Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. Packaging and storage of glutamate into glutamatergic neuronal vesicles requires ATP-dependent vesicular glutamate uptake systems, which utilize the electrochemical proton gradient as a driving force. VGLUT1, the first identified vesicular glutamate transporter, is only expressed in a subset of glutamatergic neurons. We report here the molecular cloning and functional characterization of a novel glutamate transporter, VGLUT2, from mouse brain. VGLUT2 has all major functional characteristics of a synaptic vesicle glutamate transporter, including ATP dependence, chloride stimulation, substrate specificity, and substrate affinity. It has 75 and 79% amino acid identity with human and rat VGLUT1, respectively. However, expression patterns of VGLUT2 in brain are different from that of VGLUT1. In addition, VGLUT2 activity is dependent on both membrane potential and pH gradient of the electrochemical proton gradient, whereas VGLUT1 is primarily dependent on only membrane potential. The presence of VGLUT2 in brain regions lacking VGLUT1 suggests that the two isoforms together play an important role in vesicular glutamate transport in glutamatergic neurons.Neurotransmission depends on the regulated exocytotic release of vesicular transmitter molecules to the synaptic cleft, where they interact with postsynaptic receptors that subsequently transduce the information. Two types of neurotransmitter transporters have been identified based on membrane localization on plasma membrane or vesicular membrane. Removal of the transmitter from the synaptic cleft results in termination of the signal, and this requires destruction of transmitter or reuptake of transmitter back to the presynaptic terminal or glial cells via a sodium-dependent uptake system on the plasma membrane (1). Packaging and storage of neurotransmitters into specialized secretory vesicles in neurons ensures their regulated release. This storage is also crucial for protecting the neurotransmmitter molecules from leakage or intraneuronal metabolism and for protecting the neuron from possible toxic effects. This process is mediated by specific transporters on the vesicular membranes. At least four different types of vesicular transporters have been functionally identified that are specific for transport of classic neurotransmitters: monoamines, acetylcholine, ␥-aminobutyric acid (GABA), and glutamate (2, 3). Unlike the plasma membrane transporters, which rely on a sodium gradient across the plasma membrane, all of these vesicular transport processes depend on the proton electrochemical gradient (⌬ Hϩ ) 1 generated by a Mg 2ϩ -activated vacuolar H ϩ -ATPase (V-ATPase) on the vesicular membrane (4). When protons are pumped into the vesicular lumen, a proton gradient (⌬pH) and a membrane potential (⌬) occur across the membrane to form ⌬ Hϩ, which favors the exchange of luminal protons for cytoplasmic transmitter. The transport of monoamines and acetylcholine rely predominantly on ...

The current studies were designed to characterize type IIb sodium-inorganic phosphate (P(i)) cotransporter (NaP(i)-IIb) expression and to assess the effect of 1,25-(OH)(2) vitamin D(3) on NaP(i)-IIb gene expression during rat ontogeny. Sodium-dependent P(i) absorption by intestinal brush-border membrane vesicles (BBMVs) decreased with age, and NaP(i)-IIb gene expression also decreased proportionally with age. 1,25-(OH)(2) vitamin D(3) treatment increased intestinal BBMV P(i) absorption by approximately 2.5-fold in suckling rats and by approximately 2.1-fold in adult rats. 1,25-(OH)(2) vitamin D(3) treatment also increased NaP(i)-IIb mRNA abundance by approximately 2-fold in 14-day-old rats but had no effect on mRNA expression in adults. Furthermore, in rat intestinal epithelial (RIE) cells, 1,25-(OH)(2) vitamin D(3) increased NaP(i)-IIb mRNA abundance, an effect that was abolished by actinomycin D. Additionally, human NaP(i)-IIb gene promoter activity in transiently transfected RIE cells showed approximately 1.6-fold increase after 1,25-(OH)(2) vitamin D(3) treatment. In conclusion, we demonstrate that the age-related decrease in intestinal sodium-dependent P(i) absorption correlates with decreased NaP(i)-IIb mRNA expression. Our data also suggest that the effect of 1,25-(OH)(2) vitamin D(3) on NaP(i)-IIb expression is at least partially mediated by gene transcription in suckling rats.

Among water channel proteins (aquaporins), aquaporin-collecting duct (AQP-CD) is the vasopressin-regulated water channel. Vasopressin causes cAMP production in the renal collecting duct cells, and this is believed to lead to exocytic insertion of water channel into the apical membrane (shuttle hypothesis). AQP-CD contains a consensus sequence for cAMP-dependent protein kinase, residues at positions 253-256 (Arg-Arg-Gln-Ser). To determine the role of this site, Ser-256 was substituted for Ala, Leu, Thr, Asp, or Glu by site-directed mutagenesis. In Xenopus oocytes injected with wild-type or mutated AQP-CD cRNAs, osmotic water permeability (Pf) was 4.8-7.7 times higher than Pf of water-injected oocytes. Incubation with cAMP plus forskolin or direct cAMP injection into the oocytes increased Pf of wild-type, but not mutated, AQP-CD-expressing oocytes, whereas the amounts of AQP-CD expression were similar in wild and mutated types as identified by Western blot analysis. In vitro phosphorylation studies of AQP-CD proteins expressed in oocyte showed that cAMP-dependent protein kinase phosphorylated wild-type, but not mutated, AQP-CD proteins. Phosphoamino acid analysis revealed that this phosphorylation occurred at the serine residue. Moreover, phosphorylation of AQP-CD protein in intact rat kidney medulla tissues was stimulated by incubation with cAMP. Our data suggest that cAMP stimulates water permeability of AQP-CD by phosphorylation. This process may contribute to the vasopressin-regulated water permeability of collecting duct in addition to the apical insertion of AQP-CD by exocytosis.

The SLC20 family transport proteins were originally identified as retroviral receptors (called Glvr-1 and Ram-1). Since then, they have been shown to function as sodium-phosphate (Na/P(i)) cotransporters, and have subsequently been classified as type III Na/P(i) cotransporters (now called Pit-1 and Pit-2). The Pit cotransporters share approximately 60% sequence homology, they have a high affinity for P(i), they are electrogenic with a coupling stoichiometry of >1 Na(+) per P(i) ion cotransported, and are inhibited by alkaline pH and phosphonoformic acid (PFA). Pit-1 and Pit-2 expression and/or activity has also been shown to be regulated by P(i) deprivation in some, but not all cells and tissues examined. The Pit-1 and Pit-2 cotransporters are widely expressed, but cell-type specific expression has only been investigated in bone, kidney and intestine. Both proteins are likely expressed on the basolateral membranes of polarized epithelial cells, where they are likely involved in cellular P(i) homeostasis. The Pit-1 and Pit-2 gene promoters have been cloned and characterized. While the exact roles of the Pit cotransporters in different cell types has not been definitively determined, they may be involved in important physiological pathways in bone, aortic smooth muscle cells, parathyroid glands, kidney and intestine.

No abstract