreceptors, cGMP phosphodiesterase (PDE6), is a catalytic heterodimer (alphabeta) to which low molecular weight inhibitory gamma subunits bind to form the nonactivated PDE holoenzyme (alphabetagamma(2)). Although it is known that gamma binds tightly to alphabeta, the binding affinity for each gamma subunit to alphabeta, the domains on gamma that interact with alphabeta, and the allosteric interactions between gamma and the regulatory and catalytic regions on alphabeta are not well understood. We show here that the gamma subunit binds to two distinct sites on the catalytic alphabeta dimer (K(D)(1) < 1 pm, K(D)(2) = 3 pm) when the regulatory GAF domains of bovine rod PDE6 are occupied by cGMP. Binding heterogeneity of gamma to alphabeta is absent when cAMP occupies the noncatalytic sites. Two major domains on gamma can interact independently with alphabeta with the N-terminal half of gamma binding with 50-fold greater affinity than its C-terminal, inhibitory region. The N-terminal half of gamma is responsible for the positive cooperativity between gamma and cGMP binding sites on alphabeta but has no effect on catalytic activity. Using synthetic peptides, we identified regions of the amino acid sequence of gamma that bind to alphabeta, restore high affinity cGMP binding to low affinity noncatalytic sites, and retard cGMP exchange with both noncatalytic sites. Subunit heterogeneity, multiple sites of gamma interaction with alphabeta, and positive cooperativity of gamma with the GAF domains are all likely to contribute to precisely controlling the activation and inactivation kinetics of PDE6 during visual transduction in rod photoreceptors.     

Specific and nonspecific effects of protein kinase C on the epithelial Na (+) channel

The Xenopus oocyte expression system was used to explore the mechanisms of inhibition of the cloned rat epithelial Na(+) channel (rENaC) by PKC (Awayda, M.S., I.I. Ismailov, B.K. Berdiev, C.M. Fuller, and D.J. Benos. 1996. J. Gen. Physiol. 108:49-65) and to determine whether human ENaC exhibits similar regulation. Effects of PKC activation on membrane and/or channel trafficking were determined using impedance analysis as an indirect measure of membrane area. hENaC-expressing oocytes exhibited an appreciable activation by hyperpolarizing voltages. This activation could be fit with a single exponential, described by a time constant (tau) and a magnitude (DeltaI (V)). A similar but smaller magnitude of activation was also observed in oocytes expressing rENaC. This activation likely corresponds to the previously described effect of hyperpolarizing voltage on gating of the native Na(+) channel (Palmer, L.G., and G. Frindt. 1996. J. Gen. Physiol. 107:35-45). Stimulation of PKC with 100 nM PMA decreased DeltaI(V) in hENaC-expressing oocytes to a plateau at 57.1 +/- 4.9% (n = 6) of baseline values at 20 min. Similar effects were observed in rENaC-expressing oocytes. PMA decreased the amiloride-sensitive hENaC slope conductance (g(Na)) to 21.7 +/- 7.2% (n = 6) of baseline values at 30 min. This decrease was similar to that previously reported for rENaC. This decrease of g (Na) was attributed to a decrease of membrane capacitance (C (m)), as well as the specific conductance (g(m)/C(m )). The effects on g(m)/C(m) reached a plateau within 15 min, at approximately 60% of baseline values. This decrease is likely due to the specific ability of PKC to inhibit ENaC. On the other hand, the decrease of C(m) was unrelated to ENaC and is likely an effect of PKC on membrane trafficking, as it was observed in ENaC-expressing as well as control oocytes. At lower PMA concentrations (0.5 nM), smaller changes of C(m) were observed in rENaC- and hENaC-expressing oocytes, and were preceded by larger changes of g(m ) and by changes of g(m)/C(m), indicating specific effects on ENaC. These findings indicate that PKC exhibits multiple and specific effects on ENaC, as well as nonspecific effects on membrane trafficking. Moreover, these findings provide the electrophysiological basis for assessing channel-specific effects of PKC in the Xenopus oocyte expression system.