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.     

Reaccumulation of [K+]o in the toad retina during maintained illumination

Using K+-selective microelectrodes, [K+]o was measured in the subretinal space of the isolated retina of the toad, Bufo marinus. During maintained illumination, [K+]o fell to a minimum and then recovered to a steady level that was approximately 0.1 mM below its dark level. Spatial buffering of [K+]o by Müller (glial) cells could contribute to this reaccumulation of K+. However, superfusion with substances that might be expected to block glial transport of K+ had no significant effect upon the reaccumulation of K+. These substances included blockers of gK (TEA+, Cs+, Rb+, 4-AP) and a gliotoxin (alpha AAA). Progressive slowing of the rods' Na+/K+ pump (perhaps caused by a light-evoked decrease in [Na+]i) also could contribute to this reaccumulation of K+ by reducing the uptake of K+ from the subretinal space. As evidence for a major contribution by this mechanism, treatments designed to prevent such slowing of the pump reversibly blocked reaccumulation. These treatments included superfusion with 2 microM ouabain, or lowering [K+]o, PO2, or temperature. It is likely that such treatments inhibit the pump, increase [Na+]i, and attenuate any light-evoked decrease in [Na+]i. The results are consistent with the following hypothesis. At light onset, the decrease in rod gNa will reduce the Na+ influx and the resulting rod hyperpolarization will reduce the K+ efflux. In combination with these reduced passive fluxes, the continuing active fluxes will lower both [K+]o and [Na+]i, which in turn will inhibit the pump. In support of this hypothesis, the solutions to a pair of coupled differential equations that model changes in both [K+]o and [Na+]i match quantitatively the time course of the observed changes in [K+]o during and after maintained illumination for all stimuli examined.     

Functional interaction of the K-Cl cotransporter (KCC1) with the Na-K-Cl cotransporter in HEK-293 cells

We have studiedthe regulation of the K-Cl cotransporter KCC1 and its functionalinteraction with the Na-K-Cl cotransporter. K-Cl cotransporter activitywas substantially activated in HEK-293 cells overexpressing KCC1(KCC1-HEK) by hypotonic cell swelling, 50 mM external K, andpretreatment with N-ethylmaleimide(NEM). Bumetanide inhibited ⁸⁶Rbefflux in KCC1-HEK cells after cell swelling [inhibition constant (K_i) ~190µM] and pretreatment with NEM(K_i ~60 µM).Thus regulation of KCC1 is consistent with properties of the red cellK-Cl cotransporter. To investigate functional interactions between K-Cland Na-K-Cl cotransporters, we studied the relationship between Na-K-Clcotransporter activation and intracellular Cl concentration([Cl]_i). Without stimulation, KCC1-HEK cells had greater Na-K-Cl cotransporter activitythan controls. Endogenous Na-K-Cl cotransporter of KCC1-HEK cells wasactivated <2-fold by low-Cl hypotonic prestimulation, compared with10-fold activation in HEK-293 cells and >20-fold activation in cellsoverexpressing the Na-K-Cl cotransporter (NKCC1-HEK). KCC1-HEK cellshad lower resting[Cl]_i than HEK-293cells; cell volume was not different among cell lines. We found a steeprelationship between[Cl]_i and Na-K-Clcotransport activity within the physiological range, supporting aprimary role for [Cl]_iin activation of Na-K-Cl cotransport and in apical-basolateral crosstalk in ion-transporting epithelia.     

Sources and sinks of light-evoked delta [K+]o in the vertebrate retina

In the vertebrate retina, recordings of light-evoked changes in extracellular K+ concentration delta [K+]o are of particular interest because this tissue is complex and multilayered, yet can be activated routinely with its "natural" stimulus (i.e., light). This review identifies the components of the spatiotemporal profile of retinal light-evoked delta [K+]o and then presents evidence concerning the specific neural origins of these components as well as the mechanisms by which these delta [K+]o are dispersed from extracellular space. Finally, to gain improved resolution of K+ sources and sinks, the technique of ion source density is introduced and applied to both model and real spatiotemporal distributions of delta [K+]o.     

Glycolysis and brain function: [K+]o stimulation of protein synthesis and K+ uptake require glycolysis

Glucose is essentially the sole energy substrate in the normally functioning brain. There are, however, situations in which other substrates can partially substitute for glucose and maintain an apparently normal brain function. However, in no case has it been possible to completely substitute other substrates for glucose and maintain normal brain function. Studies on insulin-induced hypoglycemia suggest that this glucose dependence does not result from its involvement in ATP generation. Two explantation that have been offered are that toxic catabolites arise if nonglucose substrates are oxidized or that glycolysis is necessary to maintain neurotransmitter metabolism. We consider a third basis for the glucose requirement: our past studies have shown that hippocampal slice protein synthesis is activated by small increases in extracellular [K+] ([K+]o), and that this results from activation of K+ uptake into brain cells. We find that this process specifically requires aerobic glycolysis. The basis for the requirement appears to be that [K+]o activation of the Na+-K+ pump is specifically dependent on glycolytically generated energy. Thus, it is possible that glucose is required to maintain normal K+ clearance from the extracellular space during neural activity. This could partially account for the dependence of brain function on glycolysis.     

Overdrive excitation in the guinea pig sinoatrial node superfused in high [K+]o

The aim of the present experiments was to study the characteristics and mechanisms of the rhythm induced by overdrive (overdrive excitation, ODE) in the sinoatrial node (SAN) superfused in high [K⁺]_o (8–14 mM). It was found that: (1) overdrive may induce excitation in quiescent SAN and during a slow drive; (2) in spontaneously active SAN, overdrive may accelerate the spontaneous discharge; (3) immediately after the end of overdrive, a pause generally precedes the onset of the induced rhythm; (4) during the pause, an oscillatory potential (V_os) may be superimposed on the early diastolic depolarization (DD); (5) during the subsequent late DD, a different kind of oscillatory potential appears near the threshold for the upstroke (ThV_os) which is responsible for the initiation of spontaneous activity; (6) once started, the induced rhythm is fastest soon after overdrive; (7) faster drives induce longer and faster spontaneous rhythms; (8) the induced action potentials are slow responses followed by DD with a superimposed V_os, but ThV_os is responsible for ODE; (9) the induced rhythm subsides when ThV_os miss the threshold and gradually decay; (10) low [Ca²⁺]_o abolishes ODE; (11) in quiescent SAN, high [Ca²⁺]_o induces spontaneous discharge through ThV_os and increases its rate by enhancing V_os and shifting the threshold to more negative values, and (12) tetrodotoxin abolishes ODE as well as the spontaneous discharge induced by high [Ca²⁺]_o. In conclusion, in K⁺-depolarized SAN, ODE may be present in the apparent absence of calcium overload, is Ca²⁺- and Na⁺-dependent and is mediated by ThV_os and not by V_os.     

Molecular cloning and functional characterization of KCC3, a new K-Cl cotransporter

We isolated and characterized a novelK-Cl cotransporter, KCC3, from human placenta. The deduced proteincontains 1,150 amino acids. KCC3 shares 75-76% identity at theamino acid level with human, pig, rat, and rabbit KCC1 and 67%identity with rat KCC2. KCC3 is 40 and 33% identical to twoCaenorhabditis elegans K-Cl cotransporters and ~20%identical to other members of the cation-chloride cotransporter family(CCC), two Na-K-Cl cotransporters (NKCC1, NKCC2), and the Na-Clcotransporter (NCC). Hydropathy analysis indicates a typical KCCtopology with 12 transmembrane domains, a large extracellular loopbetween transmembrane domains 5 and 6 (unique to KCCs), and largeNH₂ and COOH termini. KCC3 is predominantly expressed inkidney, heart, and brain, and is also expressed in skeletal muscle,placenta, lung, liver, and pancreas. KCC3 was localized to chromosome15. KCC3 transiently expressed in human embryonic kidney (HEK)-293cells fulfilled three criteria for increased expression of K-Clcotransport: stimulation of cotransport by swelling, treatment withN-ethylmaleimide, or treatment with staurosporine.

     

Mouse K-Cl cotransporter KCC1: cloning, mapping, pathological expression, and functional regulation

Although K-Cl cotransporter (KCC1) mRNA is expressed in manytissues, K-Cl cotransport activity has been measured in few cell types,and detection of endogenous KCC1 polypeptide has not yet been reported.We have cloned the mouse erythroid KCC1 (mKCC1) cDNA and its flankinggenomic regions and mapped the mKCC1 gene to chromosome 8. Threeanti-peptide antibodies raised against recombinant mKCC1 function asimmunoblot and immunoprecipitation reagents. The tissue distributionsof mKCC1 mRNA and protein are widespread, and mKCC1 RNA isconstitutively expressed during erythroid differentiation of ES cells.KCC1 polypeptide or related antigen is present in erythrocytes ofmultiple species in which K-Cl cotransport activity has beendocumented. Erythroid KCC1 polypeptide abundance is elevated inproportion to reticulocyte counts in density-fractionated cells, inbleeding-induced reticulocytosis, in mouse models of sickle celldisease and thalassemia, and in the corresponding human disorders.mKCC1-mediated uptake of ⁸⁶Rb intoXenopus oocytes requires extracellularCl, is blocked by thediureticR(+)-[2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-indenyl-5-yl-)oxy]acetic acid, and exhibits an erythroid pattern of acute regulation, with activation by hypotonic swelling,N-ethylmaleimide, and staurosporine and inhibition by calyculin and okadaic acid. These reagents and findings will expedite studies of KCC1 structure-function relationships and of the pathobiology of KCC1-mediated K-Cl cotransport.

     

Force development at elevated [Mg2+]o and [K+]o in myocardium from the freshwater turtle (Trachemys scripta) and influence of factors associated with hibernation

The effects of high [Mg(2+)](o) on force development were examined for heart muscle of freshwater turtle. Plasma [Mg(2+)] during hibernation may increase drastically and like plasma [K(+)] approach values as high as 10 mM. Each experiment performed at either 20 or 5 degrees C involved four ventricular preparations of which one pair was exposed to 10, and one to 1 mMMg(2+). One preparation of each pair was furthermore exposed to 10 mM K(+), whereas the other was maintained at 2.5 mM K(+). During oxygenation, high relative to low [Mg(2+)](o) displayed a weak tendency to reduce twitch force; a tendency that was not reduced by elevations of [Ca(2+)](o). Severe hypoxia accentuated the negative effect of high [Mg(2+)](o). This effect disappeared, however, when hypoxia was combined with acidosis obtained by 24 mM lactic acid. In comparison to [Mg(2+)](o), high [K(+)](o) strongly depressed force development under both oxygenation and hypoxia, but no consistent interplay between the two ions was revealed. The negative inotropic effects of both high [Mg(2+)](o) and high [K(+)](o) were reduced or eliminated by 10 muM adrenaline. In conclusion the cardiac effects of elevations in [Mg(2+)](o) appear to be small during hibernation, in particular when considering the concomitant adrenergic stimulation and acidosis.     

Comparison of changes evoked by GABA (gamma-aminobutyric acid) and anoxia in [K+]o, [Cl-]o, and [Na+]o in stratum pyramidale and stratum radiatum of the guinea pig hippocampus

Ion-selective microelectrode recordings were made to assess a possible contribution of extracellular gamma-aminobutyric acid (GABA) accumulation to early responses evoked in the brain by anoxia and ischemia. Changes evoked by GABA or N2 in [K+]o, [Cl-]o, [Na+]o, and [TMA+]o were recorded in the cell body and dendritic regions of the stratum pyramidale (SP) and stratum radiatum (SR), respectively, of pyramidal neurons in CA1 of guinea pig hippocampal slices. Bath application of GABA (1-10 mM) for approximately 5 min evoked changes in [K+]o and [Cl-]o with respective EC50 levels of 3.8 and 4.1 mM in SP, and 4.7 and 5.6 mM in SR. In SP 5 mM GABA reversibly increased [K+]o and [Cl-]o and decreased [Na+]o; replacement of 95% O2 -5% CO2 by 95% N2 -5% CO2 for a similar period of time evoked changes which were for each ion in the same direction as those with GABA. In SR both GABA and N2 caused increases in [K+]o and decreases in [Cl-]o and [Na+]. The reduction of extracellular space, estimated from levels of [TMA+]o during exposures to GABA and N2, was 5-6% and insufficient to cause the observed changes in ion concentration. Ion changes induced by GABA and N2 were reversibly attenuated by the GABA(A) receptor antagonist bicuculline methiodide (BMI, 100 microM). GABA-evoked changes in [K+]o in SP and SR and [Cl-]o in SP were depressed by > or =90%, and of [Cl-]o in SR by 50%; N2-evoked changes in [K+]o in SP and SR were decreased by 70% and those of [Cl-]o by 50%. BMI blocked delta [Na+]o with both GABA and N2 by 20-30%. It is concluded that during early anoxia: (i) accumulation of GABA and activation of GABA(A) receptors may contribute to the ion changes and play a significant role, and (ii) responses in the dendritic (SR) regions are greater than and (or) differ from those in the somal (SP) layers. A large component of the [K+]o increase may involve a GABA-evoked Ca2+-activated gk, secondary to [Ca2+]i increase. A major part of [Cl-]o changes may arise from GABA-induced g(Cl) and glial efflux, with strong stimulation of active outward transport and anion exchange at SP, and inward Na+/K+/2Cl- co-transport at SR. Na+ influx is attributable mainly to Na+-dependent transmitter uptake, with only a small amount related to GABA(A) receptor activation. Although the release and (or) accumulation of GABA during anoxia might be viewed as potentially protectant, the ultimate role may more likely be an important contribution to toxicity and delayed neuronal death.     

Changes of action potentials and force at lowered [Na+]o in mouse skeletal muscle: implications for fatigue

We examined 1) whether the effects of lowered trans-sarcolemmal Na⁺ gradient on force differed between nonfatigued fast- and slow-twitch muscles of mice and 2) whether effects on action potentials could explain the decrease of force. The Na⁺ gradient was reduced by lowering the extracellular [Na⁺] ([Na⁺]_o). The peak force-[Na⁺]_o relationships for the twitch and tetanus were the same in nonfatigued extensor digitorum longus and soleus muscles: force was maintained over a large range of [Na⁺]_o and then decreased abruptly over a much smaller range. However, fatigue was significantly exacerbated at a lowered [Na⁺]_o that had little effect in nonfatigued soleus muscle. This finding suggests that substantial differences exist in the Na⁺ effect on force between nonfatigued and fatigued muscle. The reduced contractility in nonfatigued muscles at lowered [Na⁺]_o was largely due to 1) an increased number of inexcitable fibers and threshold for action potentials, 2) a reduction of action potential amplitude, and 3) a reduced capacity to generate action potentials throughout trains. sodium gradient; muscle contraction; action potential train; extensor digitorum longus; soleus     

The neuron-specific K-Cl cotransporter, KCC2. Antibody development and initial characterization of the protein

The neuron-specific K-Cl cotransporter (KCC2) is hypothesized to function as an active Cl- extrusion pathway important in postsynaptic inhibition mediated by ligand-gated anion channels, like gamma-aminobutyric acid type A (GABAA) and glycine receptors. To understand better the functional role of KCC2 in the nervous system, we developed polyclonal antibodies to a KCC2 fusion protein and used these antibodies to characterize and localize KCC2 in the rat cerebellum. The antibodies specifically recognized the KCC2 protein which is an approximately 140-kDa glycoprotein detectable only within the central nervous system. The KCC2 protein displayed a robust and punctate distribution in primary cultured retinal amacrine cells known to form exclusively GABAAergic synapses in culture. In immunolocalization studies, KCC2 was absent from axons and glia but was highly expressed at neuronal somata and dendrites, indicating a specific postsynaptic distribution of the protein. In the granule cell layer, KCC2 exhibited a distinct colocalization with the beta2/beta3-subunits of the GABAA receptor at the plasma membrane of granule cell somata and at cerebellar glomeruli. KCC2 lightly labeled the plasma membrane of Purkinje cell somata. Within the molecular layer, KCC2 exhibited a distinctly punctate distribution along dendrites, indicating it may be highly localized at inhibitory synapses along these processes. The distinct postsynaptic localization of KCC2 and its colocalization with GABAA receptor in the cerebellum are consistent with the putative role of KCC2 in neuronal Cl- extrusion and postsynaptic inhibition.     

Synaptic transmission at high pressure: Effects of [Ca2+]o

• 1.1. The effects of pressure on synaptic currents were examined in crayfish abdominal muscles.
• 2.2. Helium pressure (10.1 MPa) considerably decreased extracellulariy-recorded excitatory junctional potentials associated with increased short-term facilitation.
• 3.3. These effects could be mimicked by a reduction of [Ca²⁺]_o, and partially compensated by an increase in [Ca²⁺]_o.
• 4.4. Pressure also reduced the amplitude of the extracellular nerve terminal potentials (ENTP) by up to 25%, and significantly increased synaptic delay in a [Ca²⁺]_o-dependent manner.
• 5.5. The interaction between compression and various [Ca²⁺]_o were analysed in terms of an existing model of transmitter release. The results were consistent with the hypothesis that high pressure decreases the maximal Ca²⁺ influx into nerve terminals.
• 6.6. The decreased ENTP and increased synaptic delay suggest that additional processes may be involved in pressure effects on synaptic transmission.

     

A dominant negative mutant of the KCC1 K-Cl cotransporter: both N- and C-terminal cytoplasmic domains are required for K-Cl cotransport activity

K-Cl cotransport regulates cell volume and chloride equilibrium potential. Inhibition of erythroid K-Cl cotransport has emerged as an important adjunct strategy for the treatment of sickle cell anemia. However, structure-function relationships among the polypeptide products of the four K-Cl cotransporter (KCC) genes are little understood. We have investigated the importance of the N- and C-terminal cytoplasmic domains of mouse KCC1 to its K-Cl cotransport function expressed in Xenopus oocytes. Truncation of as few as eight C-terminal amino acids (aa) abolished function despite continued polypeptide accumulation and surface expression. These C-terminal loss-of-function mutants lacked a dominant negative phenotype. Truncation of the N-terminal 46 aa diminished function. Removal of 89 or 117 aa (Delta(N)117) abolished function despite continued polypeptide accumulation and surface expression and exhibited dominant negative phenotypes that required the presence of the C-terminal cytoplasmic domain. The dominant negative loss-of-function mutant Delta(N)117 was co-immunoprecipitated with wild type KCC1 polypeptide, and its co-expression did not reduce wild type KCC1 at the oocyte surface. Delta(N)117 also exhibited dominant negative inhibition of human KCC1 and KCC3 and, with lower potency, mouse KCC4 and rat KCC2.     

A novel N-terminal isoform of the neuron-specific K-Cl cotransporter KCC2

The neuronal K-Cl cotransporter KCC2 maintains the low intracellular chloride concentration required for the hyperpolarizing actions of inhibitory neurotransmitters gamma-aminobutyric acid and glycine in the central nervous system. This study shows that the mammalian KCC2 gene (alias Slc12a5) generates two neuron-specific isoforms by using alternative promoters and first exons. The novel KCC2a isoform differs from the only previously known KCC2 isoform (now termed KCC2b) by 40 unique N-terminal amino acid residues, including a putative Ste20-related proline alanine-rich kinase-binding site. Ribonuclease protection and quantitative PCR assays indicated that KCC2a contributes 20-50% of total KCC2 mRNA expression in the neonatal mouse brain stem and spinal cord. In contrast to the marked increase in KCC2b mRNA levels in the cortex during postnatal development, the overall expression of KCC2a remains relatively constant and makes up only 5-10% of total KCC2 mRNA in the mature cortex. A rubidium uptake assay in human embryonic kidney 293 cells showed that the KCC2a isoform mediates furosemide-sensitive ion transport activity comparable with that of KCC2b. Mice that lack both KCC2 isoforms die at birth due to severe motor defects, including disrupted respiratory rhythm, whereas mice with a targeted disruption of the first exon of KCC2b survive for up to 2 weeks but eventually die due to spontaneous seizures. We show that these mice lack KCC2b but retain KCC2a mRNA. Thus, distinct populations of neurons show a differential dependence on the expression of the two isoforms: KCC2a expression in the absence of KCC2b is presumably sufficient to support vital neuronal functions in the brain stem and spinal cord but not in the cortex.     

Functional characterization of thioesterase superfamily member 1/Acyl-CoA thioesterase 11: implications for metabolic regulation

Thioesterase superfamily member 1 (Them1; synonyms acyl-CoA thioesterase 11 and StarD14) is highly expressed in brown adipose tissue and limits energy expenditure in mice. Them1 is a putative fatty acyl-CoA thioesterase that comprises tandem hot dog-fold thioesterase domains and a lipid-binding C-terminal steroidogenic acute regulatory protein-related lipid transfer (START) domain. To better define its role in metabolic regulation, this study examined the biochemical and enzymatic properties of Them1. Purified recombinant Them1 dimerized in solution to form an active fatty acyl-CoA thioesterase. Dimerization was induced by fatty acyl-CoAs, coenzyme A (CoASH), ATP, and ADP. Them1 hydrolyzed a range of fatty acyl-CoAs but exhibited a relative preference for long-chain molecular species. Thioesterase activity varied inversely with temperature, was stimulated by ATP, and was inhibited by ADP and CoASH. Whereas the thioesterase domains of Them1 alone were sufficient to yield active recombinant protein, the START domain was required for optimal enzyme activity. An analysis of subcellular fractions from mouse brown adipose tissue and liver revealed that Them1 contributes principally to the fatty acyl-CoA thioesterase activity of microsomes and nuclei. These findings suggest that under biological conditions, Them1 functions as a lipid-regulated fatty acyl-CoA thioesterase that could be targeted for the management of metabolic disorders.     

On the inhibition of [Na+,K+]-ATPases by the components of Naja mossambica mossambica venom: evidence for two distinct rat brain [Na+,K+]-ATPase activities

We have compared the effects of highly purified preparations of cardiotoxins and phospholipases A2 from Naja mossambica mossambica venom on rat brain [Na+,K+]-ATPase activity. The results were the following: (i) micromolar concentrations of cardiotoxin preparations were required to inhibit [Na+,K+]-ATPase activity to the extent achieved by picomolar concentrations of phospholipases A2; i.e., the inhibitory effect of cardiotoxins appeared to be related to the contamination of the preparations by trace amounts of phospholipase A2; (ii) comparing phospholipases A2 from varied origins, a correlation was observed between [Na+,K+]-ATPase inhibition, isoelectric point, and toxicity for mice; (iii) when rat brain membranes were used, incubation for extended times with the most basic N. mossambica mossambica phospholipase A2 resulted in a biphasic [Na+,K+]-ATPase inhibition, suggesting that two distinct [Na+,K+]-ATPases were affected differentially. In contrast, incubation of rat brain membranes with either porcine pancreatic phospholipase A2, notexin, or beta-bungarotoxin and also incubation of erythrocyte membranes with the most basic N. mossambica mossambica phospholipase A2 produced monophasic [Na+,K+]-ATPase inhibitions. We discuss a possible specific action of toxic, basic phospholipase A2 on one of the [Na+,K+]-ATPase isoforms of excitable membranes.