Calcineurin regulates aldosterone production via dephosphorylation of NFATC4.

The mineralocorticoid aldosterone, secreted by the adrenal zona glomerulosa (ZG), is critical for life, maintaining ion homeostasis and blood pressure. Therapeutic inhibition of protein phosphatase 3 (calcineurin, Cn) results in inappropriately low plasma aldosterone levels despite concomitant hyperkalemia and hyperreninemia. We tested the hypothesis that Cn participates in the signal transduction pathway regulating aldosterone synthesis.

Specific disruption of calcineurin-signaling in the distal convoluted tubule impacts the transcriptome and proteome, and causes hypomagnesemia and metabolic acidosis.

Adverse effects of calcineurin inhibitors (CNI), such as hypertension, hyperkalemia, acidosis, hypomagnesemia and hypercalciuria, have been linked to dysfunction of the distal convoluted tubule (DCT). To test this, we generated a mouse model with an inducible DCT-specific deletion of the calcineurin regulatory subunit B alpha (CnB1-KO). Three weeks after CnB1 deletion, these mice exhibited hypomagnesemia and acidosis, but no hypertension, hyperkalemia or hypercalciuria.

A five amino acids deletion in NKCC2 of C57BL/6 mice affects analysis of NKCC2 phosphorylation but does not impact kidney function.

Aim: The phosphorylation level of the furosemide-sensitive Na -K -2Cl cotransporter (NKCC2) in the thick ascending limb (TAL) is used as a surrogate marker for NKCC2 activation and TAL function. However, in mice, analyses of NKCC2 phosphorylation with antibodies against phosphorylated threonines 96 and 101 (anti-pT96/pT101) give inconsistent results. We aimed (a) to elucidate these inconsistencies and (b) to develop a phosphoform-specific antibody that ensures reliable detection of NKCC2 phosphorylation in mice.

Collecting system-specific deletion of Kcnj10 predisposes for thiazide- and low-potassium diet-induced hypokalemia.

The basolateral potassium channel KCNJ10 (Kir4.1), is expressed in the renal distal convoluted tubule and controls the activity of the thiazide-sensitive sodium chloride cotransporter. Loss-of-function mutations of KCNJ10 cause EAST/SeSAME syndrome with salt wasting and severe hypokalemia.

Protein Phosphatase 1 Inhibitor-1 Mediates the cAMP-Dependent Stimulation of the Renal NaCl Cotransporter.

Background: A number of cAMP-elevating hormones stimulate phosphorylation (and hence activity) of the NaCl cotransporter (NCC) in the distal convoluted tubule (DCT). Evidence suggests that protein phosphatase 1 (PP1) and other protein phosphatases modulate NCC phosphorylation, but little is known about PP1's role and the mechanism regulating its function in the DCT.

Methods: We used mouse kidney preparations to test whether a DCT-enriched inhibitor of PP1, protein phosphatase 1 inhibitor-1 (I1), mediates cAMP's effects on NCC, and conducted yeast two-hybrid and coimmunoprecipitation experiments in NCC-expressing MDCK cells to explore protein interactions.

C-terminally truncated, kidney-specific variants of the WNK4 kinase lack several sites that regulate its activity.

WNK lysine-deficient protein kinase 4 (WNK4) is an important regulator of renal salt handling. Mutations in its gene cause pseudohypoaldosteronism type II, mainly arising from overactivation of the renal Na/Cl cotransporter (NCC). In addition to full-length WNK4, we have observed faster migrating bands (between 95 and 130 kDa) in Western blots of kidney lysates.

Extracellular K rapidly controls NaCl cotransporter phosphorylation in the native distal convoluted tubule by Cl -dependent and independent mechanisms.

Key Points: High dietary potassium (K ) intake dephosphorylates and inactivates the NaCl cotransporter (NCC) in the renal distal convoluted tubule (DCT). Using several ex vivo models, we show that physiological changes in extracellular K , similar to those occurring after a K rich diet, are sufficient to promote a very rapid dephosphorylation of NCC in native DCT cells. Although the increase of NCC phosphorylation upon decreased extracellular K appears to depend on cellular Cl fluxes, the rapid NCC dephosphorylation in response to increased extracellular K is not Cl -dependent.

Coupled RNA polymerase II transcription and 3' end formation with yeast whole-cell extracts.

RNA polymerase II (RNAP II) transcription and pre-mRNA 3' end formation are linked through physical and functional interactions. We describe here a highly efficient yeast in vitro system that reproduces both transcription and 3' end formation in a single reaction. The system is based on simple whole-cell extracts that were supplemented with a hybrid Gal4-VP16 transcriptional activator and supercoiled plasmid DNA templates encoding G-less cassette reporters.

Cotranslational assembly of the yeast SET1C histone methyltransferase complex.

While probing the role of RNA for the function of SET1C/COMPASS histone methyltransferase, we identified SET1RC (SET1 mRNA-associated complex), a complex that contains SET1 mRNA and Set1, Swd1, Spp1 and Shg1, four of the eight polypeptides that constitute SET1C. Characterization of SET1RC showed that SET1 mRNA binding did not require associated Swd1, Spp1 and Shg1 proteins or RNA recognition motifs present in Set1. RNA binding was not observed when Set1 protein and SET1 mRNA were derived from independent genes or when SET1 transcripts were restricted to the nucleus.

Cordycepin interferes with 3' end formation in yeast independently of its potential to terminate RNA chain elongation.

Cordycepin (3' deoxyadenosine) is a biologically active compound that, when incorporated during RNA synthesis in vitro, provokes chain termination due to the absence of a 3' hydroxyl moiety. We were interested in the effects mediated by this drug in vivo and analyzed its impact on RNA metabolism of yeast. Our results support the view that cordycepin-triphosphate (CoTP) is the toxic component that is limiting cell growth through inhibition of RNA synthesis.

Cordycepin-hypersensitive growth links elevated polyphosphate levels to inhibition of poly(A) polymerase in Saccharomyces cerevisiae.

To identify genes involved in poly(A) metabolism, we screened the yeast gene deletion collection for growth defects in the presence of cordycepin (3'-deoxyadenosine), a precursor to the RNA chain terminating ATP analog cordycepin triphosphate. Deltapho80 and Deltapho85 strains, which have a constitutively active phosphate-response pathway, were identified as cordycepin hypersensitive. We show that inorganic polyphosphate (poly P) accumulated in these strains and that poly P is a potent inhibitor of poly(A) polymerase activity in vitro.