Transport and membrane binding of the glutamine analogue 6-diazo-5-oxo-L-norleucine (DON) in Xenopus laevis oocytes

Summary We have examined transport and membrane binding of 6-diazo-5-oxo-l-norleucine (DON, a photoactive diazo-analogue of glutamine) and their relationships to glutamine transport in Xenopus laevis oocytes. DON uptake was stereospecific and saturable (V _max of 0.44 pmol/oocyte · min and a K _m of 0.065 mm). DON uptake was largely Na^u+ dependent (80% at 50 m DON) and inhibited (>75%) by glutamine and arginine (substrates of the System B^0,+ transporter) at 1 mm. Glutamine and DON show mutual competitive inhibition of Na⁺-dependent transport. Preincubation of oocytes in medium containing 0.1 mm DON for 24 or 48 hr depressed the V _max for System B^0,+ transport (as measured by Na⁺-dependent glutamine uptake), this effect was highly specific (neither d-DON nor the System B^0,+ substrates glutamine and d-alanine showed any independent effect) and required Na⁺ ions. Glutamine (1 mm in preincubation medium) protected transport from inhibition by DON. The possibility that specific inactivation of System B^0,+ by DON reflects attachment of DON to the transporter was tested by examining the binding of [¹⁴C]DON to Xenopus oocyte membranes. Oocytes incubated in 100 mm NaCl in the presence of [¹⁴C]DON for up to 48 hr showed 2.4-fold higher ¹⁴C-binding to membranes than oocytes incubated in choline chloride. Na⁺-dependent DON binding (31 ± 11 fmol/g membrane protein) was suppressed by external glutamine, arginine or alanine and was largely confined to a membrane protein fraction of 48–65 kDa (as assessed by SDS-polyacrylamide gel electrophoresis). The present studies indicate that DON and glutamine uptake in oocytes are both mediated by System B^0,+ and demonstrate that DON binding to a particular membrane protein fraction is associated with inactivation of the transporter, offering the prospect of using [¹⁴C]DON as a covalent label for the transport protein in order to facilitate its isolation and subsequent biochemical characterization.This work was supported by The Wellcome Trust, Action Research for the Crippled Child, Ajinomoto GmbH, Pfrimmer GmbH, the Rank Prize Funds, the Medical Research Council and the University of Dundee. We are grateful to Dr. C.I. Pogson (Wellcome Research Laboratories) and Drs. J.C. Ellory and B. Elford (University of Oxford) for gifts of [¹⁴C]DON.     

Expression of catfish amino acid taste receptors inXenopus oocytes

We demonstrate that poly (A⁺)RNA isolated from catfish barbels directs the expression of functional amino acid taste receptors in theXenopus oocyte. The activity of these receptors is monitored in ovo by the two electrode voltage clamp technique. Specific conductance changes recorded in response to amino acid stimulation are analogous to those recorded electrophysiologically from intact catfish barbels. These responses exhibit specificity, reproducibility, rapid onset and termination, and desensitization to repetitive stimulation. A functional assay system that encompasses the full complement of transduction events from the ligand-receptor interaction to subsequent conductance changes is necessary to identify molecular components responsible for these events. Our results demonstrate that theXenopus oocyte can be used to characterize and identify clones coding for amino acid taste receptors analogous to its use in studying receptor molecules for other neuroactive compounds.Special issue is dedicated to Dr. Sidney Udenfriend.     

Transport of branched-chain amino acids in Corynebacterium glutamicum

The transport of branched-chain amino acids was characterized in intact cells of Corynebacterium glutamicum ATCC 13032. Uptake and accumulation of these amino acids occur via a common specific carrier with slightly different affiniteis for each substrate (K _m[Ile]=5.4 M, K _m[Leu]=9.0 M, K _m[Val]=9.5 M). The maximal uptake rates for all three substrates were very similar (0.94–1.30 nmol/mg dw · min). The optimum of amino acid uptake was at pH 8.5 and the activation energy was determined to be 80 kJ/mol. The transport activity showed a marked dependence on the presence of Na⁺ ions and on the membrane potential, but was independent of an existing proton gradient. It is concluded, that uptake of branched-chain amino acid transport proceeds via a secondary active Na⁺-coupled symport mechanism.Abbreviations CCCP Carboxyl cyanide m-chlorophenylhydrazone - dw dry weight - MES 2[N-morpholino]ethanesulfonic acid - mon monensin - nig nigericin - TPP tetraphenylphosphonium bromide - Tris tris[hydroxymethyl]aminomethane - val valinomycin     

Endogenousd-glucose transport in oocytes ofXenopus laevis

Summary Endogenous glucose uptake by the oocytes ofXenopus laevis consists of two distinct components: one that is independent of extracellular Na⁺, and the other one that represents Na⁺-glucose cotransport. The latter shows similar characteristics as 2 Na⁺-1 glucose cotransport of epithelial cells: The similarities include the dependencies on external concentrations of Na⁺, glucose, and phlorizin, and on pH. As in epithelial cells, the glucose uptake in oocytes can also be stimulated by lanthanides. Both the electrogenic cotransport and the inhibition by phlorizin are voltage-dependent; the data are compatible with the assumption that the membrane potential acts as a driving force for the reaction cycle of the transport process. In particular, hyperpolarization seems to stimulat transport by recruitment of substrate binding sites to the outer membrane surface. The results described pertain to oocytes arrested in the prophase of the first meiotic division; maturation of the oocytes leads to a downregulation of both the Na⁺-independent and the Na⁺-dependent transport systems. The effect on the Na⁺-dependent cotransport is the consequence of a change of driving force due to membrane depolarization associated with the maturation process.     

Voltage dependence of the rheogenic Na+/K+ ATPase in the membrane of oocytes ofXenopus laevis

Summary Electrophysiological experiments were performed to analyze the Na⁺/K⁺-ATPase in full-grown prophase-arrested oocytes ofXenopus laevis. If the Na⁺/K⁺-ATPase is inhibited by dihydroouabain (DHO), the resting potential of the membrane of Na⁺-loaded oocytes may depolarize by nearly 50 mV. This hyperpolarizing contribution to the resting potential depends on the degree of activation of the Na⁺/K⁺-ATPase and varies with intra-cellular Na⁺ activity (a _Na ⁱ ), and extracellular K⁺ (K ₀ ⁺ ) It is concluded that variations ofa _Na ⁱ among different oocytes are primarily responsible for the variations of resting potentials measured in oocytes ofX. laevis. Under voltage-clamp conditions, the DHO-sensitive current also exhibits dependence ona _Na ⁱ that may be described by a Hill equation with a coefficient of 2. This current will be shown to be identical with the electrogenic current generated by the 3Na⁺/2K⁺ pump. The voltage dependence of the pump current was investigated at saturating values ofa _Na ⁱ (33 mmol/liter) and of K ₀ ⁺ (3 mmol/liter) in the range from –200 to +100 mV. The current was found to exhibit a characteristic maximum at about +20 mV. This is taken as evidence that in the physiological range at least two steps within the cycle of the pump are voltage dependent and are oppositely affected by the membrane potential.     

Regulation of endogenous and expressed Na+/K+ pumps in Xenopus oocytes by membrane potential and stimulation of protein kinases

Modulation of the current generated by the Na+/K+ pump by membrane potential and protein kinases was investigated in oocytes of Xenopus laevis. In addition to a positive slope region in the current-voltage (I-V) relationship of the Na+/K+ pump, a negative slope region has been described in these cells (Lafaire & Schwarz, 1986) and has been attributed to a voltage-dependent apparent Km value for pump stimulation by external [K+] (Rakowski et al., 1991). To study this feature in more detail, Xenopus oocytes were used for comparative analysis of the negative slope of the I-V relationship of the endogenous Na+/K+ pump and of the Na+/K+ pump of the electric organ of Torpedo californica expressed in the oocytes. The effects of stimulation of protein kinases A and C on the negative slope were also analyzed. To investigate the negative slope over a wide potential range, experiments were performed in Na(+)-free solution and in the presence of high concentrations of Ba2+ and tetraethylammonium, to block all nonpump related K(+)-sensitive currents. Pump currents and pump-mediated fluxes were determined as differences of currents or fluxes in solutions with and without extracellular K+. The voltage dependence of the Km value for stimulation of the Na+/K+ pump by external [K+] shows significant species differences. Over the entire voltage range from -140 to +20 mV, the Km value for the Na+/K+ pump of Torpedo electroplax is substantially higher than for the endogenous pump and exhibits more pronounced voltage dependence. For the Xenopus pump, the voltage dependence can be described by voltage-dependent stimulation by external [K+] and can be interpreted by voltage-dependent K+ binding, assuming that an effective charge between 0.37 and 0.56 of an elementary charge is moved in the electrical field. An analogous evaluation of the voltage dependence of the Torpedo pump requires the assumption of movement of two effective charges of 0.16 and 1.0 of an elementary charge. Application of 1,2-dioctanoyl-sn-glycerol (diC8, 10-50 microM), which is known to stimulate protein kinase C, reduces the maximum activity of the Xenopus pumps in the oocyte membrane by 40% and modulates the voltage dependence of K+ stimulation. For the endogenous Xenopus pump, the apparent effective charge increased from 0.37 to 0.51 of elementary charge and the apparent Km at 0 mV increased from 0.46 to 0.83 mM. For the Torpedo pump, one of the apparent effective charges increased from 1.0 to 2.5 of elementary charge.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mutational analysis of potential pore-lining amino acids in TM IV of the Na/H exchanger

The Na⁺/H⁺ exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH by extruding an intracellular H⁺ in exchange for one extracellular Na⁺. In this study we examined the effect of site-specific mutagenesis on the pore-lining amino acid Phe161 and effects of mutagenesis on the charged amino acids Asp159 and Asp172. There was no absolute requirement for a carboxyl side chain at amino acid Asp159 or Asp172. Mutation of Asp159 to Asn or Gln maintained or increased the activity of the protein. Similarly, for Asp172, substitution with a Gln residue maintained activity of the protein, even though substitution with an Asn residue was inhibitory. The Asp172Glu mutant possessed normal activity after correction for its aberrant expression and surface targeting. Replacement of Phe161 with a Leu demonstrated that it was not irreplaceable in NHE1 function. However, the mutation Phe161lys inhibited NHE1 function, while the Phe161Ala mutation caused altered NHE1 targeting and expression levels. Our results show that these three amino acids, while being important in NHE1 function, are not irreplaceable. This study demonstrates that multiple substitutions at a single amino acid residue may be necessary to get a clearer picture membrane protein function.     

Endogenous lactate transport in Xenopus laevis oocyte: dependence on cytoskeleton and regulation by protein kinases

Carbon flux in Xenopus laevis oocyte is glycogenic and an endogenous monocarboxylate transporter is responsible for intracellular lactate uptake. The aim of the present study was to determine if direct activation of protein kinases C and A modulates the activity of lactate transporter, as well as to investigate the possible role of cytoskeleton in these regulatory phenomena. The modulation was studied in isolated Xenopus oocytes of stage V–VI by measuring ¹⁴C-lactate uptake, both in the absence and in the presence of cytoskeletal-perturbing toxins. We found that the basal lactate transporter activity depends on the integrity of the cytoskeleton since it is partially inhibited by cytoskeleton disorganisation. Both PKA and PKC activation caused a significant decrease in transport activity and this decrease could be blocked by specific protein kinase inhibitors. The evidenced effects were not additive. Transport inhibition was annulled by agents that destabilize actin filaments or microtubules. We conclude that both protein kinases A and C, whose effects are mediated by cytoskeleton, negatively regulate the endogenous lactate transporter of Xenopus oocyte, suggesting that these kinases may have a role in the control of cytosolic pyruvate/lactate pool in the oocyte. All the experiments in this study comply with the current laws of Italy.     

Functional expression of renal organic anion transport in Xenopus laevis oocytes

Secretion of organic anions by the kidney plays a critical role in the elimination of toxic agents from the body. Recent findings in isolated membranes and intact tissue have demonstrated the participation of multiple transport proteins in this process. As a first step toward molecular characterization of these proteins through expression cloning, the studies reported below demonstrate functional expression of both fumarate- and lithium-sensitive glutarate and probenecid-sensitive p-aminohippurate transport in Xenopus oocytes injected with rat kidney poly(A)⁺RNA. Maximal increase in substrate uptake over buffer-injected controls was reached by 5 days after mRNA injection. Expression of size-fractionated mRNA indicated that the active species with respect to both transport activities were in the range of 1.8 to 3.5 kb.     

Mobilization of newly synthesized RNAs into polysomes inXenopus laevis embryos

Summary The mobilization of newly synthesized 18S and 28S rRNAs, 4S RNA and poly(A)⁺ RNA into polysomes was studied in isolated cells ofXenopus laevis embryos between cleavage and neurula stages. Throughout these stages, 4S RNA and poly(A)⁺ RNA were mobilized immediately following their appearance in the cytoplasm. 18S rRNA however, stayed in the ribosomal subunit fraction for about 30 min until the 28S rRNA appeared, when the two rRNAs were mobilized together at an equimolar ratio. This mobilization, at a 1:1 molar ratio, appeared to be realized at initiation monome formation. Thus, the efficiency of the mobilization of two newly synthesized rRNAs, shortly after their arrival at the cytoplasm, differed considerably but difference disappeared once steady state was reached.The contribution of newly synthesized 18S and 28S rRNAs to polysomes remains small throughout early development. around 3% of newly synthesized 4S RNA is polysomal which is the same distribution observed for unlabeled 4S RNA. Less than 10% of the newly synthesized cytoplasmic poly(A)⁺ RNA was mobilized into polysomes during cleavage, but in later stages the proportion increased to around 20%–25%. These results show that newly synthesized RNAs are utilized for protein synthesis at characteristic rates soon after they are synthesized during early embryonic development. On the basis of the data presented here and elsewhere we discuss quantitative aspects of the utilization of newly synthesized and maternal RNAs during early embryogenesis.     

Transport of Na+ inside the giant axon of squid

The transport mechanism of Na ions within the nerve cell was studied by measuring the radioactivity distribution profile of²²Na that had been intracellularly injected into the giant axon. Specifically, we tested whether or not the movement of Na ions is coupled with the process of “fast axonal transport.” Results of our measurements indicate that the intracellular transport of Na⁺ and the fast axonal transport are two independent processes. Very few Na ions are irreversibly sequestered into the axoplasmic vesicles involved in axonal transport. The movement of Na⁺ inside the axon can be modeled by a one-dimension diffusion. The effective diffusion coefficient of the intracellular Na⁺ was determined in this study.     

Evidence for coupling between Na+ pump activity and TEA-sensitive K+ currents in Xenopus laevis oocytes

Using the two-microelectrode voltage clamp technique in Xenopus laevis oocytes, we estimated Na⁺-K⁺-ATPase activity from the dihydroouabain-sensitive current (I _DHO) in the presence of increasing concentrations of tetraethylammonium (TEA⁺; 0, 5, 10, 20, 40 mm), a well-known blocker of K⁺ channels. The effects of TEA⁺ on the total oocyte currents could be separated into two distinct parts: generation of a nonsaturating inward current increasing with negative membrane potentials (V _M) and a saturable inhibitory component affecting an outward current easily detectable at positive V _M. The nonsaturating component appears to be a barium-sensitive electrodiffusion of TEA⁺ which can be described by the Goldman-Hodgkin-Katz equation, while the saturating component is consistent with the expected blocking effect of TEA⁺ on K⁺ channels. Interestingly, this latter component disappears when the Na⁺-K⁺-ATPase is inhibited by 10 m DHO. Conversely, TEA⁺ inhibits a component of I _DHO with a k _d of 25±4 mm at +50 mV. As the TEA⁺-sensitive current present in I _DHO reversed at –75 mV, we hypothesized that it could come from an inhibition of K⁺ channels whose activity varies in parallel with the Na⁺-K⁺-ATPase activity. Supporting this hypothesis, the inward portion of this TEA⁺-sensitive current can be completely abolished by the addition of 1 mm Ba²⁺ to the bath. This study suggests that, in X. laevis oocytes, a close link exists between the Na-K-ATPase activity and TEA⁺-sensitive K⁺ currents and indicates that, in the absence of effective K⁺ channel inhibitors, I _DHO does not exclusively represent the Na⁺-K⁺-ATPase-generated current.     

Mitochondrial Calcium Transport Systems: Properties, Regulation, and Taxonomic Features

Currently available information on properties and regulation of mitochondrial Ca2+ transporting systems in eukaryotic cells is summarized. We describe in detail kinetic properties and effects of inhibitors and modulators on the energy-dependent Ca2+ uptake through the Ca2+ uniporter, as well as on Na+-dependent and Na+-independent pathways for Ca2+ release in mammalian mitochondria. Special emphasis is placed on Ca2+ transport systems (for ion uptake and release) in mitochondria of higher plants, algae, and yeasts. Potential physiological implications of mitochondrial Ca2+ fluxes (influx and efflux), e.g., regulation of activity of Ca2+-dependent enzymes of the Krebs cycle, maintaining of cellular Ca2+ homeostasis, and engagement in pathophysiological processes, are discussed.     

The effects of amiloride on electrolyte transport and acid-base balance in the larval salamander,Ambystoma tigrinum

Summary The responses of net and unidirectional fluxes of Na⁺ and acid-base balance to the drug amiloride were assessed during normocapnia and hypercapnia in larval salamanders, Ambystoma tigrinum. Isotope flux measurements demonstrated that 10^-4 M amiloride in the external medium inhibits Na⁺ influx during normocapnia and reverses the usual increase in influx of this ion during hypercapnia, causing a significant decrease instead. Measurements of blood-gas/acid-base balance conditions of artcrially cannulated salamanders demonstrated a significant metabolic acidosis in amiloridetreated animals that did not occur in untreated animals over the same period. the same concentration of amiloride also blocked the normal compensatory increase in [HCO _- ³ ] that follows a respiratory acidosis produced by a hypercapnic environment.Abbreviations IU international nnits - J _in influx - J _net net flux - PCO ₂ parial pressure of carbon dioxide     

Functional analysis of amino acids of the Na+/H+ exchanger that are important for proton translocation

The Na⁺/H⁺ exchanger is an integral membrane protein found in the plasma membrane of eukaryotic and prokaryotic cells. In eukaryotes it functions to exchange one proton for a sodium ion. In mammals it removes intracellular protons while in plants and fungal cells the plasma membrane form removes intracellular sodium in exchange for extracellular protons. In this study we used the Na⁺/H⁺ exchanger of Schizosaccharomyces pombe (Sod2) as a model system to study amino acids critical for activity of the protein. Twelve mutant forms of the Na⁺/H⁺ exchanger were examined for their ability to translocate protons as assessed by a cytosensor microphysiometer. Mutation of the amino acid Histidine 367 resulted in defective proton translocation. The acidic residues Asp145, Asp178, Asp266 and Asp267 were important in the proton translocation activity of the Na⁺/H⁺ exchanger. Mutation of amino acids His98, His233 and Asp241 did not significantly impair proton translocation by the Na⁺/H⁺ exchanger. These results confirm that polar amino acids are important in proton flux activity of Na⁺/H⁺ exchangers.     

Progesterone-induced down-regulation of an electrogenic Na+, K+-ATPase during the first meiotic division in amphibian oocytes

Summary Progesterone initiates the resumption of the meiotic divisions in the amphibian oocyte. Depolarization of theRana pipiens oocyte plasma membrane begins 6–10 hr after exposure to progesterone (1–2 hr before nuclear breakdown). The oocyte cytoplasm becomes essentially isopotential with the medium by the end of the first meiotic division (20–22 hr). Voltage-clamp studies indicate that the depolarization coincides with the disappearance of an electrogenic Na⁺, K⁺-pump, and other electrophysiological studies indicate a decrease in both K⁺ and Cl^– conductances of the oocyte plasma membrane. Measurement of [³H]-ouabain binding to the plasma-vitelline membrane complex indicates that there are high-affinity (K _d-4.2×10^–8 m), K⁺-sensitive ouabain-binding sites on the unstimulated (prophase-arrest) oocyte and that ouabain binding virtually disappears during membrane depolarization. [³H]-Leucine incorporation into the plasma-vitelline membrane complex increased ninefold during depolarization with no significant change in uptake or incorporation into cytoplasmic proteins or acid soluble pool(s). This together with previous findings suggests that progesterone acts at a translational level to produce a cytoplasmic factor(s) that down-regulates the membrane Na⁺, K⁺-ATPase and alters the ion permeability and transport properties of both nuclear and plasma membranes.     

The expression of Na+/K+-ATPase β-subunit cRNA injected into Xenopus oocytes is affected by coinjection with α-subunit cRNA

The cRNA for Torpedo californica Na⁺/K⁺-ATPase -subunit (cRNA) was injected into Xenopus oocytes alone or with the cRNA for the Na⁺/K⁺-ATPase -subunit (cRNA). When cRNA was injected alone, the amount of the -subunit that accumulated in oocytes increased with increasing amounts of injected cRNA. When cRNA and cRNA were injected simultaneously, less -subunit accumulated than when cRNA was injected alone, whereas the Na⁺/K⁺-ATPase activity increased markedly. The decrease in the accumulation of the -subunit was dose-dependent upon the cRNA. The mutant -subunit unable to assemble with the -subunit accumulated in oocytes independently of cRNA, suggesting that post-translational control mechanisms may serve to reduce the accumulation of the -subunit.This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (No. 05259226, No. 06454149).     

Increases in transepithelial vectorial Na+ transport facilitates Na+-dependent L-DOPA transport in renal OK cells

The present study evaluated the hypothesis of whether increases in vectorial Na+ transport translate into facilitation of Na+-dependent L-DOPA uptake in cultured renal epithelial tubular cells. Increases in vectorial Na+ transport were obtained in opossum kidney (OK) cells engineered to overexpress Na+-K+-ATPase after transfection of wild type OK cells with the rodent Na+-K+-ATPase alpha1 subunit. The most impressive differences between wild type and transfected OK cells are that the latter overexpressed Na+-K+-ATPase accompanied by an increased activity of the transporter. Non-linear analysis of the saturation curve for l-DOPA uptake revealed a Vmax value (in nmol mg protein/6 min) of 62 and 80 in wild type and transfected cells, respectively. The uptake of a non-saturating concentration (0.25 microM) of [14C]-L-DOPA in OK-WT cells was not affected by Na+ removal, whereas in OK-alpha1 cells accumulation of [14C]-L-DOPA was clearly dependent on the presence of extracellular Na+. When Na+ was replaced by choline, the inhibitory profile of neutral l-amino acids, but not of basic and acidic amino acids, upon [14C]-L-DOPA uptake in both cell types, was significantly greater than that observed in the presence of extracellular Na+. It is concluded that enhanced ability of OK cells overexpressing Na+-K+-ATPase to translocate Na+ from the apical to the basal cell side correlates positively with their ability to accumulate L-DOPA, which is in agreement with the role of Na+ in taking up the precursor of renal dopamine.     

Amiloride-sensitive Na+ transport in human red cells: Evidence for a Na/H exchange system

Summary The role of transmembrane pH gradients on the ouabain, bumetanide and phloretin-resistant Na⁺ transport was studied in human red cells. Proton equilibration through the Jacobs-Stewart cycle was inhibited by the use of DIDS (125 m) and methazolamide (400 m). Red cells with different internal pH (pH _i =6.4, 7.0 and 7.8) were prepared and Na⁺ influx was measured at different external pH (pH _o =6.0, 7.0, 8.0). Na⁺ influx into acid-loaded cells (pH _i =6.4) markedly increased when pH _o was raised from 6.0 to 8.0. Amiloride, a well-known inhibitor of Na⁺/H⁺ exchange systems blocked about 60% of the H⁺-induced Na⁺ entry, while showing small inhibitory effects in the absence of pH gradients. When pH₀ was kept at 8.0, the amiloride-sensitive Na⁺ entry was abolished as pH _i was increased from 6.4 to 7.8. Moreover, measurements of H⁺ efflux into lightly buffered media indicated that the imposition of an inward Na⁺ gradient stimulated a net H⁺ efflux which was sensitive to the amiloride analog 5-N-methyl-N-butyl-amiloride. Furthermore, in the absence of a chemical gradient for Na⁺ (Na _i ⁺ =Na ₀ ⁺ =15mm,Em=+6.7 mV), an outward H⁺ gradient (pH _i =6.4, pH₀=8.0) promoted a net amiloride-sensitive Na⁺ uptake which was abolished at an external pH of 6.0. These findings are consistent with the presence of an amiloride-sensitive Na⁺/H⁺ exchange system in human red cells.