Loss of cell viability by histidine substitution of leucine 325 of the glutamate transporter EAAT1

Although glutamate transporters and neutral amino acid transporters have 55% amino acid identity in the transmembrane domains, many residues are still unique to individual transporters, providing for structural stability or substrate binding. In this study, the mutant protein L325H, which replaced a leucine 325 of the glutamate transporter EAAT1 by a histidine, was evaluated. When expressed in Xenopus oocytes, L325H caused oocytes to weaken pigmentation in the animal pole, accompanied by patches of colorless spots. Oocytes finally oozed cytoplasm. The resting membrane potential in L325H oocytes was -18.9 +/- 2.5 mV, significantly more positive than -37.3 +/- 2.5 mV of oocytes expressing EAAT1. The holding current at -60 mV was 283.1 +/- 48.3 nA in L325H oocytes and 92.2 +/- 12.6 nA in EAAT1 oocytes. These results suggest that even though glutamate and neutral amino acid transporters have strong overall homology, the local structure in the transmembrane domains may be different.     

Thrombin-stimulated glutamate uptake in human platelets is predominantly mediated by the glial glutamate transporter EAAT2

Glutamate toxicity has been implicated in the pathogenesis of various neurological diseases. Glial glutamate transporters play a key role in the regulation of extracellular glutamate levels in the brain by removing glutamate from the extracellular fluid. Since human blood platelets possess an active glutamate uptake system, they have been used as a peripheral model of glutamate transport in the central nervous system (CNS). The present study is aimed at identifying the glutamate transporter on blood platelets, and to asses the influence of platelet activation on glutamate uptake. Platelets from healthy donors showed Na+-dependent glutamate uptake (Km, 3.5+/-0.9 microM; Vmax, 2.8+/-0.2 pmol glutamate/75 x 10(6)platelets/30 min), which could be blocked dose-dependently by the EAAT specific inhibitors DL-threo-E-benzyloxyaspartate (TBOA), L-trans-pyrrolidine-2,4-dicarboxylic acid (tPDC) and high concentrations of the EAAT2 inhibitor dihydrokainate (DHK). Analysis of platelet homogenates on Western blots showed EAAT2 as the predominant glutamate transporter. Platelet activation by thrombin caused an increase in glutamate uptake, which could be inhibited by TBOA and the EAAT2 inhibitor DHK. Kinetic analysis showed recruitment of new transporters to the membrane. Indeed, Western blot analysis of subcellular fractions revealed that alpha-granules, which fuse with the membrane upon thrombin stimulation, contained significant EAAT2 immunoreactivity. Inhibition of the second messengers involved in alpha-granule secretion (protein kinase C, phosphatidylinositol-3-kinase) inhibited thrombin-stimulated uptake, but not basal uptake. These data show that the glial EAAT2 is the predominant glutamate transporter on blood platelets and suggest, that thrombin increases glutamate uptake capacity by recruiting new transporters (EAAT2) from alpha-granules.     

Alteration of the transmembrane K+ gradient during development of delayed rectifier in isolated rat pulmonary arterial cells

The properties of the tail current associated with the delayed rectifier K+ current (IK) in isolated rat pulmonary artery smooth muscle cells were examined using the whole cell patch clamp technique. The tail currents observed upon repolarization to -60 mV after brief (e.g., 20 ms) or small (i.e. to potentials negative of 0 mV) depolarizations were outwardly directed, as expected given the calculated K+ reversal potential of -83 mV. The tail currents seen upon repolarization after longer (e.g., 500 ms) and larger (e.g., to +60 mV) depolarizations tended to be inwardly directed. Depolarizations of intermediate strength and/or duration were followed by biphasic tail currents, which were inwardly directed immediately upon repolarization, but changed direction and became outwardly directed before deactivation was complete. When cells were depolarized to +60 mV for 500 ms both IK and the subsequent inward tail current at -60 mV were similarly blocked by phencyclidine. Both IK and the inward tail current were also blocked by 4-aminopyridine. Application of progressively more depolarized 30 s preconditioning potentials inactivated IK, and reduced the inward tail current amplitude with a similar potential dependency. These results indicated that the inward tail current was mediated by IK. The reversal potential of the tail current became progressively more positive with longer depolarizations to +60 mV, shifting from -76.1 +/- 2.2 mV (n = 10) after a 20-ms step to -57.7 +/- 3.5 mV (n = 9) after a 500-ms step. Similar effects occurred when extracellular K+ and Na+ were replaced by choline. When extracellular K+ was raised to 50 mM, the tail current was always inwardly directed at -60 mV, but showed little change in amplitude as the duration of depolarization was increased. These observations are best explained if the dependencies of tail current direction and kinetics upon the duration of the preceding depolarization result from an accumulation of K+ at the external face of the membrane, possibly in membrane invaginations. A mathematical model which simulates the reversal potential shift and the biphasic kinetics of the tail current on this basis is presented.     

Preconditioning with cortical spreading depression decreases intraischemic cerebral glutamate levels and down-regulates excitatory amino acid transporters EAAT1 and EAAT2 from rat cerebal cortex plasma membranes

We previously reported a 50% reduction in cortical infarct volume following transient focal cerebral ischemia in rats preconditioned 3 days earlier with cortical spreading depression (CSD). The mechanism of the protective effect of prior CSD remains unknown. Recent studies demonstrate reversal of excitatory amino acid transporters (EAATs) to be a principal cause for elevated extracellular glutamate levels during cerebral ischemia. The present study measured the effect of CSD preconditioning on (a) intraischemic glutamate levels and (b) regulation of glutamate transporters within the ischemic cortex of the rat. Three days following either CSD or sham preconditioning, rats were subjected to 200 min of focal cerebral ischemia, and extracellular glutamate concentration was measured by in vivo microdialysis. Cortical glutamate exposure decreased 70% from 1,772.4 +/- 1,469.2 microM-min in sham-treated (n = 8) to 569.0 +/- 707.8 microM-min in CSD-treated (n = 13) rats (p <0.05). The effect of CSD preconditioning on glutamate transporter levels in plasma membranes (PMs) prepared from rat cerebral cortex was assessed by western blot analysis. Down-regulation of the glial glutamate transporter isoforms EAAT2 and EAAT1 from the PM fraction was observed at 1, 3, and 7 days but not at 0 or 21 days after CSD. Semiquantitative lane analysis showed a maximal decrease of 90% for EAAT2 and 50% for EAAT1 at 3 days post-CSD. The neuronal isoform EAAT3 was unaffected by CSD. This period of down-regulation coincides with the time frame reported for induced ischemic tolerance. These data are consistent with reversal of glutamate transporter function contributing to glutamate release during ischemia and suggest that down-regulation of these transporters may contribute to ischemic tolerance induced by CSD.     

Regulation of glial glutamate transporters by C-terminal domains

Excitatory amino acid transporter 2 (EAAT2) is a high affinity glutamate transporter predominantly expressed in astroglia. Human EAAT2 encompasses eight transmembrane domains and a 74-amino acid C-terminal domain that resides in the cytoplasm. We examined the role of this region by studying various C-terminal truncations and mutations using heterologous expression in mammalian cells, whole-cell patch clamp recording and confocal imaging. Removal of the complete C terminus (K498X EAAT2) results in loss of function because of intracellular retention of truncated proteins in the cytoplasm. However, a short stretch of amino acids (E500X EAAT2) within the C terminus results in correctly processed transporters. E500X reduced glutamate transport currents by 90%. Moreover, the voltage and substrate dependence of E500X EAAT2 anion currents was significantly altered. WT and mutant EAAT2 anion channels are modified by external Na(+) in the presence as well as in the absence of L-glutamate. Whereas Na(+) stimulates EAAT2 anion currents in the presence of L-glutamate, increased [Na(+)] reduces such currents without glutamate. In cells internally dialyzed with Na(+), WT, and truncated EAAT2 display comparable Na(+) dependence. With K(+) as main internal cation, E500X drastically increased the apparent dissociation constant for external Na(+). The effects of E500X can be represented by a kinetic model that allows translocation of the empty transporter from the outward- to the inward-facing conformation and stabilization of the inward-facing conformation by internal K(+). Our results demonstrate that the C terminus modifies the glutamate uptake cycle, possibly affecting the movements of the translocation domain of EAAT2 glutamate transporter.     

A Conserved Aspartate Determines Pore Properties of Anion Channels Associated with Excitatory Amino Acid Transporter 4 (EAAT4)

Excitatory amino acid transporter (EAAT) glutamate transporters function not only as secondary active glutamate transporters but also as anion channels. Recently, a conserved aspartic acid (Asp¹¹²) within the intracellular loop near to the end of transmembrane domain 2 was proposed as a major determinant of substrate-dependent gating of the anion channel associated with the glial glutamate transporter EAAT1. We studied the corresponding mutation (D117A) in another EAAT isoform, EAAT4, using heterologous expression in mammalian cells, whole cell patch clamp, and noise analysis. In EAAT4, D117A modifies unitary conductances, relative anion permeabilities, as well as gating of associated anion channels. EAAT4 anion channel gating is characterized by two voltage-dependent gating processes with inverse voltage dependence. In wild type EAAT4, external l-glutamate modifies the voltage dependence as well as the minimum open probabilities of both gates, resulting in concentration-dependent changes of the number of open channels. Not only transport substrates but also anions affect wild type EAAT4 channel gating. External anions increase the open probability and slow down relaxation constants of one gating process that is activated by depolarization. D117A abolishes the anion and glutamate dependence of EAAT4 anion currents and shifts the voltage dependence of EAAT4 anion channel activation by more than 200 mV to more positive potentials. D117A is the first reported mutation that changes the unitary conductance of an EAAT anion channel. The finding that mutating a pore-forming residue modifies gating illustrates the close linkage between pore conformation and voltage- and substrate-dependent gating in EAAT4 anion channels.     

Interaction of Ca2+ agonist and depolarization on Ca2+ channel current in guinea pig detrusor cells [published erratum appears in J Gen Physiiol 1996 Jun;107(6):755]

The interaction of large depolarization and dihydropyridine Ca2+ agonists, both of which are known to enhance L-type Ca2+ channel current, was examined using a conventional whole-cell clamp technique. In guinea pig detrusor cells, only L-type Ca2+ channels occur. A second open state (long open state: O2) of the Ca2+ channels develops during large depolarization (at +80 mV, without Ca2+ agonists). This was judged from lack of inactivation of the Ca2+ channel current during the large depolarizing steps (5 s) and slowly deactivating inward tail currents (= 10-15 ms) upon repolarization of the cell membrane to the holding potential (-60 mV). Application of Bay K 8644 (in 2.4 mM Ca(2+)- containing solutions) increased the amplitude of the Ca2+ currents evoked by simple depolarizations, and made it possible to observe inward tail currents (= 2.5-5 ms at -60 mV). The open state induced by large depolarization (O2*) in the Bay K 8644 also seemed hardly to inactivate. After preconditioning with large depolarizing steps, the decay time course of the inward tail currents upon repolarization to the holding potential (-60 mV) was significantly slowed, and could be fitted reasonably with two exponentials. The fast and slow time constants were 10 and 45 ms, respectively, after 2 s preconditioning depolarizations. Qualitatively the same results were obtained using Ba2+ as a charge carrier. Although the amplitudes of the inward currents observed in the test step and the subsequent repolarization to the holding potential were decreased in the same manner by additional application of nifedipine (in the presence of Bay K 8644), the very slow deactivation time course of the tail current was little changed. The additive enhancement by large depolarization and Ca2+ agonists of the inward tail current implies that two mechanisms separately induce long opening of the Ca2+ channels: i.e., that there are four open states.     

Functional Properties of the Retinal Glutamate Transporters GLT-1c and EAAT5

In the mammalian retina, glutamate uptake is mediated by members of a family of glutamate transporters known as “excitatory amino acid transporters (EAATs).” Here we cloned and functionally characterized two retinal EAATs from mouse, the GLT-1/EAAT2 splice variant GLT-1c, and EAAT5. EAATs are glutamate transporters and anion-selective ion channels, and we used heterologous expression in mammalian cells, patch-clamp recordings and noise analysis to study and compare glutamate transport and anion channel properties of both EAAT isoforms. We found GLT-1c to be an effective glutamate transporter with high affinity for Na⁺ and glutamate that resembles original GLT-1/EAAT2 in all tested functional aspects. EAAT5 exhibits glutamate transport rates too low to be accurately measured in our experimental system, with significantly lower affinities for Na⁺ and glutamate than GLT-1c. Non-stationary noise analysis demonstrated that GLT-1c and EAAT5 also differ in single-channel current amplitudes of associated anion channels. Unitary current amplitudes of EAAT5 anion channels turned out to be approximately twice as high as single-channel amplitudes of GLT-1c. Moreover, at negative potentials open probabilities of EAAT5 anion channels were much larger than for GLT-1c. Our data illustrate unique functional properties of EAAT5, being a low-affinity and low-capacity glutamate transport system, with an anion channel optimized for anion conduction in the negative voltage range.     

Extracellular ATP-induced inward current in isolated epithelial cells of the endolymphatic sac.

Using whole-cell patch-clamp technique and Fura-2 fluorescence measurement, the presence of ATP-activated ion channels and its dependence on intracellular Ca2+ concentration ([Ca2+]i) in the epithelial cells of the endolymphatic sac were investigated. In zero current-clamp configuration, the average resting membrane potential was -66.8+/-1.3 mV (n=18). Application of 30 microM ATP to the bath induced a rapid membrane depolarization by 43.1+/-2.4 mV (n=18). In voltage-clamp configuration, ATP-induced inward current at holding potential (VH) of -60 mV was 169.7+/-6.3 pA (n=18). The amplitude of ATP-induced currents increased in sigmoidal fashion over the concentration range between 0.3 and 300 microM with a Hill coefficient (n) of 1.2 and a dissociation constant (Kd) of 11.7 microM. The potency order of purinergic analogues in ATP-induced current, which was 2MeSATP>ATPgammas>/=ATP>alpha, beta-ATP>ADP=AMP>/=adenosine=UTP, was consistent with the properties of the P2Y receptor. The independence of the reversal potential of the ATP-induced current from Cl- concentration suggests that the current is carried by a cation channel. The relative ionic permeability ratio of the channel modulated by ATP for cations was Ca2+>Na+>Li+>Ba2+>Cs+=K+. ATP (10 microM) increased [Ca2+]i in an external Ca2+-free solution to a lesser degree than that in the external solution containing 1.13 mM CaCl2. ATP-induced increase in [Ca2+]i can be mimicked by application of ionomycin in a Ca2+-free solution. These results indicate that ATP increases [Ca2+]i through the P2Y receptor with a subsequent activation of the non-selective cation channel, and that these effects of ATP are dependent on [Ca2+]i and extracellular Ca2+.     

Benzodiazepines differently modulate EAAT1/GLAST and EAAT2/GLT1 glutamate transporters expressed in CHO cells.

It has been described recently that low concentrations of benzodiazepines stimulate the transport activity of the neuronal glutamate transporter EAAT3, whereas high concentrations inhibit it. The present study is aimed to investigate whether benzodiazepines have similar effects on the two glial glutamate transporter, EAAT1 and EAAT2. To this end, the transporters were transiently expressed in CHO cells and transport activity was determined by isotope fluxes using D-aspartate as non-metabolizable homologue of L-glutamate. At low D-aspartate concentrations (1 micromol/l) EAAT1-mediated uptake was reduced significantly by low concentrations of oxazepam (1 micromol/l) and diazepam (1 and 10 micromol/l). At 100 micromol/l D-aspartate oxazepam stimulated EAAT1-mediated uptake up to 150% in a dose dependent manner, whereas the inhibition by low concentrations of diazepam was attenuated. In contrast, a significant effect of diazepam on EAAT2-mediated uptake was only observed at 1000 micromol/l where uptake was inhibited by 60%. A similar inhibition was observed for EAAT1. These studies demonstrate a different modulation of EAAT1 and EAAT2 by benzodiazepines. Furthermore the glial transporters differ from the neuronal glutamate transporter. Thus, a complex in vivo response of the various transporters to benzodiazepines can be expected.     

Enhancement of substrate-gated Cl- currents via rat glutamate transporter EAAT4 by PMA

Glutamate transporters (also called excitatory amino acid transporters, EAAT) are important in extracellular homeostasis of glutamate, a major excitatory neurotransmitter. EAAT4, a neuronally expressed EAAT in cerebellum, has a large portion (95% of the total L-aspartate-induced currents in human EAAT4) of substrate-gated Cl^– currents, a distinct feature of this EAAT. We cloned EAAT4 from rat cerebellum. This molecule was predicted to have eight putative transmembrane domains. L-Glutamate induced an inward current in oocytes expressing this EAAT4 at a holding potential –60 mV. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, significantly increased the magnitude of L-glutamate-induced currents but did not affect the apparent affinity of EAAT4 for L-glutamate. This PMA-enhanced current had a reversal potential –17 mV at extracellular Cl^– concentration ([Cl^–]_o) 104 mM with an 60-mV shift per 10-fold change in [Cl^–]_o, properties consistent with Cl^–-selective conductance. However, PMA did not change EAAT4 transport activity as measured by [³H]-L-glutamate. Thus PMA-enhanced Cl^– currents via EAAT4 were not thermodynamically coupled to substrate transport. These PMA-enhanced Cl^– currents were partially blocked by staurosporine, chelerythrine, and calphostin C, the three PKC inhibitors. Ro-31-8425, a PKC inhibitor that inhibits conventional PKC isozymes at low concentrations (nM level), partially inhibited the PMA-enhanced Cl^– currents only at a high concentration (1 µM). Intracellular injection of BAPTA, a Ca²⁺-chelating agent, did not affect the PMA-enhanced Cl^– currents. 4-Phorbol-12,13-didecanoate, an inactive analog of PMA, did not enhance glutamate-induced currents. These data suggest that PKC, possibly isozymes other than conventional ones, modulates the substrate-gated Cl^– currents via rat EAAT4. Our results also suggest that substrate-gated ion channel activity and glutamate transport activity, two EAAT4 properties that could modulate neuronal excitability, can be regulated independently. oocytes; protein kinase C     

Single K+ channels in endocrine cells dispersed from the cricket (Gryllus bimaculatus) corpora allata

Single channel currents were recorded from cell-attached patches of endocrine cells of the adult male cricket corpora allata. Three distinct types of K+ channels were identified; a weak inward rectifier (Type 1), a strong inward rectifier (Type 2) and a weak outward rectifier (Type 3). The type 1 channel had a slope conductance of 191 +/- 9 pS (n = 4) at negative membrane potentials (Vm) and 101 +/- 6 pS (n = 6) at positive Vm. In addition, the channel showed fast open-closed kinetics at negative Vm and slow open-closed kinetics at positive Vm. The open probability (Po) of this channel was strongly voltage-dependent at positive Vm, but less voltage-dependent at negative Vm. The reversal potential was not modified significantly by the substitution of gluconate for external Cl- but was modified after N-methyl-D-glucamine (NMDG+) was substituted for external K+, according to the Nernst equation for a K+-selective channel. The type 2 channel had a slope conductance of 44 +/- 2 pS (n = 5) at negative Vm, but no detectable outward current was observed at positive Vm. This channel showed very slow open-closed kinetics at negative Vm and its Po was not voltage-dependent. The type 3 channel had a limit conductance of 55 +/- 12 pS (n = 3) at negative Vm and 88 +/- 10 pS (n = 3) at positive Vm. This channel showed slow open-closed kinetics at negative Vm and fast open-closed kinetics at positive Vm. The Po for the channel was voltage-dependent at positive Vm but was voltage-independent at negative Vm. These three types of K+ channels may be important for the control of the resting membrane potential, and may thus participate in the regulation of Ca2+ influx and juvenile hormone secretion in corpora allata cells.     

The glutamate transporter subtypes EAAT4 and EAATs 1-3 transport glutamate with dramatically different kinetics and voltage dependence but share a common uptake mechanism

Here, we report the application of glutamate concentration jumps and voltage jumps to determine the kinetics of rapid reaction steps of excitatory amino acid transporter subtype 4 (EAAT4) with a 100-micros time resolution. EAAT4 was expressed in HEK293 cells, and the electrogenic transport and anion currents were measured using the patch-clamp method. At steady state, EAAT4 was activated by glutamate and Na+ with high affinities of 0.6 microM and 8.4 mM, respectively, and showed kinetics consistent with sequential binding of Na(+)-glutamate-Na+. The steady-state cycle time of EAAT4 was estimated to be >300 ms (at -90 mV). Applying step changes to the transmembrane potential, V(m), of EAAT4-expressing cells resulted in the generation of transient anion currents (decaying with a tau of approximately 15 ms), indicating inhibition of steady-state EAAT4 activity at negative voltages (<-40 mV) and activation at positive V(m) (>0 mV). A similar inhibitory effect at V(m) < 0 mV was seen when the electrogenic glutamate transport current was monitored, resulting in a bell-shaped I-V(m) curve. Jumping the glutamate concentration to 100 muM generated biphasic, saturable transient transport and anion currents (K(m) approximately 5 microM) that decayed within 100 ms, indicating the existence of two separate electrogenic reaction steps. The fast electrogenic reaction was assigned to Na+ binding to EAAT4, whereas the second reaction is most likely associated with glutamate translocation. Together, these results suggest that glutamate uptake of EAAT4 is based on the same molecular mechanism as transport by the subtypes EAATs 1-3, but that its kinetics and voltage dependence are dramatically different from the other subtypes. EAAT4 kinetics appear to be optimized for high affinity binding of glutamate, but not rapid turnover. Therefore, we propose that EAAT4 is a high-affinity/low-capacity transport system, supplementing low-affinity/high-capacity synaptic glutamate uptake by the other subtypes.     

Identification of the hyperpolarization-activated inward current in young embryonic chick heart myocytes

Whole-cell voltage-clamp experiments were performed to examine the underlying currents flowing during the pacemaker potential of spontaneously-beating embryonic chick ventricles. The holding potential was -30 mV. Long-duration (3 s) hyperpolarizing pulses were applied to -40 to -120 mV, in increments of 10 mV. A marked hyperpolarization-activated inward current (If) was produced. In cells from 3-day-old hearts, the threshold potential for the inward current was -50 to -60 mV. In 17-day-old cells, there was almost no If current. At -120 mV, the inward current was -93.8 +/- 6.3 pA (n = 5) in 3-day-old cells and -15.7 +/- 2.8 pA (n = 5) in 17-day-old cells. The average capacitances were 10.1 +/- 2.0 pF (n = 17) in 3-day-old cells, and 6.9 +/- 1.2 pF (n = 14) in 17-day-old cells. The reduction of If paralleled the decrease in spontaneous activity. In the presence of 3 mM CsCl, the inward current was blocked completely, and the tail current was reduced. In addition, 3 mM CsCl depressed the spontaneous action potentials and had a negative chronotropic effect. These results indicate that the hyperpolarization-activated inward If current exists in young embryonic chick heart cells, and decreases during development. This If current may contribute somewhat to the electrogenesis of the pacemaker potential.     

Chemosensitive conductance and inositol 1,4,5-trisphosphate-induced conductance in snake vomeronasal receptor neurons

Snake vomeronasal receptor neurons in slice preparations were studied using the patch-clamp technique in the conventional and nystatin-perforated whole-cell configurations. The mean resting potential was approximately -70 mV; the average input resistance was 3 GOmega. Neurons required current injection of only 1-10 pA to display a variety of spiking patterns. Intracellular dialysis of 100 microM inositol 1,4,5-trisphosphate (IP(3)) evoked an inward current in 38% of neurons, with an average peak amplitude of 16.4 +/- 2.8 pA at a holding potential of -70mV. Application of 100 microM 3-deoxy-3-fluoro-D-myo-inositol 1,4,5-trisphosphate (F-IP(3)), a derivative of IP(3), also evoked an inward current in 4/8 (50%) neurons (32.6 +/- 58 pA at -70 mV, n = 4). The reversal potentials of the induced components were estimated to be -14 +/- 5 mV for IP(3) and -17 +/- 3 mV for F-IP(3). Bathing the neurons in 10 microM ruthenium red solution greatly reduced the IP(3)-evoked inward current to 1.6 +/- 1.1 pA at -70 mV (n = 6). With Cs(+)-containing internal solution, neither the Ca(2+)-ATPase inhibitor thapsigargin (1-50 microM) nor the Ca(2+)-ionophore ionomycin (10 microM) evoked a significant current response, suggesting that IP(3) can elicit current response in the neurons without mediation by intracellular Ca(2+) stores. Intracellular application of 1 mM cAMP evoked no detectable current response. Extracellular application of chemoattractant for snakes evoked a very large inward current. The reversal potential of the chemoattractant-induced current was similar to that of the IP(3)-induced current. The present results suggest that IP(3) may act as a second messenger in the transduction of chemoattractants in the garter snake vomeronasal organ.     

Na(+)-dependent glutamate transporters (EAAT1, EAAT2, and EAAT3) of the blood-brain barrier. A mechanism for glutamate removal.

Na(+)-dependent transporters for glutamate exist on astrocytes (EAAT1 and EAAT2) and neurons (EAAT3). These transporters presumably assist in keeping the glutamate concentration low in the extracellular fluid of brain. Recently, Na(+)-dependent glutamate transport was described on the abluminal membrane of the blood-brain barrier. To determine whether the above-mentioned transporters participate in glutamate transport of the blood-brain barrier, total RNA was extracted from bovine cerebral capillaries. cDNA for EAAT1, EAAT2, and EAAT3 was observed, indicating that mRNA was present. Western blot analysis demonstrated all three transporters were expressed on abluminal membranes, but none was detectable on luminal membranes of the blood-brain barrier. Measurement of transport kinetics demonstrated voltage dependence, K(+)-dependence, and an apparent K(m) of 14 microM (aggregate of the three transporters) at a transmembrane potential of -61 mV. Inhibition of glutamate transport was observed using inhibitors specific for EAAT2 (kainic acid and dihydrokainic acid) and EAAT3 (cysteine). The relative activity of the three transporters was found to be approximately 1:3:6 for EAAT1, EAAT2, and EAAT3, respectively. These transporters may assist in maintaining low glutamate concentrations in the extracellular fluid.     

Post-translational regulation of EAAT2 function by co-expressed ubiquitin ligase Nedd4-2 is impacted by SGK kinases

The human excitatory amino acid transporter (EAAT)2 is the major glutamate carrier in the mammalian CNS. Defective expression of the transporter results in neuroexcitotoxicity that may contribute to neuronal disorders such as amyotrophic lateral sclerosis (ALS). The serum and glucocorticoid inducible kinase (SGK) 1 is expressed in the brain and is known to interact with the ubiquitin ligase Nedd4-2 to modulate membrane transporters and ion channels. The present study aimed to investigate whether SGK isoforms and the related kinase, protein kinase B (PKB), regulate EAAT2. Expression studies in Xenopus oocytes demonstrated that glutamate-induced inward current (IGLU) was stimulated by co-expression of SGK1, SGK2, SGK3 or PKB. IGLU is virtually abolished by Nedd4-2, an effect abrogated by additional co-expression of either kinase. The kinases diminish the effect through Nedd4-2 phosphorylation without altering Nedd4-2 protein abundance. SGKs increase the transporter maximal velocity without significantly affecting substrate affinity. Similar to glutamate-induced currents, [3H] glutamate uptake and cell surface abundance of the transporter were increased by the SGK isoforms and down-regulated by the ubiquitin ligase Nedd4-2. In conclusion, all three SGK isoforms and PKB increase EAAT2 activity and plasma membrane expression and thus, may participate in the regulation of neuroexcitability.     

T-type Ca2+ channel lowers the threshold of spike generation in the newt olfactory receptor cell

Mechanisms underlying action potential generation in the newt olfactory receptor cell were investigated by using the whole-cell version of the patch-clamp technique. Isolated olfactory cells had a resting membrane potential of -70 +/- 9 mV. Injection of a depolarizing current step triggered action potentials under current clamp condition. The amplitude of the action potential was reduced by lowering external Na+ concentration. After a complete removal of Na+, however, cells still showed action potentials which was abolished either by Ca2+ removal or by an application of Ca2+ channel blocker (Co2+ or Ni2+), indicating an involvement of Ca2+ current in spike generation of newt olfactory receptor cells. Under the voltage clamp condition, depolarization of the cell to -40 mV from the holding voltage of -100 mV induced a fast transient inward current, which consisted of Na+ (INa) and T-type Ca2+ (ICa.T) currents. The amplitude of ICa,T was about one fourth of that of INa. Depolarization to more positive voltages also induced L-type Ca2+ current (ICa,L). ICa,L was as small as a few pA in normal Ringer solution. The activating voltage of ICa,T was approximately 10 mV more negative than that of INa. Under current clamp, action potentials generated by a least effective depolarization was almost completely blocked by 0.1 mM Ni2+ (a specific T-type Ca2+ channel blocker) even in the presence of Na+. These results suggest that ICa,T contributes to action potential in the newt olfactory receptor cell and lowers the threshold of spike generation.