Functional characterization of the Betaine/gamma-aminobutyric acid transporter BGT-1 expressed in Xenopus oocytes.

Betaine is an osmolyte accumulated in cells during osmotic cell shrinkage. The canine transporter mediating cellular accumulation of the osmolyte betaine and the neurotransmitter gamma-aminobutyric acid (BGT-1) was expressed in Xenopus oocytes and analyzed by two-electrode voltage clamp and tracer flux studies. Exposure of oocytes expressing BGT-1 to betaine or gamma-aminobutyric acid (GABA) depolarized the cell membrane in the current clamp mode and induced an inward current under voltage clamp conditions. At 1 mM substrate the induced currents decreased in the following order: betaine = GABA > diaminobutyric acid = beta-alanine > proline = quinidine > dimethylglycine > glycine > sarcosine. Both the Vmax and Km of GABA- and betaine-induced currents were voltage-dependent, and GABA- and betaine-induced currents and radioactive tracer uptake were strictly Na+-dependent but only partially dependent on the presence of Cl-. The apparent affinity of GABA decreased with decreasing Na+ concentrations. The Km of Na+ also depended on the GABA and Cl- concentration. A decrease of the Cl- concentration reduced the apparent affinity for Na+ and GABA, and a decrease of the Na+ concentration reduced the apparent affinity for Cl- and GABA. A comparison of 22Na+-, 36Cl--, and 14C-labeled GABA and 14C-labeled betaine fluxes and GABA- and betaine-induced currents yielded a coupling ratio of Na+/Cl-/organic substrate of 3:1:1 or 3:2:1. Based on the data, a transport model of ordered binding is proposed in which GABA binds first, Na+ second, and Cl- third. In conclusion, BGT-1 displays significant functional differences from the other members of the GABA transporter family.     

Electrogenic uptake of nucleosides and nucleoside-derived drugs by the human nucleoside transporter 1 (hCNT1) expressed in Xenopus laevis oocytes

The concentrative pyrimidine-preferring nucleoside transporter 1 (hCNT1), cloned from human fetal liver, was expressed in Xenopus laevis oocytes. Using the two-electrode voltage-clamp technique, it is shown that translocation of nucleosides by this transporter generates sodium inward currents. Membrane hyperpolarization (from -50 to -150 mV) did not affect the K(0.5) for uridine, although it increased the transport current approximately 3-fold. Gemcitabine (a pyrimidine nucleoside-derived drug) but not fludarabine (a purine nucleoside-derived drug) induced currents in oocytes expressing the hCNT1 transporter. The K(0.5) value for gemcitabine at -50 mV membrane potential was lower than that for natural substrates, although this drug induced a lower current than uridine and cytidine, thus suggesting that the affinity binding of the drug transporter is high but that translocation occurs more slowly. The analysis of the currents generated by the hCNT1-mediated transport of nucleoside-derived drugs used in anticancer and antiviral therapies will be useful in the characterization of the pharmacological profile of this family of drug transporters and will allow rapid screening for uptake of newly developed nucleoside-derived drugs.     

Expression of a cloned gamma-aminobutyric acid transporter in mammalian cells.

The cDNA clone GAT-1, which encodes a Na(+)- and Cl(-)-coupled GABA transporter from rat brain, has been expressed in mammalian cells using three different systems: (1) transient expression upon transfection of mouse Ltk- cells with a eukaryotic expression vector containing GAT-1; (2) stable expression in L-cells transfected with the same vector; (3) transfection of HeLa cells infected with a recombinant vaccinia virus expressing T7 RNA polymerase. Similar results both qualitatively and quantitatively were obtained with all systems. The GABA transporter expressed in HeLa and L-cells retains all the properties described previously for GABA transport into synaptosomes and synaptic plasma membrane vesicles. It was fully inhibited by cis-3-aminocyclohexanecarboxylic acid (ACHC) and not by beta-alanine. The KM for GABA transport and the IC50 for ACHC inhibition were similar to the presynaptic transporter. Accumulated [3H]GABA was released from transfected cells by dissipating the transmembrane Na+ gradient with nigericin or by exchange with unlabeled external GABA. Accumulation was stimulated by both Na+ and Cl- in the external medium. However, in the absence of external Cl-, a small amount of GABA transport remained which was dependent on GAT-1 transfection. Functional expression of the GABA transporter was abolished by tunicamycin. An antitransporter antibody specifically immunoprecipitates a polypeptide with an apparent molecular mass of about 70 kDa from GAT-1-transfected cells. When cells were grown in the presence of tunicamycin, only a faint band of apparent mass of about 60 kDa was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inactivation of cloned Na channels expressed in Xenopus oocytes

This study investigates the inactivation properties of Na channels expressed in Xenopus oocytes from two rat IIA Na channel cDNA clones differing by a single amino acid residue. Although the two cDNAs encode Na channels with substantially different activation properties (Auld, V. J., A. L. Goldin, D. S. Krafte, J. Marshall, J. M. Dunn, W. A. Catterall, H. A. Lester, N. Davidson, and R. J. Dunn. 1988. Neuron. 1:449-461), their inactivation properties resemble each other strongly but differ markedly from channels induced by poly(A+) rat brain RNA. Rat IIA currents inactivate more slowly, recover from inactivation more slowly, and display a steady-state voltage dependence that is shifted to more positive potentials. The macroscopic inactivation process for poly(A+) Na channels is defined by a single exponential time course; that for rat IIA channels displays two exponential components. At the single-channel level these differences in inactivation occur because rat IIA channels reopen several times during a depolarizing pulse; poly(A+) channels do not. Repetitive stimulation (greater than 1 Hz) produces a marked decrement in the rat IIA peak current and changes the waveform of the currents. When low molecular weight RNA is coinjected with rat IIA RNA, these inactivation properties are restored to those that characterize poly(A+) channels. Slow inactivation is similar for rat IIA and poly(A+) channels, however. The data suggest that activation and inactivation involve at least partially distinct regions of the channel protein.     

Amino acid uptake in Xenopus laevis oocytes.

The isolated oocytes from Xenopus laevis are able to take up radioactive amino acids from the exogenous medium. Most amino acids tested are taken up to reach concentrations higher than the extracellular medium. The initial uptake velocities vary with the external amino acid concentration in a Michaelis-Menten fashion. Aspartic acid requires concentrations an order of magnitude higher than the five other amino acids tested to reach half the maximal uptake velocity. The uptake mechanism seems to be specific for groups of analogous amino acids, as can be determined by competition studies. The amino acid groups for which there is some evidence of uptake specificity would be aromatic, aliphatic, acidic and basic. Amino acid pools of oocytes show that these cells can concentrate amino acids from Xenopus blood, as well as from artificial media.     

Heteropolymeric potassium channels expressed in Xenopus oocytes from cloned subunits

Voltage-dependent potassium currents were measured in Xenopus oocytes previously injected with RNAs generated in vitro from each of three cloned cDNAs (RBK1, RBK2, and RGK5). The currents differed in their sensitivities to blockade by tetraethylammonium (TEA; respective KDs 0.3, greater than 100, and 10 mM) and in their inactivation during a depolarizing pulse. Injections of RNA combinations (RBK1/RBK2 and RBK1/RGK5) caused currents that had TEA sensitivities different from those expected from the sum, in any proportion, of the two native channels. It is concluded that novel potassium channels are formed by the oocytes injected with two RNAs, presumably by heteropolymerization of subunits; such heteropolymerization would contribute functional diversity to voltage-dependent potassium channels in addition to that provided by a large gene family.     

Effects of interferon-alpha on cloned opioid receptors expressed in Xenopus oocytes

Interferon-α (IFNα) affects the opioid system. However, the direct action of IFNα on cloned opioid receptors remains unknown. Taking advantage of the functional coupling of cloned opioid receptors to G protein-activated inwardly rectifying K⁺ (GIRK) channels in a Xenopus oocyte expression system, we investigated the effects of recombinant IFNα on cloned μ-, δ- and κ-opioid receptors. In oocytes co-injected with mRNAs for either the δ- or κ-opioid receptor and for GIRK channel subunits, IFNα at high concentrations induced small GIRK currents that were abolished by naloxone, an opioid-receptor antagonist, compared with the control responses to each selective opioid agonist. Additionally, IFNα induced no significant current response in oocytes injected with mRNA(s) for either opioid receptor alone or GIRK channels. In oocytes expressing the μ-opioid receptor and GIRK channels, IFNα had little or no effect. Moreover, in oocytes expressing each opioid receptor and GIRK channels, GIRK current responses to each selective opioid agonist were not affected by the presence of IFNα, indicating no significant antagonism of IFNα toward the opioid receptors. Furthermore, IFNα had little or no effect on the μ/δ-, δ/κ- or μ/κ-opioid receptors expressed together with GIRK channels in oocytes. Our results suggest that IFNα weakly activates the δ and κ-opioid receptors. The direct activation of the δ- and κ-opioid receptors by IFNα may partly contribute to some of the IFNα effects under its high-dose medication.     

Expression of cloned aromatic L-amino acid decarboxylase in Xenopus laevis oocytes.

Sense mRNA coding for bovine adrenal medulla aromatic L-amino acid decarboxylase (AADC) was expressed following microinjection into Xenopus laevis oocytes. The expressed enzyme activity was stereoselective for L-5-hydroxytryptophan and L-DOPA and blocked by NSD-1015 an inhibitor of AADC. Heating the expressed enzyme at 55 degrees C resulted in a parallel loss of activity towards both substrates. Our findings are consistent with the prevailing notion that a single enzyme is able to decarboxylate both substrates in vivo.     

A physiological study on acetylcholine receptor expressed in Xenopus oocytes from cloned cDNAs

Nicotinic ACh receptor was expressed in Xenopus oocytes by injecting mRNAs produced from cloned cDNAs encoding the four subunits of ACh receptor of Torpedo californica. ACh responses recorded from oocytes 3 days after injection of the mRNAs were reversibly blocked by d-tubocurarine (1-2 microM), indicating that the newly synthesized receptor is of nicotinic type. The reversal potential of ACh response was found at around -1 - -5 mV. The reversal potential was not changed by removal of extracellular C1-, suggesting that the ionic channel of the newly expressed ACh receptor is permeable only to cations. Repetitive applications of ACh caused desensitization of the receptor. The rate of the desensitization was greater when the membrane potential was more negative. Subunit deletion studies showed that all four subunits are required for the formation of ACh receptors with normal ACh sensitivity. However, ACh receptors without delta subunit responded to ACh with low sensitivity. Studies on ACh receptor mutants with -subunits altered by site directed mutagenesis of the cDNA suggest that the anphipathic segment is involved in the channel function of the receptor as well as the four hydrophobic segments since partial deletion of amino acids in these segments essentially abolished ACh sensitivity with relatively little change in 125I-alpha-bungarotoxin binding activity.     

Surface Charges of K channels. Effects of strontium on five cloned channels expressed in Xenopus oocytes

The effects of strontium (Sr2+; 7-50 mM) on five different cloned rat K channels (Kv1.1, Kv1.5, Kv1.6, Kv2.1, and Kv3.4), expressed in oocytes of Xenopus laevis, were investigated with a two-electrode voltage clamp technique. The main effect was a shift of the Gk(V) curve along the potential axis, different in size for the different channels. Kv1.1 was shifted most and Kv3.4 least, 21 and 8 mV, respectively, at 50 mM. The effect was interpreted in terms of screening of fixed surface charges. The estimated charge densities ranged from -0.37 (Kv1.1) to -0.11 (Kv3.4) e nm-2 and showed good correlation with the total net charge of the extracellularly located amino acid residues of the channel as well as with the charge of a specific region (the loop between the S5 segment and the pore forming segment). The estimated surface potentials were found to be linearly related to the activation midpoint potential, suggesting a functional role for the surface charges.     

Ethanol potentiates the function of the human dopamine transporter expressed in Xenopus oocytes

Ethanol alters a variety of properties of brain dopaminergic neurons including firing rate, synthesis, release, and metabolism. Recent studies suggest that ethanol's action on central dopamine systems may also involve modulation of dopamine transporter (DAT) activity. The human DAT was expressed in Xenopus oocytes to examine directly the effects of ethanol on transporter function. [3H]Dopamine (100 nM) accumulation into DAT-expressing oocytes increased significantly in response to ethanol (10 min; 10-100 mM). In two-electrode voltage-clamp experiments, DAT-mediated currents were also enhanced significantly by ethanol (10-100 mM). The magnitude of the ethanol-induced potentiation of DAT function depended on ethanol exposure time and substrate concentration. Cell surface DAT binding ([3H]WIN 35,428; 4 nM) also increased as a function of ethanol exposure time. Thus, the increase in dopamine uptake was associated with a parallel increase in the number of DAT molecules expressed at the cell surface. These experiments demonstrate that DAT-mediated substrate translocation and substrate-associated ionic conductances are sensitive to intoxicating concentrations of ethanol and suggest that DAT may represent an important site of action for ethanol's effects on central dopaminergic transmission. A potential mechanism by which ethanol acts to enhance DAT function may involve regulation of DAT expression on the cell surface.     

Substrate-induced changes in the density of peptide transporter PEPT1 expressed in Xenopus oocytes

The adaptation of the capacity of the intestinal peptide transporter PEPT1 to varying substrate concentrations may be important with respect to its role in providing bulk quantities of amino acids for growth, development, and other nutritional needs. In the present study, we describe a novel phenomenon of the regulation of PEPT1 in the Xenopus oocyte system. Using electrophysiological and immunofluorescence methods, we demonstrate that a prolonged substrate exposure of rabbit PEPT1 (rPEPT1) caused a retrieval of transporters from the membrane. Capacitance as a measure of membrane surface area was increased in parallel with the increase in rPEPT1-mediated transport currents with a slope of approximately 5% of basal surface per 100 nA. Exposure of oocytes to the model peptide Gly-l-Gln for 2 h resulted in a decrease in maximal transport currents with no change of membrane capacitance. However, exposure to substrate for 5 h decreased transport currents but also, in parallel, surface area by endocytotic removal of transporter proteins from the surface. The reduction of the surface expression of rPEPT1 was confirmed by presteady-state current measurements and immunofluorescent labeling of rPEPT1. A similar simultaneous decrease of current and surface area was also observed when endocytosis was stimulated by the activation of PKC. Cytochalasin D inhibited all changes evoked by either dipeptide or PKC stimulation, whereas the PKC-selective inhibitor bisindolylmaleimide only affected PKC-stimulated endocytotic processes but not substrate-dependent retrieval of rPEPT1. Coexpression experiments with human Na(+)-glucose transporter 1 (hSGLT1) revealed that substrate exposure selectively affected PEPT1 but not the activity of hSGLT1.     

Expression of a functional glucose transporter in Xenopus oocytes

A cDNA encoding the rat brain glucose transporter was inserted between the 5' and 3' untranslated regions from the Xenopus globin gene and downstream of an SP6 RNA polymerase start site. RNA synthesized from this vector was microinjected into oocytes from Xenopus laevis; this resulted in expression of the glucose transporter, as determined by both immunoblotting and the appearance of transport activity. The properties of the transporter were those expected from previous studies: it was glycosylated, and its activity, measured by 3-O-methylglucose transport, was inhibited by D-glucose and cytochalasin B, but not by L-glucose. The low level of endogenous glucose transport activity found in water-injected oocytes makes this a useful system in which to determine the kinetic parameters of transport. The Km for 3-O-methylglucose was found to be 20 mM under equilibrium exchange conditions. Despite the fact that oocytes exhibit insulin-dependent responses, insulin did not stimulate 3-O-methylglucose transport by injected oocytes.     

Corticosterone decreases the activity of rat glutamate transporter type 3 expressed in Xenopus oocytes

Glucocorticoids can increase the extracellular concentrations of glutamate, the major excitatory neurotransmitter. We investigated the effects of corticosterone on the activity of a glutamate transporter, excitatory amino acid carrier 1 (EAAC1; also called excitatory amino acid transporter type 3 [EAAT3]), and the roles of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) in regulating these effects. Rat EAAC1 was expressed in Xenopus oocytes by injecting mRNA. l-Glutamate (30 μM)-induced membrane currents were measured using the two-electrode voltage clamp technique. Exposure of these oocytes to corticosterone (0.01–1 μM) for 72 h decreased EAAC1 activity in a dose-dependent fashion, and this inhibition was incubation time-dependent. Corticosterone (0.01 μM for 72 h) significantly decreased the V_max, but not the K_m, of EAAC1 for glutamate. Furthermore, pretreatment of oocytes with staurosporine, a PKC inhibitor, significantly decreased EAAC1 activity (1.00 ± 0.06 to 0.70 ± 0.05 μC; P < 0.05). However, no statistical differences were observed between oocytes treated with staurosporine, corticosterone, or corticosterone plus staurosporine. Similar patterns of responses were achieved by chelerythrine or calphostin C, other PKC inhibitors. Phorbol-12-myristate-13-acetate (PMA), a PKC activator, inhibited corticosterone-induced reduction in EAAC1 activity. Pretreating oocytes with wortmannin or LY294002, PI3K inhibitors, also significantly reduced EAAC1 activity, but no difference was observed between oocytes treated with wortmannin, corticosterone, or wortmannin plus corticosterone. The above results suggest that corticosterone exposure reduces EAAC1 activity and this effect is PKC- and PI3K-dependent.     

SLC36A4 (hPAT4) is a high affinity amino acid transporter when expressed in Xenopus laevis oocytes

The SLC36 family of transporters consists of four genes, two of which, SLC36A1 and SLC36A2, have been demonstrated to code for human proton-coupled amino acid transporters or hPATs. Here we report the characterization of the fourth member of the family, SLC36A4 or hPAT4, which when expressed in Xenopus laevis oocytes also encodes a plasma membrane amino acid transporter, but one that is not proton-coupled and has a very high substrate affinity for the amino acids proline and tryptophan. hPAT4 in Xenopus oocytes mediated sodium-independent, electroneutral uptake of [(3)H]proline, with the highest rate of uptake when the uptake medium pH was 7.4 and an affinity of 3.13 μM. Tryptophan was also an excellently transported substrate with a similarly high affinity (1.72 μM). Other amino acids that inhibited [(3)H]proline were isoleucine (K(i) 0.23 mM), glutamine (0.43 mM), methionine (0.44 mM), and alanine (1.48 mM), and with lower affinity, glycine, threonine, and cysteine (K(i) >5 mM for all). Of the amino acids directly tested for transport, only proline, tryptophan, and alanine showed significant uptake, whereas glycine and cysteine did not. Of the non-proteogenic amino acids and drugs tested, only sarcosine produced inhibition (K(i) 1.09 mM), whereas γ-aminobutyric acid (GABA), β-alanine, L-Dopa, D-serine, and δ-aminolevulinic acid were without effect on [(3)H]proline uptake. This characterization of hPAT4 as a very high affinity/low capacity non-proton-coupled amino acid transporter raises questions about its physiological role, especially as the transport characteristics of hPAT4 are very similar to the Drosophila orthologue PATH, an amino acid "transceptor" that plays a role in nutrient sensing.     

Potentiation of GABAA receptors expressed in Xenopus oocytes by perfume and phytoncid.

To study the effects of perfume and phytoncid on GABAA receptors, ionotropic GABAA receptors were expressed in Xenopus oocytes by injecting mRNAs that had been prepared from rat whole brain. Essential oil, perfume and such phytoncid as leaf alcohol, hinokitiol, pinene, eugenol, citronellol and citronellal potentiated the response in the presence of GABA at low concentrations (10 and 30 microM), possibly because they bound to the potentiation-site in GABAA receptors and increased the affinity of GABA to the receptors. Since it is known that the potentiation of GABAA receptors by benzodiazepine, barbiturate, steroids and anesthetics induces the anxiolytic, anticonvulsant and sedative activity or anesthetic effect, these results suggest the possibility that the intake of perfume or phytoncid through the lungs, the skin or the intestines modulates the neural transmission in the brain through ionotropic GABAA receptors and changes the frame of the human mind, as alcohol or tobacco does.