Uphill Transport Research Articles

Uphill and downhill water transport in rat glioma cells

The cell water content determines the cell volume, which in turn controls numerous cellular functions. The mean volume of rat glioma cells was electronically measured under isotonic and anisotonic conditions. Two types of isotonic solutions were used containing either high or low concentrations of NaCl, KCl or N-methylglucamineCl. In low salt solutions, osmolarity was maintained constant by the addition of sucrose or mannitol. Anisotonicity was induced by changing the concentration of electrolytes. As expected, the cell volume increased when the concentration of electrolytes was decreased from a high (165 mM) monovalent cation concentration. In contrast, the cell volume decreased when the concentration of electrolytes was decreased from a low (85 mM) monovalent cation concentration. Reciprocally and unexpectedly, the cell volume increased during a hyperosmotic challenge when the initial cation concentration was low, whereas it decreased when the initial cation concentration was high. These opposite volume changes observed during similar anisotonic challenges but starting from different electrolyte concentrations provide the first evidence that H2O is not only passively transported (downhill) through aquaporins but also follows ion fluxes (uphill).

Transport of salicylic acid through supported liquid membrane based on ionic liquids

Transport of salicylic acid (SA) through flat-sheet supported liquid membrane (SLM) was investigated using as liquid membrane the ionic liquids 1-hexyl-3-methylimidazolium hexafluorophosphate ([C 6mim][PF 6]) or ethylammonium nitrate (EAN). Using [C 6mim][PF 6], it has been observed that the transport efficiency decreases with increasing pH, indicating that the un-dissociated form of SA is mainly extracted. On the other hand, the ionic dissociated form of salicylic acid is mostly extracted via the anion exchange mechanism between nitrate and salicylate anions when EAN is used as liquid membrane. Parameters such as nature and concentration of the strippant in the receiving phase and concentration of the SA in the feed phase were studied. By comparing the SLM transport efficiency of SA (initial flux) of the two used ionic liquids, EAN appears to be slight efficient than [C 6mim][PF 6]. Despite the use of different stripping solutions (NaCl, NaOH and Na 2CO 3) and even with pH maintenance around initial values, uphill transport driven by pH difference was not observed using both ionic liquids. The absence of uphill transport has been attributed to the formation, along the course of the experiment, of water microenvironments (aggregates) inside the ionic liquid. SA transport through these water microenvironments inside the liquid membrane becomes the main mechanism. The main feature of SLMs based on ionic liquids is their higher stability compared to classical SLMs. In fact, our SLM system retained its stability and initial performance during the 9 days long experiment.

Plasticity of lipid-protein interactions in the function and topogenesis of the membrane protein lactose permease from Escherichia coli

Phosphatidylcholine (PC) has been widely used in place of naturally occurring phosphatidylethanolamine (PE) in reconstitution of bacterial membrane proteins. However, PC does not support native structure or function for several reconstituted transport proteins. Lactose permease (LacY) of Escherichia coli, when reconstituted in E. coli phospholipids, exhibits energy-dependent uphill and energy-independent downhill transport function and proper conformation of periplasmic domain P7, which is tightly linked to uphill transport function. LacY expressed in cells lacking PE and containing only anionic phospholipids exhibits only downhill transport and lacks native P7 conformation. Reconstitution of LacY in the presence of E. coli-derived PE, but not dioleoyl-PC, results in uphill transport. We now show that LacY exhibits uphill transport and native conformation of P7 when expressed in a mutant of E. coli in which PC completely replaces PE even though the structure is not completely native. E. coli-derived PC and synthetic PC species containing at least one saturated fatty acid also support the native conformation of P7 dependent on the presence of anionic phospholipids. Our results demonstrate that the different effects of PE and PC species on LacY structure and function cannot be explained by differences in the direct interaction of the lipid head groups with specific amino acid residues alone but are due to more complex effects of the physical and chemical properties of the lipid environment on protein structure. This conclusion is supported by the effect of different lipids on the proper folding of domain P7, which indirectly influences uphill transport function.

Identification of the Minimal Functional Unit of the Homo-oligomeric Human Reduced Folate Carrier

The reduced folate carrier (RFC) is the major transport system for folates in mammals. We previously demonstrated the existence of human RFC (hRFC) homo-oligomers and established the importance of these higher order structures to intracellular trafficking and carrier function. In this report, we examined the operational significance of hRFC oligomerization and the minimal functional unit for transport. In negative dominance experiments, multimeric transporters composed of different ratios of active (either wild type (WT) or cysteine-less (CLFL)) and inactive (either inherently inactive (Y281L and R373A) due to mutation, or resulting from inactivation of the Y126C mutant by (2-sulfonatoethyl) methanethiosulfonate (MTSES)) hRFC monomers were expressed in hRFC-null HeLa (R5) cells, and residual WT or CLFL activity was measured. In either case, residual transport activity with increasing levels of inactive mutant correlated linearly with the fraction of WT or CLFL hRFC in plasma membranes. When active covalent hRFC dimers, generated by fusing CLFL and Y126C monomers, were expressed in R5 cells and treated with MTSES, transport activity of the CLFL-CLFL dimer was unaffected, whereas Y126C-Y126C was potently (64%) inhibited; heterodimeric CLFL-Y126C and Y126C-CLFL were only partly (27 and 23%, respectively) inhibited by MTSES. In contrast to Y126C-Y126C, trans-stimulation of methotrexate uptake by intracellular folates for Y126C-CLFL and CLFL-Y126C was nominally affected by MTSES. Collectively, these results strongly support the notion that each hRFC monomer comprises a single translocation pathway for anionic folate substrates and functions independently of other monomers (i.e. despite an oligomeric structure, hRFC functions as a monomer).

Uphill Transport of Rare-Earth Metals through a Highly Stable Supported Liquid Membrane Based on an Ionic Liquid

We have developed a highly stable supported liquid membrane based on ionic liquids (ILs) for the separation of rare-earth metals, employing N,N-dioctyldiglycol amic acid as a mobile carrier. The quantitative transport of Y and Eu through the membrane was successfully attained, and separation from metal impurities, Zn, was efficiently accomplished. A membrane stable enough for long-term operation was constructible from imidazolium-based ILs having a longer alkyl chain, such as octyl or dodecyl groups in an imidazolium cation.

Water-Transporting Proteins

Transport through lipids and aquaporins is osmotic and entirely driven by the difference in osmotic pressure. Water transport in cotransporters and uniporters is different: Water can be cotransported, energized by coupling to the substrate flux by a mechanism closely associated with protein. In the K(+)/Cl(-) and the Na(+)/K(+)/2Cl(-) cotransporters, water is entirely cotransported, while water transport in glucose uniporters and Na(+)-coupled transporters of nutrients and neurotransmitters takes place by both osmosis and cotransport. The molecular mechanism behind cotransport of water is not clear. It is associated with the substrate movements in aqueous pathways within the protein; a conventional unstirred layer mechanism can be ruled out, due to high rates of diffusion in the cytoplasm. The physiological roles of the various modes of water transport are reviewed in relation to epithelial transport. Epithelial water transport is energized by the movements of ions, but how the coupling takes place is uncertain. All epithelia can transport water uphill against an osmotic gradient, which is hard to explain by simple osmosis. Furthermore, genetic removal of aquaporins has not given support to osmosis as the exclusive mode of transport. Water cotransport can explain the coupling between ion and water transport, a major fraction of transepithelial water transport and uphill water transport. Aquaporins enhance water transport by utilizing osmotic gradients and cause the osmolarity of the transportate to approach isotonicity.

Selective Facilitated Transport of Uranium(VI) Across a Bulk Liquid Membrane Containing Benzoyltrifluoroacetone as Extractant-Carrier

A study has been made on carrier-mediated transport of uranium(VI) using a bulk liquid membrane prepared by dissolving benzoyltrifluoroacetone (HBTA) in carbon tetrachloride. The source phase comprised of a solution of UO2 2+ or its binary mixture with other cations such as Th4+, Hf4+, Zr4+, Fe3+, La3+, Cu2+, Co2+, Mn2+, Ni2+, and Zn2+ in aqueous solutions of pH 6.0, while 0.1 M hydrochloric acid was serving as a stripping agent in the receiving compartment. The interference from Th4+ and a few other cations could be eliminated by using trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (DCTA) as a proper masking agent in the feed solution. Various factors influencing the transport process have been studied and an uphill transport (>99%) of uranium(VI) from the source phase could be accomplished under optimum conditions.

Analysis of Extraction of Cu(II) by Strip Dispersion Hybrid Liquid Membrane (SDHLM) using PMBP as Carrier

A liquid membrane system, denoted a strip dispersion hybrid liquid membrane (SDHLM) containing 1‐phenyl‐3‐methyl‐4‐benzoyl‐pyrazolone –5 as carrier in xylene, was reported for the transport and separation of Cu(II) from Zn (II) ions. The effects of various factors on the transport of copper(II) ions through SDHLM were systematically investigated by orthogonal tests. The optimum transport conditions of copper ions were summarized. In the overall mass transfer process the mass transfer resistance due to the aqueous boundary layer diffusion and diffusion in the microporous membrane is dominant. The accumulation of the Cu(II)‐carrier coordination compound in the membrane shows that the transfer in SDHLM possesses the characteristic of nonequlibrium mass transfer in this study. The rheologic experiments verified that the organic phase in the SDHLM system was the non‐Newtonian fluid and the organic phase after transport of 6 hr was a system of thixotropy in our experimental conditions. The lag ring experiments proved that the thixotropy of the organic phase in the SDHLM system was relevant to the composition of the membrane. In the experimental comparison of two types of liquid membrane, SDHLM has superiority over SLM in respect of transport flux, permeability coefficient, recovery percentage or concentration of solute in the stripping solution, efficiency of uphill transport, loss of membrane solution, and the separation efficiency of the membrane.

Role of the glutamate 185 residue in proton translocation mediated by the proton-coupled folate transporter SLC46A1

The proton-coupled folate transporter (PCFT) SLC46A1 mediates uphill folate transport into enterocytes in proximal small intestine coupled to the inwardly directed proton gradient. Hereditary folate malabsorption is due to loss-of-function mutations in the PCFT gene. This study addresses the functional role of conserved charged amino acid residues within PCFT transmembrane domains with a detailed analysis of the PCFT E185 residue. D156A-, E185A-, E232A-, R148A-, and R376A-PCFT mutants lost function at pH 5.5, as assessed by transient transfection in folate transport-deficient HeLa cells. At pH 7.4, function was preserved only for E185A-PCFT. Loss of function for E185A-PCFT at pH 5.5 was due to an eightfold decrease in the [(3)H]methotrexate (MTX) influx V(max); the MTX influx K(t) was identical to that of wild-type (WT)-PCFT (1.5 microM). Consistent with the intrinsic functionality of E185A-PCFT, [(3)H]MTX influx at pH 5.5 or 7.4 was trans-stimulated in cells preloaded with nonlabeled MTX or 5-formyltetrahydrofolate. Replacement of E185 with Leu, Cys, His, or Gln resulted in a phenotype similar to E185A-PCFT. However, there was greater preservation of activity (approximately 38% of WT) for the similarly charged E185D-PCFT at pH 5.5. All E185 substitution mutants were biotin accessible at the plasma membrane at a level comparable to WT-PCFT. These observations suggest that the E185 residue plays an important role in the coupled flows of protons and folate mediated by PCFT. Coupling appears to have a profound effect on the maximum rate of transport, consistent with augmentation of a rate-limiting step in the PCFT transport cycle.

Coupling of Na+/α-ketoglutarate symport and PAH/α-ketoglutarate antiport in epithelial cells. Estimation of probability of the anion exchanger spontaneous re-orientation

In this work, dynamics of uptake ofp-aminohippurate (PAH) by basolateral membrane vesicles isolated from rat kidney proximal tubules was studied. The uphill PAH transport into the basolateral membrane vesicles in the presence of alpha-ketoglutarate and Na+-gradient was shown. Based on mathematical model of symport and antiport cooperation the mechanism of energy coupling of PAH transport by the way of exchanger with Na+-dicarboxylate symport is discussed. Based on comparison of our own and literature data, the data analysis shows adequacy of the proposed mathematical model to describe the symport and antiport cooperation. This model was demonstrated to enable estimation of re-orientation probability of the empty anion exchanger (without substrate) from one membrane side to the other.

Thallium Ions Can Replace both Sodium and Potassium Ions in the Glutamate Transporter Excitatory Amino Acid Carrier 1

The excitatory amino acid carrier EAAC1 belongs to a family of glutamate transporters that use the electrochemical transmembrane gradients of sodium and potassium to mediate uphill transport of glutamate into the cell. While the sites of cation interaction with EAAC1 are unknown, two cation binding sites were observed in the crystal structure of the bacterial glutamate transporter homologue GltPh. Although occupied by Tl(+) in the crystal structure, these sites were proposed to be Na(+) binding sites. Therefore, we tested whether Tl(+) has the ability to replace Na(+) also in the mammalian transporters. Our data demonstrate that Tl(+) can bind to EAAC1 with high affinity and mediate a host of different functions. Tl(+) can functionally replace potassium when applied to the cytoplasm and can support glutamate transport current. When applied extracellularly, Tl(+) induces some behavior that mimics that of the Na(+)-bound transporter, such as activation of the cation-induced anion conductance and creation of a substrate binding site, but it cannot replace Na(+) in supporting glutamate transport current. Moreover, our data show a differential effect of mutations to two acidic amino acids potentially involved in cation binding (D367 and D454) on Na(+) and Tl(+) affinity. Overall, our results demonstrate that the ability of the glutamate transporters to interact with Tl(+) is conserved between GltPh and a mammalian member of the transporter family. However, in contrast to GltPh, which does not bind K(+), Tl(+) is more efficient in mimicking K(+) than Na(+) when interacting with the mammalian protein.

Design and Preparation of a Novel Temperature-Responsive Ionic Gel. 3. Valence Selective Control of Transport Modes of Ions in Response to Temperature

We propose a novel model dialysis system that can valence-selectively control the transport modes of ions in response to temperature change. In a dialysis system consisting of an anionic gel membrane and mixed solutions containing a driving electrolyte and electrolytes with uni-, bi-, and trivalent cations, the dependence of the charge density of the gel and the valence of the ions on the transport modes of the ions through the gel membrane was investigated by computer simulations. The simulations show that the system has four transport types in the transport modes of the cations according to their valence [downhill (transport along their own concentration gradient in the system) and uphill (transport against their own concentration gradient)] in response to the charge density changes: (A) downhill transport of all the cations; (B) uphill transport of trivalent cations, downhill transport of the other cations; (C) uphill transport of bi- and trivalent cations, downhill transport of univalent cations; and (D) uphill transport of all the cations except for the driving cations. To examine the prediction of the simulations, a temperature-responsive anionic gel membrane was prepared from a modified poly(vinyl alcohol) (PVA) containing 2 mol % of sulfonic acid groups and another modified PVA prepared by in situ polymerization of N-isopropylacrylamide in a PVA solution. Permeation experiments in a dialysis system consisting of the membrane and mixed electrolyte solutions of NaCl, LiCl, CaCl2, and LaCl3 indicate that the system valence-selectively controls the transport modes of the cations in response to temperature change as predicted in our simulations.

A Proton-mediated Conformational Shift Identifies a Mobile Pore-lining Cysteine Residue (Cys-561) in Human Concentrative Nucleoside Transporter 3

The concentrative nucleoside transporter (CNT) protein family in humans is represented by three members, hCNT1, hCNT2, and hCNT3. Belonging to a CNT subfamily phylogenetically distinct from hCNT1/2, hCNT3 mediates transport of a broad range of purine and pyrimidine nucleosides and nucleoside drugs, whereas hCNT1 and hCNT2 are pyrimidine and purine nucleoside-selective, respectively. All three hCNTs are Na(+)-coupled. Unlike hCNT1/2, however, hCNT3 is also capable of H(+)-mediated nucleoside cotransport. Using site-directed mutagenesis in combination with heterologous expression in Xenopus oocytes, we have identified a C-terminal intramembranous cysteine residue of hCNT3 (Cys-561) that reversibly binds the hydrophilic thiol-reactive reagent p-chloromercuribenzene sulfonate (PCMBS). Access of this membrane-impermeant probe to Cys-561, as determined by inhibition of hCNT3 transport activity, required H(+), but not Na(+), and was blocked by extracellular uridine. Although this cysteine residue is also present in hCNT1 and hCNT2, neither transporter was affected by PCMBS. We conclude that Cys-561 is located in the translocation pore in a mobile region within or closely adjacent to the nucleoside binding pocket and that access of PCMBS to this residue reports a specific H(+)-induced conformational state of the protein.

The Peptide Transporter PEPT2 is Targeted by the Protein Kinase SGK1 and the Scaffold Protein NHERF2

PEPT1 and PEPT2 are members of the family of proton-dependent oligopeptide transporters that mediate electrogenic uphill transport of small peptides and peptidomimetics into a variety of cells. Kinetic properties and substrate recognition sites of those transporters have been well defined previously. Little is known, however, about regulation of those transporters. Both PEPT isoforms contain putative phosphorylation sites for the serum and glucocorticoid inducible kinase SGK1 and a C-terminal PDZ binding motif that might be recognized by PDZ domains of the Na⁺/H⁺ exchanger regulatory factors NHERF1 and NHERF2. Thus, the present study attempted to clarify the role of SGK1 and NHERFs in the modulation of PEPT isoforms. Expression studies in Xenopus oocytes with subsequent electrophysiology and immunoassays revealed that SGK1 and NHERF2, but not the NHERF1 isoform specifically enhance PEPT2 function and surface abundance. The kinase is effective through phosphorylation of ¹⁸⁵Ser within the SGK1 consensus site, since disruption of this site prevented transporter modulation by the kinase. NHERF2 failed to regulate the C-terminal deletion mutant (PEPT2ΔC) indicating that the C-terminal PDZ-binding motif in PEPT2 governs transport modulation by NHERF2. Coexpression of NHE3 stimulates PEPT2 activity to a similar extent as coexpression of NHERF2. Dynasore experiments demonstrated that SGK1 and NHERF2 activate PEPT2 by stabilizing the transporter at the cell surface. In conclusion, the present results reveal two novel PEPT2 posttranslational modulators, SGK1 and NHERF2, which might regulate transport of oligopeptides and peptidomimetic drugs.

When Can the Fokker-Planck Equation Describe Anomalous or Chaotic Transport?

The Fokker-Planck equation, applied to transport processes in fusion plasmas, can model several anomalous features, including uphill transport, scaling of confinement time with system size, and convective propagation of externally induced perturbations. It can be justified for generic particle transport provided that there is enough randomness in the Hamiltonian describing the dynamics. Then, except for 1 degree of freedom, the two transport coefficients are largely independent. Depending on the statistics of interest, the same dynamical system may be found diffusive or dominated by its Lévy flights.

Extraction Equilibrium and Membrane Transport of Copper(II) with New N-6-(t-Dodecylamido)-2-Pyridinecarboxylic Acid in Polymer Inclusion Membrane

A plasticized cellulose triacetate (CTA) membrane consisting of N-6-(t-dodecylamido)-2-pyridine carboxylic acid (t-DAPA) as a new carrier to facilitate membrane transport of copper(II) has been prepared to develop the selective recovery system for copper(II) from other divalent metal ions. First of all, the solid (CTA)−liquid extraction equilibrium of copper(II) was examined to obtain information concerning Cu(II)-t-DAPA complex stoichiometry and its extraction constant in the CTA membrane. Membrane transport studies were performed in a two-compartment cell. The CTA−t-DAPA membrane exhibited uphill transport of copper(II) against the concentration gradient. The influences of the aqueous and membrane components on the permeability of copper(II) were studied to elucidate its transport mechanism. Their results suggest that the transport mechanism consists of a diffusion process through an aqueous diffusion film, a fast interfacial chemical reaction, and diffusion through the membrane itself. The mass-transfe...

Selective and Efficient Liquid Membrane Transport of Thallium (III) Ion by Potassium‐dicyclohexyl‐18‐crown‐6 as Specific Carrier

Potassium‐dicyclohexyl‐18‐crown‐6 was used as a selective and efficient carrier for the uphill transport of thallium (III) ion as [TlCl4]− complex ion through a chloroform bulk liquid membrane. By using oxalate anion as a metal ion acceptor in the receiving phase, the amount of thallium (III) transported across the liquid membrane after 120 min was 96±2%. The selectivity and efficiencies of thallium transport from aqueous solutions containing Cu2+, Zn2+, Ni2+, Cd2+, Pb2+, Co3+, Mn2+ , Cr3+, Mg2+, Ca2+, K+, Na+, and Fe3+ ions were investigated. In the presence of Na3PO4 (0.01 M) at pH=3 as a suitable precipitation agent in the source phase, the interfering effect of Pb2+ ion were diminished drastically.

PH‐controlled uphill transport of boric acid through a poly(vinyl alcohol) (PVA) membrane

AbstractThe uphill transport of boric acid in aqueous solutions through a thermal‐crosslinked poly(vinyl alcohol) (PVA) membrane was investigated. A normal permeation caused by the concentration difference of the boron along the PVA membrane was observed for equal pH conditions at both sides of the membrane, and higher flux was observed under an acidic condition at pH = 5.0 than under a basic condition at pH = 10.0. When the pH of one side is kept pH = 5.0 (acid side) and the other side was kept at pH = 10.0 (base side), uphill transport of boric acid from the acid side to the base side was observed under an equal initial concentration of both sides. Such an uphill transport was also observed against the concentration difference under the condition in which the initial concentration of the base side was higher than that of the acid side. The uphill transport could be explained by the difference in the permeation rates through the PVA membrane between B(OH)3, the dominant form under lower pH, and B(OH)4−, the dominant form under higher pH, which makes a complex with diols in PVA. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 104: 1451–1455, 2007

PH and Ionic Strength Effects on Amino Acid Transport through Au-Nanotubule Membranes Charged with Self-Assembled Monolayers

The transport of the amino acids tyrosine and phenylalanine through Au-nanotubule membranes with self-assembled monolayers of alkanethiols is theoretically and experimentally studied. The membranes are prepared by electroless deposition of gold on porous polycarbonate track-etched membranes with control of the inner pore size, followed by self-assembly of acid-functionalized thiols (final mean average pore radius: 7 nm). The capability of switchable ion-transport selectivity by external control (e.g., by changing the pH, ionic strength, and amino acid concentration) is discussed. The flux changes with the pH and the ionic strength of the solution clearly show that electrical charges play a key role in the amino acid transport through the nanopores. Remarkably, an uphill transport of the amino acid is observed when a pH difference is imposed in the external solutions. The theoretical approach based on the Nernst−Planck flux equations considers all of the charged species present in the system (the cationic, anionic, and zwitterionic forms of the amino acid, the hydrogen and hydroxide ions, and the two salt ions) and allows a qualitative understanding of the transport phenomena in the charged nanopores.

Separation study of silver(I) ion through a bulk liquid membrane containing meloxicam

The uphill transport of Ag+ through a chloroform bulk liquid membrane containing meloxicam as an ion carrier was studied. Different experimental conditions for the increasing selectivity, sensitivity and kinetics of transport, e. g., the effect of meloxicam concentration in the chloroform membrane phase, pH of donors and acceptor phases, types and concentration of various stripping agents were examined. In the presence of thiocyanate as a suitable stripping agent in the acceptor phase, the amount of silver transported across the liquid membrane after 120 min was 97.8 ± 1.2%. The selectivity and efficiency of Ag+ transport from the aqueous solution containing other cations, such as: Co2+, Cr3+, Ni2+, Cd2+, Mn2+, Cu2+, Pb2+, Ca2+, Mg2+ and Zn2+ were investigated. Except Cr3+ and Pb2+ ions, none of the cations used interfere the silver transport, even at a Mn+/Ag+ molar ratio of 500. The interfering effect of the two mentioned cations was diminished in the presence of EDTA at pH 6.