The influence of uncouplers on facilitated diffusion of sorbose in Saccharomyces cerevisiae

Sorbose uptake in Saccharomyces cerevisiae, strain Delft 1, proceeds via mediated passive transport. In the cell sorbose is distributed in at least two compartments. Efflux studies showed that sorbose uptake in one of these compartments is not readily reversible. Uncouplers of oxidative phosphorylation inhibit both transport velocity and steady-state uptake level. It could be shown that these two effects are caused by different modes of action of the uncouplers. None of these two effects could be ascribed to changes of the electrochemical H+ gradient or of the intracellular pH. It is suggested that the inhibition of uptake velocity is caused by binding of the uncoupler to the sorbose translocator, thus lowering the transport activity. The uncoupler binding site is probably located at the intracellular fragment of the carrier. The second effect, reduction of the steady-state uptake level, is probably due to blocking of sorbose influx into the compartment that exhibits poor reversibility.     

Kinetic analysis of simultaneously occurring proton-sorbose symport and passive sorbose transport in Saccharomyces fragilis

Sorbose transport in Saccharomyces fragilis takes place both via an active sugar-H⁺ symport system and via facilitated diffusion.To establish whether the two modes of transport proceed via the same transporter or via two different carriers, the kinetic consequences of both models were investigated. The kinetic equations for initial transport were derived for three possible reaction sequences with respect to sugar and H⁺ binding to the symport carrier: random binding and obligatory ordered binding with either sugar or H⁺ binding first, yielding six sets of kinetic parameters.Analysis of experimental data of sorbose transport in S. fragilis showed the existence of separate carriers for active, sorbose-H⁺ symport and facilitated diffusion. Furthermore, it could be concluded that the symport carrier shows random binding of sugar and H⁺.In recent literature, a similar combination of active and passive sugar transport in Rhodotorula gracilis and Chlorella vulgaris was interpreted as two modes of action of the same carrier, viz., active symport via the protonated, and facilitated diffusion via the unprotonated carrier. Analysis of the experimental data according to the criteria presented in this paper showed, however, that this supposition is untenable and that two different carriers must also be involved in these micro-organisms.     

Towards an Understanding of Mesocestoides vogae Fatty Acid Binding Proteins’ Roles

Two fatty acid binding proteins, MvFABPa and MvFABPb were identified in the parasite Mesocestoides vogae (Platyhelmithes, Cestoda). Fatty acid binding proteins are small intracellular proteins whose members exhibit great diversity. Proteins of this family have been identified in many organisms, of which Platyhelminthes are among the most primitive. These proteins have particular relevance in flatworms since de novo synthesis of fatty acids is absent. Fatty acids should be captured from the media needing an efficient transport system to uptake and distribute these molecules. While HLBPs could be involved in the shuttle of fatty acids to the surrounding host tissues and convey them into the parasite, FABPs could be responsible for the intracellular trafficking. In an effort to understand the role of MvFABPs in fatty acid transport of M. vogae larvae, we analysed the intracellular localization of both MvFABPs and the co-localization with in vivo uptake of fatty acid analogue BODIPY FL C₁₆. Immunohistochemical studies on larvae sections using specific antibodies, showed a diffuse cytoplasmic distribution of each protein with some expression in nuclei and mitochondria. MvFABPs distribution was confirmed by mass spectrometry identification from 2D-electrophoresis of larvae subcellular fractions. This work is the first report showing intracellular distribution of MvFABPs as well as the co-localization of these proteins with the BODIPY FL C₁₆ incorporated from the media. Our results suggest that fatty acid binding proteins could target fatty acids to cellular compartments including nuclei. In this sense, M. vogae FABPs could participate in several cellular processes fulfilling most of the functions attributed to vertebrate’s counterparts.     

Uptake of mercury by Chlorella and its effect on potassium regulation

Summary Addition of mercuric chloride at concentrations which resulted in an overall binding level of about 8 mmoles Hg/l packed cells and above caused a breakdown in the permeability of the cell membrane as indicated by a net efflux of internal K⁺. Below this level in region of 2 mmoles Hg/l packed cells the rate of K⁺ transfer across the cell surface was stimulated without affecting the internal K⁺ level. Maintainence of the stimulation was dependent both on time and dose. Enhancement of the rate of K⁺ turnover was associated with a fast component of the inorganic mercury uptake which could be removed by washing with cysteine. The mercury stimulated K⁺/K⁺ exchange was inhibited by low temperature, by the uncoupler CCCP and the energy transfer inhibitor DCCD. Overall binding concentrations of inorganic mercury below 0.5 mmoles/l packed cells had no effect on the K⁺ transport system. In contrast to mercuric chloride, methyl mercuric chloride over similar concentration ranges did not seem to induce a breakdown in the permeability barrier or directly interact with the K⁺/K⁺ exchange but more likely influenced the latter by inhibiting intracellular processes.     

Transport of methotrexate in L1210 cells: Effect of ions on the rate and extent of uptake

Transport of methotrexate (MTX) in L1210 cells is highly dependent upon the ionic composition of the external medium. Half-maximal rates of MTX transport (K_t values) vary from 0.9 μm in cells suspended in potassium-Hepes buffer containing Mg²⁺ (Hepes-Mg), to 10 μm in phosphate-buffered saline (PBS). At saturating levels of substrate, however, transport rates approach the same maximum velocity (V) regardless of the buffering medium. The increased K_t value for MTX in PBS is due to the presence of the competitive inhibitors, phosphate (K_i = 0.87 mM) and Cl^? (K_i = 46 mM). Concentration gradients for MTX at the steady state are also much lower (about 20-fold) in PBS than in Hepes-Mg; the components of PBS that reduce this uptake parameter are phosphate, Cl^?, Ca²⁺, and Na⁺. Ions that decrease the influx rate or the steady-state level also produce an increase in MTX efflux. Glucose (which increases ATP levels) reduces influx rates and steady-state levels of MTX, and induces efflux in both PBS and Hepes-Mg. Conversely, the combination of azide plus iodoacetate (which reduces ATP levels) stimulates MTX uptake in PBS, but has little effect on MTX transport parameters in Hepes-Mg. The unusually high sensitivity of MTX transport to various anions is consistent with the hypothesis that this system catalyzes the exchange of external MTX for an intracellular anion, and that efflux of the anion down a concentration gradient provides the driving force for active transport of MTX.     

ATP depletion promotes deactivation of insulin-stimulated sugar transport in rat soleus muscle

It has been reported that deactivation of insulin-stimulated sugar transport in adipocytes is an energy-dependent process (F. V. Vega, R. J. Key, J. E. Jordan, and T. Kono (1980) Arch. Biochem. Biophys. 203, 167-173). The stimulatory effect of insulin (0.1 U/ml) on the uptake of D-[U-14C]xylose by rat soleus muscle was rapidly reversed when muscle ATP was depleted by exposure to 2,4-dinitrophenol (0.5 mM). Insulin action was not completely eliminated by ATP depletion; there was a small, residual stimulatory effect of the hormone which persisted for about 30 min after muscle ATP had been lowered to an unmeasurable level. The extent of deactivation was not altered when the rate of ATP depletion was accelerated, either by increasing the 2,4-dinitrophenol concentration, or by inducing leakiness by incubating muscles for 90 min at 37 degrees C prior to the addition of the uncoupler. 2,4-Dinitrophenol lowered steady-state 125I-insulin binding. These differences between the effect of ATP depletion on insulin-stimulated sugar transport in muscle and adipose tissue may be related to the action of the uncoupler in lowering steady-state insulin binding in muscle. Such a fall in bound insulin could be expected to promote deactivation during the period of ATP depletion. However, at present the possibility that these differences may represent some more fundamental difference in deactivation between muscle and adipose tissue cannot be excluded.     

Copper Uptake and Its Effect on Metal Distribution in Root Growth Zones of Commelina communis Revealed by SRXRF

To explore the copper uptake mechanisms by the Cu-tolerant plant Commelina communis, the contents of Cu and other metals (including Fe, Zn, and Mn) in roots were detected using atomic absorption spectrometer under transporter inhibitors, partial element deficiency, or Cu excess treatments, while distribution characters of Cu and other metals in root growth zones were investigated by synchrotron radiation X-ray fluorescence spectroscopy (SRXRF). Cu uptake was inhibited by the uncoupler DNP and P-type ATPase inhibitor Na₃VO₄, not by the Ca²⁺ ion channel inhibitor LaCl₃, suggesting that Cu could probably be assimilated actively by root and be related with P-type ATPase, but not through Ca²⁺ ion channel. Fe or Zn deficiency could enhance Cu uptake, while 100 μM Cu inhibited Fe, Zn, and Mn accumulation in roots significantly. Metal distribution under 100 μM Cu treatment was investigated by SRXRF. High level of Cu was found in the root meristem, and higher Cu concentrations were observed in the vascular cylinder than those in the endodermis, further demonstrating the initiative Cu transport in the root of C. communis. Under excess Cu stress, most Fe was located in the epidermis, and Fe concentrations in the endodermis were higher than those in the vascular cylinder, suggesting Cu and Fe competition not only in the epidermal cells but also for the intercellular and intracellular transport in roots. Zn was present in the meristem and the vascular cylinder similar to Cu. Cu and Zn showed a similar pattern. Mn behaves as Zn does, but not like Fe.     

Effects of monovalent cations on Ca uptake by skeletal and cardiac muscle sarcoplasmic reticulum

Ca²⁺ transport by the sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase (SERCA) is sensitive to monovalent cations. Possible K⁺ binding sites have been identified in both the cytoplasmic P-domain and the transmembrane transport-domain of the protein. We measured Ca²⁺ transport into SR vesicles and SERCA ATPase activity in the presence of different monovalent cations. We found that the effects of monovalent cations on Ca²⁺ transport correlated in most cases with their direct effects on SERCA. Choline⁺, however, inhibited uptake to a greater extent than could be accounted for by its direct effect on SERCA suggesting a possible effect of choline on compensatory charge movement during Ca²⁺ transport. Of the monovalent cations tested, only Cs⁺ significantly affected the Hill coefficient of Ca²⁺ transport (n_H). An increase in n_H from ∼2 in K⁺ to ∼3 in Cs⁺ was seen in all of the forms of SERCA examined. The effects of Cs⁺ on the maximum velocity of Ca²⁺ uptake were also different for different forms of SERCA but these differences could not be attributed to differences in the putative K⁺ binding sites of the different forms of the protein.     

Uncoupler and anaerobic resistant transport of phosphate in Escherichia coli

Active transport of inorganic phosphate into whole cells of a strain (AB3311) derived from $\text{Escherichia coli}$ K12 was found to be partially resistant to 50 μM carbonyl cyanide $\text{m}$-chlorophenyl hydrazone (CCCP), a powerful uncoupler of oxidative phosphorylation. The presence of 10 mM dithiothreitol (DTT) before the addition of CCCP completely prevented the inhibition of phosphate uptake caused by the uncoupler. The addition of DTT to the CCCP-inhibited system restored phosphate uptake to the control rate even when added 5 min after the phosphate transport assay was started. This uncoupler resistant transport is insensitive to anaerobiosis, or the addition of 10 mM KCN which reduces oxygen consumption to less than 1% that of aerobic controls. Additional studies of transport in a mutant (CBT302) deficient in membranebound Ca²⁺-, Mg²⁺-ATPase activity also demonstrated the retention of appreciable inorganic phosphate uptake under anaerobic conditions.     

Membrane changes in yeast cells caused by sulfhydryl reagents and accompanied by a selective release of sugar

Summary Iodoacetic acid or N-ethylmaleimide included in cell suspensions during measurements of sorbose exit from yeast cells caused sorbose efflux to occur at a uniform rate in contrast to the usual two-phase exit. Cells pretreated with these agents were still capable of sugar uptake, but the entire efflux now occurred at the usual initial rate. Microscopically, the vacuoles of treated cells were observed to be altered or disrupted. Vacuolar effects occurred before methylene blue was able to penetrate the external cell membrane and stain the cells. Vacuoleless cells also allowed a single rate of sorbose efflux. The selective effect upon intracellular membranes is interpreted as a disruption of the boundaries of an internal sugar compartment with the result that sugar exits from the cell at a rate controlled only by the external membrane.     

Ca2+ transport against its electrochemical gradient in cytochrome oxidase vesicles reconstituted with mitochondrial hydrophobic proteins

Cytochrome oxidase vesicles have recently been shown to accumulate Ca²⁺ in an energy-dependent manner. Energization of these vesicles with internally trapped cytochrome c and externally added ascorbate and phenazine methylsulfate generated an internally positive membrane potential and prevented Ca²⁺ influx (R. N. Rosier and T. E. Gunter, 1980, FEBS Lett.109, 99–103). In contradistinction, when cytochrome oxidase vesicles were reconstituted with complex V, a mitochondrial protein fraction containing the uncoupler binding site (Y. Hatefi, D. L. Stiggall, Y. Galante and W. G. Hanstein, 1974, Biochem. Biophys. Res. Commun.61, 313–321), both Ca²⁺ uptake and generation of an internally positive membrane potential were observed. The uptake was specifically dependent on energization of electron transport. Control experiments verified that the energization conditions used produced appropriately oriented membrane potentials. Other partially purified hydrophobic mitochondrial protein complexes were found to be less effective than complex V. The reconstituted system showed cation selectivity since Ca²⁺, Mn²⁺, and Rb⁺ were transported, while Na⁺ was not. Low levels of uncoupler, which did not affect oxidation rates, were found to partially inhibit Ca²⁺ uptake regardless of the membrane potential polarity. Uncoupling levels of uncoupler markedly inhibited Ca²⁺ uptake in internally negative cytochrome oxidase vesicles; however, inhibition in internally positive cytochrome oxidase vesicles was less relative to that at lower levels of uncoupler. The uncoupling combination of nigericin, valinomycin, and K⁺ was inhibitory to uptake regardless of membrane potential polarity. A reconstituted system of oxidative phosphorylation, which contains a hydrophobic protein fraction, energized with cytochrome oxidase similarly accumulated Ca²⁺ despite formation of an internally positive membrane potential. The results suggest that cytochrome oxidase, when coupled to appropriate hydrophobic mitochondrial proteins, can act as an electrogenic Ca²⁺ pump deriving its energy directly from electron transport.     

Effect of temperature on glucocorticoid uptake and glucocorticoidreceptor translocation in rat thymocytes

The temperature dependence of uptake of [³H]dexamethasone by rat thymocytes in suspension and of the intracellular distribution of the bound hormone was studied as a function of time of incubation. The transport of [³H]dexamethasone was found to obey a simple solubility-diffusion mechanism. The permeability coefficient for glucocorticoid transport corresponded to values reported for other nonelectrolytes of a similar size through biological membranes. At temperatures ranging from 0 to 42 °C, the permeability coefficient increased with temperature and no maximum was observed. However, the maximum cellular uptake of the hormone varied depending on the temperature and time of incubation. Maximal uptake of [³H]dexamethasone was observed at 30 min when the reaction mixture was incubated at 30 °C; when incubated at 20 °C, maximum uptake of [³H]dexamethasone was observed at 3 h. These data were interpreted to mean that there was competition between two temperature-dependent processes, namely steroid transport and inactivation of intracellular binding sites. Intracellular hormone was observed to bind to specific sites as well as to nonspecific, presumably membranal sites. Two independent methods, one of which is based on a linear plot of uptake versus extracellular hormone concentration, gave similar values for the amount of specifically bound hormone, estimated to be 3300 molecules per cell. The binding results are in accord with the sequence of events previously proposed for the interaction of glucocorticoids with thymocytes. These events include nonspecific uptake, specific cytoplasmic binding, a highly temperature-dependent translocation into the nucleus, intranuclear binding, as well as receptor inactivation and regeneration. The amount of intracellular bound hormone and its distribution between the cytoplasmic and nuclear fractions showed no equilibrium or steady-state phenomenon throughout extended periods of incubation up to 28 h. The experiments verified kinetic equations which predicted maximum nuclear binding of the hormone at a given time, followed by an appreciable and progressive reduction in the binding of the hormone to cytoplasmic and nuclear fractions.     

The effect of CCCP on ion fluxes in the stele and cortex of maize roots

Summary The accumulation of ⁸⁶Rb labelled potassium by isolated stelar and cortical tissues from 7-day-old roots of Zea mays has been compared with the levels accumulated by these tissues in the intact root. Cortical tissues have similar uptake eapacities in these two conditions whereas stelar tissues only exhibit an uptake capacity in the intact root system. The uncoupler carbonylcyanide m-chlorophenylhydrazone caused a considerable decrease in the uptake of potassium by these tissues. In the intact root system it prevented ions from the bathing medium reaching the stelar tissues. The efflux pattern from preloaded isolated stelar and cortical tissues was considerably altered by the inhibitor, a promotion of the efflux occurring in both of these tissues.It is concluded that stelar tissues only accumulated ions when these are supplied through the root symplasm and that the stelar plasmalemma has only a limited uptake capacity per se. Stelar uptake is thus a reflection of vacuolar accumulation across the tonoplast. There is no evidence in the present study of a carrier-mediated active secretion of ions across the stelar plasmalemma. The fact that the efflux was promoted rather than depressed by the uncoupler supports the postulate that a passive leakage is the final stage in the transport of ions across the plant root.     

Sugar transport in immature internodal tissue of sugarcane: I. Mechanism and kinetics of accumulation

Transmembrane sugar transport into immature internodal parenchyma tissue of sugarcane (Saccharum officinarum L.) is a metabolically regulated process as evidenced by its sensitivity to pH, temperature, anaerobiosis, and metabolic inhibitors. All sugars studied—glucose, fructose, galactose, sorbose, glucose 6-phosphate, 3-O-methylglucose, and 2-deoxy-d-glucose—were apparently transported via the same carrier sites since they competed with each other for uptake. External concentrations of these sugars at one-half V_max were in the range of 3.9 to 8.4 nm. Preliminary data indicated that phosphorylation may be closely associated with glucose transport. The dominant intracellular sugar after 4-hours incubation was sucrose when glucose, glucose-6-P, or fructose was the exogenously supplied sugar; but when galactose was supplied, only 28% of intracellular radioactivity was in sucrose. Sorbose, 3-O-methylglucose, and 2-deoxy-d-glucose were not metabolized. Thus, by using these analogs, transport could be studied independently of subsequent metabolism, effectively eliminating a complicating factor in previous studies.     

Comprehensive understanding of multiple actions of anticancer drug tamoxifen in isolated mitochondria

Tamoxifen has been widely used in the treatment of estrogen receptor (ER)-positive breast cancer, whereas it also exhibits ER-independent anticancer effects in various cancer cell types. As one of the convincing mechanisms underlying the ER-independent effects, induction of apoptosis through mitochondrial dysfunction has been advocated. However, the mechanism of action of tamoxifen even at the isolated mitochondrial level is not fully understood and remains controversial. Here, we attempted to comprehensively understand tamoxifen's multiple actions in isolated rat liver mitochondria through not only revisiting the actions hitherto reported but also conducting originally designed experiments. Using submitochondrial particles, we found that tamoxifen has potential as an inhibitor of both respiratory complex I and ATP synthase. However, these inhibitory effects were not elicited in intact mitochondria, likely because penetration of tamoxifen across the inner mitochondrial membrane is highly restricted owing to its localized positive charge (-N⁺H(CH₃)₂). This restricted penetration may also explain why tamoxifen is unable to function as a protonophore-type uncoupler in mitochondria. Moreover, tamoxifen suppressed opening of the mitochondrial permeability transition pore induced by Ca²⁺ overload through enhancing phosphate uptake into the matrix. The photoaffinity labeling experiments using a photolabile tamoxifen derivative (pTAM1) indicated that pTAM1 specifically binds to voltage-dependent anion channels (VDACs) 1 and 3, which regulate transport of various substances into mitochondria. The binding of tamoxifen to VDAC1 and/or VDAC3 could be responsible for the enhancement of phosphate uptake. Taking all the results together, we consider the principal impairment of mitochondrial functions caused by tamoxifen.     

Regulatory function of sorbose-resistance gene C in sugar transport of Neurospora

Summary A sorbose-resistant double mutant sor ^r A-10/sor ^r C-17 produces larger colonies in sorbose containing test-medium than the respective single mutants; wildtype colonies remain very small. Resistance of the single mutants was shown to be connected with a decreased rate of sorbose-uptake into their conidia; however, sorbose uptake of the double mutant had not been measured. To check, whether the improved performance of the double mutant on test medium is correlated with a further decrease of sorbose uptake in this strain, studies on the uptake of fructose, sorbose and deoxyglucose by ungerminated conidia of the two single mutants, the double mutant and the wildtype were conducted, using C¹⁴-marked sugars, the millipore filter technique, and conidia either untreated or pretreated with 1% sorbose for 4 hours.If sorbose uptake is referred to that of fructose as basis of calculations, as in the earlier studies, the sorbose uptake by cells of the double mutant is smaller than that of both single mutants for conidia not pretreated with sorbose (Fig. 7a). However, for conidia pretreated with sorbose, this correlation does not hold. Rather, cells of the double mutant take up less sorbose than those of the C-mutant, but as much or slightly more than those of the A-mutant (Fig. 7b). If sorbose uptake is referred to that of deoxyglucose for an independent point of reference, cells of the double mutant take up less sorbose than those of the C-mutant, but much more than those of the A-mutant. This holds for untreated and sorbose pretreated cells (Fig. 5 a and b). These data rule out a correlation between colony size and transport defect for at least one of the strains used here, i.e. the C-mutant.The following data suggest a new interpretation: In contrast to the earlier findings with germinated conidia, ungerminated untreated cells of the C-mutant take up much more fructose and sorbose than those of the wildtype (Fig. 3 a and 1a). The uptake of fructose by cells of the C-mutant can not be improved by sorbose pretreatement (Fig. 3 b), but in both wildtype and A-mutant it is increased (Figs. 1b and 2b). Uptake of deoxyglucose was nearly equal for all three strains either untreated or pretreated. Untreated cells of the A-mutant take up as much sorbose as those of the wildtype (Figs. 2 a and 1 a). On pretreatment their sorbose uptake remains nearly constant (Figs. 2b), in contrast to wildtype cells, where it increases drastically and without an increase of fructose uptake by an equivalent amount (Fig. 1b).The new interpretation suggests that gene C is of the regulator type. Mutation of it in the C-strain used here has lead to the simultaneous de-repression of a system for fructose and sorbose uptake. Deoxyglucose uptake is not served by this system. Gene A is a structural gene, harbouring the information for the inducible synthesis of a carrier or permease specifically engaged in sorbose uptake. It is not under the controll of gene C.This interpretation is supported by results on untreated cells of the double mutant. However, fructose uptake of such cells is roughly equal to that of C-mutant cells (Fig. 6a) and sorbose uptake is less (Fig. 5a). Hence, a secondary effect of the A-gene, i.e. on the amount of de-repression of sorbose uptake by mutation in gene C, is indicated.     

Transport kinetics of ectoine, an osmolyte produced by Brevibacterium epidermis

Brevibacterium epidermis DSM 20659 is a halotolerant Gram-positive bacterium which can synthesize the osmolyte, ectoine, but prefers to take it up from its environment. The present study revealed that B. epidermis is equipped with at least one transport system for ectoine, with a maximal transport velocity of 15.7 ± 4.3 nmol/g CDW·min. The transport requires energy (ATP) and is completely inhibited by the proton uncoupler, CCCP. The ectoine uptake system is constitutively expressed at a basal level of activity and its activity is immediately 10-fold increased by hyper-osmotic stress. Initial uptake rates are not influenced by the intensity of the hyper-osmotic shock but the duration of the increased activity of the uptake system could be directly related to the osmotic strength of the assay solution. Competition assays indicate that betaine, but not proline, is also transported by the ectoine uptake system.     

Insulin absorption in renal proximal tubules: A quantitative immunocytochemical study

The purposes of the present study are mainly biological concerning proximal tubular handling of insulin: we will study the intracellular transport to subcellular compartments involved in insulin degradation, the specificity and saturability of the luminal endocytic absorption of insulin, the visualization of transtubular transport, and finally, if possible, the evaluation of the relative distribution (accumulation) of insulin in endocytic vacuoles and lysosomes. The second part is methodological: application of quantitative immunocytochemistry to endocytosis, quantitation of the effect of particle size and antigen density on labeling density on tissue sections, labeling at very low antigen densities, and effect of fish gelatin on background. Isolated renal proximal tubules were perfused with native insulin, ¹²⁵I-insulin, or [leucine^B-25]-insulin (2% receptor-binding ability and full immunoreactivity) or exposed to native insulin from the basolateral membranes. In conclusion, the luminal uptake of insulin is of low specificity, as native and [leucine^B-25]-insulin were accumulated to the same extent. Endocytic uptake is of high capacity and the mechanism is saturable. Insulin accumulated in endocytic vacuoles and lysosomes, thus following the classical degradation pathway. No other subcellular compartment is associated with insulin degradation. It was not possible to detect the basolateral uptake, indicating loss of immunoreactivity after binding to its receptor. Absolute quantitative immunocytochemistry is applicable in studying endocytosis. The labeling density increases nonproportionally with antigen density probably caused by steric hindrances. Reduction of the particle size (16 to 6 nm) increased the labeling density 17.6 times.     

Energetics of sodium-dependent α-aminoisobutyric acid transport in the moderate halophile Vibrio costicola

The energetics of α-aminoisobutyric acid transport were examined in Vibrio costicola grown in a medium containing the NaCl content (1 M) optimal for growth. Respiration rate, the membrane potential (Δψ) and α-aminoisobutyric acid transport had similar pH profiles, with optima at 8.5–9.0. Cells specifically required Na⁺ ions to transport α-aminoisobutyric acid and to maintain the highest Δψ (150–160 mV). Sodium was not required to sustain high rates of O₂-uptake. Δψ (and α-aminoisobutyric acid transport) recovered fully upon addition of Na⁺ to Na⁺-deficient cells, showing that Na⁺ is required in formation or maintenance of the transmembrane gradients of ions. Inhibitions by protonophores, monensin, nigericin and respiratory inhibitors revealed a close correlation between the magnitudes of Δψ and α-aminoisobutyric acid transport. Also, dissipation of Δψ with triphenylmethylphosphonium cation abolished α-aminoisobutyric acid transport without affecting respiration greatly. On the other hand, alcohols which stimulated respiration showed corresponding increases in α-aminoisobutyric acid transport, without affecting Δψ. Similarly, N,N′-dicyclohexylcarbodiimide (10 μM) stimulated respiration and α-aminoisobutyric acid transport and did not affect Δψ, but caused a dramatic decline in intracellular ATP content. From these, and results obtained with artificially established energy sources (Δψ and Na⁺ chemical potential), we conclude that Δψ is obligatory for α-aminoisobutyric acid transport, and that for maximum rates of transport an Na⁺ gradient is also required.     

Dual Action of Zn2+ on the Transport Cycle of the Dopamine Transporter

The dopamine transporter shapes dopaminergic neurotransmission by clearing extracellular dopamine and by replenishing vesicular stores. The dopamine transporter carries an endogenous binding site for Zn²⁺, but the nature of the Zn²⁺-dependent modulation has remained elusive: both, inhibition and stimulation of DAT have been reported. Here, we exploited the high time resolution of patch-clamp recordings to examine the effects of Zn²⁺ on the transport cycle of DAT: we recorded peak currents associated with substrate translocation and steady-state currents reflecting the forward transport mode of DAT. Zn²⁺ depressed the peak current but enhanced the steady-state current through DAT. The parsimonious explanation is preferential binding of Zn²⁺ to the outward facing conformation of DAT, which allows for an allosteric activation of DAT, in both, the forward transport mode and substrate exchange mode. We directly confirmed that Zn²⁺ dissociated more rapidly from the inward- than from the outward-facing state of DAT. Finally, we formulated a kinetic model for the action of Zn²⁺ on DAT that emulated all current experimental observations and accounted for all previous (in part contradictory) findings. Importantly, the model predicts that the intracellular Na⁺ concentration determines whether substrate uptake by DAT is stimulated or inhibited by Zn²⁺. This prediction was directly verified. The mechanistic framework provided by the current model is of relevance for the rational design of allosteric activators of DAT. These are of interest for treating de novo loss-of-function mutations of DAT associated with neuropsychiatric disorders such as attention deficit hyperactivity disorder (ADHD).