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 γ‐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. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 558–568, 1999     

Apoptosis-associated tyrosine kinase scaffolding of protein phosphatase 1 and SPAK reveals a novel pathway for Na-K-2C1 cotransporter regulation

Previous work from our laboratory and others has established that Ste-20-related proline-alanine-rich kinase (SPAK/PASK) is central to the regulation of NKCC1 function. With no lysine (K) kinase (WNK4) has also been implicated in the regulation of NKCC1 activity through upstream activation of SPAK. Because previous studies from our laboratory also demonstrated a protein-protein interaction between SPAK and apoptosis-associated tyrosine kinase (AATYK), we explore here the possibility that AATYK is another component of the regulation of NKCC1. Heterologous expression of AATYK1 in NKCC1-injected Xenopus laevis oocytes markedly inhibited cotransporter activity under isosmotic conditions. Interestingly, mutation of key residues in the catalytic domain of AATYK1 revealed that the kinase activity does not play a role in the suppression of NKCC1 function. However, mutagenesis of the two SPAK-binding motifs in AATYK1 completely abrogated this effect. As protein phosphatase 1 (PP1) also plays a central role in the dephosphorylation and inactivation of NKCC1, we investigated the possibility that AATYK1 interacts with the phosphatase. We identified a PP1 docking motif in AATYK1 and demonstrated interaction using yeast-2-hybrid analysis. Mutation of a key valine residue (V1175) within this motif prevented protein-protein interaction. Furthermore, the physical interaction between PP1 and AATYK was required for inhibition of NKCC1 activity in Xenopus laevis oocytes. Taken together, our data are consistent with AATYK1 indirectly inhibiting the SPAK/WNK4 activation of the cotransporter by scaffolding an inhibitory phosphatase in proximity to a stimulatory kinase. ion fluxes; Xenopus laevis oocytes; yeast-2 hybrid; phosphorylation     

Short Forms of Ste20-related Proline/Alanine-rich Kinase (SPAK) in the Kidney Are Created by Aspartyl Aminopeptidase (Dnpep)-mediated Proteolytic Cleavage

The Ste20-related kinase SPAK regulates sodium, potassium, and chloride transport in a variety of tissues. Recently, SPAK fragments, which lack the catalytic domain and are inhibitory to Na⁺ transporters, have been detected in kidney. It has been hypothesized that the fragments originate from alternative translation start sites, but their precise origin is unknown. Here, we demonstrate that kidney lysate possesses proteolytic cleavage activity toward SPAK. Ion exchange and size exclusion chromatography combined with mass spectrometry identified the protease as aspartyl aminopeptidase. The presence of the protease was verified in the active fractions, and recombinant aspartyl aminopeptidase recapitulated the cleavage pattern observed with kidney lysate. Identification of the sites of cleavage by mass spectrometry allowed us to test the function of the smaller fragments and demonstrate their inhibitory action toward the Na⁺-K⁺-2Cl⁻ cotransporter, NKCC2.     

Expression of Lex antigen in Schistosoma japonicum and S.haematobium and immune responses to Lex in infected animals: lack of Lex expression in other trematodes and nematodes

Adults of the human parasitic trematode Schistosoma mansoni, which causes hepatosplenic/intestinal complications in humans, synthesize glycoconjugates containing the Lewis x (Lex) Galbeta1-->4(Fucalpha1-- >3)GlcNAcbeta1-->R, but not sialyl Lewis x (sLex), antigen. We now report on our analyses of Lexand sLexexpression in S.haematobium and S.japonicum, which are two other major species of human schistosomes that cause disease, and the possible autoimmunity to these antigens in infected individuals. Antigen expression was evaluated by both ELISA and Western blot analyses of detergent extracts of parasites using monoclonal antibodies. Several high molecular weight glycoproteins in both S. haematobium and S. japonicum contain the Lexantigen, but no sialyl Lexantigen was detected. In addition, sera from humans and rodents infected with S.haematobium and S.japonicum contain antibodies reactive with Lex. These results led us to investigate whether Lexantigens are expressed in other helminths, including the parasitic trematode Fasciola hepatica , the parasitic nematode Dirofilaria immitis (dog heartworm), the ruminant nematode Haemonchus contortus , and the free-living nematode Caenorhabditis elegans . Neither Lexnor sialyl-Lexis detectable in these other helminths. Furthermore, none of the helminths, including schistosomes, express Lea, Leb, Ley, or the H- type 1 antigen. However, several glycoproteins from all helminths analyzed are bound by Lotus tetragonolobus agglutinin , which binds Fucalpha1-->3GlcNAc, and Wisteria floribunda agglutinin, which binds GalNAcbeta1-->4GlcNAc (lacdiNAc or LDN). Thus, schistosomes may be unique among helminths in expressing the Lexantigen, whereas many different helminths may express alpha1,3-fucosylated glycans and the LDN motif.      

A critical role for GluN2B-containing NMDA receptors in cortical development and function

The subunit composition of N-methyl D-aspartate receptors (NMDARs) is tightly regulated during cortical development. NMDARs are initially dominated by GluN2B (NR2B), whereas GluN2A (NR2A) incorporation increases after birth. The function of GluN2B-containing NMDARs during development, however, is incompletely understood. We generated a mouse in which we genetically replaced GluN2B with GluN2A (2B→2A). Although this manipulation restored NMDAR-mediated currents at glutamatergic synapses, it did not rescue GluN2B loss of function. Protein translation-dependent homeostatic synaptic plasticity is occluded in the absence of GluN2B, and AMPA receptor contribution is enriched at excitatory cortical synapses. Our experiments indicate that specificity of GluN2B-mediated signaling is due to its unique interaction with the protein effector alpha calcium-calmodulin kinase II and the regulation of the mTOR pathway. Homozygous 2B→2A mice exhibited high rates of lethality, suppressed feeding, and depressed social exploratory behavior. These experiments indicate that GluN2B-containing NMDARs activate unique cellular processes that cannot be rescued by replacement with GluN2A.     

NKCC1 transporter facilitates seizures in the developing brain

During development, activation of Cl(-)-permeable GABA(A) receptors (GABA(A)-R) excites neurons as a result of elevated intracellular Cl(-) levels and a depolarized Cl(-) equilibrium potential (E(Cl)). GABA becomes inhibitory as net outward neuronal transport of Cl(-) develops in a caudal-rostral progression. In line with this caudal-rostral developmental pattern, GABAergic anticonvulsant compounds inhibit motor manifestations of neonatal seizures but not cortical seizure activity. The Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) facilitates the accumulation of Cl(-) in neurons. The NKCC1 blocker bumetanide shifted E(Cl) negative in immature neurons, suppressed epileptiform activity in hippocampal slices in vitro and attenuated electrographic seizures in neonatal rats in vivo. Bumetanide had no effect in the presence of the GABA(A)-R antagonist bicuculline, nor in brain slices from NKCC1-knockout mice. NKCC1 expression level versus expression of the Cl(-)-extruding transporter (KCC2) in human and rat cortex showed that Cl(-) transport in perinatal human cortex is as immature as in the rat. Our results provide evidence that NKCC1 facilitates seizures in the developing brain and indicate that bumetanide should be useful in the treatment of neonatal seizures.     

A Novel Ste20-related Proline/Alanine-rich Kinase (SPAK)-independent Pathway Involving Calcium-binding Protein 39 (Cab39) and Serine Threonine Kinase with No Lysine Member 4 (WNK4) in the Activation of Na-K-Cl Cotransporters

Na⁺-dependent chloride cotransporters (NKCC1, NKCC2, and NCC) are activated by phosphorylation to play critical roles in diverse physiological responses, including renal salt balance, hearing, epithelial fluid secretion, and volume regulation. Serine threonine kinase WNK4 (With No K = lysine member 4) and members of the Ste20 kinase family, namely SPAK and OSR1 (Ste20-related proline/alanine-rich kinase, Oxidative stress-responsive kinase) govern phosphorylation. According to present understanding, WNK4 phosphorylates key residues within SPAK/OSR1 leading to kinase activation, allowing SPAK/OSR1 to bind to and phosphorylate NKCC1, NKCC2, and NCC. Recently, the calcium-binding protein 39 (Cab39) has emerged as a binding partner and enhancer of SPAK/OSR1 activity, facilitating kinase autoactivation and promoting phosphorylation of the cotransporters. In the present study, we provide evidence showing that Cab39 differentially interacts with WNK4 and SPAK/OSR1 to switch the classic two kinase cascade into a signal kinase transduction mechanism. We found that WNK4 in association with Cab39 activates NKCC1 in a SPAK/OSR1-independent manner. We discovered that WNK4 possesses a domain that bears close resemblance to the SPAK/OSR1 C-terminal CCT/PF2 domain, which is required for physical interaction between the Ste20 kinases and the Na⁺-driven chloride cotransporters. Modeling, yeast two-hybrid, and functional data reveal that this PF2-like domain located downstream of the catalytic domain in WNK4 promotes the direct interaction between the kinase and NKCC1. We conclude that in addition to SPAK and OSR1, WNK4 is able to anchor itself to the N-terminal domain of NKCC1 and to promote cotransporter activation.     

Kinetics of DIDS inhibition of swelling-activated K-Cl contrasport in low K sheep erythrocytes

Summary The inhibitory effect of various stilbene disulfonates was examined on the swelling-activated Cl-dependent K transport (K-Cl cotransport) in low K sheep erythrocytes. Both diisothiocy-anatostilbenes H₂DIDS and DIDS were found to be potent inhibitors. The DIDS concentration yielding 50% inhibition (IC₅₀) of KCl cotransport was 60 m in the absence of external K and 3 m at physiological K concentration. Other stilbene derivatives, such as SITS (4-acetamido-4 isothiocyanatostilbene-2,2-disulfonic acid), were only effective in the presence of external K, whereas DNDS (4,4-dinitrostilbene-2,2-disulfonic acid) and ISA (4-sulfophenyl isothiocyanate) had only slight effects at a concentration of 1 mm. The augmenting effect of external K is due to a second K site, distinguishable from the K transport site by its much higher affinity. No inhibition occurred in the absence of external Cl, whether or not external Rb(K) was present. Additionally, DIDS inhibited K-Cl cotransport activated by thiol alkylation with N-ethylmaleimide (NEM) as well as by Mg depletion in the presence of A23187 and a chelator. We conclude that allosteric sites affect the stilbene binding. When these sites are saturated, changes in external K or Cl concentration do not affect the affinity for DIDS (noncompetitive inhibition).This work was supported by grants in aid from the American Heart Association.