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