Identification of key functional domains in the C terminus of the K+-Cl- cotransporters

The K+-Cl- cotransporter (KCC) isoforms constitute a functionally heterogeneous group of ion carriers. Emerging evidence suggests that the C terminus (Ct) of these proteins is important in conveying isoform-specific traits and that it may harbor interacting sites for 4beta-phorbol 12-myristate 13-acetate (PMA)-induced effectors. In this study, we have generated KCC2-KCC4 chimeras to identify key functional domains in the Ct of these carriers and single point mutations to determine whether canonical protein kinase C sites underlie KCC2-specific behaviors. Functional characterization of wild-type (wt) and mutant carriers in Xenopus laevis oocytes showed for the first time that the KCCs do not exhibit similar sensitivities to changes in osmolality and that this distinguishing feature as well as differences in transport activity under both hypotonic and isotonic conditions are in part determined by the residue composition of the distal Ct. At the same time, several mutations in this domain and in the proximal Ct of the KCCs were found to generate allosteric-like effects, suggesting that the regions analyzed are important in defining conformational ensembles and that isoform-specific structural configurations could thus account for variant functional traits as well. Characterization of the other mutants in this work showed that KCC2 is not inhibited by PMA through phosphorylation of its canonical protein kinase C sites. Intriguingly, however, the substitutions N728S and S940A were seen to alter the PMA effect paradoxically, suggesting again that allosteric changes in the Ct are important determinants of transport activity and, furthermore, that the structural configuration of this domain can convey specific functional traits by defining the accessibility of cotransporter sites to regulatory intermediates such as PMA-induced effectors.     

Cloning, characterization, and chromosomal location of a novel human K+-Cl- cotransporter

Differential display polymerase chain reaction has been used to isolate genes regulated in vascular endothelial cells by the angiogenic factor vascular endothelial cell growth factor (VEGF). Analysis of one of the bands consistently up-regulated by VEGF led us to the identification of a cDNA from a human umbilical vein endothelial cell library that is 77% identical to the human K+-Cl- cotransporter1 (KCC1). We have referred to the predicted protein as K+-Cl- cotransporter 3 (KCC3). Hydrophobicity analysis of the KCC3 amino acid sequence showed an almost identical pattern to KCC1, suggesting 12 membrane-spanning segments, a large extracellular loop with potential N-glycosylation sites, and cytoplasmic N- and C-terminal regions. The KCC3 mRNA was highly expressed in brain, heart, skeletal muscle, and kidney, showing a distinct pattern and size from KCC1 and KCC2. The KCC3 mRNA level in endothelial cells increased on treatment with VEGF and decreased with the proinflammatory cytokine tumor necrosis factor alpha, whereas KCC1 mRNA levels remained unchanged. Stable overexpression of KCC3 cDNA in HEK293 cells produced a glycoprotein of approximately 150 kDa, which was reduced to 120 kDa by glycosidase digestion. An increased initial uptake rate of 86Rb was seen in clones with high KCC3 expression, which was dependent on extracellular Cl- but not Na+ and was inhibitable by the loop diuretic agent furosemide. The KCC3 genomic localization was shown to be 15q13 by fluorescence in situ hybridization. Radiation hybrid analysis placed KCC3 within an area associated with juvenile myoclonic epilepsy. These results suggest KCC3 is a new member of the KCC family that is under distinct regulation from KCC1.     

Interaction of neuron-specific K+-Cl- cotransporter, KCC2, with brain-type creatine kinase

gamma-Aminobutyric acid, a major inhibitory neurotransmitter within the adult central nervous system, is also known to be excitatory at early developmental stages due to the elevated intracellular Cl(-) concentration. This functional change is primarily attributable to a K(+)-Cl(-) cotransporter, KCC2, the expression of which is developmentally regulated in neurons. However, little detail information is available concerning the intracellular regulation of KCC2 function. Here, we identify an interaction between KCC2 and brain-type creatine kinase by means of yeast two-hybrid screening. This interaction, which was also detected in cultured cells and brain extracts, might contribute to KCC2-mediated modulation of Cl(-) homeostasis.     

BDNF-induced TrkB activation down-regulates the K+-Cl- cotransporter KCC2 and impairs neuronal Cl- extrusion

Pathophysiological activity and various kinds of traumatic insults are known to have deleterious long-term effects on neuronal Cl- regulation, which can lead to a suppression of fast postsynaptic GABAergic responses. Brain-derived neurotrophic factor (BDNF) increases neuronal excitability through a conjunction of mechanisms that include regulation of the efficacy of GABAergic transmission. Here, we show that exposure of rat hippocampal slice cultures and acute slices to exogenous BDNF or neurotrophin-4 produces a TrkB-mediated fall in the neuron-specific K+-Cl- cotransporter KCC2 mRNA and protein, as well as a consequent impairment in neuronal Cl- extrusion capacity. After kindling-induced seizures in vivo, the expression of KCC2 is down-regulated in the mouse hippocampus with a spatiotemporal profile complementary to the up-regulation of TrkB and BDNF. The present data demonstrate a novel mechanism whereby BDNF/TrkB signaling suppresses chloride-dependent fast GABAergic inhibition, which most likely contributes to the well-known role of TrkB-activated signaling cascades in the induction and establishment of epileptic activity.     

A C-terminal domain in KCC2 confers constitutive K+-Cl- cotransport

The neuron-specific K(+)-Cl(-) cotransporter KCC2 plays a crucial role in determining intracellular chloride activity and thus the neuronal response to gamma-aminobutyric acid and glycine. Of the four KCCs, KCC2 is unique in mediating constitutive K(+)-Cl(-) cotransport under isotonic conditions; the other three KCCs are exclusively swelling-activated, with no isotonic activity. We have utilized a series of chimeric cDNAs to localize the determinant of isotonic transport in KCC2. Two generations of chimeric KCC4-KCC2 cDNAs initially localized this characteristic to within a KCC2-specific expansion of the cytoplasmic C terminus, between residues 929 and 1043. This region of KCC2 is rich in prolines, serines, and charged residues and encompasses two predicted PEST sequences. Substitution of this region in KCC2 with the equivalent sequence of KCC4 resulted in a chimeric KCC that was devoid of isotonic activity, with intact swelling-activated transport. A third generation of chimeras demonstrated that a domain just distal to the PEST sequences confers isotonic transport on KCC4. Mutagenesis of this region revealed that residues 1021-1035 of KCC2 are sufficient for isotonic transport. Swelling-activated K(+)-Cl(-) cotransport is abrogated by calyculin A, whereas isotonic transport mediated by KCC chimeras and KCC2 is completely resistant to this serine-threonine phosphatase inhibitor. In summary, a 15-residue C-terminal domain in KCC2 is both necessary and sufficient for constitutive K(+)-Cl(-) cotransport under isotonic conditions. Furthermore, unlike swelling-activated transport, constitutive K(+)-Cl(-) cotransport mediated by KCC2 is completely independent of serine-threonine phosphatase activity, suggesting that these two modes of transport are activated by distinct mechanisms.     

K+-Cl- Cotransporter-3a Up-regulates Na+,K+-ATPase in Lipid Rafts of Gastric Luminal Parietal Cells

Gastric parietal cells migrate from the luminal to the basal region of the gland, and they gradually lose acid secretory activity. So far, distribution and function of K+-Cl(-) cotransporters (KCCs) in gastric parietal cells have not been reported. We found that KCC3a but not KCC3b mRNA was highly expressed, and KCC3a protein was predominantly expressed in the basolateral membrane of rat gastric parietal cells located in the luminal region of the glands. KCC3a and the Na+,K+-ATPase alpha1-subunit (alpha1NaK) were coimmunoprecipitated, and both of them were highly localized in a lipid raft fraction. The ouabain-sensitive K+-dependent ATP-hydrolyzing activity (Na+,K+-ATPase activity) was significantly inhibited by a KCC inhibitor (R-(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]acetic acid (DIOA)). The stable exogenous expression of KCC3a in LLC-PK1 cells resulted in association of KCC3a with endogenous alpha1NaK, and it recruited alpha1NaK in lipid rafts, accompanying increases of Na+,K+-ATPase activity and ouabain-sensitive Na+ transport activity that were suppressed by DIOA, whereas the total expression level of alpha1NaK in the cells was not significantly altered. On the other hand, the expression of KCC4 induced no association with alpha1NaK. In conclusion, KCC3a forms a functional complex with alpha1NaK in the basolateral membrane of luminal parietal cells, and it up-regulates alpha1NaK in lipid rafts, whereas KCC3a is absent in basal parietal cells.     

Brain-type creatine kinase activates neuron-specific K+-Cl- co-transporter KCC2

GABA, a major inhibitory neurotransmitter in the adult CNS, is excitatory at early developmental stages as a result of the elevated intracellular Cl- concentration ([Cl-]i). This functional switch is primarily attributable to the K+-Cl- co-transporter KCC2, the expression of which is developmentally regulated in neurons. Previously, we reported that KCC2 interacts with brain-type creatine kinase (CKB). To elucidate the functional significance of this interaction, HEK293 cells were transfected with KCC2 and glycine receptor alpha2 subunit, and gramicidin-perforated patch-clamp recordings were performed to measure the glycine reversal potential (Egly), giving an estimate of [Cl-]i. KCC2-expressing cells displayed the expected changes in Egly following alterations in the extracellular K+ concentration ([K+]o) or administration of an inhibitor of KCCs, suggesting that the KCC2 function was being properly assessed. When added into KCC2-expressing cells, dominant-negative CKB induced a depolarizing shift in Egly and reduced the hyperpolarizing shift in Egly seen in response to a lowering of [K+]o compared with wild-type CKB. Moreover, 2,4-dinitrofluorobenzene (DNFB), an inhibitor of CKs, shifted Egly in the depolarizing direction. In primary cortical neurons expressing CKB, the GABA reversal potential was also shifted in the depolarizing direction by DNFB. Our findings suggest that, in the cellular micro-environment, CKB activates the KCC2 function.     

Effect of intracellular magnesium and oxygen tension on K+-Cl- cotransport in normal and sickle human red cells.

In red cells from normal individuals (HbA cells), the K+-Cl- cotransporter (KCC) is inactivated by low O2 tension whilst in those from sickle cell patients (HbS cells), it remains fully active. Changes in free intracellular [Mg2+] have been proposed as a mechanism. In HbA cells, KCC activity was stimulated by Mg2+ depletion and inhibited by Mg2+ loading but the effect of O2 was independent of Mg2+. At all [Mg2+]is, the transporter was stimulated in oxygenated cells, minimally active in deoxygenated ones. By contrast, the stimulatory effects of O2 was abolished by inhibitors of protein (de)phosphorylation. HbS cells had elevated KCC activity, which was of similar magnitude in oxygenated and deoxygenated cells, regardless of Mg2+ clamping. In deoxygenated cells, the antisickling agent dimethyl adipimidate inhibited sickling, Psickle and KCC. Results indicate a role for protein phosphorylation in O2 dependence of KCC, with different activities of the relevant enzymes in HbA and HbS cells, probably dependent on Hb.     

Electroneutral Cation-Chloride Cotransporters in the Central Nervous System

Several members of the cation-chloride cotransporter (solute carrier family 12, SLC12) gene family are expressed within the central nervous system, with one family member, the K+-Cl- cotransporter KCC2, exclusive to neurons. These transporters are best known for their roles in cell volume regulation and epithelial salt transport, but are increasingly receiving attention in neuroscience. In particular, intracellular chloride activity and hence the neuronal response to GABA and glycine appears to be determined by a balance between chloride efflux and influx through KCC2 and the Na+-K+-2Cl- cotransporter NKCC1, respectively. This relationship has important implications for neuronal development, sensory perception, neuronal excitability, and the response to neuronal injury. Finally, the association between loss of function in the K+-Cl- cotransporter KCC3, with a severe peripheral neuropathy associated with agenesis of the corpus callosum, has revealed an unexpected role for K+-Cl- cotransport in the development and/or maintenance of both the central and peripheral nervous systems.     

Functional comparison of the K+-Cl- cotransporters KCC1 and KCC4

The K(+)-Cl(-) cotransporters (KCCs) are members of the cation-chloride cotransporter gene family and fall into two phylogenetic subgroups: KCC2 paired with KCC4 and KCC1 paired with KCC3. We report a functional comparison in Xenopus oocytes of KCC1 and KCC4, widely expressed representatives of these two subgroups. KCC1 and KCC4 exhibit differential sensitivity to transport inhibitors, such that KCC4 is much less sensitive to bumetanide and furosemide. The efficacy of these anion inhibitors is critically dependent on the concentration of extracellular K(+), with much higher inhibition in 50 mm K(+) versus 2 mm K(+). KCC4 is also uniquely sensitive to 10 mm barium and to 2 mm trichlormethiazide. Kinetic characterization reveals divergent affinities for K(+) (K(m) values of approximately 25.5 and 17.5 mm for KCC1 and KCC4, respectively), probably due to variation within the second transmembrane segment. Although the two isoforms have equivalent affinities for Cl(-), they differ in the anion selectivity of K(+) transport (Cl(-) > SCN(-) = Br(-) > PO(4)(-3) > I(-) for KCC1 and Cl(-) > Br(-) > PO(4)(-3) = I(-) > SCN(-) for KCC4). Both KCCs express minimal K(+)-Cl(-) cotransport under isotonic conditions, with significant activation by cell swelling under hypotonic conditions. The cysteine-alkylating agent N-ethylmaleimide activates K(+)-Cl(-) cotransport in isotonic conditions but abrogates hypotonic activation, an unexpected dissociation of N-ethylmaleimide sensitivity and volume sensitivity. Although KCC4 is consistently more volume-sensitive, the hypotonic activation of both isoforms is critically dependent on protein phosphatase 1. Overall, the functional comparison of these cloned K(+)-Cl(-) cotransporters reveals important functional, pharmacological, and kinetic differences with both physiological and mechanistic implications.     

Expression of K+-Cl- cotransporters in the alpha-cells of rat endocrine pancreas

The expression of K+-Cl- cotransporters (KCC) was examined in pancreatic islet cells. mRNA for KCC1, KCC3a, KCC3b and KCC4 were identified by RT-PCR in islets isolated from rat pancreas. In immunocytochemical studies, an antibody specific for KCC1 and KCC4 revealed the expression of KCC protein in alpha-cells, but not pancreatic beta-cells nor delta-cells. A second antibody which does not discriminate among KCC isoforms identified KCC expression in both alpha-cell and beta-cells. Exposure of isolated alpha-cells to hypotonic solutions caused cell swelling was followed by a regulatory volume decrease (RVD). The RVD was blocked by 10 microM [dihydroindenyl-oxy] alkanoic acid (DIOA; a KCC inhibitor). DIOA was without effect on the RVD in beta-cells. NEM (0.2 mM), a KCC activator, caused a significant decrease of alpha-cell volume, which was completely inhibited by DIOA. By contrast, NEM had no effects on beta-cell volume. In conclusion, KCCs are expressed in pancreatic alpha-cells and beta-cells. However, they make a significant contribution to volume homeostasis only in alpha-cells.     

Self-interacting domains in the C terminus of a cation-Cl- cotransporter described for the first time

The first isoform of the Na+-K+-Cl- cotransporter (NKCC1), a widely distributed member of the cation-Cl- cotransporter superfamily, plays key roles in many physiological processes by regulating the ion and water content of animal cells and by sustaining electrolyte secretion across various epithelia. Indirect studies have led to the prediction that NKCC1 operates as a dimer assembled through binding domains that are distal to the amino portion of the carrier. In this study, evidence is presented that NKCC1 possesses self-interacting properties that result in the formation of a large complex between the proximal and the distal segment of the cytosolic C terminus. Elaborate mapping studies of these segments showed that the contact sites are dispersed along the entire C terminus, and they also led to the identification of a critical interacting residue that belongs to a putative forkhead-associated binding domain. In conjunction with previous findings, our results indicate that the uncovered interacting domains are probably a major determinant of the NKCC1 conformational landscape and assembly into a high order structure. A model is proposed in which the carrier could alternate between monomeric and homo-oligomeric units via chemical- or ligand-dependent changes in conformational dynamics.     

Functional association between K+-Cl- cotransporter-4 and H+,K+-ATPase in the apical canalicular membrane of gastric parietal cells

We studied whether K+-Cl(-) cotransporters (KCCs) are involved in gastric HCl secretion. We found that KCC4 is expressed in the gastric parietal cells more abundantly at the luminal region of the gland than at the basal region. KCC4 was found in the stimulation-associated vesicles (SAV) derived from the apical canalicular membrane but not in the intracellular tubulovesicles, whereas H+,K+-ATPase was expressed in both of them. In contrast, KCC1, KCC2, and KCC3 were not found in either SAV or tubulovesicles. KCC4 coimmunoprecipitated with H+,K+-ATPase in the lysate of SAV. Interestingly the MgATP-dependent uptake of (36)Cl(-) into the SAV was suppressed by either the H+,K+-ATPase inhibitor (SCH28080) or the KCC inhibitor ((R)-(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]acetic acid). The KCC inhibitor suppressed the H+ uptake into SAV and the H+,K+-ATPase activity of SAV, but the inhibitor had no effects on these activities in the freeze-dried leaky SAV. These results indicate that the K+-Cl(-) cotransport by KCC4 is tightly coupled with H+/K+ antiport by H+,K+-ATPase, resulting in HCl accumulation in SAV. In the tetracycline-regulated expression system of KCC4 in the HEK293 cells stably expressing gastric H+,K+-ATPase, KCC4 was coimmunoprecipitated with H+,K+-ATPase. The rate of recovery of intracellular pH in the KCC4-expressing cells after acid loading through an ammonium pulse was significantly faster than that in the KCC4-non-expressing cells. Our results suggest that KCC4 and H+,K+-ATPase are the main machineries for basal HCl secretion in the apical canalicular membrane of the resting parietal cell. They also may contribute in part to massive acid secretion in the stimulated state.     

Effect of peroxynitrite on passive K+ transport in human red blood cells.

Peroxynitrite is generated in vivo by the reaction between nitric oxide, from endothelial and other cells, and the superoxide anion. It is therefore pertinent to examine its effects on the membrane permeability of red blood cells. Treatment of human red blood cells with peroxynitrite (nominally 1 mM) markedly stimulated passive K+ permeability. The main effect was on a Cl(-)-independent K+ pathway, which remains unidentified. Although K+-Cl- cotransport (KCC) was stimulated, this was dependent on saline composition, being inhibited by physiological levels of glucose (IC50 4 mM), and also by sucrose and MOPS. Effects on the Cl(-)-independent K+ pathway were less dependent on saline composition, and were not inhibited by amiloride, ethylisopropylamiloride, dimethylamiloride or gadolinium. Na+-K+-2Cl- cotransporter was inhibited whilst there was little effect on the Gardos channel (Ca2+-activated K+ channel). Peroxynitrite was markedly more effective in oxygenated cells than deoxygenated ones. Treatment with peroxynitrite per se did not affect initial cell volume. Anisotonic swelling modestly increased the Cl(-)-independent K+ influx, but did not affect peroxynitrite-stimulated KCC. Decreasing extracellular pH from 7.4 to 7.2 or 7.0 increased KCC stimulation, whilst the Cl(-)-independent component of K+ transport was lowest at pH 7.2. Finally, protein phosphatase inhibition with calyculin A (100 nM) inhibited KCC, implying that, as with other KCC stimuli, peroxynitrite acts via decreased protein phosphorylation; pre-treatment with calyculin A also inhibited the Cl(-)-independent component of K+ transport. These findings are relevant to the actions of peroxynitrite in vivo.     

Allopregnanolone enhancement of GABAergic transmission in rat medial preoptic area neurons

Gamma-aminobutyric acid (GABA)-mediated transmission in the medial preoptic area (MPOA) of the hypothalamus plays an important role in functions such as sex steroid hormone dynamics and control of body temperature. The action of allopregnanolone, the primary metabolite of progesterone, on GABAergic transmission was investigated by employing patch clamp whole cell recording on acutely dissociated rat MPOA neurons with the functional connection of presynaptic terminals. Allopregnanolone enhanced spontaneous GABA release on the MPOA neurons and induced prolonged decay of miniature GABAergic-inhibitory postsynaptic currents (mIPSCs). The facilitation of GABA release from the presynaptic terminals by allopregnanolone disappeared in Ca2+-free extracellular solution. The presynaptic action of this neurosteroid was also blocked by bumetanide, a blocker of cation-Cl- cotransporters, and by removal of extracellular Na+. The results suggest that allopregnanolone enhances GABAergic transmission at the MPOA neurons by pre- and postsynaptic mechanisms. The enhancement of GABA release by allopregnanolone might require a high Cl- concentration in the presynaptic terminal maintained by Na+-dependent, bumetanide-sensitive mechanisms (e.g., Na+-K+-Cl- cotransporter) and might be mediated by Ca2+ influx into presynaptic terminal.     

Genetic selection of mutations in the high affinity K+ transporter HKT1 that define functions of a loop site for reduced Na+ permeability and increased Na+ tolerance

Potassium is an important macronutrient required for plant growth, whereas sodium (Na+) can be toxic at high concentrations. The wheat K+ uptake transporter HKT1 has been shown to function in yeast and oocytes as a high affinity K+-Na+ cotransporter, and as a low affinity Na+ transporter at high external Na+. A previous study showed that point mutations in HKT1, which confer enhancement of Na+ tolerance to yeast, can be isolated by genetic selection. Here we report on the isolation of mutations in new domains of HKT1 showing further large increases in Na+ tolerance. By selection in a Na+ ATPase deletion mutant of yeast that shows a high Na+ sensitivity, new HKT1 mutants at positions Gln-270 and Asn-365 were isolated. Several independent mutations were isolated at the Asn-365 site. N365S dramatically increased Na+ tolerance in yeast compared with all other HKT1 mutants. Cation uptake experiments in yeast and biophysical characterization in Xenopus oocytes showed that the mechanisms underlying the Na+ tolerance conferred by the N365S mutant were: reduced inhibition of high affinity Rb+ (K+) uptake at high Na+ concentrations, reduced low affinity Na+ uptake, and reduced Na+ to K+ content ratios in yeast. In addition, the N365S mutant could be clearly distinguished from less Na+-tolerant HKT1 mutants by a markedly decreased relative permeability for Na+ at high Na+ concentrations. The new mutations contribute to the identification of new functional domains and an amino acid in a loop domain that is involved in cation specificity of a plant high affinity K+ transporter and will be valuable for molecular analyses of Na+ transport mechanisms and stress in plants.     

Diversity of neuron-specific K+-Cl- cotransporter expression and inhibitory postsynaptic potential depression in rat motoneurons.

Motoneurons receive a robust recurrent synaptic inhibition by gamma-aminobutyric acid and glycine, which activate Cl(-) channels. Thus, Cl(-) homeostasis determines the efficacy of synaptic inhibition in the motoneurons. In situ hybridization reveals that the neuronal K(+)-Cl(-) cotransporter isoform 2 (KCC2), a major mechanism in maintaining a low Cl(-) concentration in neurons, is abundantly expressed in the facial, hypoglossal (XII), and spinal motoneurons innervating striated muscle, whereas the dorsal vagal motoneurons (DMVs) controlling smooth muscle exhibited little expression of KCC2. This raises a general interest in the correlation between KCC2 expression and inhibitory postsynaptic potential (IPSP) performance in the native circuits. Intracellular and whole-cell patch recordings revealed that an activity-dependent depression of IPSPs and positive shift of IPSP reversal potentials were more prominent in the DMV than in the XII. Cl(-) influx through Cl(-) channels was extruded more potently in the XII than in the DMV, suggesting that differences in Cl(-) extrusion account for these dynamic differences of IPSP. Cl(-) extrusion was inhibited by either furosemide or an increase in extracellular potassium concentrations. Thus, the rigid maintenance of IPSP and rapid Cl(-) extrusion in the XII reflects an intense expression of KCC2. KCC2 expression may strongly influence the IPSP depression and functional properties of the motoneurons innervating striated muscles.     

Taurine inhibits K+-Cl- cotransporter KCC2 to regulate embryonic Cl- homeostasis via with-no-lysine (WNK) protein kinase signaling pathway

GABA inhibits mature neurons and conversely excites immature neurons due to lower K(+)-Cl(-) cotransporter 2 (KCC2) expression. We observed that ectopically expressed KCC2 in embryonic cerebral cortices was not active; however, KCC2 functioned in newborns. In vitro studies revealed that taurine increased KCC2 inactivation in a phosphorylation-dependent manner. When Thr-906 and Thr-1007 residues in KCC2 were substituted with Ala (KCC2T906A/T1007A), KCC2 activity was facilitated, and the inhibitory effect of taurine was not observed. Exogenous taurine activated the with-no-lysine protein kinase 1 (WNK1) and downstream STE20/SPS1-related proline/alanine-rich kinase (SPAK)/oxidative stress response 1 (OSR1), and overexpression of active WNK1 resulted in KCC2 inhibition in the absence of taurine. Phosphorylation of SPAK was consistently higher in embryonic brains compared with that of neonatal brains and down-regulated by a taurine transporter inhibitor in vivo. Furthermore, cerebral radial migration was perturbed by a taurine-insensitive form of KCC2, KCC2T906A/T1007A, which may be regulated by WNK-SPAK/OSR1 signaling. Thus, taurine and WNK-SPAK/OSR1 signaling may contribute to embryonic neuronal Cl(-) homeostasis, which is required for normal brain development.     

Chromosomal localization of SLC12A5/Slc12a5, the human and mouse genes for the neuron-specific K(+)-Cl(-) cotransporter (KCC2) defines a new region of conserved homology.

K(+)-Cl(-) cotransporters (KCCs) constitute a branch of the cation-chloride cotransporter (CCC) family. To date, four KCC isoforms (KCC1-KCC4) have been identified and they all mediate obligatorily coupled, electroneutral transmembrane movement of K(+) and Cl(-) ions. KCC2 (gene symbol SLC12A5) is expressed exclusively in neurons within the central nervous system and abnormalities in its expression have been proposed to play a role in pathological conditions such as epilepsy and neuronal trauma. Here we have determined chromosome location of both the human and the mouse genes encoding KCC2, which may assist in future efforts to determine the contribution of KCC2 to inherited human disorders. We assigned human SLC12A5 to 20q12-->q13.1 and its murine homolog, Slc12a5, to 5G2-G3 by fluorescence in situ hybridization (FISH). These mapping data are contradictory to the previously reported human-mouse conserved synteny relationships disrupting an exceptionally well-conserved homology segment between human Chr 20 and mouse Chr 2. We hence suggest the first region of conserved homology between human Chr 20 and mouse Chr 5.