Interaction of syntaxins with the amiloride-sensitive epithelial sodium channel.

Amiloride-sensitive sodium channels mediate sodium entry across the apical membrane of epithelial cells in variety of tissues. The rate of Na(+) entry is controlled by the regulation of the epithelial sodium channel (ENaC) complex. Insertion/retrieval of the ENaC complex into the apical membrane as well as direct kinetic effects at the single channel level are recognized mechanisms of regulation. Recent data suggest that the syntaxin family of targeting proteins interact with and functionally regulate a number of ion channels and pumps. To evaluate the role of these proteins in regulating ENaC activity, we co-expressed rat ENaC cRNA (alpha, beta, gamma subunits) with syntaxin 1A or 3 cRNAs in Xenopus oocytes. Basal ENaC currents were inhibited by syntaxin 1A and stimulated by syntaxin 3. Both syntaxin 1A and syntaxin 3 could be co-immunoprecipitated with ENaC subunit proteins, suggesting physical interaction. Interestingly, immunofluorescence data suggest that with either syntaxin isoform the ENaC-associated epifluorescence on the oocyte surface is enhanced. These data indicate that (i) both syntaxin isoforms increase the net externalization of the ENaC channel complex, (ii) that the functional regulation is isoform specific, and (iii) suggest that ENaC may be regulated through mechanisms involving protein-protein interactions.     

ClC-5 Chloride Channel Alters Expression of the Epithelial Sodium Channel (ENaC)

ClC-5 chloride channels and epithelial sodium channels (ENaC) are present in many cell types including airway and retinal epithelia. Since ENaC activity is known to be affected by chloride transport, we co-injected Xenopus oocytes with cRNAs encoding ENaC and ClC-5 to investigate whether channel currents are impacted by heterologous co-expression of these proteins. ClC-5 currents were not detectably affected by co-expression with ENaC, whereas amiloride-sensitive ENaC currents were significantly lower compared to control oocytes expressing ENaC alone. Co-expression of ENaC with cRNA sequences encoding non-conducting fragments of ClC-5 revealed that the amino acid sequence region between positions 347 and 647 was sufficient for inhibition of ENaC currents. Co-expression of ENaC and another transport protein, the sodium dicarboxylate co-transporter (NaDC-1), did not affect ENaC currents. To test whether the inhibitory effects of ClC-5 were specific for ENaC, ClC-5 was also co-expressed with CFTR. CFTR currents were also inhibited by co-expression with ClC-5, whereas ClC-5 currents were unaffected. Western blot analysis of biotinylated oocyte surface membranes revealed that the co-expression of ClC-5 with ENaC, CFTR, or NaDC-1 decreased the abundance of these proteins at the surface membrane. We conclude that overexpression of ClC-5, specifically amino acids 347–647, can alter the normal translation or trafficking of ENaC and other ion transport proteins by a mechanism that is independent of the chloride conductance of ClC-5.     

Rab11b regulates the trafficking and recycling of the epithelial sodium channel (ENaC)

Expression of the epithelial sodium channel (ENaC) at the apical membrane of cortical collecting duct (CCD) principal cells is modulated by regulated trafficking mediated by vesicle insertion and retrieval. Small GTPases are known to facilitate vesicle trafficking, recycling, and membrane fusion events; however, little is known about the specific Rab family members that modify ENaC surface density. Using a mouse CCD cell line that endogenously expresses ENaC (mpkCCD), the channel was localized to both Rab11a- and Rab11b-positive endosomes by immunoisolation and confocal fluorescent microscopy. Expression of a dominant negative (DN) form of Rab11a or Rab11b significantly reduced the basal and cAMP-stimulated ENaC-dependent sodium (Na(+)) transport. The greatest reduction in Na(+) transport was observed with the expression of DN-Rab11b. Furthermore, small interfering RNA-mediated knockdown of each Rab11 isoform demonstrated the requirement for Rab11b in ENaC surface expression. These data indicate that Rab11b, and to a lesser extent Rab11a, is involved in establishing the constitutive and cAMP-stimulated Na(+) transport in mpkCCD cells.     

Distinct domain-dependent effect of syntaxin1A on amiloride-sensitive sodium channel (ENaC) currents in HT-29 colonic epithelial cells

The amiloride-sensitive epithelial sodium channel (ENaC), a plasma membrane protein mediates sodium reabsorption in epithelial tissues, including the distal nephron and colon. Syntaxin1A, a trafficking protein of the t-SNARE family has been reported to inhibit ENaC in the Xenopus oocyte expression and artificial lipid bilayer systems. The present report describes the regulation of the epithelial sodium channel by syntaxin1A in a human cell line that is physiologically relevant as it expresses both components and also responds to aldosterone stimulation. In order to evaluate the physiological significance of syntaxin1A interaction with natively expressed ENaC, we over-expressed HT-29 with syntaxin1A constructs comprising various motifs. Unexpectedly, we observed the augmentation of amiloride-sensitive currents with wild-type syntaxin1A full-length construct (1-288) in this cell line. Both gammaENaC and neutralizing syntaxin1A antibodies blocked native expression as amiloride-sensitive sodium currents were inhibited while munc18-1 antibody reversed this effect. The coiled-coiled domain H3 (194-266) of syntaxin1A inhibited, however the inclusion of the transmembrane domain to this motif (194-288) augmented amiloride sensitive currents. More so, data suggest that ENaC interacts with multiple syntaxin1A domains, which differentially regulate channel function. This functional modulation is the consequence of the physical enhancement of ENaC at the cell surface in cells over-expressed with syntaxin(s). Our data further suggest that syntaxin1A up-regulates ENaC function by multiple mechanisms that include PKA, PLC, PI3 and MAP Kinase (p42/44) signaling systems. We propose that syntaxin1A possesses distinct inhibitory and stimulatory domains that interact with ENaC subunits, which critically determines the overall ENaC functionality/regulation under distinct physiological conditions.     

ENaC contribution to epithelial wound healing is independent of the healing mode and of any increased expression in the channel

Previous work from our laboratory and others has shown that, in some epithelia, the epithelial sodium channel (ENaC) increases its expression during wound healing. In these cases, inhibition of the channel determines a decrease in the healing rate, a result suggesting a role for ENaC in the overall healing process. To understand further this role of ENaC in epithelia, we explored the participation of ENaC in wound healing in four cultured epithelial cell lines selected on the basis of their different embryonic origins, function and modality of healing, i.e., by lamellipodial cell crawling or by actin cable formation. Three of the cell lines (bovine corneal endothelial cells, rabbit corneal epithelial cells and Madin-Darby canine kidney cells) exhibited an increase in ENaC expression and consequent membrane potential depolarization and an increase in cytosolic sodium and calcium, whereas one line (bovine aortal endothelial cells, BAEC) did not exhibit any of these changes. In all of the cell lines, however, ENaC inhibition determined a similar decrease in the rate of wound healing. In BAEC monolayers, the increase in ENaC activity produced plasma membrane depolarization, increased cytosolic sodium and calcium, and augmented the velocity of healing. These novel findings contribute to the idea that ENaC plays a critical role in wound healing in various epithelia, independently of the modality of healing and of any increase in the expression of the channel.     

Regulation of renal NaCl and water transport by the ATP/UTP/P2Y2 receptor system

Extracellular nucleotides (e.g., ATP) activate ionotropic P2X and metabotropic P2Y receptors in the plasma membrane to regulate and maintain cell function and integrity. This includes the renal tubular and collecting duct system, where the locally released nucleotides act in a paracrine and autocrine way to regulate transport of electrolytes and water and maintain cell volume. A prominent role has been assigned to Gq-coupled P2Y(2) receptors, which are typically activated by both ATP and UTP. Studies in gene knockout mice revealed an antihypertensive activity of P2Y(2) receptors that is linked to vasodilation and an inhibitory influence on renal salt reabsorption. Flow induces apical ATP release in the thick ascending limb, and first evidence indicates an inhibitory influence of P2Y(2) receptor tone on the expression and activity of the Na-K-2Cl cotransporter NKCC2 in this segment. The apical ATP/UTP/P2Y(2) receptor system in the connecting tubule/cortical collecting duct mediates the inhibitory effect of dietary salt on the open probability of the epithelial sodium channel ENaC and inhibits ENaC activity during aldosterone escape. Connexin 30 has been implicated in the luminal release of the ATP involved in the regulation of ENaC. An increase in collecting duct cell volume in response to manipulating water homeostasis increases ATP release. The subsequent activation of P2Y(2) receptors inhibits vasopressin-induced cAMP formation and water reabsorption, which facilitates water excretion and stabilizes cell volume. Thus recent studies have established the ATP/UTP/P2Y(2) receptor system as a relevant regulator of renal salt and water homeostasis and blood pressure regulation. The pathophysiological relevance and therapeutic potential remains to be determined, but dual effects of P2Y(2) receptor activation on both the vasculature and renal salt reabsorption implicate these receptors as potential therapeutic targets in hypertension.     

The Lhs1/GRP170 Chaperones Facilitate the Endoplasmic Reticulum-associated Degradation of the Epithelial Sodium Channel

The epithelial sodium channel, ENaC, plays a critical role in maintaining salt and water homeostasis, and not surprisingly defects in ENaC function are associated with disease. Like many other membrane-spanning proteins, this trimeric protein complex folds and assembles inefficiently in the endoplasmic reticulum (ER), which results in a substantial percentage of the channel being targeted for ER-associated degradation (ERAD). Because the spectrum of factors that facilitates the degradation of ENaC is incomplete, we developed yeast expression systems for each ENaC subunit. We discovered that a conserved Hsp70-like chaperone, Lhs1, is required for maximal turnover of the ENaC α subunit. By expressing Lhs1 ATP binding mutants, we also found that the nucleotide exchange properties of this chaperone are dispensable for ENaC degradation. Consistent with the precipitation of an Lhs1-αENaC complex, Lhs1 holdase activity was instead most likely required to support the ERAD of αENaC. Moreover, a complex containing the mammalian Lhs1 homolog GRP170 and αENaC co-precipitated, and GRP170 also facilitated ENaC degradation in human, HEK293 cells, and in a Xenopus oocyte expression system. In both yeast and higher cell types, the effect of Lhs1 on the ERAD of αENaC was selective for the unglycosylated form of the protein. These data establish the first evidence that Lhs1/Grp170 chaperones can act as mediators of ERAD substrate selection.     

An Alu cassette in the human epithelial sodium channel

Here, we report the presence of two splice variants of the human epithelial sodium channel alpha subunit (h alpha ENaC) containing Alu cassette, namely h alpha ENaC+22 and h alpha ENaC+Alu, in various tissues. Functional expression of these splice variants with hENaC beta and gamma subunits produced loss-of-channel activity in the Xenopus oocyte expression system. Interestingly, coexpression of h alpha ENaC+22 or h alpha ENaC+Alu, respectively, with wild type hENaC alpha, beta, and gamma subunits enhanced the expression of amiloride-sensitive current in oocytes. The presence of Alu sequences in the 3'-untranslated region of h gamma ENaC was also identified.     

Rapid stimulation of human renal ENaC by cAMP in Xenopus laevis oocytes

Among the compensatory mechanisms restoring circulating blood volume after severe haemorrhage, increased vasopressin secretion enhances water permeability of distal nephron segments and stimulates Na⁺ reabsorption in cortical collecting tubules via epithelial sodium channels (ENaC). The ability of vasopressin to upregulate ENaC via a cAMP-dependent mechanism in the medium to long term is well established. This study addressed the acute regulatory effect of cAMP on human ENaC (hENaC) and thus the potential role of vasopressin in the initial compensatory responses to haemorrhagic shock. The effects of raising intracellular cAMP (using 5 mmol/L isobutylmethylxanthine (IBMX) and 50 μmol/L forskolin) on wild-type and Liddle-mutated hENaC activity expressed in Xenopus oocytes and hENaC localisation in oocyte membranes were evaluated by dual-electrode voltage clamping and immunohistochemistry, respectively. After 30 min, IBMX + forskolin had stimulated amiloride-sensitive Na⁺ current by 52 % and increased the membrane density of Na⁺ channels in oocytes expressing wild-type hENaC. These responses were prevented by 5 μmol/L brefeldin A, which blocks antegrade vesicular transport. By contrast, IBMX + forskolin had no effects in oocytes expressing Liddle-mutated hENaC. cAMP stimulated rapid, exocytotic recruitment of wild-type hENaC into Xenopus oocyte membranes, but had no effect on constitutively over-expressed Liddle-mutated hENaC. Extrapolating these findings to the early cAMP-mediated effect of vasopressin on cortical collecting tubule cells, they suggest that vasopressin rapidly mobilises ENaC to the apical membrane of cortical collecting tubule cells, but does not enhance ENaC activity once inserted into the membrane. We speculate that this stimulatory effect on Na⁺ reabsorption (and hence water absorption) may contribute to the early restoration of extracellular fluid volume following severe haemorrhage.     

Nongenomic regulation of ENaC by aldosterone

Aldosterone isinvolved in salt and water homeostasis. The main effect is thought toinvolve genomic mechanisms. However, the existence of plasma membranesteroid receptors has been postulated. We used whole cell patch clampto test the hypothesis that epithelial sodium channels (ENaC) expressedby renal collecting duct principal cells can be regulatednongenomically by aldosterone. In freshly isolated principal cells fromrabbit, aldosterone (100 nM) rapidly (<2 min) increased ENaC sodiumcurrent specifically. The aldosterone-activated current was completelyinhibited by amiloride. Aldosterone also activated ENaC in cellstreated with the mineralocorticoid receptor blocker spiranolactone.Nongenomic activation was inhibited by inclusion ofS-adenosyl-L-homocysteine in the pipettesolution, which inhibits methylation reactions. Also, the nongenomicactivation required 2 mM ATP supplementation in the pipette solution.Aldosterone did not activate any ENaC current in whole cell clamped ratcollecting duct principal cells. These functional studies areconsistent with aldosterone membrane binding studies, suggesting thepresence of a plasma membrane steroid receptor that affects cellularprocesses by mechanisms unrelated to altered gene expression.

     

Filamin Interacts with Epithelial Sodium Channel and Inhibits Its Channel Function

Epithelial sodium channel (ENaC) in the kidneys is critical for Na⁺ balance, extracellular volume, and blood pressure. Altered ENaC function is associated with respiratory disorders, pseudohypoaldosteronism type 1, and Liddle syndrome. ENaC is known to interact with components of the cytoskeleton, but the functional roles remain largely unclear. Here, we examined the interaction between ENaC and filamins, important actin filament components. We first discovered by yeast two-hybrid screening that the C termini of ENaC α and β subunits bind filamin A, B, and C, and we then confirmed the binding by in vitro biochemical assays. We demonstrated by co-immunoprecipitation that ENaC, either overexpressed in HEK, HeLa, and melanoma A7 cells or natively expressed in LLC-PK1 and IMCD cells, is in the same complex with native filamin. Furthermore, the biotinylation and co-immunoprecipitation combined assays showed the ENaC-filamin interaction on the cell surface. Using Xenopus oocyte expression and two-electrode voltage clamp electrophysiology, we found that co-expression of an ENaC-binding domain of filamin substantially reduces ENaC channel function. Western blot and immunohistochemistry experiments revealed that the filamin A C terminus (FLNAC) modestly reduces the expression of the ENaC α subunit in oocytes and A7 cells. After normalizing the current by plasma membrane expression, we found that FLNAC results in ∼50% reduction in the ENaC channel activity. The inhibitory effect of FLNAC was confirmed by lipid bilayer electrophysiology experiments using purified ENaC and FLNAC proteins, which showed that FLNAC substantially reduces ENaC single channel open probability. Taken together, our study demonstrated that filamin reduces ENaC channel function through direct interaction on the cell surface.     

Stimulative effect of guanylylimidodiphosphate on the vasopressin-induced increment of osmotic water flow of the toad bladder.

The effect of guanylylimidodiphosphate [Gpp(NH)p] on vasopressin-induced osmotic water flow across the bladder of the toad, $\text{Bufo}\text{bufo}\text{japonicus}$ was examined. Gpp(NH)p significantly enhanced vasopressin-induced osmotic water flow of the bladder at a concentration of 1 × 10^?5M, while it showed no effect on the water flow without vasopressin. Gpp(NH)p alone could not enhance cyclic AMP-induced osmotic water flow of the toad bladder. Adenylylimidodiphosphate [App(NH)p] could not enhance vasopressin-induced osmotic water flow of the bladder at a concentration of 1 × 10^?5M. The results suggest that Gpp(NH)p can enhance the physiological effect of vasopressin by stimulating vasopressin activation of adenylate cyclase during substrate and hormone depletion of the toad bladder.     

Cl- channel activity in Xenopus oocytes expressing the cystic fibrosis gene.

The expression of the cystic fibrosis (CF) gene on its introduction into nonepithelial somatic cells has recently been shown to result in the appearance of distinctive low conductance chloride channels stimulated by cyclic AMP (Kartner, N., Hanrahan, J.W., Jensen, T.J., Naismith, A.L., Sun, S., Ackerley, C.A., Reyes, E.F., Tsui, L.-C., Rommens, J.M., Bear, C.E., and Riordan, J.R. (1991) Cell 64, 681-691; Anderson, M. P., Rich, D.P., Gregory, R.J., Smith, A.E., and Welsh, M.J. (1991) Science 251, 679-682). Since Xenopus oocytes provide a powerful system for ion channel characterization, we have examined whole cell and single channel currents in them after injection of cRNA to program the synthesis of the cystic fibrosis transmembrane conductance regulator (CFTR). This has enabled the direct demonstration that the cyclic AMP activation is mediated by protein kinase A and that CFTR is without effect on the endogenous calcium-activated chloride channels of the oocyte, which have been well characterized previously and widely used as reporters of the expression of G-protein-coupled receptors. These findings strengthen the argument that the CF gene codes for a novel regulated chloride channel rather than a regulatory protein which can modulate separate chloride channel molecules.     

Aldosterone regulates rapid trafficking of epithelial sodium channel subunits in renal cortical collecting duct cells via protein kinase D activation

Aldosterone elicits rapid physiological responses in target tissues such as the distal nephron through the stimulation of cell signaling cascades. We identified protein kinase D (PKD1) as an early signaling response to aldosterone treatment in the M1-cortical collecting duct (M1-CCD) cell line. PKD1 activation was blocked by the PKC inhibitor chelerythrine chloride and by rottlerin, a specific inhibitor of PKCdelta. The activation of PKCdelta and PKCepsilon coincided with PKD1 activation and while a complex was formed between PKD1 and PKCepsilon after aldosterone treatment, there was a concurrent reduction in PKD1 association with PKCdelta. A stable PKD1 knockdown M1-CCD-derrived clone was developed in which PKD1 expression was 90% suppressed by gene silencing with a PKD1-specific siRNA. The effect of aldosterone treatment on the subcellular distribution of enhanced cyan fluorescent protein (eCFP)-tagged epithelial sodium channel (ENaC) subunits in wild type (WT) and PKD1 suppressed cells was examined using confocal microscopy. In an untreated confluent monolayer of M1-CCD cells, alpha, beta, and gamma ENaC subunits were evenly distributed throughout the cytoplasm of WT and PKD1-suppressed cells. After 2 min treatment, aldosterone stimulated the localization of each of the ENaC subunits to discrete regions within the cytoplasm of WT cells. The translocation of eCFP-ENaC subunits in WT cells was inhibited by rottlerin and the mineralocorticoid receptor (MR) antagonist spironolactone. No subcellular translocation of eCFP-ENaC subunits was observed in PKD1-suppressed cells treated with aldosterone. These data demonstrate the involvement of a novel MR/PKCdelta /PKD1 signaling cascade in the earliest ENaC subunit intracellular trafficking events that follow aldosterone treatment.     

Non-coordinate regulation of endogenous epithelial sodium channel (ENaC) subunit expression at the apical membrane of A6 cells in response to various transporting conditions

In many epithelial tissues in the body (e.g. kidney distal nephron, colon, airways) the rate of Na(+) reabsorption is governed by the activity of the epithelial Na(+) channel (ENaC). ENaC activity in turn is regulated by a number of factors including hormones, physiological conditions, and other ion channels. To begin to understand the mechanisms by which ENaC is regulated, we have examined the trafficking and turnover of ENaC subunits in A6 cells, a polarized, hormonally responsive Xenopus kidney cell line. As previously observed by others, the half-life of newly synthesized ENaC subunits was universally short ( approximately 2 h). However, the half-lives of alpha- and gamma-ENaC subunits that reached the apical cell surface were considerably longer (t(12) > 24 h), whereas intriguingly, the half-life of cell surface beta-ENaC was only approximately 6 h. We then examined the effects of various modulators of sodium transport on cell surface levels of individual ENaC subunits. Up-regulation of ENaC-mediated sodium conductance by overnight treatment with aldosterone or by short term incubation with vasopressin dramatically increased cell surface levels of beta-ENaC without affecting alpha- or gamma-ENaC levels. Conversely, treatment with brefeldin A selectively decreased the amount of beta-ENaC at the apical membrane. Short term treatment with aldosterone or insulin had no effect on cell surface amounts of any subunits. Subcellular fractionation revealed a selective loss of beta-ENaC from early endosomal pools in response to vasopressin. Our data suggest the possibility that trafficking and turnover of individual ENaC subunits at the apical membrane of A6 cells is non-coordinately regulated. The selective trafficking of beta-ENaC may provide a mechanism for regulating sodium conductance in response to physiological stimuli.     

Regulation of the amiloride-sensitive epithelial sodium channel by syntaxin 1A.

The first step in transepithelial sodium absorption lies at the apical membrane where the amiloride-sensitive, epithelial sodium channel, ENaC, facilitates sodium entry into the cell. Here we report that the vesicle traffic regulatory (SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)) protein, syntaxin 1A (S1A), inhibits ENaC mediated sodium entry. This inhibitory effect is selective for S1A and is not reproduced by syntaxin 3. The inhibition does not require the membrane anchoring domain of syntaxin 1A. It was reversed by the S1A-binding protein, Munc-18, but not by a Munc-18 mutant, which lacks syntaxin affinity. Immunostaining of epitope-tagged ENaC subunits showed that syntaxin 1A decreases ENaC current by reducing the number of ENaC channels in the plasma membrane; S1A does not interfere with ENaC protein expression. Immunoprecipitation of syntaxin 1A from the sodium-transporting epithelial cell line, A6, co-precipitates ENaC. These findings indicate that syntaxin 1A and other members of the SNARE machinery are involved in the control of plasma membrane ENaC content, and they suggest that SNARE proteins participate in the regulation of sodium absorption in relation to agonist mediated vesicle insertion-retrieval processes.