Mechanisms of Regulation of Epithelial Sodium Channel by SGK1 in A6 Cells (original) (raw)
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Pflügers Archiv - European Journal of Physiology
How phosphorylation of the epithelial sodium channel (ENaC) contributes to its regulation is incompletely understood. Previously, we demonstrated that in outside-out patches ENaC activation by serum- and glucocorticoid-inducible kinase isoform 1 (SGK1) was abolished by mutating a serine residue in a putative SGK1 consensus motif RXRXX(S/T) in the channel’s α-subunit (S621 in rat). Interestingly, this serine residue is followed by a highly conserved proline residue rather than by a hydrophobic amino acid thought to be required for a functional SGK1 consensus motif according to invitro data. This suggests that this serine residue is a potential phosphorylation site for the dual-specificity tyrosine phosphorylated and regulated kinase 2 (DYRK2), a prototypical proline-directed kinase. Its phosphorylation may prime a highly conserved preceding serine residue (S617 in rat) to be phosphorylated by glycogen synthase kinase 3 β (GSK3β). Therefore, we investigated the effect of DYRK2 on ENaC...
WNK1 activates SGK1 to regulate the epithelial sodium channel
WNK (with no lysine [K]) kinases are serine–threonine protein kinases with an atypical placement of the catalytic lysine. Intronic deletions increase the expression of WNK1 in humans and cause pseudohypoaldosteronism type II, a form of hypertension. WNKs have been linked to ion carriers, but the underlying regulatory mechanisms are unknown. Here, we report a mechanism for the control of ion permeability by WNK1. We show that WNK1 activates the serum- and glucocorticoid-inducible protein kinase SGK1, leading to activation of the epithelial sodium channel. Increased channel activity induced by WNK1 depends on SGK1 and the E3 ubiquitin ligase Nedd4-2. This finding provides compelling evidence that this molecular mechanism contributes to the pathogenesis of hypertension in pseudohypoaldosteronism type II caused by WNK1 and, possibly, in other forms of hypertension.
Journal of Biological Chemistry, 1999
The serum-and glucocorticoid-induced kinase (sgk) is a serine and threonine kinase that stimulates amiloridesensitive sodium transport in Xenopus oocytes. Because aldosterone induces phosphorylation on serine/threonine (Ser/Thr) residues in the carboxyl termini of  and ␥ subunits of epithelial sodium channels (ENaCs) and causes an increase in the sgk transcript in mammalian and amphibian renal epithelial cells, it seems likely that sgk mediates the action of aldosterone to stimulate sodium transport. Experiments were performed in Xenopus oocytes to determine the mechanism by which sgk increases sodium conductance by examining its effect on phosphorylation, kinetics, and membrane abundance of ENaC. Our results demonstrate that deletions of the carboxyl termini of the three subunits do not inhibit sgk-induced sodium current, indicating that the effect of sgk is not mediated via phosphorylation within the carboxyl termini of ENaC. They also show no evidence that sgk reduces the removal of ENaC from the plasma membrane because mutations of tyrosine residues in the sequences necessary for endocytosis and degradation did not affect the response to sgk. Further studies performed with the patch-clamp technique indicated that sgk did not increase the open probability or changed the kinetics of ENaC. These studies, however, showed a 3-fold increase in the abundance of ENaC in the plasma membrane in the presence of sgk compared with control. Together, the experiments indicate that sgk stimulates electrogenic sodium transport by increasing the number of ENaCs at the cell surface and suggest that sgk may mediate the early increase in aldosterone-induced sodium current.
Journal of Biological Chemistry, 2004
In many epithelial tissues in the body, the rate of Na ؉ reabsorption is governed by the activity of the epithelial sodium channel (ENaC). The assembly, trafficking, and turnover of the three ENaC subunits (␣, , and ␥) is complex and not well understood. Recent experiments suggest that ENaC must be proteolytically cleaved for maximal activity and may explain the discrepancies reported in prior biochemical approaches focused on quantitating the trafficking and half-life of full-length subunits. As an alternative approach to examining the dynamics of ENaC subunits, we have generated doxycycline-repressible replication-defective recombinant adenoviruses encoding individual epitope-tagged mouse ENaC subunits and expressed these in polarized MDCK I cells. Co-infection with these viruses encoding all three subunits generates robust amiloride-sensitive currents in polarized MDCK cells. Significant current was also observed in cells expressing ␣and ␥-mENaC in the absence of -mENaC. These currents did not appear to result from association with endogenous canine -ENaC. Treatment of ␣␥-expressing cells with cycloheximide (CHX) resulted in the rapid inhibition (within 3 h) of ϳ50-80% of the initial current; however, a sizable fraction of the initial current remained even after 6 h of CHX. By contrast, CHX addition to cells expressing only ␣and ␥-mENaC resulted in rapid decay in current with no residual fraction. Our data suggest that ENaC channels of differing stoichiometries are differentially trafficked and degraded and provide support for the possibility that noncoordinate trafficking of ENaC subunits may function in vivo as a mechanism to modulate ENaC activity.
Epithelial sodium channel (ENaC) in GtoPdb v.2021.2
IUPHAR/BPS Guide to Pharmacology CITE, 2021
The epithelial sodium channels (ENaC) are located on the apical membrane of epithelial cells in the kidney tubules, lung, respiratory tract, male and female reproductive tracts, sweat and salivary glands, placenta, colon, and some other organs [9, 13, 22, 21, 42]. In these epithelia, Na+ ions flow from the extracellular fluid into the cytoplasm of epithelial cells via ENaC. The Na+ ions are then pumped out of the cytoplasm into the interstitial fluid by the Na+/K+ ATPase located on the basolateral membrane [36]. As Na+ is one of the major electrolytes in the extracellular fluid (ECF), osmolarity change initiated by the Na+ flow is accompanied by a flow of water accompanying Na+ ions [6]. Thus, ENaC has a central role in regulating ECF volume and blood pressure, primarily via its function in the kidney [37]. The expression of ENaC subunits, hence its activity, is regulated by the renin-angiotensin-aldosterone system, and other factors involved in electrolyte homeostasis [37, 30]. In ...
The epithelial sodium channel δ-subunit: new notes for an old song
American Journal of Physiology-Renal Physiology, 2012
Amiloride-sensitive epithelial Na+ channels (ENaCs) can be formed by different combinations of four homologous subunits, named α, β, γ, and δ. In addition to providing an apical entry pathway for transepithelial Na+ reabsorption in tight epithelia such as the kidney distal tubule and collecting duct, ENaCs are also expressed in nonepithelial cells, where they may play different functional roles. The δ-subunit of ENaC was originally identified in humans and is able to form amiloride-sensitive Na+ channels alone or in combination with β and γ, generally resembling the canonical kidney ENaC formed by α, β, and γ. However, δ differs from α in its tissue distribution and channel properties. Despite the low sequence conservation between α and δ (37% identity), their similar functional characteristics provide an excellent model for exploring structural correlates of specific ENaC biophysical and pharmacological properties. Moreover, the study of cellular mechanisms modulating the activity ...
Role of SGK in hormonal regulation of epithelial sodium channel in A6 cells
AJP: Cell Physiology, 2003
The purpose of this study was to examine the role of the serum-and glucocorticoidinduced kinase (SGK) in the activation of the epithelial sodium channel (ENaC) by aldosterone, arginine vasopressin (AVP), and insulin. We used a tetracycline-inducible system to control the expression of wild-type (SGK wt T ), constitutively active (S425D mutation; SGK S425D
Journal of Biological …, 2010
The four WNK (with no lysine (K)) protein kinases affect ion balance and contain an unusual protein kinase domain due to the unique placement of the active site lysine. Mutations in two WNKs cause a heritable form of ion imbalance culminating in hypertension. WNK1 activates the serum-and glucocorticoidinduced protein kinase SGK1; the mechanism is noncatalytic. SGK1 increases membrane expression of the epithelial sodium channel (ENaC) and sodium reabsorption via phosphorylation and sequestering of the E3 ubiquitin ligase neural precursor cell expressed, developmentally down-regulated 4-2 (Nedd4-2), which otherwise promotes ENaC endocytosis. Questions remain about the intrinsic abilities of WNK family members to regulate this pathway. We find that expression of the N termini of all four WNKs results in modest to strong activation of SGK1. In reconstitution experiments in the same cell line all four WNKs also increase sodium current blocked by the ENaC inhibitor amiloride. The N termini of the WNKs also have the capacity to interact with SGK1. More detailed analysis of activation by WNK4 suggests mechanisms in common with WNK1. Further evidence for the importance of WNK1 in this process comes from the ability of Nedd4-2 to bind to WNK1 and the finding that endogenous SGK1 has reduced activity if WNK1 is knocked down by small interfering RNA.