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.

Fang L, Li D, Welling PA. Hypertension resistance polymorphisms in ROMK (Kir1.1) alter channel function by different mechanisms.

A cDNA encoding a putative aquaporin was cloned from the ovaries of the American dog tick, Dermacentor variabilis (Say) (Acari: Ixodidae). The encoded protein is most similar to the vertebrate aquaporin 9 protein family. Localization by reverse transcription-polymerase chain reaction (RT-PCR) shows expression in the gut and ovaries of adult females but not in the synganglion, Malpighian tubules, or salivary glands. Quantitative RT-PCR indicates that it is primarily expressed in the ovaries, with approximately 146 times more transcript than in the gut. When expressed in Xenopus oocytes, the aquaporin-like protein localized to the plasma membrane.

We used the patch-clamp technique to study the effect of insulin-like growth factor I (IGF-I) on the apical 70-pS K channel in the isolated thick ascending limb (TAL) of the rat kidney. The isolated TAL was cut open to gain access to the apical membrane. Addition of 25 nM IGF-I stimulates the apical 70-pS K channel and increases channel activity, defined by the product of channel open probability and channel number, from 0.31 to 1.21. The stimulatory effect of IGF-I is not mediated by nitric oxide- or protein tyrosine phosphatase-dependent mechanisms, because inhibition of nitric oxide synthase or blocking protein tyrosine phosphatase did not abolish the stimulatory effect of IGF-I on the 70-pS K channel. In contrast, inhibition of mitogen-activated protein (MAP) kinase with PD-98059 or U0126 abolished the stimulatory effect of IGF-I. This suggests that MAP kinase is responsible for mediating the effect of IGF-I on the apical K channels. Moreover, the effect of IGF-I on the apical 70-pS K channel is biphasic because high concentrations (>200 nM) inhibit apical 70-pS K channels. Application of 400 nM IGF-I decreased channel activity from 1.45 to 0.2. The inhibitory effect of IGF-I is not blocked by calphostin C (an inhibitor of PKC), but inhibition of protein tyrosine kinase with herbimycin A abolished the IGF-induced inhibition. We conclude that IGF-I has a dual effect on the apical 70-pS K channel in the TAL: low concentrations of IGF-I stimulate, whereas high concentrations inhibit the channel activity. The stimulatory effect of IGF-I is mediated by a MAP kinase-dependent pathway, whereas the inhibitory effect is the result of stimulation of protein tyrosine kinase.

A multiplex family was identified with biochemical and clinical features suggestive of Bartter's syndrome (BS). The eldest sibling presented with developmental delay and rickets at 4 years of age with evidence of hypercalciuria and hypokalemia. The second sibling presented at 1 year of age with urinary tract infections, polyuria, and polydipsia. The third child was born after a premature delivery with a history of polyhydramnios and neonatal hypocalcemia. Following corrective treatment she also developed hypercalciuria and a hypokalemic metabolic alkalosis. There was evidence of secondary hyperreninemia and hyperaldosteronism in all three siblings consistent with BS. Known BS genes were screened and functional assays of ROMK (alias KCNJ1, Kir1.1) were carried out in Xenopus oocytes. We detected compound heterozygous missense changes in KCNJ1, encoding the potassium channel ROMK. The S219R/L220F mutation was segregated from father and mother, respectively. In silico modeling of the missense mutations suggested deleterious changes. Studies in Xenopus oocytes revealed that both S219R and L220F had a deleterious effect on ROMK-mediated potassium currents. Coinjection to mimic the compound heterozygosity produced a synergistic decrease in channel function and revealed a loss of PKA-dependent stabilization of PIP2 binding. In conclusion, in a multiplex family with BS, we identified compound heterozygous mutations in KCNJ1. Functional studies of ROMK confirmed the pathogenicity of these mutations and defined the mechanism of channel dysfunction.

A cDNA encoding a putative aquaporin was cloned from the ovaries of the American dog tick, Dermacentor variabilis (Say) (Acari: Ixodidae). The encoded protein is most similar to the vertebrate aquaporin 9 protein family. Localization by reverse transcription-polymerase chain reaction (RT-PCR) shows expression in the gut and ovaries of adult females but not in the synganglion, Malpighian tubules, or salivary glands. Quantitative RT-PCR indicates that it is primarily expressed in the ovaries, with approximately 146 times more transcript than in the gut. When expressed in Xenopus oocytes, the aquaporin-like protein localized to the plasma membrane.

Background: The urinary potassium excretion machinery is upregulated with increasing dietary potassium, but the role of accompanying dietary anions remains inadequately characterized. Poorly absorbable anions, including HCO3-, are thought to increase K+ secretion through a transepithelial voltage effect. Here we test if they also influence the potassium secretion machinery. Methods: Wild-type mice, aldosterone synthase knockout (AS-KO), or pendrin knockout mice were randomized to control, high KCl, or high KHCO3 diets. The potassium secretory capacity was assessed in balance studies. Protein abundance, modification, and localization of the potassium-secretory transporters were evaluated by western blot and confocal microscopy. Results: Feeding the high KHCO3 diet increased urinary K+ excretion and the trans-tubular K+ gradient significantly more than the high KCl diet, coincident with more pronounced upregulation of ENaC and ROMK and apical localization in the distal nephron. Studies in AS-KO mice revealed that the enhanced effects of bicarbonate were aldosterone-independent. The KHCO3 diet also uniquely increased the BK potassium channel b4 subunit, stabilizing BKa on the apical membrane, the Cl/HCO3 exchanger, pendrin, and the apical KCl cotransporter (KCC3a), all of which are expressed specifically in pendrin-positive IC cells. Studies in pendrin KO mice revealed that pendrin was required to increase K+excretion with the KHCO3 diet. In summary, bicarbonate stimulates potassium excretion beyond a poorly-absorbable anion effect, upregulating ENaC and ROMK in principal cells and BK, pendrin, and KCC3a in PP-ICs. The adaptive mechanism prevents hyperkalemia and alkalosis with the consumption of alkaline-ash-rich diets but may drive potassium wasting and hypokalemia in alkalosis.

The ROMK potassium channel plays a critical role in renal sodium handling. Genome sequencing efforts in the Framingham Heart Study cohort recently revealed an association between suspected loss‐of‐function polymorphisms in ROMK and resistance to hypertension, suggesting that ROMK activity is also a determinant of blood pressure in the general population. Here, we examine whether these sequence variants do, in fact, alter ROMK function and explore the mechanisms. As assessed by two‐microelectrode voltage clamp in Xenopus Oocytes, 3/5 of the variants (R193P, H251Y and T313FS) displayed an almost complete attenuation of whole‐cell ROMK channel activity. Surface antibody binding to external epitope tagged channels and analysis of glycosylation‐state maturation revealed that these variants prevent channel expression at the plasmalemma, likely as a consequence of ER retention. The other variants (P166S, R169H) had no obvious effect on the basal channel activity or surface expression but, instead, displayed a regulated‐gating defect. Apparent PIP2 binding affinity of the variants was reduced, causing channels to become susceptible to inhibition by GPCR‐mediated stimulation of PIP2 hydrolysis. In summary, hypertension resistance sequence variants inhibit ROMK channel function by different mechanisms, providing new insights into the role of the channel in blood pressure control.