The Kir1.1 (ROMK) subtypes of inward rectifier K ؉ channels mediate potassium secretion and regulate sodium chloride reabsorption in the kidney. The density of ROMK channels on the cortical collecting duct apical membrane is exquisitely regulated in concert with physiological demands. Although protein kinase A-dependent phosphorylation of one of the three phospho-acceptors in Kir1.1, Ser-44, also a canonical serum-glucocorticoid-regulated kinase (SGK-1) phosphorylation site, controls the number of active channels, it is unknown whether this involves activating dormant channels already residing on the plasma membrane or recruiting new channels to the cell surface. Here we explore the mechanism and test whether SGK-1 phosphorylation of ROMK regulates cell surface expression. Removal of the phosphorylation site by point mutation (Kir1.1, S44A) dramatically attenuated the macroscopic current density in Xenopus oocytes. As measured by antibody binding of external epitope-tagged forms of Kir1.1, surface expression of Kir1.1 S44A was inhibited, paralleling the reduction in macroscopic current. In contrast, surface expression and macroscopic current density was augmented by a phosphorylation mimic mutation, Kir1.1 S44D. In vitro phosphorylation assays revealed that Ser-44 is a substrate of SGK-1 phosphorylation, and expression of SGK-1 with the wild type channel increased channel density to the same level as the phosphorylation mimic mutation. Moreover, the stimulatory effect of SGK-1 was completely abrogated by mutation of the phosphorylation site. In conclusion, SGK-1 phosphorylation of Kir1.1 drives expression on the plasmalemma. Because SGK-1 is an early aldosterone-induced gene, our results suggest a possible molecular mechanism for aldosterone-dependent regulation of the secretory potassium channel in the kidney.Extracellular potassium homeostasis, maintained by the regulation of renal potassium excretion, is dependent on the activity of weakly inward rectifying ''small conductance'' potassium channels (SK) 1 that are expressed on the apical membrane of epithelial cells in the distal nephron (1, 2). Encoded by the ROMK (Kir 1.1 or KCNJ1) gene (3, 4), these Kir channels are thought to be the major, but not exclusive (5, 6), route for potassium transport into the tubule lumen and constitute a final regulated component of the potassium secretory machinery of the kidney (7,8). Indeed, aldosterone, vasopressin, and other factors precisely regulate SK activity, controlling potassium excretion in accord with the demands of potassium balance. Because ROMK channels normally exhibit a very high open probability, near unity, physiologic augmentation of channel activity, as controlled by hormones and dietary potassium (9), is achieved largely by regulated changes in the number of active channels on the plasmalemma.Although the precise molecular mechanisms responsible for physiological augmentation of ROMK channel surface density have remained unclear, a growing body of evidence has pointed to an important role of protein kina...

Members of the WNK family of serine͞threonine kinases have been implicated as important modulators of salt homeostasis, regulating the balance between renal sodium reabsorption and potassium excretion. Gain-of-expression mutations in the WNK1 gene uncouple Na ؉ and K ؉ balance and cause a familial disorder of diminished renal potassium excretion, excessive sodium retention, and hypertension (pseudohypoaldosteronism type II or Gordon's syndrome). Alternative splicing of the WNK1 gene produces a kidney-specific short form of WNK1 (KS-WNK1) and a more ubiquitous long form (L-WNK1), but it is not clear how either of these isoforms influence renal potassium excretion. Here we demonstrate that KS-WNK1 and L-WNK1 converge in a pathway to regulate the renal outermedullary K ؉ channel, Kir1.1. Reconstitution studies in Xenopus oocytes reveal that L-WNK1 significantly inhibits Kir1.1 by reducing cell surface localization of the channel.

Tonicity-responsive enhancer binding protein (TonEBP) is the transcription factor that regulates tonicity-responsive expression of the genes for the sodium-myo-inositol cotransporter (SMIT) and the sodium-chloride-betaine cotransporter (BGT1). Hypertonicity stimulates the activity of TonEBP due to a combination of increased protein abundance and increased nuclear distribution (proportion of TonEBP that is in the nucleus). We found that inhibitors of proteasome activity markedly reduce the induction of SMIT and BGT1 mRNA in response to hypertonicity. These inhibitors also reduce hypertonicity-induced stimulation of expression of a reporter gene controlled by the tonicity-responsive enhancer. Western and immunohistochemical analyses revealed that the proteasome inhibitors reduce the hypertonicity-induced increase of TonEBP in the nucleus by inhibiting its nuclear redistribution without affecting its abundance. Although the nuclear distribution of TonEBP is sensitive to inhibition of proteasome activity as is that of nuclear factor (NF)-kappaB, the signaling pathways appear to be different in that hypertonicity does not affect the nuclear distribution of NF-kappaB. Conversely, treatment with tumor necrosis factor-alpha increases the nuclear distribution of NF-kappaB but not TonEBP.

Tonicity-responsive enhancer binding protein (TonEBP)- nuclear factor of activated T cell family 5 is a DNA binding protein that plays a key role in the response of cells to hypertonicity. However, TonEBP is expressed and active in tissues that are in an isotonic milieu. To explore the biological role of TonEBP, we cloned mouse TonEBP that shares 92% of amino acids with the human counterpart. TonEBP is expressed in embryonic stem cells and throughout the stages of fetal development. Immunohistochemical analysis shows expression of TonEBP in most, if not all, developing tissues, including the brain, colon, heart, muscle, and eyes. Widespread alternative splicing in exons 2-4 was detected throughout development and in different adult tissues. As a result, four different polypeptides are produced with different lengths at the NH(2) terminus. Two of the isoforms differ in their ability to stimulate transcription. In conclusion, the presence of TonEBP mRNA during mouse embryogenesis suggests that TonEBP functions at all stages of mouse development, as well as in isotonic adult tissues.