Frog Oocytes Research Articles

The Rana catesbeiana rcr Gene Encoding a Cytotoxic Ribonuclease: TISSUE DISTRIBUTION, CLONING, PURIFICATION, CYTOTOXICITY, AND ACTIVE RESIDUES FOR RNase ACTIVITY

Rana catesbeiana ribonuclease (RC-RNase) is a pyrimidine-guanine sequence-specific ribonuclease found in R. catesbeiana (bullfrog) oocytes. It possesses both ribonuclease activity and cytotoxicity against tumor cells. We report here for the first time the cloning of RC-RNase cDNA from liver rather than from oocytes where RC-RNase is stored. An internal fragment of cDNA was obtained by reverse transcription-PCR using deduced oligonucleotides as primers. Full-length cDNA was obtained by 5'- and 3'-RACE technique. The cDNA clone, named rcr gene, contained a 5'-untranslated region, a putative signal peptide (22 amino acids), a mature protein (111 amino acids), a 3'-untranslated region, and a polyadenylation site. The cDNA which encoded the mature protein was fused upstream with a modified pelB signal peptide DNA and inserted into pET11d for expression in Escherichia coli strain BL21(DE3). The secretory RC-RNase in the culture medium was enzymatically active and was purified to homogeneity. The recombinant RC-RNase had the same amino acid sequence, specific activity, substrate specificity, antigenicity, and cytotoxicity as that of native RC-RNase from frog oocytes. Amino acid residues His-10, Lys-35, and His-103 are involved in RC-RNase catalytic activity. Ribonucleolytic activity was involved in and may be essential for RC-RNase cytotoxicity. DNA sequence analysis showed that RC-RNase had approximately 45% identity to that of RNase superfamily genes. This indicates that RC-RNase is a distinct ribonuclease gene in the RNase superfamily.

Characterization of a receptor subtype-selective lysophosphatidic acid mimetic.

Despite an intriguing cell biology and the suggestion of a role in pathophysiological responses, the mechanism of action of such lipid phosphoric acid mediators as lysophosphatidic acid (LPA) remains obscure, in part because of an underdeveloped medicinal chemistry. We report now the agonist activity of a synthetic phospholipid in which the glycerol backbone of LPA is replaced by L-serine. Like LPA, the L-serine-based lipid mobilizes calcium and inhibits activation of adenylyl cyclase in the human breast cancer cell line MDA MB231. Treatment with LPA desensitizes MDA MB231 cells to subsequent application of the L-serine compound; when the order of application is reversed, however, the L-serine compound does not prevent calcium mobilization by LPA, which might indicate the existence of two LPA receptors in these cells. The analogous D-serine-based phospholipid was distinctly less potent than the L-isomer in those assays; this finding demonstrates stereoselectivity by an LPA receptor. Unlike LPA, the L-serine-based lipid does not evoke a chloride conductance in Xenopus laevis oocytes, but injection of poly(A)+ RNA from HEK 293 cells confers this phenotype on the oocyte. The latter result has practical importance in that it allows use of the frog oocyte for expression cloning of an LPA receptor DNA, an assay system made problematic by the oocyte's strong endogenous response to LPA.

A potassium channel mutation in neonatal human epilepsy.

Benign familial neonatal convulsions (BFNC) is an autosomal dominant epilepsy of infancy, with loci mapped to human chromosomes 20q13.3 and 8q24. By positional cloning, a potassium channel gene (KCNQ2) located on 20q13.3 was isolated and found to be expressed in brain. Expression of KCNQ2 in frog (Xenopus laevis) oocytes led to potassium-selective currents that activated slowly with depolarization. In a large pedigree with BFNC, a five-base pair insertion would delete more than 300 amino acids from the KCNQ2 carboxyl terminus. Expression of the mutant channel did not yield measurable currents. Thus, impairment of potassium-dependent repolarization is likely to cause this age-specific epileptic syndrome.

Fast- and slow-gating modes of the sodium channel are altered by a paramyotonia congenita-linked mutation.

We have studied the expression in frog oocytes of the alpha subunit of the rat skeletal muscle sodium channel mutation T1306M, homologous to the mutation T1313M of the human isoform that causes the muscular hereditary disease paramyotonia congenita. Wild-type (WT) channels show a bimodal behavior, with two gating modes characterized by inactivation time constants that differ at least by one order of magnitude and with voltage dependencies shifted by +27 mV in the slow mode (M2) relative to the fast (M1) mode. In the myopathy-linked mutant the propensity of the channel for the mode M2 is increased fourfold and the kinetics and voltage dependence of inactivation in both modes are altered. In mode M1, the onset of inactivation is faster and the recovery from inactivation is slower whereas both processes are slowed in mode M2. The half-inactivation potential of both modes is shifted by the mutation to positive potentials. Coexpression of beta subunit causes a threefold reduction of the M2 propensity of both WT and T1306M channels, with small changes in the voltage dependency and kinetic properties of inactivation. All the changes are consistent with the hyperexcitability of the muscle fibers observed in patients affected by potassium-aggrevated myotonia (PAM).

Regulatory Role of Fructose-2,6-bisP on Glucose Metabolism in Frog Oocytes:In VivoInhibition of Glycogen Synthesis

Glycogen synthesis following glucose microinjection in frog oocytes proceeds preferentially by an indirect pathway involving gluconeogenesis from triose compounds. Because of the known regulatory role of fructose-2,6-bisP on glucose utilization in most vertebrate tissues we coinjected [U-14C]glucose and fructose-2,6-bisP into oocytes and observed a marked inhibition of label incorporation into glycogen, with anI50value of 2 μM, which is similar to the value measured for thein vitroinhibition of oocyte fructose-1,6-bisphosphatase. Other hexoses-bisP were tested: 2,5-anhydromannitol-1,6-bisP was as effective as inhibitor as fructose-2,6-bisP; glucose-1,6-bisP showed some effect although 50% inhibition was obtained at a concentration 10 times higher than with fructose-2,6-bisP; fructose-1,6-bisP had no effect at all. The inhibition pattern for thein vivoglycogen synthesis by these analogs closely matched the one obtained with partially purified oocyte fructose-1,6-bisphosphatase. The intracellular concentration of fructose-2,6-bisP in unperturbed oocytes was found to be between 0.1 and 0.2 μM. Fructose-6-phosphate,2-kinase levels measured in oocyte homogenates were between 0.02 and 0.06 mU per gram of ovary. After 60 min incubation, fructose-2,6-bisP microinjected into the oocytes was almost completely degraded, suggesting that fructose-2,6-bisphosphatase is activein vivo.The results presented in this paper indicate that fructose-2,6-bisP plays an important role in thein vivoregulation of glucose utilization in frog-grown oocytes.

MAP and cdc2 Kinase Activities at Germinal Vesicle Breakdown inChaetopterus

In frog oocytes, activation of mitogen-activated protein kinase (MAPK, ERK) leads to activation of cdc2 and germinal vesicle breakdown (GVBD). By contrast, in starfish, MAPK is activated after GVBD. Here we have examined the relative involvements of MAPK and cdc2 in GVBD ofChaetopterusoocytes. MAPK was rapidly tyrosine-phosphorylated and activated (within 1–2 min) in response to exposure of the oocytes either to natural seawater (the normal trigger of GVBD in this organism) or to the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), which can also elicit GVBD. This response preceded the tyrosine dephosphorylation and activation of cdc2 by several minutes. MAPK phosphorylation and activation were transient, lasting only until GVBD occurred and the spindle migrated to the cortex. The enzyme was not phosphorylated again as a result of egg activation. These results are consistent with the hypothesis that the activation of MAPK has a role in GVBD. However, PD 98059, a potent and selective inhibitor of MEK, the protein kinase that phosphorylates and activates MAPK, blocked the phosphorylation of MAPK but did not block GVBD, the dephosphorylation and activation of cdc2, or spindle formation and migration. Oocytes that underwent GVBD in PD 98059 could be fertilized and cleaved normally. Ionophore A23187, although it caused germinal vesicles to disappear and caused transient phosphorylation of MAPK, did not cause dephosphorylation of cdc2, and therefore this disappearance is artifactual. These results suggest that MAPK activation is neither obligatory nor sufficient for either GVBD or meiotic metaphase arrest inChaetopterusand that activation of MAPK and cdc2 occur on independent, parallel pathways.

Polyadenylation-mediated translational regulation of maternal P450(11beta) mRNA in frog oocytes.

Northern blot analysis of bullfrog tissues using a cDNA probe of cytochrome P450(11beta) showed that a large amount of message was present in the ovary as well as in the adrenal tissue. Two kinds of mRNA of different sizes were found in the ovary. Sequence determination of the two cDNAs and analysis by reverse-transcription polymerase chain reaction indicated that the protein encoded by the larger mRNA was identical to the adrenal enzyme, while the protein encoded by the smaller had a truncated sequence lacking an extension peptide necessary for the protein transport to the mitochondria. The mRNAs were present in the oocytes but not in the follicular cells, and their content in an oocyte varied little during its maturation. Immunoblot analyses of the mitochondrial fraction of oocytes failed to demonstrate the presence of P450(11beta) protein. In contrast the eggs were found to contain a large amount of enzymatically active protein. Interestingly the mRNA has a cis-element called cytoplasmic polyadenylation element at its 3' untranslated region. When poly(A) tails of the message prepared from eggs and oocytes were examined by RNase H digestion or reverse-transcription polymerase chain reaction, those of eggs were about 150 nucleotides longer than those of oocytes. These results suggest that translation of the message is stimulated during the oocyte maturation as a result of enhanced polyadenylation at its 3'-end. Finally a finding is presented that progesterone was converted to 11beta-hydroxyprogesterone by the frog P450(11beta), implying that the enzyme expressed in eggs may control a level of progesterone which is needed to initiate the oocyte maturation.

Evidence That Cysteine String Proteins Regulate an Early Step in the Ca2+-Dependent Secretion of Neurotransmitter atDrosophilaNeuromuscular Junctions

Previous work indicated that the temperature-dependent block of synaptic transmission in cysteine string protein (csp) mutants of Drosophila was attributable to a failure of nerve impulses to trigger transmitter release. The current investigations were undertaken to resolve in more detail the mechanism of this transmission deficit. Our studies reveal that the spider venom toxin alpha-latrotoxin can trigger a sustained discharge of quanta at neuromuscular junctions of csp mutant larvae at nonpermissive temperature. The same is true of the calcium ionophore ionomycin. However, solutions with an elevated concentration of K or Ca ions fail to circumvent the block of quantal secretion in these mutants. Likewise, 4-aminopyridine, which augments transmitter release at permissive temperature in csp mutants, fails to reverse the inhibition of impulse-evoked transmitter release at elevated temperature. These data are consistent with the hypothesis that there is a deficit either in Ca ion entry or in the ability of Ca ions to trigger exocytosis in csp mutants at nonpermissive temperatures. In part, because of previous work showing that csps are important for the functional expression of N-type Ca channels in frog oocytes, we favor the idea that csps participate in a regulatory interaction involving presynaptic Ca channels.

The Major Soluble Tubulins are Found in Mega Dalton (MDa) Fractions in Fully-Grown Oocytes and Eggs But Not in Brain of the Frog, Rana pipiens

During progesterone-induced oocyte maturation, the cell is converted from prophase I to metaphase II, which is known to involve microtubule changes. We hypothesize that progesterone affects the polymeric state of tubulins, isoform dynamics, as well as tubulin synthesis and degradation. To test the former, leopard frog (Rana pipiens) oocytes were treated with progesterone, homogenized and centrifuged at 16,000 × g. The resulting pellet and supernatant fractions were analyzed using immunoblots probed with anti-α-tubulin antibody DM1A and anti-β-tubulin antibody DM1B. The results indicate that the majority of tubulins are in supernatant fractions (soluble cellular component) and further indicate changes in size of pelletable tubulins with time postprogesterone treatment. To characterize the supernatant tubulins, Superose-6b columns were used to fractionate 16,000 × g supernatant materials. The results indicate tubulins in fractions of up to 5 MDa (equivalent in size to c. 50 tubulin dimers or 5,000 kDa) predominate, while tubulin dimers and monomers of 110 kDa and 55 kDa, respectively, are relatively minor components. The tubulin in MDa fraction pools in the oocyte soluble component seem to be tissue specific, since the reverse patterns were found in either frog brain or rat (Rattus norvegicus) brain tissue under the same conditions. Microtubule poisons, taxol and nocodazole, were used to test the dynamics of tubulin in MDa fractions and the results were opposite to that expected, i.e., taxol, a microtubule stabilizer, decreased tubulin in large MDa fractions while nocodazole, a microtubule destabilizer, increased it. Preliminary data also indicated that progesterone treatment alters the tubulin size classes (size-shift) in comparison with that in immature oocytes. These results, in combination with other research reports, suggest that tubulin in MDa fractions may either be associated with other cellular components or act as intermediates in microtubule dynamics, perhaps via oligomerization, during oocyte maturation and early development.

Nerve cell lesions caused by 3-hydroxyglutaric acid: a possible mechanism for neurodegeneration in glutaric acidaemia I.

Glutaric acidaemia type I (GA I) may be associated with elevated concentrations of glutaric, 3-hydroxyglutaric and glutaconic acids in blood and urine. Elevated concentrations of glutaric acid (about I mmol/L) (Goodman et al 1977) and 3-hydroxyglutaric acid (Land et al 1992) were also detected in brain tissue. Morphological examinations of the brain reveal atrophy of frontal and temporal lobes, the nucleus caudatus and putamen. The concept of excitotoxicity describes the potential cell damage induced by excitatory amino acids like glutamate (via N-methyl-D-aspartate (NMDA) and non-NMDA receptors). Excessive activation of glutamate receptors can be mediated by either elevated agonist concentrations or cellular energy depletion even in the presence of normal agonist concentrations (Olney 1980). Excitotoxic mechanisms may be suspected to lead to neuronal degeneration in GA I as glutaric acid inhibits glutamate uptake into synaptosomes (Bennett et al 1973) and glutaric, 3-hydroxyglutaric, and glutaconic acids inhibit glutamate decarboxylase, the enzyme which produces γ-aminobutyric acid (GABA) (Stokke et al 1976). In addition, post-synaptic vacuolization, characteristic of excitotoxic neuronal death, has been described in post-mortem examination of brain tissue (cortex and striatum) (Goodman et al 1977). In organotypic slice cultures from rat brain expressing different glutamate receptor subtypes (Gahwiler 1981; Vornov et al 1991), we tested whether glutaric, 3-hydroxyglutaric, or glutaconic acids are neurotoxic (normal and energy deprived conditions) and if this neurotoxicity is related to excitotoxic mechanisms. In addition, the effect of glutaric, 3-hydroxyglutaric and glutaconic acids on glutamate receptor-mediated membrane currents was investigated in frog oocytes expressing different glutamate receptor subtypes (Musshoff et al 1994).

Intracellular localization and unique conserved sequences of three small nucleolar RNAs.

Three human small nucleolar RNAs (snoRNAs), E1, E2 and E3, were reported earlier that have unique sequences, interact directly with unique segments of pre-rRNA in vivo and are encoded in introns of protein genes. In the present report, human and frog E1, E2 and E3 RNAs injected into the cytoplasm of frog oocytes migrated to the nucleus and specifically to the nucleolus. This indicates that the nucleolar and nuclear localization signals of these snoRNAs reside within their evolutionarily conserved segments. Homologs of these snoRNAs from several vertebrates were sequenced and this information was used to develop RNA secondary structure models. These snoRNAs have unique phylogenetically conserved sequences.

Mitogen-activated protein kinase activation down-regulates a mechanism that inactivates cyclin B-cdc2 kinase in G2-arrested oocytes.

The G2 arrest of oocytes from frogs, clams, and starfish requires that preformed cyclin B-cdc2 complexes [prematuration-promoting factor (MPF)] be kept in an inactive form that is largely due to inhibitory phosphorylation of this pre-MPF. We have investigated the role of mitogen-activated protein (MAP) kinase in the activation of this pre-MPF. The cytoplasm of both frog and starfish oocytes contains an activity that can rapidly inactivate injected MPF. When the MAP kinase of G2-arrested starfish or Xenopus oocytes was prematurely activated by microinjection of c-mos or Ste-11 delta N fusion proteins, the rate and extent of MPF inactivation was much reduced. Both effects were suppressed by expression of the specific MAP kinase phosphatase Pyst 1. These results show that MAP kinase down-regulates a mechanism that inactivates cyclin B-cdc2 kinase in Xenopus oocytes. In starfish oocytes, however, MAP kinase activation occurs only after germinal vesicle breakdown, much after MPF activation. In this case, down-regulation of the cyclin B-cdc2 inhibiting pathway is a sensitive response to hormonal stimulation that does not require MAP kinase activation.

Low temperature induces oocytes p34cdc2 synthesis and accumulation- the acquisition of competence to resume meiosis in toad oocytes

Low temperature induces oocytes p34 cdc2 synthesis and accumulation- the acquisition of competence to resume meiosis in toad oocytes 1

Cleavage Arrest of Early Frog Embryos by the G Protein-activated Protein Kinase PAK I

PAK I is a member of the PAK (p21-activated protein kinase) family and is activated by Cdc42 (Jakobi, R., Chen, C.-J., Tuazon, P. T., and Traugh, J. A. (1996) J. Biol. Chem. 271, 6206-6211). To examine the effects of PAK I on cleavage arrest, subfemtomole amounts of endogenously active (58 kDa) and inactive (60 kDa) PAK I and a tryptic peptide (37 kDa) containing the active catalytic domain were injected into one blastomere of 2-cell frog embryos. Active PAK I resulted in cleavage arrest in the injected blastomere at mitotic metaphase, whereas the uninjected blastomere progressed through mid- to late cleavage. Injection of other protein kinases at similar concentrations had no effect on cleavage. Endogenous PAK I was highly active in frog oocytes, and antibody to PAK I reacted specifically with protein of 58-60 kDa. PAK I protein was decreased at 60 min post-fertilization, with little or no PAK I protein or activity detectable at 80 min post-fertilization or in 2-cell embryos. At the 4-cell stage PAK I protein increased, but the protein kinase was present primarily as an inactive form. Rac2 and Cdc42, but not Rac 1, were identified in oocytes and throughout early embryo development. Thus, PAK I appears to be a potent cytostatic protein kinase involved in maintaining cells in a non-dividing state. PAK I activity is high in oocytes and appears to be regulated by degradation/synthesis and through autophosphorylation via binding of Cdc42. PAK I may act through regulation of the stress-activated protein kinase signaling pathway and/or by direct regulation of multiple metabolic pathways.

Glycogen synthesis in amphibian oocytes: evidence for an indirect pathway.

Glycogen is the main end product of glucose metabolism in amphibian oocytes. However, in the first few minutes after [U-14C]glucose microinjection most of the label is found in lactate. The burst of lactate production and the shape of the time curves for the labelling of glucose 6-phosphate, fructose 6-phosphate, glucose 1-phosphate and glycogen suggest a precursor-product relationship of lactate with respect to glycogen and its intermediates. Expansion (by microinjection) of the pool of glycolytic intermediates, such as dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, 3-phosphoglycerate or phosphoenolpyruvate, results in a marked decrease in [U-14C]glucose incorporation into glycogen. After co-injection of doubly labelled glucoses, extensive detritiation (93%) of the glucosyl units of glycogen was observed with [2-3H, U-14C]glucose and partial detritiation with [3-3H,U-14C]glucose (34%) or [5-3H,U-14C]glucose (46%). After injection of [6-3H,U-14C]glucose, a small but significant and reproducible detritiation (13%) in glycogen was observed. Co-injection of [U-14C]glucose and 3-mercaptopicolinate resulted in marked inhibition of glycogen labelling. Half-maximal inhibition was observed at 0.58 mM 3-mercaptopicolinate, which agrees with the IC50 value (0.47 mM) for the inhibition in vitro of phosphoenolpyruvate carboxykinase activity. We concluded that in frog oocytes most of the glucosyl units are incorporated into glycogen by an indirect pathway involving breakdown of glucose to lactate, which is then converted into glycogen via gluconeogenesis. Both processes, glycolytic degradation of glucose to lactate and subsequent reconversion of the latter into hexose phosphates and glycogen, occur in the same cell.

Functional reconstitution of KCl-evoked, Ca2+-dependent acetylcholine release system inXenopus oocytes microinjected with presynaptic plasma membranes and synaptic vesicles

We have developed a new method for the generation of functionally active presynaptic chimeras in Xenopus laevis oocytes. Frog oocytes injected with presynaptic subcellular fractions extracted from the electric organ of Torpedo marmorata release acetylcholine in a calcium-dependent manner upon chemical stimulation. Neither oocytes injected without presynaptic plasma membranes nor oocytes injected with ghost erythrocyte plasma membrane instead of presynaptic plasma membrane release acetylcholine. This suggests that specific presynaptic components necessary for KCl-evoked, Ca(2+)-dependent acetylcholine release become functionally integrated in the Xenopus laevis oocytes. Moreover, rhodaminated presynaptic plasma membranes and the synaptic vesicle protein synaptophysin are detected on the oocyte surface by fluorescence or immunofluorescence, respectively, showing that the injected presynaptic components are incorporated into the membrane of the frog oocyte. Furthermore, Botulinum neurotoxin type A, a specific blocker of acetylcholine release in the neuromuscular junction, inhibits the neurotransmitter release from the chimerical oocytes. This suggests that targets for toxin action are also functionally incorporated in the oocyte upon injection of membranous presynaptic components. Our results show that oocytes injected with presynaptic components behave as cholinergic nerve ending chimeras, at least in terms of neurotransmitter release and toxin targets. The system bypasses some problems associated with messenger RNA expression because not only proteins, but native presynaptic components are incorporated. This new technique may provide a useful approach for electrophysiological and pharmacological studies in order to characterize the synaptic transmission.

Amphibian transcription factor IIIA proteins contain a sequence element functionally equivalent to the nuclear export signal of human immunodeficiency virus type 1 Rev.

The human immunodeficiency virus type 1 (HIV-1) Rev protein is required for nuclear export of late HIV-1 mRNAs. This function is dependent on the mutationally defined Rev activation domain, which also forms a potent nuclear export signal. Transcription factor IIIA (TFIIIA) binds to 5S rRNA transcripts and this interaction has been proposed to play a role in the efficient nuclear export of 5S rRNA in amphibian oocytes. Here it is reported that amphibian TFIIIA proteins contain a sequence element with homology to the Rev activation domain that effectively substitutes for this domain in inducing the nuclear export of late HIV-1 mRNAs. It is further demonstrated that this TFIIIA sequence element functions as a protein nuclear export signal in both human cells and frog oocytes. Thus, this shared protein motif may play an analogous role in mediating the nuclear export of both late HIV-1 RNAs and 5S rRNA transcripts.

Sodium-dependent GABA-induced currents in GAT1-transfected HeLa cells.

1. HeLa cells were infected with recombinant vaccinia virus containing the T7 RNA polymerase gene and transfected with the cDNA for a rat GABA transporter, GAT1, cloned downstream of a T7 RNA polymerase promoter. Six to sixteen hours after transfection, whole-cell recording with a voltage ramp in the range -90 to 50 mV revealed GABA-induced currents (approximately -100 pA at -60 mV in 100 microM GABA, 16 h after transfection at room temperature). No GABA-induced currents were observed in parental HeLa cells or in mock-transfected cells. 2. GABA-induced currents were suppressed by extracellular perfusion with GABA-free solutions or addition of GAT1 inhibitors SKF89976-A or SKF100330-A. At fixed voltage the GABA dependence of the inward current fitted the Michaelis-Menten equation with a Hill coefficient, n, near unity and an equilibrium constant, K(m), near 3 microM. The Na+ dependence of the inward currents fitted the Michaelis-Menten equation with n approximately equal to 2 and K(m) approximately equal to 10 mM. The constants n and K(m) for GABA and Na+ were independent of voltage in the range -90 to -30 mV. 3. GABA-induced currents reverse direction in the range 5-10 mV. The implication of this result is that GAT1 can mediate electrogenic (electrophoretic) influx or efflux of GABA depending on the membrane voltage. The presence of an outward current in our experiments is consistent with radioactive-labelled flux data from resealed vesicle studies. However, it is inconsistent with frog oocyte expression experiments using the sample clone. In oocytes, GAT1 generates no outward current in a similar voltage range. Smaller intracellular volume or higher turnover rates in the mammalian expression system may explain the outward currents. 4. External GABA induces inward current, and internal GABA induces outward current. However, in cells initially devoid of internal GABA, external GABA can also facilitate an outward current. This GAT1-mediated outward current occurs only after applying negative potentials to the cell. These data are consistent with the concept that negative potentials drive GABA and Na+ into the cell, which then leads to electrogenic efflux through GAT1 at positive voltages. 5. Assuming coupled transport, we estimate the number of transporters, N, times the turnover rate, r, to be Nr approximately 10(9) s-1 under nominal conditions (V = -60 mV, 30 microM GABA, 130 mM Na+ and room temperature). This indicates either very high levels of expression (approximately 10(4) microns-2), assuming published turnover rates (approximately 10 s-1), or turnover rates that are significantly greater than previously reported. As an alternative, a channel may exist in the GAT1 protein that is gated by GABA and Na+ and blocked by GAT1 antagonists. The channel mode of conduction would exist in addition to the coupled, fixed-stoichiometry transporter mode of conduction.

Potassium Currents Expressed from Drosophila and Mouse eag cDNAs in Xenopus Oocytes

The ether-a-go-go (eag) gene family encodes a set of related ion channel polypeptides expressed in the excitable cells of organisms ranging from invertebrates to mammals. Earlier studies demonstrated that eag mutations in Drosophila cause an increase in membrane excitability in the nervous system. Mutations in the human eag-related gene (HERG) have been implicated in cardiac arrhythmia, and recent studies show that HERG subunits contribute to the channels mediating IKr and the terminal repolarization of the cardiac action potential. A physiological role for M-EAG, the mouse counterpart to Drosophila eag, has not been determined. Here, we describe basic properties of Eag and M-EAG channels expressed in frog oocytes, using two-electrode voltage clamp and patch clamp techniques. Both Eag and M-EAG channels are voltage-dependent, outwardly rectifying and highly selective for K+ over Na+ions. In contrast to previous reports, we found no evidence for Ca2+ flux through Eag channels. The most notable difference between these closely related channels is that Eag currents exhibit partial inactivation, whereas M-EAG currents are sustained for the duration of an activating voltage command. In addition, Eag currents run down more rapidly than do M-EAG currents in excised macropatches. Rundown is reversible by inserting the patch into the interior of the oocyte, indicating that a cytosolic factor regulates channel activity or stability. These studies should facilitate identification of currents mediated by Eag and M-EAG channels in vivo. Copyright © 1996 Published by Elsevier Science Ltd

YKC1 encodes the depolarization-activated K + channel in the plasma membrane of yeast

Our previous patch-clamp studies showed that depolarization activates a K +-specific current in the plasma membrane of the binding yeast, Saccharomyces cerevisiae [Gustin et al. (1986) Science 233, 1195–1197]. The yeast Genome Sequencing Project has now uncovered on the left arm of chromosome X an open reading frame (ORF) that predicts a 77-kDa protein reminiscent of a shaker-like α subunit with 6 membrane spans followed by a subunit with 2 spans. We found that deleting this ORF removes the yeast K + current. Furnishing the ORF from plasmids restores or even greatly amplifies this current. These manipulations have no effects on the 40-pS mechanosensitive conductance also native to this membrane. Thus, this ORF, named YKC1 here, likely encodes a structure for the K +-specific channel of the yeast plasma membrane. This and other K + channel subunits are compared and the possible uses of this gene in research are discussed. YKC1 has recently been shown by others to induce in frog oocytes a K + current. Its activation is coupled to E K + and its outward rectification depends on external divalent cations. We found the YKC1 channel in its native membrane activates at low voltages largely independent of E K + and it remains so despite removal of divalents by chelation.