Genetic, pharmacological and lesion analyses reveal a selective role for corticohippocampal GLUN2B in a novel repeated swim stress paradigm.

Glutamate and N-methyl-d-aspartate receptor (NMDAR) dysfunction is strongly implicated in the pathophysiology of mood and anxiety disorders. Treatment with NMDAR antagonists has antidepressant efficacy in treatment-resistant depressives. In preclinical rodent models, NMDAR antagonist administration reduces anxiety- and stress-related behaviors in concert with increases in prefrontal cortical (PFC) dendritic spinogenesis and synaptic proteins. While these effects have been attributed to actions at the NMDAR GluN2B subunit, the precise role of cortical GluN2B in mediating emotional behaviors and stress-responsivity is not fully understood. Here, we employed a novel mutant model in which the GluN2B subunit is postnatally deleted in principal neurons in the cortex and the dorsal CA1 subregion of the hippocampus. GluN2BKO mice were phenotyped on a battery of tests for anxiety-related (light/dark exploration, stress-induced hyperthermia) and antidepressant-sensitive (sucrose preference, novelty-induced hypophagia, single-trial forced swim) behaviors. A novel repeated inescapable forced swim paradigm (riFS) was developed to assess behavioral responses to repeated stress in the GluN2BKO mice. For comparison, non-mutant C57BL/6J mice were tested for single-trial forced swim behavior after systemic Ro 25-6981 treatment and for riFS behavior after lesions of the ventromedial prefrontal cortex. riFS-induced alterations in corticolimbic GluN2B expression were also examined in C57BL/6J mice. We found that GluN2BKO mice reduced "despair-like" behavior in the riFS procedure, as compared to GluN2BFLOX controls. By contrast, GluN2BKO mice showed minimal alterations on anxiety-like or antidepressant-sensitive assays, including the single-trial forced swim test. In C57BL/6J mice, induction of "despair-like" responses in the riFS test was attenuated by vmPFC lesions, and was associated with changes in limbic GluN2B expression. Collectively, these data suggest that cortical GluN2B plays a major role in modulating adaptive responses to stress. Current findings provide further support for GluN2B as a key mechanism underlying stress responsivity, and a novel pharmacotherapeutic target for stress-related neuropsychiatric disorders.

SPAK and OSR1, key kinases involved in the regulation of chloride transport.

Reversible phosphorylation by protein kinases is probably one of the most important examples of post-translational modification of ion transport proteins. Ste20-related proline alanine-rich kinase (SPAK) and oxidative stress response kinase (OSR1) are two serine/threonine kinases belonging to the germinal centre-like kinase subfamily VI. Genetic analysis suggests that OSR1 evolved first, with SPAK arising following a gene duplication in vertebrate evolution. SPAK and OSR1 are two recently discovered kinases which have been linked to several key cellular processes, including cell differentiation, cell transformation and proliferation, cytoskeleton rearrangement, and most recently, regulation of ion transporters. Na-K-2Cl cotransporter activity is regulated by phosphorylation. Pharmacological evidence has identified several kinases and phosphatases which alter cotransporter function, however, no direct linkage between these enzymes and the cotransporter has been demonstrated. This article will review some of the physical and physiological properties of SPAK and OSR1, and present new evidence of a direct interaction between the Na-K-Cl cotransporter and the stress kinases.

Localization of the KCC4 potassium-chloride cotransporter in the nervous system.

Potassium-chloride cotransporters (KCCs) collectively play a crucial role in the function and development of both the peripheral and central nervous systems. KCC4 is perhaps the least abundant KCC in the adult mammalian brain, where its localization is unknown. In the embryonic brain, KCC4 mRNA is found in the periventricular zone, cranial nerves and choroid plexus [Eur J Neurosci 16 (2002) 2358]. To investigate the distribution of KCC4 protein in the nervous system we developed a rabbit polyclonal antibody directed against a short N-terminal peptide. Western blot analysis of brain microsomal protein using purified antibody revealed the presence of a band at approximately 145 kDa, consistent with the size of a glycosylated K-Cl cotransporter. Western blot analysis of brain, spinal cord and peripheral nerves revealed high expression levels in peripheral nerves and spinal cord, with low levels in whole brain. Within the brain, the cerebral cortex, hippocampus, and cerebellum revealed minimal KCC4 expression, whereas midbrain and brainstem demonstrated higher levels. In the adult mouse brain, KCC4 staining was observed on the apical membrane of choroid plexus epithelial cells as well as in cranial nerves. All other brain structures, e.g. cortex, hippocampus, cerebellum showed no KCC4 immunoreactivity, suggesting very low or absent expression of the cotransporter in these regions. Co-staining of KCC4 with anti-MAP2, GFAP and CNPase revealed that KCC4 is expressed in peripheral neurons. Thus, KCC4 is expressed on the apical membrane of the choroid plexus, where it likely participates to K(+) reabsorption. KCC4 is also expressed in peripheral neurons, where its function remains to be determined.

K-Cl cotransport: immunohistochemical and ion flux studies in human embryonic kidney (HEK293) cells transfected with full-length and C-terminal-domain-truncated KCC1 cDNAs.

Coupled K and Cl movements are mediated by several isoforms of the K-Cl cotransporter (COT) encoded by the KCC genes. The ubiquitous KCC1 isoform, important for cell volume and ion homeostasis, has 12 transmembrane domains (Tmds), and cytoplasmic N- and C-terminal domains (Ntd and Ctd). This study investigates the cellular localization of KCC1 by confocal microscopy, activation of K-Cl COT by various non-osmotic and osmotic interventions with net unidirectional K and Rb fluxes at 37( degrees )C, and the effect of Ctd deletion on K-Cl COT regulation. Human embryonic kidney (HEK293) cells were transfected with full-length (fl) rabbit (rb)KCC1 and - CtdKCC1 cDNAs obtained after truncation at nucleotide 2011. Normal cells exposed to polyclonal anti-Ctd antibodies against Ctd epitopes within a 77 amino acid sequence (a.a.943-1020) revealed granular membrane and cytoplasmic immunostaining, presumably endogenous KCC1. Additional diffuse membrane and cytoplasmic immunofluorescence in flKCC1-transfected cells was absent in -CtdKCC1-transfected cells. Monoclonal antibodies against a c-myc epitope at the protein Ntd showed both membrane and cytosolic fluorescence. Basal and N-ethylmaleimide (NEM)-stimulated Rb influxes through K-Cl COT, calculated as Cl-dependent Rb fluxes, were 2-3-fold higher in flKCC1-transfected than in normal cells. NEM stimulation of K-Cl COT was highest in flKCC1-transfected cells, significantly lower in stably and abrogated in transiently -CtdKCC1-transfected cells. Furosemide, calyculin and genistein inhibited basal and NEM-stimulated K-Cl COT in normal and transfected cells. Staurosporine and hydroxylamine were ineffective stimulators. No effect of pH(0) changes (6.3-8.4) was observed in basal or NEM-stimulated K-Cl COT, in both normal and transfected cells. However, inhibition by NEM occurred at pH(0) 8.4. Furthermore, in a Cl-independent manner, NEM lowered cell K content by >30% and hypotonicity (210-70mOsM) stimulated furosemide-sensitive Rb influx and K loss. Thus, in cultured normal and KCC1-transfected cells, K-Cl COT shows significant differences from erythrocytes, and NEM and cell swelling open furosemide-sensitive and Cl-independent K/Rb channels. Failure of K-Cl COT in cells transfected with Ctd-truncated KCC1 to respond to NEM suggests a role of the Ctd for signal transduction.

Localization of the K(+)-Cl(-) cotransporter, KCC3, in the central and peripheral nervous systems: expression in the choroid plexus, large neurons and white matter tracts.

Na(+)-independent K(+)-Cl(-) cotransporters function in the regulation of cell volume, control of CNS excitability and epithelial ion transport. Several K(+)-Cl(-) cotransporter isoforms are expressed in the nervous system, and KCC3 in particular is expressed at significant levels in both the brain and spinal cord. The cellular localization of this transporter has, however, not been determined. In this study, we generated a polyclonal antibody against the KCC3 cotransporter in order to characterize and localize this protein in the brain. Western blot analysis of mouse kidney and brain demonstrated KCC3 proteins of different size, 150 and 170kDa, respectively; this disparity remained after deglycosylation. Northern blot confirmed the presence of two distinct forms of KCC3, KCC3a and KCC3b, generated by the inclusion of different first coding exons. KCC3a predominates in the brain, whereas KCC3b is more abundant in the kidney. Western blots with membrane protein from dissected mouse brain revealed abundant expression in all brain regions examined: the cerebral cortex, hippocampus, diencephalon, brainstem and cerebellum. The spinal cord showed the highest levels of KCC3 expression, whereas peripheral nerves did not contain immunoreactive KCC3 protein. Western blot analysis of whole brain from rats of various ages indicated increasing expression in the postnatal period, concurrent with CNS maturation and myelination. Immunofluorescence studies demonstrated strong signal in myelinated tracts of the spinal cord, consistent with individual myelin sheaths. Brain sections also showed white matter enhancement, but also cellular signal consistent with pyramidal neurons and Purkinje cells. The base of the choroid plexus epithelium was also strongly labeled. These data demonstrate the specificity and diversity of KCC3 expression in the mouse CNS.

Neuronal restrictive silencing element is found in the KCC2 gene: molecular basis for KCC2-specific expression in neurons.

KCC2 is one of four known isoforms of the K-Cl cotransporter with an expression pattern restricted to neurons. It mediates efflux of Cl- across neuronal membranes and plays an important role in GABAergic and glycinergic neurotransmission. To understand the molecular basis for neuronal specificity of KCC2 expression, we isolated and sequenced portions of the KCC2 gene, including some of its 5' flanking (control) region. We found a 21-bp sequence, within intron 1, that shares 80% homology to the consensus site for neuronal-restrictive silencing factor binding. We demonstrated that this specific sequence of the KCC2 gene promotes transcriptional regulation by showing that nuclear proteins isolated from a mouse neural progenitor cell line interact with this 21-bp element and by establishing that this element silences reporter gene expression in nonneuronal cells.

K-Cl co-transport: immunocytochemical and functional evidence for more than one KCC isoform in high K and low K sheep erythrocytes.

K-Cl co-transport (COT) is significantly higher in low K (LK), L-antigen (L) positive, than in high K (HK), M-antigen (M) positive, sheep red blood cells (SRBCs) and is inhibited by sheep allo-anti-L1 antibody. To answer the question of whether this difference in K-Cl co-transport activity resides at the level of the transporter or its regulation, a combined immunocytochemical and functional approach was taken. At least four isoforms of K-Cl COT encoded by different KCC genes are known, with 12 transmembrane domains and cytoplasmic C- and N-terminal domains (Ctd and Ntd, respectively). Polyclonal anti-rat (rt)KCC1 antibodies against a fusion peptide with 77 amino acids from the Ctd of rtKCC1 and anti-human (h)KCC3 against an 18-aa peptide from the Ntd of hKCC3, were prepared in rabbits (rb). Two distinctly separate protein bands of 180 and 145 kDa molecular mass were detected in hemoglobin-free ghosts from RBCs of two LK (one homozygous LL and one heterozygous LM) and one HK (homozygous MM) sheep by Western blots with rb anti-rtKCC1 and rb anti-hKCC3. Confocal microscopy showed specific immunostaining of KCC1 with rb anti-rtKCC1, and of KCC3 with rb anti-hKCC3, in white ghosts from both LK and HK SRBCs. To test the functional heterogeneity of K-Cl COT, the effect of the anti-L1 antibody was assessed on K-Cl COT activated by the kinase inhibitor staurosporine. Incubation of LK SRBCs with anti-L1 serum inhibited by 30% staurosporine-stimulated K-Cl COT suggesting that approximately two-thirds of the transport activity is independent of the L1 antigen. That staurosporine altered the L1 antigen/antibody reaction is unlikely since the action of another antibody, anti-Lp, stimulating the Na/K pump flux, was not modified. The present results, in conjunction with earlier work, lead to the hypothesis that the partial anti-L1 inhibition of K-Cl COT may be related to the molecular KCC dimorphism, seen in these cells with anti-KCC1 and anti-KCC3 antibodies.

Abnormal GABAA receptor-mediated currents in dorsal root ganglion neurons isolated from Na-K-2Cl cotransporter null mice.

We have recently disrupted Slc12a2, the gene encoding the secretory Na-K-2Cl cotransporter in mice (NKCC1) (Delpire et al., 1999). Gramicidin perforated-patch and whole-cell recordings were performed to study GABA-induced currents in dorsal root ganglion (DRG) neurons isolated from wild-type and homozygote NKCC1 knock-out mice. In wild-type DRG neurons, strong GABA-evoked inward current was observed at the resting membrane potential, suggesting active accumulation of Cl(-) in these cells. This GABA-induced current was blocked by picrotoxin, a GABA(A) receptor blocker. The strong Cl(-) accumulation that gives rise to depolarizing GABA responses is caused by Na-K-2Cl cotransport because reduction of external Cl(-) or application of bumetanide induced a decrease in [Cl(-)](i), whereas an increase in external K(+) caused an apparent [Cl(-)](i) accumulation. In contrast to control neurons, little or no net current was observed at the resting membrane potential in homozygote NKCC1 mutant DRG neurons. E(GABA) was significantly more negative, demonstrating the absence of Cl(-) accumulation in these cells. Application of bumetanide induced a positive shift of E(GABA), suggesting the presence of an outward Cl(-) transport mechanism. In agreement with an absence of GABA depolarization in DRG neurons, behavioral analysis revealed significant alterations in locomotion and pain perception in the knock-out mouse. Our results clearly demonstrate that the Na-K-2Cl cotransporter is responsible for [Cl(-)](i) accumulation in DRG neurons and that via regulation of intracellular Cl(-), the Na-K-2Cl cotransporter participates in the modulation of GABA neurotransmission and sensory perception.

Dependence of KCC2 K-Cl cotransporter activity on a conserved carboxy terminus tyrosine residue.

K-Cl cotransporters (KCC) play fundamental roles in ionic and osmotic homeostasis. To date, four mammalian KCC genes have been identified. KCC2 is expressed exclusively in neurons. Injection of Xenopus oocytes with KCC2 cRNA induced a 20-fold increase in Cl(-)-dependent, furosemide-sensitive K(+) uptake. Oocyte swelling increased KCC2 activity 2-3 fold. A canonical tyrosine phosphorylation site is located in the carboxy termini of KCC2 (R1081-Y1087) and KCC4, but not in other KCC isoforms. Pharmacological studies, however, revealed no regulatory role for phosphorylation of KCC2 tyrosine residues. Replacement of Y1087 with aspartate or arginine dramatically reduced K(+) uptake under isotonic and hypotonic conditions. Normal or near-normal cotransporter activity was observed when Y1087 was mutated to phenylalanine, alanine, or isoleucine. A tyrosine residue equivalent to Y1087 is conserved in all identified KCCs from nematodes to humans. Mutation of the Y1087 congener in KCC1 to aspartate also dramatically inhibited cotransporter activity. Taken together, these results suggest that replacement of Y1087 and its congeners with charged residues disrupts the conformational state of the carboxy terminus. We postulate that the carboxy terminus plays an essential role in maintaining the functional conformation of KCC cotransporters and/or is involved in essential regulatory protein-protein interactions.

Cation-Chloride Cotransporters in Neuronal Communication.

Two isoforms of the cation-Cl(-) cotransporter family are expressed in neurons and modulate neurotransmission. NKCC1, a Na(+)-K(+)-2Cl(-) cotransporter, by raising internal Cl(-), is responsible for excitatory GABAergic activity in immature brain and in adult sensory neurons. KCC2, a neuronal-specific isoform of the K(+)-Cl(-) cotransporter, by lowering internal Cl(-), is critical for inhibitory GABA responses in mature central nervous system neurons.

Gastrin-mediated effects of omeprazole on rat colon mucosa.

BACKGROUND: Omeprazole increases circulating gastrin levels, which in turn may affect the growth and differentiation of colon mucosa. Chloride transport mechanisms in normal colon were analyzed as markers for possible trophic actions of endogenous hypergastrinemia. METHODS: Four groups of Fischer rats were studied for 10 days. Group 1 (baseline) received no treatment. Group 2 received omeprazole only. Group 3 received omeprazole plus vehicle. Group 4 received omeprazole plus CCK-B gastrin receptor antagonist (GRA) L740,093 in vehicle. On day 10 serum gastrin was assayed. Colon mucosa was analyzed for protein and DNA content. Semiquantitative Northern analysis measured levels of messenger RNA (mRNA) encoding for key Cl- transporters: Na-K-Cl cotransporter (Cl- secretion in crypts), Cl-/HCO3- exchanger (Cl- absorption in villi), and Na/K adenosine triphosphatase (not directly involved in Cl- transport). RESULTS: Omeprazole increased gastrin levels, which were not altered by vehicle or GRA. Omeprazole increased protein, DNA, and Na/K adenosine triphosphatase mRNA levels, with no effect by GRA. In contrast, omeprazole decreased Na-K-Cl and Cl-/HCO3- mRNA levels, effects that were partly reversed by GRA. CONCLUSIONS: Omeprazole augments growth index values of colon mucosa independent of serum gastrin. Against a background of omeprazole-induced achlorhydria hypergastrinemia appears to influence differentiation rather than growth of normal colon mucosa.

Developmental regulation of the neuronal-specific isoform of K-Cl cotransporter KCC2 in postnatal rat brains.

We examined the expression of the KCC2 isoform of the K-Cl cotransporter in the developing and adult brain, using an affinity-purified antibody directed against a unique region of the KCC2 protein. Expression was shown to be limited to neurons at the cell bodies and cell processes in the hippocampus and cerebellum. Expression seemed to be the highest at the end of processes that originated from the CA1 pyramidal cells. Developmental up-regulation of KCC2 expression was demonstrated in the entire rat brain by Northern and Western blot analyses, and in the hippocampus by immunofluorescence. Level of KCC2 expression was minimal at birth and increased significantly during postnatal development. This pattern of expression was opposite to the one of the Na-K-2Cl cotransporter that is highly expressed in immature brain and decreases during development. The up-regulation of the K-Cl cotransporter expression is consistent with the developmental down-regulation of the intracellular Cl- concentration in neurons. The level of intracellular Cl-, in turn, determines the excitatory versus inhibitory response of the neurotransmitter gamma-aminobutyric acid in the immature versus mature brain. Finally, KCC2 expression was shown in dorsal root ganglion neurons, demonstrating that expression of the cotransporter is not strictly confined to central nervous system neurons.

Deafness and imbalance associated with inactivation of the secretory Na-K-2Cl co-transporter.

Deafness can result from a variety of gene defects. Some genes involved in the physiology of hearing encode membrane transporters that regulate the ionic composition of the fluid bathing the inner ear. The endolymph is an extracellular fluid with an atypical composition that resembles the intracellular milieu, high in K+ and low in Na+. Recent studies have emphasized the prominent role of K+ channels in endolymph secretion and mechanical transduction. Coupled electroneutral transport of Na+, K+ and Cl- is mediated by two isoforms of the Na-K-2Cl co-transporter: the absorptive isoform BSC1 (also called NKCC2, encoded by Slc12a1 in mouse) that is exclusively expressed in kidney; and BSC2/NKCC1 (encoded by Slc12a2 in mouse), the secretory isoform which has a wider pattern of expression including epithelia, muscle cells, neurons and red blood cells. These co-transporters share 57% homology at the amino acid level and are pharmacologically inhibited by loop diuretics. There is functional and histochemical evidence for the presence of the secretory isoform of the Na-K-2Cl co-transporter in gerbil, rat and rabbit inner ear. We disrupted mouse Slc12a2 and report here that Slc12a2-/- mice are deaf and exhibit classic shaker/waltzer behaviour, indicative of inner-ear defects. We localized the co-transporter to key secreting epithelia of the mouse inner ear and show that absence of functional co-transporter leads to structural damages in the inner ear consistent with a decrease in endolymph secretion.

Cloning and characterization of KCC3 and KCC4, new members of the cation-chloride cotransporter gene family.

The K+-Cl- cotransporters (KCCs) belong to the gene family of electroneutral cation-chloride cotransporters, which also includes two bumetanide-sensitive Na+-K+-2Cl- cotransporters and a thiazide-sensitive Na+-Cl- cotransporter. We have cloned cDNAs encoding mouse KCC3, human KCC3, and human KCC4, three new members of this gene family. The KCC3 and KCC4 cDNAs predict proteins of 1083 and 1150 amino acids, respectively. The KCC3 and KCC4 proteins are 65-71% identical to the previously characterized transporters KCC1 and KCC2, with which they share a predicted membrane topology. The four KCC proteins differ at amino acid residues within key transmembrane domains and in the distribution of putative phosphorylation sites within the amino- and carboxyl-terminal cytoplasmic domains. The expression of mouse KCC3 in Xenopus laevis oocytes reveals the expected functional characteristics of a K+Cl- cotransporter: Cl--dependent uptake of 86Rb+ which is strongly activated by cell swelling and weakly sensitive to furosemide. A direct functional comparison of mouse KCC3 to rabbit KCC1 indicates that KCC3 has a much greater volume sensitivity. The human KCC3 and KCC4 genes are located on chromosomes 5p15 and 15q14, respectively. Although widely expressed, KCC3 transcripts are the most abundant in heart and kidney, and KCC4 is expressed in muscle, brain, lung, heart, and kidney. The unexpected molecular heterogeneity of K+-Cl- cotransport has implications for the physiology and pathophysiology of a number of tissues.

Selective increase in gastric mucosal mRNA encoding basolateral Na-K-2C1 cotransporter following ileostomy in the rat.

Results of previous studies suggest that major surgical resections or reconstructions of the distal small intestine can alter morphologic and functional properties of the stomach. Little is known about the effect of lesser surgical alterations such as construction of an ileostomy, on the morphology and transport properties of the gastric mucosa. To evaluate the effects of ileostomy, Sprague-Dawley rats underwent sham laparotomy (n = 10) or loop ileostomy construction (n = 10). After body weights had stabilized ( approximately 21 days) the animals were killed. Gastric mucosal scrapings were prepared for Northern blot analysis of messenger RNA levels for (1) H/K ATPase, found in parietal cells; (2) Na-K-2C1 cotransporter, found in both parietal and surface cells; and (3)Na/K ATPase, found in all gastric mucosal cells. Gastric mucosa from ileostomy animals was visibly hypertrophied compared to sham-operated animals. There was a 145% increase in the mRNA levels of the Na-K-2Cl cotransporter in gastric mucosa of the ileostomy group but no significant changes in H/K ATPase or Na/K ATPase mRNA levels. Construction of an ileostomy selectively enhances expression of the Na-K-C1 cotransporter in the gastric mucosa. Further studies are required to understand the neurohumoral stimuli underlying this selective response.

Functional demonstration of Na+-K+-2Cl- cotransporter activity in isolated, polarized choroid plexus cells.

The function of the apical Na+-K+-2Cl- cotransporter in mammalian choroid plexus (CP) is uncertain and controversial. To investigate cotransporter function, we developed a novel dissociated rat CP cell preparation in which single, isolated cells maintain normal polarized morphology. Immunofluorescence demonstrated that in isolated cells the Na+-K+-ATPase, Na+-K+-2Cl- cotransporter, and aquaporin 1 water channel remained localized to the brush border, whereas the Cl-/HCO-3 (anion) exchanger type 2 was confined to the basolateral membrane. We utilized video-enhanced microscopy and cell volume measurement techniques to investigate cotransporter function. Application of 100 microM bumetanide caused CP cells to shrink rapidly. Elevation of extracellular K+ from 3 to 6 or 25 mM caused CP cells to swell 18 and 33%, respectively. Swelling was blocked completely by Na+ removal or by addition of 100 microM bumetanide. Exposure of CP cells to 5 mM BaCl2 induced rapid swelling that was inhibited by 100 microM bumetanide. We conclude that the CP cotransporter is constitutively active and propose that it functions in series with Ba2+-sensitive K+ channels to reabsorb K+ from cerebrospinal fluid to blood.

The electroneutral cation-chloride cotransporters.

Electroneutral cation-chloride cotransporters are widely expressed and perform a variety of physiological roles. A novel gene family of five members, encompassing a Na+-Cl- transporter, two Na+-K+-2Cl- transporters and two K+-Cl- cotransporters, encodes these membrane proteins; homologous genes have also been identified in a prokaryote and a number of lower eukaryotes. The cotransporter proteins share a common predicted membrane topology, with twelve putative transmembrane segments flanked by long hydrophilic N- and C-terminal cytoplasmic domains. The molecular identification of these transporters has had a significant impact on the study of their function, regulation and pathophysiology.

Partial cloning and characterization of Slc12a2: the gene encoding the secretory Na+-K+-2Cl- cotransporter.

The Slc12a2 gene encodes a widely expressed bumetanide-sensitive Na+-K+-2Cl- cotransporter that participates in various functions such as Cl- secretion and cell volume regulation. We isolated and characterized 75 kilobases of the murine gene encoding the cotransporter. The cotransport protein is encoded by 27 exons. Ribonuclease protection assay and primer extension demonstrated tissue-specific transcription initiation sites located within 270 base pairs upstream of the start codon. Nucleotide sequence analysis of the proximal 5'-flanking region revealed the presence of a weak TATA box, multiple Sp1/GC consensus sites, and the consensus sequence of a putative transcriptional initiator. Transfection of luciferase reporter gene constructs in mouse inner medullary collecting duct (mIMCD-3) cells confirmed the location of the minimal promoter within a 120-base pair fragment upstream of the cDNA. We also report the identification of an alternatively spliced variant of the cotransporter, expressed primarily in brain. This new spliced variant lacks exon 21, which encodes a 16-amino acid peptide located in the COOH-terminal tail of the protein. The absence of this exon causes the loss of the single protein kinase A consensus site of the cotransport protein.

Expression of the Na-K-2Cl cotransporter is developmentally regulated in postnatal rat brains: a possible mechanism underlying GABA's excitatory role in immature brain.

An inhibitory neurotransmitter in mature brain, gamma-aminobutyric acid (GABA) also appears to be excitatory early in development. The mechanisms underlying this shift are not well understood. In vitro studies have suggested that Na-K-Cl cotransport may have a role in modulating immature neuronal and oligodendrocyte responses to the neurotransmitter GABA. An in vivo developmental study would test this view. Therefore, we examined the expression of the BSC2 isoform of the Na-K-2Cl cotransporter in the postnatal developing rat brain. A comparison of sections from developing rat brains by in situ hybridization revealed a well-delineated temporal and spatial pattern of first increasing and then diminishing cotransporter expression. Na-K-2Cl mRNA expression in the cerebral cortex and hippocampus was highest in the first week of postnatal life and then diminished from postnatal day (PND) 14 to adult. Cotransporter signal in white-matter tracts of the cerebrum, cerebellum, peaked at PND 14. Expression was detected in cerebellar progenitor cells of the external granular layer, in internal granular layer cells at least as early as PND 7, and in Purkinje cells beginning at PND 14. Double-labeling immunofluorescence of brain sections with anti-BSC2 antibody and cell type-specific antibodies confirmed expression of the cotransporter gene product in neurons and oligodendrocytes in the white matter in a pattern similar to that determined by in situ hybridization. The temporal pattern of expression of the Na-K-2Cl cotransporter in the postnatal rat brain supports the hypothesis that the cotransporter is the mechanism of intracellular Cl- accumulation in immature neurons and oligodendrocytes.

Expression of the Na(+)-K(+)-2Cl- cotransporter BSC2 in the nervous system.

We used in situ hybridization and immunocytochemistry with polyclonal antibodies against the mouse bumetanide-sensitive Na(+)-K(+)-2Cl- cotransporter (mBSC2) to determine the location of this cotransporter in rat brain. Northern blots and in situ hybridization showed the presence of cotransporter mRNA in the brain, with an especially high level of expression in the choroid plexus (CP). Affinity-purified anti-BSC2 antibody identified proteins of 145-155 kDa on Western blot analysis and immunoprecipitation of brain and CP membrane protein. Indirect immunofluorescence demonstrated that BSC2 protein is located on the apical surface of the CP and is heterogeneously distributed in cell bodies and dendrites of neurons in the central and peripheral nervous system. The apical localization of BSC2 in the CP was confirmed by 86Rb+ uptakes in primary cultures of CP cells grown on permeable filters and confocal immunofluorescence microscopy. The apical localization of the cotransporter in CP epithelium suggests a role for the cotransporter in cerebrospinal fluid K+ homeostasis. In neurons, the cotransporter may help regulate intracellular Cl- concentration and thereby affect neuronal response to gamma-aminobutyric acid.