No abstract

Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are IP3-gated Ca 2؉ channels that are located on intracellular Ca 2؉ stores. We previously identified an IP 3R binding protein, termed IP3R binding protein released with IP3 (IRBIT). Because IRBIT is released from IP3R by physiological concentrations of IP3, we hypothesized that IRBIT is a signaling molecule that is released from IP3R and regulates downstream target molecules in response to the production of IP3. Therefore, in this study, we attempted to identify the target molecules of IRBIT, and we succeeded in identifying Na ؉ ͞HCO3 ؊ cotransporter 1 (NBC1) as an IRBIT binding protein. Of the two major splicing variants of NBC1, pancreas-type NBC1 (pNBC1) and kidney-type NBC1 (kNBC1), IRBIT was found to bind specifically to pNBC1 and not to bind to kNBC1. IRBIT binds to the N-terminal pNBC1-specific domain, and its binding depends on the phosphorylation of multiple serine residues of IRBIT. Also, an electrophysiological analysis in Xenopus oocytes revealed that pNBC1 requires coexpression of IRBIT to manifest substantial activity comparable with that of kNBC1, which displays substantial activity independently of IRBIT. These results strongly suggest that pNBC1 is the target molecule of IRBIT and that IRBIT has an important role in pH regulation through pNBC1. Also, our findings raise the possibility that the regulation through IRBIT enables NBC1 variants to have different physiological roles.pH ͉ acidosis ͉ phosphorylation I nositol 1,4,5-trisphosphate (IP 3 ) receptors (IP 3 Rs) are intracellular Ca 2ϩ -release channels that are located on intracellular Ca 2ϩ -storage organelles, mainly the endoplasmic reticulum (ER) (1). IP 3 Rs release Ca 2ϩ from the ER into the cytoplasm and increase the cytoplasmic concentration of Ca 2ϩ in response to the binding of a second messenger, IP 3 . This IP 3 -Ca 2ϩ pathway regulates many biological processes, including cell growth, cell differentiation, apoptosis, synaptic plasticity, secretion, and fertilization (1).We identified (2) an IP 3 R binding protein, termed IP 3 R binding protein released with IP 3 (IRBIT). IRBIT consists of an N-terminal domain (residues 1-104), which contains a serine͞threonine-rich region, and a C-terminal domain (residues 105-530), which has homology with the methylation pathway enzyme S-adenosylhomocysteine hydrolase. We found (2) that the N-terminal amino acids 1-277 of IRBIT are sufficient for the interaction with the IP 3 R and that the interaction between IRBIT and the IP 3 R is inhibited by physiological concentrations of IP 3 , indicating that IRBIT interacts with the IP 3 R in the resting state and dissociates from the IP 3 R when IP 3 production is induced by extracellular stimuli. Therefore, we speculated that IRBIT acts as a signaling molecule that dissociates from the IP 3 R and regulates target proteins in response to IP 3 production, raising the possibility of the existence of an unidentified pathway, the IP 3 -IRBIT pathway.The Na ϩ ͞HCO 3 Ϫ cotransporter 1 (NBC1) is a membrane...

Mutations in the Na؉ -HCO 3 ؊ co-transporter (NBC1) cause permanent proximal renal tubular acidosis (pRTA) with ocular abnormalities. However, little has been known about the relationship between the degree of NBC1 inactivation and the severity of pRTA. This study identified three new homozygous mutations (T485S, A799V, and R881C) in the common coding regions of NBC1. T he Na ϩ -HCO 3 Ϫ co-transporter (NBC1) has multiple functions (1,2). Whereas the kidney-type transporter (kNBC1) plays an essential role in bicarbonate absorption from renal proximal tubules, the pancreatic-type transporter (pNBC1) is involved in bicarbonate secretion from pancreatic duct cells (3-6). We showed recently that mutations in NBC1 cause proximal renal tubular acidosis (pRTA) with ocular abnormalities (7-9). Because NBC1 is widely expressed in several ocular tissues (10), its inactivation may potentially explain the occurrence of ocular abnormalities. However, only a limited number of NBC1 mutations have been identified so far (7-9,11-13), and several important questions remain unanswered. For example, the exact relationship between the degree of NBC1 inactivation and the severity of pRTA has not been established. A study in NHE3-deficient mice suggests that acid secretion from distal tubules is greatly enhanced in pRTA (14).How effectively such compensatory mechanism works in human, however, is largely unknown. In addition, the effects of individual mutations on the transport properties of NBC1 have not been investigated intensively. These issues would be important to clarify further the molecular mechanism of pRTA as well as the physiologic roles of NBC1. In our study, we identified three new missense mutations in kNBC1 from patients with pRTA and ocular abnormalities. The functional analysis of these new as well as the known mutants was performed in Xenopus oocytes and cultured cells. Materials and Methods PatientsPatient 1 (T485S) is a boy from consanguineous parents. He had a history of failure to thrive and received a diagnosis of severe pRTA and band keratopathy at 2 yr of age. At 3 yr of age, his height (90 cm) and weight (11.8 kg) both were less than the third percentile. BP was 79/49 mmHg. Bilateral corneas were cloudy, and a red reflex was bilaterally absent, suggesting the presence of cataracts. Bone survey was normal with no evidence of rickets. While he was taking sodium bicarbonate (9.75 g/d), serum analysis revealed Na ϩ 140 mEq/L, K ϩ 3.8 mEq/L, Cl Ϫ 116 mEq/L, creatinine 0.5 mg/dl, and HCO 3 Ϫ 13 mmol/L. He had a thyroid function test that was initially suggestive of mild hypothyroidism. However, during the follow-up, thyroid function tests were

Homozygous mutations in SLC4A4 , encoding the electrogenic Na + -HCO 3 − cotransporter NBCe1, have been known to cause proximal renal tubular acidosis (pRTA) and ocular abnormalities. In this study, we report two sisters with pRTA, ocular abnormalities, and hemiplegic migraine. Genetic analysis ruled out pathological mutations in the known genes for familial hemiplegic migraine, but identified a homozygous 65-bp deletion (Δ65bp) in the C terminus of NBCe1, corresponding to the codon change S982NfsX4. Several heterozygous members of this family also presented glaucoma and migraine with or without aura. Despite the normal electrogenic activity in Xenopus oocytes, the Δ65bp mutant showed almost no transport activity due to a predominant cytosolic retention in mammalian cells. Furthermore, coexpression experiments uncovered a dominant negative effect of the mutant through hetero-oligomer formation with wild-type NBCe1. Among other pRTA pedigrees with different NBCe1 mutations, we identified four additional homozygous patients with migraine. The immunohistological and functional analyses of these mutants demonstrate that the near total loss of NBCe1 activity in astrocytes can cause migraine potentially through dysregulation of synaptic pH.

No abstract

The evolutionary loss of hepatic urate oxidase (uricase) has resulted in humans with elevated serum uric acid (urate). Uricase loss may have been beneficial to early primate survival. However, an elevated serum urate has predisposed man to hyperuricemia, a metabolic disturbance leading to gout, hypertension, and various cardiovascular diseases. Human serum urate levels are largely determined by urate reabsorption and secretion in the kidney. Renal urate reabsorption is controlled via two proximal tubular urate transporters: apical URAT1 (SLC22A12) and basolateral URATv1/GLUT9 (SLC2A9). In contrast, the molecular mechanism(s) for renal urate secretion remain unknown. In this report, we demonstrate that an orphan transporter hNPT4 (human sodium phosphate transporter 4; SLC17A3) was a multispecific organic anion efflux transporter expressed in the kidneys and liver. hNPT4 was localized at the apical side of renal tubules and functioned as a voltage-driven urate transporter. Furthermore, loop diuretics, such as furosemide and bumetanide, substantially interacted with hNPT4. Thus, this protein is likely to act as a common secretion route for both drugs and may play an important role in diuretics-induced hyperuricemia. The in vivo role of hNPT4 was suggested by two hyperuricemia patients with missense mutations in SLC17A3. These mutated versions of hNPT4 exhibited reduced urate efflux when they were expressed in Xenopus oocytes. Our findings will complete a model of urate secretion in the renal tubular cell, where intracellular urate taken up via OAT1 and/or OAT3 from the blood exits from the cell into the lumen via hNPT4.Urate is the end product of purine metabolism in humans and certain primates as a result of uricase genetic loss (urate oxidase degrades urate to allantoin) (1). Two independent nonsense mutations in this gene, found in human, chimpanzee, and orangutan but not in the gibbon, indicate that this loss had evolutionary advantages for early primates (2). Because urate has powerful antioxidant properties, uricase loss resulting in elevated serum urate may have been beneficial to early primate survival (1). In addition, Watanabe et al. (3) hypothesized that elevated serum urate levels provided a survival advantage by helping to maintain blood pressure under the low salt dietary conditions that prevailed during the middle to late Miocene period. Despite its beneficial role and given the fact that more than half of uricase-deficient mice die from urate nephropathy within 4 weeks of age, elevation in serum urate level produces a burden on the body (4). To circumvent this problem, the human body had to develop a urate excretion system.The kidney plays a dominant role in maintaining serum urate levels (1, 5). Renal urate excretion is a function of the balance between reabsorption and secretion. Recently it was demonstrated that luminal urate is taken up by a urate-anion exchanger (URAT1; SLC22A12) 3 (6) into the renal proximal tubular cell and that intracellular urate exits the cell into the interstitium/blood ...

Background & Aims The cAMP and Ca2+ signaling pathways synergize to regulate many physiological functions. However, little is known about the mechanisms by which these pathways interact. We investigated the synergy between these signaling pathways in mouse pancreatic and salivary gland ducts. Methods We created mice with disruptions in genes encoding the solute carrier family 26, member 6 (Slc26a6−/− mice) and inositol 1,4,5-triphosphate (InsP3) receptor-binding protein released with InsP3 (Irbit−/− mice). We investigated fluid secretion by sealed pancreatic ducts and the function of Slc26a6 and the cystic fibrosis transmembrane conductance regulator (CFTR) in HeLa cells and in ducts isolated from mouse pancreatic and salivary glands. Slc26a6 activity was assayed by measuring intracellular pH, and CFTR activity by measuring Cl− current. Protein interactions were determined by immunoprecipitation analyses. Results Irbit mediated the synergistic activation of CFTR and Slc26a6 by Ca2+ and cAMP. In resting cells, Irbit was sequestered by InsP3 receptors (IP3Rs) in the endoplasmic reticulum. Stimulation of Gs-coupled receptors led to phosphorylation of IP3Rs, which increased their affinity for InsP3 and reduced their affinity for Irbit. Subsequent weak stimulation of Gq-coupled receptors, which led to production of low levels of IP3, caused dissociation of Irbit from IP3Rs and allowed translocation of Irbit to CFTR and Slc26a6 in the plasma membrane. These processes stimulated epithelial secretion of electrolytes and fluid. These pathways were not observed in pancreatic and salivary glands from Irbit−/− or Slc26a6−/− mice, or in salivary gland ducts expressing mutant forms of IP3Rs that could not undergo protein kinase A-mediated phosphorylation. Conclusions Irbit promotes synergy between the Ca2+ and cAMP signaling pathways in cultured cells and in pancreatic and salivary ducts from mice. Defects in this pathway could be involved in CF, pancreatitis, or Sjögren’s syndrome.

Mutations in the Na(+)-HCO(3)(-) cotransporter NBC1 cause severe proximal tubular acidosis (pRTA) associated with ocular abnormalities. Recent studies have suggested that at least some NBC1 mutants show abnormal trafficking in the polarized cells. This study identified a new homozygous NBC1 mutation (G486R) in a patient with severe pRTA. Functional analysis in Xenopus oocytes failed to detect the G486R activity due to poor surface expression. In ECV304 cells, however, G486R showed the efficient membrane expression, and its transport activity corresponded to approximately 50% of wild-type (WT) activity. In Madin-Darby canine kidney (MDCK) cells, G486R was predominantly expressed in the basolateral membrane domain as observed for WT. Among the previously identified NBC1 mutants that showed poor surface expression in oocytes, T485S showed the predominant basolateral expression in MDCK cells. On the other hand, L522P was exclusively retained in the cytoplasm in ECV304 and MDCK cells, and functional analysis in ECV304 cells failed to detect its transport activity. These results indicate that G486R, like T485S, is a partial loss of function mutation without major trafficking abnormalities, while L522P causes the clinical phenotypes mainly through its inability to reach the plasma membranes. Multiple experimental approaches would be required to elucidate potential disease mechanism by NBC1 mutations.