Mathematical model for cooperation of ion pump, symport, and antiport in epithelial cells

Transcellular transport in epithelial cells plays an important role in providing such physiological functions as excretion of cytotoxic substances or reabsorption of metabolites useful for the body life activity. These functions have been shown to be performed by the mechanisms - symport, antiport, ion pumps, and channels - that often function cooperatively. Kinetic models of the substrate transport with the aid of the above mechanisms are widely described in the literature. Much less attention is paid to modeling of cooperative activity of transporters that have different transport mechanisms. In this work we propose a mathematical model for flux coupling of three transporters - the ion pump, symporter, and antiporter as well as of two substrates, one of which (A) can be transported simultaneously by the symport and antiport mechanisms, while the other (B) - only by the latter mechanisms. Analysis of the model has shown that for the pair of substrates (A and B) the flux coupling becomes possible if the following conditions are met: (1) the substrate A flux into the internal cell volume using the symport mechanism is to exceed its antiporter-realized flux in the opposite direction; (2) probability of reorientation from one side of membrane to the other side for the antiporter loaded with the substrate is to be essentially higher than that for empty transporter. The proposed model can be used for comparing efficiency both of excretion and of reabsorption of cell metabolites in representatives of different taxa.     

Coupling of Na<Superscript>+</Superscript>/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 was studied of uptake of p-aminohippurate by basolateral membrane vesicles isolated from rat kidney proximal tubules. The uphill PAH transport into the basolateral membrane vesicles was shown to occur in the presence of α-ketoglutarate and Na⁺-gradient. Based on mathematical model of symport and antiport cooperation, the mechanism of energy coupling of PAH transport via 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 has been shown to enable estimation of re-orientation probability of the empty anion exchanger (without substrate) from one membrane side to the other.     

Role of N-linked oligosaccharides in the transport activity of the Na+/H+ antiporter in rat renal brush-border membrane

The role of N-linked oligosaccharide side chains in the biogenesis and function of Na+-coupled transporters in renal luminal brush-border membrane (BBM) is not known. We examined the question of how in vivo inhibition by alkaloid swainsonine of alpha-mannosidase, a key enzyme in processing of glycoproteins in the Golgi apparatus, affects Na+/H+ antiport and Na+/Pi symport as well as activities of other transporters and enzymes in rat renal BBM. Administration of swainsonine to thyroparathyroidectomized rats, control or treated with 3,5,3'-triiodothyronine, markedly decreased the rate of Na+/H+ antiport, but had no effect on the rate of Na+/Pi symport across renal BBM vesicles (BBMV). Moreover, administration of swainsonine did not change activities of Na+ gradient, ([extravesicular Na+] greater than [intravesicular Na+])-dependent transport of D-glucose, L-proline, or the amiloride-insensitive 22Na+ uptake by BBMV; the activities of the BBM enzymes alkaline phosphatase, gamma-glutamyltransferase, or leucine aminopeptidase in BBMV were also not changed. The in vitro enzymatic deglycosylation of BBM by incubating freshly isolated BBMV with bacterial endoglycosidase F also resulted in a decreased rate of Na+/H+ antiport, but not Na+-coupled symports of Pi, L-proline, and D-glucose, or the activities of the BBM enzymes were not significantly affected. Similar incubation with endoglycosidase H was without effect on any of these parameters. Both the modification of BBMV glycoproteins by administration fo swainsonine in vivo as well as the in vitro incubation of BBMV with endoglycosidase F resulted in a decrease of the apparent Vmax of Na+/H+ antiport, but did not change the apparent Km of this antiporter for extravesicular Na+ and did not increase H+ conductance of BBM. Taken together, our findings suggest that intact N-linked oligosaccharide chains of the biantennary complex type in renal BBM glycoproteins are required, directly or indirectly, for the transport function of the Na+/H+ antiporter inserted into BBM of renal proximal tubules.     

Molecular characteristics of transporters of C<Subscript>4</Subscript>-dicarboxylates and mechanism of translocation

An important role in cell metabolism is played by transport of C₄-dicarboxylates (C₄-DCB). Specifically, they are intermediates of the citrate cycle. Transport of succinate across the mitochondrial membrane provides correlation between metabolism in peroxysomes and in mitochondria. There is known transport of C₄-DCB across all kinds of energy-transforming membranes of the animal, plant, fungal, and bacterial cells. This review summarizes molecular characteristics of the C₄-DCB transporters. Of particular interest are primary structures for the transporters with the known kinetic mechanism and kinetic transport parameters. For each studied group of organisms, the number of transmembrane segments in the carried molecule or the character of specificity does not correlate with the certain transport mechanism—antiport, symport with proton or symport with cation. The review describes perspective methodical approaches allowing association of peculiarities of structure with transport mechanism for individual transporters, preparation of functional hybrid transporters—“protein chimeras,” scanning of transporter transmembrane segments with aid of essential acids, probing of the transporter active center with aid of alkyl and acyl substrate derivatives used to obtain the “lipophilic profile” of the channel of the C₄-DCB transporter. It is recommended to use these approaches to one transporter that has small sizes and large substrate specificity.     

Variety of nucleotide sugar transporters with respect to the interaction with nucleoside mono- and diphosphates

Nucleotide sugar transporters have long been assumed to be antiporters that exclusively use nucleoside monophosphates as antiport substrates. Here we present evidence indicating that two other types of nucleotide sugar transporters exist that differ in their antiport substrate specificity. Biochemical studies using microsomes derived from Saccharomyces cerevisiae cells expressing either human (h) UGTrel7 or the Drosophila (d) FRC (Fringe connection) transporter revealed that (i) efflux of preloaded UDP-glucuronic acid from the yeast microsomes expressing hUGTrel7 was strongly enhanced by UDP-GlcNAc added in the external medium, but not by UMP or UDP, suggesting that hUGTrel7 may be described as a UDP-sugar/UDP-sugar antiporter, and (ii) addition of UDP-sugars, UDP, or UMP in the external medium stimulated the efflux of preloaded UDP-GlcNAc from the yeast microsomes expressing dFRC to a comparable extent, suggesting that UDP, as well as UMP, may serve as an antiport substrate of dFRC. Antiport of UDP-sugars with these specific substrates was reproduced and definitively confirmed using proteoliposomes reconstituted from solubilized and purified transporters. Possible physiological implications of these observations are discussed.     

Transport of C(4)-dicarboxylates in Wolinella succinogenes

C(4)-dicarboxylate transport is a prerequisite for anaerobic respiration with fumarate in Wolinella succinogenes, since the substrate site of fumarate reductase is oriented towards the cytoplasmic side of the membrane. W. succinogenes was found to transport C(4)-dicarboxylates (fumarate, succinate, malate, and aspartate) across the cytoplasmic membrane by antiport and uniport mechanisms. The electrogenic uniport resulted in dicarboxylate accumulation driven by anaerobic respiration. The molar ratio of internal to external dicarboxylate concentration was up to 10(3). The dicarboxylate antiport was either electrogenic or electroneutral. The electroneutral antiport required the presence of internal Na(+), whereas the electrogenic antiport also operated in the absence of Na(+). In the absence of Na(+), no electrochemical proton potential (delta p) was measured across the membrane of cells catalyzing fumarate respiration. This suggests that the proton potential generated by fumarate respiration is dissipated by the concomitant electrogenic dicarboxylate antiport. Three gene loci (dcuA, dcuB, and dctPQM) encoding putative C(4)-dicarboxylate transporters were identified on the genome of W. succinogenes. The predicted gene products of dcuA and dcuB are similar to the Dcu transporters that are involved in the fumarate respiration of Escherichia coli with external C(4)-dicarboxylates. The genes dctP, -Q, and -M probably encode a binding-protein-dependent secondary uptake transporter for dicarboxylates. A mutant (DcuA(-) DcuB(-)) of W. succinogenes lacking the intact dcuA and dcuB genes grew by nitrate respiration with succinate as the carbon source but did not grow by fumarate respiration with fumarate, malate, or aspartate as substrates. The DcuA(-), DcuB(-), and DctQM(-) mutants grew by fumarate respiration as well as by nitrate respiration with succinate as the carbon source. Cells of the DcuA(-) DcuB(-) mutant performed fumarate respiration without generating a proton potential even in the presence of Na(+). This explains why the DcuA(-) DcuB(-) mutant does not grow by fumarate respiration. Growth by fumarate respiration appears to depend on the function of the Na(+)-dependent, electroneutral dicarboxylate antiport which is catalyzed exclusively by the Dcu transporters. Dicarboxylate transport via the electrogenic uniport is probably catalyzed by the DctPQM transporter and by a fourth, unknown transporter that may also operate as an electrogenic antiporter.     

Pore-like and carrier-like properties of the mitochondrial aspartate/glutamate carrier after modification by SH-reagents: evidence for a performed channel as a structural requirement of carrier-mediated transport.

Upon modification of the reconstituted aspartate/glutamate carrier by mercury reagents the antiporter was converted into a unidirectional efflux carrier (Dierks, T., Salentin, A., Heberger, C. and Kr?mer, R. (1990) Biochim. Biophys. Acta 1028, 268). In addition to this basic change in the mechanism, the mercurials, reacting with exofacial cysteines, also affected the internal binding site of the carrier leading to an unmeasurable high Km and to a drastically reduced substrate specificity. The spectrum of efflux substrates comprised small anions from chloride to glutamate, but not cationic amino acids and ATP, hence resembling pore-like properties. However, in the efflux state important carrier properties were also observed. The activation energy (86 kJ/mol) was as high as for the antiport. Furthermore, efflux was inhibited by the presence of external substrate. This trans-inhibition strongly suggests that the external binding site of the carrier, prerequisite in the antiport mechanism, also is involved in conformational transitions during efflux function. However, antiport no longer is catalyzed after switching to the efflux state. Reversion of the induced efflux carrier to the antiport state was achieved using dithioerythritol, thereby further restoring substrate specificity and saturation kinetics. A model for antiport-efflux interconversion is presented suggesting that two reactive cysteines have to be modified in order to uncouple the inward and outward directed component of antiport. The pore-type characteristics of efflux are taken as evidence that a channel-like structure determines the selectivity of unidirectional transport. This intrinsic channel of the protein then is required for substrate translocation also during antiport function.     

Mechanisms of drug/H+ antiport: complete cysteine-scanning mutagenesis and the protein engineering approach

The notorious difficulty of elucidating structures of membrane transporters by crystallography has long prevented our understanding of active transport mechanism coupled with ion/proton transport. The determination of the first crystal structure of the drug/H+ antiporter AcrB was a breakthrough for structure-based understanding of drug/H+ antiport. However, although AcrB is a major multidrug exporter in Gram-negative organisms, the majority of bacterial drug exporters are major facilitator superfamily (MFS) drug transporters. As no crystal structures have been solved for MFS transporters, the alternative protein-engineering methods are still very useful for estimating structures and functions of drug/H+ antiporters. This review describes this alternative approach for investigating the structure and function of tetracycline/H+ antiporters.     

Cell free expression and functional reconstitution of eukaryotic drug transporters

Polyspecific organic cation and anion transporters of the SLC22 protein family are critically involved in absorption and excretion of drugs. To elucidate transport mechanisms, functional and biophysical characterization of purified transporters is required and tertiary structures must be determined. Here, we synthesized rat organic cation transporters OCT1 and OCT2 and rat organic anion transporter OAT1 in a cell free system in the absence of detergent. We solubilized the precipitates with 2% 1-myristoyl-2-hydroxy- sn-glycero-3-[phospho- rac-(1-glycerol)] (LMPG), purified the transporters in the presence of 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or octyl glucoside, and reconstituted them into proteoliposomes. From 1 mL reaction vessels 0.13-0.36 mg of transporter proteins was purified. Thus, from five to ten 1 mL reaction vessels sufficient protein for crystallization was obtained. In the presence of 1% LMPG and 0.5% CHAPS, OCT1 and OAT1 formed homo-oligomers but no hetero-oligomers. After reconstitution of OCT1, OCT2, and OAT1 into proteoliposomes, similar Michaelis-Menten K m values were measured for uptake of 1-methyl-4-phenylpyridinium and p-aminohippurate (PAH (-)) by the organic cation and anion transporters, respectively, as after expression of the transporters in cells. Using the reconstituted system, evidence was obtained that OAT1 operates as obligatory and electroneutral PAH (-)/dicarboxylate antiporter and contains a low-affinity chloride binding site that stimulates turnover. PAH (-) uptake was observed only with alpha-ketoglutarate (KG (2-)) on the trans side, and trans-KG (2-) increased the PAH (-) concentration in voltage-clamped proteoliposomes transiently above equilibrium. The V max of PAH (-)/KG (2-) antiport was increased by Cl (-) in a manner independent of gradients, and PAH (-)/KG (2-) antiport was independent of membrane potential in the absence or presence of Cl (-).     

Evolutionary ancestry and novel functions of the mammalian glucose transporter (GLUT) family

Background  

In general, sugar porters function by proton-coupled symport or facilitative transport modes. Symporters, coupled to electrochemical energy, transport nutrients against a substrate gradient. Facilitative carriers transport sugars along a concentration gradient, thus transport is dependent upon extracellular nutrient levels. Across bacteria, fungi, unicellular non-vertebrates and plants, proton-coupled hexose symport is a crucial process supplying energy under conditions of nutrient flux. In mammals it has been assumed that evolution of whole body regulatory mechanisms would eliminate this need. To determine whether any isoforms bearing this function might be conserved in mammals, we investigated the relationship between the transporters of animals and the proton-coupled hexose symporters found in other species.     

Flavone glucoside uptake into barley mesophyll and Arabidopsis cell culture vacuoles. Energization occurs by H(+)-antiport and ATP-binding cassette-type mechanisms

In many cases, secondary plant products accumulate in the large central vacuole of plant cells. However, the mechanisms involved in the transport of secondary compounds are only poorly understood. Here, we demonstrate that the transport mechanisms for the major barley (Hordeum vulgare) flavonoid saponarin (apigenin 6-C-glucosyl-7-O-glucoside) are different in various plant species: Uptake into barley vacuoles occurs via a proton antiport and is competitively inhibited by isovitexin (apigenin 6-C-glucoside), suggesting that both flavone glucosides are recognized by the same transporter. In contrast, the transport into vacuoles from Arabidopsis, which does not synthesize flavone glucosides, displays typical characteristics of ATP-binding cassette transporters. Transport of saponarin into vacuoles of both the species is saturable with a K(m) of 50 to 100 microM. Furthermore, the uptake of saponarin into vacuoles from a barley mutant exhibiting a strongly reduced flavone glucoside biosynthesis is drastically decreased when compared with the parent variety. Thus, the barley vacuolar flavone glucoside/H(+) antiporter could be modulated by the availability of the substrate. We propose that different vacuolar transporters may be responsible for the sequestration of species-specific/endogenous and nonspecific/xenobiotic secondary compounds in planta.     

The structural basis of secondary active transport mechanisms

Secondary active transporters couple the free energy of the electrochemical potential of one solute to the transmembrane movement of another. As a basic mechanistic explanation for their transport function the model of alternating access was put forward more than 40 years ago, and has been supported by numerous kinetic, biochemical and biophysical studies. According to this model, the transporter exposes its substrate binding site(s) to one side of the membrane or the other during transport catalysis, requiring a substantial conformational change of the carrier protein. In the light of recent structural data for a number of secondary transport proteins, we analyze the model of alternating access in more detail, and correlate it with specific structural and chemical properties of the transporters, such as their assignment to different functional states in the catalytic cycle of the respective transporter, the definition of substrate binding sites, the type of movement of the central part of the carrier harboring the substrate binding site, as well as the impact of symmetry on fold-specific conformational changes. Besides mediating the transmembrane movement of solutes, the mechanism of secondary carriers inherently involves a mechanistic coupling of substrate flux to the electrochemical potential of co-substrate ions or solutes. Mainly because of limitations in resolution of available transporter structures, this important aspect of secondary transport cannot yet be substantiated by structural data to the same extent as the conformational change aspect. We summarize the concepts of coupling in secondary transport and discuss them in the context of the available evidence for ion binding to specific sites and the impact of the ions on the conformational state of the carrier protein, which together lead to mechanistic models for coupling.     

Secondary active transport

Secondary active transport is defined as the transport of a solute in the direction of its increasing electrochemical potential coupled to the facilitated diffusion of a second solute (usually an ion) in the direction of its decreasing electrochemical potential. The coupling agents are membrane proteins (carriers), each of which catalyzes simultaneously the facilitated diffusion of the driving ion and the active transport of a given solute. The review starts with some considerations on the energetics followed by a presentation of the kinetics of secondary active transport. Examples of information which may be gained by such studies are discussed. In the second part, some examples of secondary transport are given; we also describe the characteristics of the corresponding carriers. The various transport systems presented are: the D-glucose/Na+ symport in brush-border membranes, the lactose/H+ symport in E. coli, the Na+/H+ antiport, the different transport systems in the inner mitochondrial membrane.     

Proton-driven sucrose symport and antiport are provided by the vacuolar transporters SUC4 and TMT1/2

The vacuolar membrane is involved in solute uptake into and release from the vacuole, which is the largest plant organelle. In addition to inorganic ions and metabolites, large quantities of protons and sugars are shuttled across this membrane. Current models suggest that the proton gradient across the membrane drives the accumulation and/or release of sugars. Recent studies have associated AtSUC4 with the vacuolar membrane. Some members of the SUC family are plasma membrane proton/sucrose symporters. In addition, the sugar transporters TMT1 and TMT2, which are localized to the vacuolar membrane, have been suggested to function in proton-driven glucose antiport. Here we used the patch-clamp technique to monitor carrier-mediated sucrose transport by AtSUC4 and AtTMTs in intact Arabidopsis thaliana mesophyll vacuoles. In the whole-vacuole configuration with wild-type material, cytosolic sucrose-induced proton currents were associated with a proton/sucrose antiport mechanism. To identify the related transporter on one hand, and to enable the recording of symporter-mediated currents on the other hand, we electrophysiologically characterized vacuolar proteins recognized by Arabidopsis mutants of partially impaired sugar compartmentation. To our surprise, the intrinsic sucrose/proton antiporter activity was greatly reduced when vacuoles were isolated from plants lacking the monosaccharide transporter AtTMT1/TMT2. Transient expression of AtSUC4 in this mutant background resulted in proton/sucrose symport activity. From these studies, we conclude that, in the natural environment within the Arabidopsis cell, AtSUC4 most likely catalyses proton-coupled sucrose export from the vacuole. However, TMT1/2 probably represents a proton-coupled antiporter capable of high-capacity loading of glucose and sucrose into the vacuole.     

Tet(L) and tet(K) tetracycline-divalent metal/H+ antiporters: characterization of multiple catalytic modes and a mutagenesis approach to differences in their efflux substrate and coupling ion preferences

The Tet(L) protein encoded in the Bacillus subtilis chromosome and the closely related Tet(K) protein from Staphylococcus aureus plasmids are multifunctional antiporters that have three cytoplasmic efflux substrates: a tetracycline-divalent metal (TC-Me(2+)) complex that bears a net single positive charge, Na+, and K+. Tet(L) and Tet(K) had been shown to couple efflux of each of these substrates to influx of H+ as the coupling ion. In this study, competitive cross-inhibition between K+ and other cytoplasmic efflux substrates was demonstrated. Tet(L) and Tet(K) had also been shown to use K+ as an alternate coupling ion in support of Na+ or K+ efflux. Here they were shown to couple TC-Me(2+) efflux to K+ uptake as well, exhibiting greater use of K+ as a coupling ion as the external pH increased. The substrate and coupling ion preferences of the two Tet proteins differed, especially in the higher preference of Tet(K) than Tet(L) for K+, both as a cytoplasmic efflux substrate and as an external coupling ion. Site-directed mutagenesis was employed to test the hypothesis that some feature of the putative "antiporter motif," motif C, of Tet proteins would be involved in these characteristic preferences. Mutation of the A157 in Tet(L) to a hydroxyamino acid resulted in a more Tet(K)-like K+ preference both as coupling ion and efflux substrate. A reciprocal S157A mutant of Tet(K) exhibited reduced K+ preference. Competitive inhibition among substrates and the parallel effects of the single mutation upon K+ preference, as both an efflux substrate and coupling ion, are compatible with a model in which a single translocation pathway through the Tet(L) and Tet(K) transporters is used both for the cytoplasmic efflux substrates and for the coupling ions, in an alternating fashion. However, the effects of the A157 and other mutations of Tet(L) indicate that even if there are a shared binding site and translocation pathway, some elements of that pathway are used by all substrates and others are important only for particular substrates.     

Dissection of mechanistic principles of a secondary multidrug efflux protein

Multidrug transporters are ubiquitous efflux pumps that provide cells with defense against various toxic compounds. In bacteria, which typically harbor numerous multidrug transporter genes, the majority function as secondary multidrug/proton antiporters. Proton-coupled secondary transport is a fundamental process that is not fully understood, largely owing to the obscure nature of proton-transporter interactions. Here we analyzed the substrate/proton coupling mechanism in MdfA, a model multidrug/proton antiporter. By measuring the effect of protons on substrate binding and by directly measuring proton binding and release, we show that substrates and protons compete for binding to MdfA. Our studies strongly suggest that competition is an integral feature of secondary multidrug transport. We identified the proton-binding acidic residue and show that, surprisingly, the substrate binds at a different site. Together, the results suggest an interesting mode of indirect competition as a mechanism of multidrug/proton antiport.     

Proposed partial beta-structures for lac permease and the Na+/H+ antiporter which use similar transport and H+ coupling mechanisms.

Most antiporters, symporters, and transporters have been represented as containing ten to 14 transmembrane helices, primarily on the basis of hydropathy plots. However, multihelix systems provide no obvious mechanism of transport and no simple way of distinguishing substrates. The models of lac permease and the Na+/H+ antiporter presented here postulate that beta-structures are involved in the transport of substrate, and in following this postulate arrive at readily understandable mechanisms for transport and for substrate specificity. The percentage of beta-structures necessary for these models is low enough that it is not in conflict with prior physical evidence for secondary structures. Immunological data also cannot rule these beta-structure mechanisms invalid. In lac permease the new model is obtained by formal representation of the C-terminal amino acids 243-405 as beta-strands. This formal representation nets two interchangeable beta-barrels which provide a simple mechanism for sugar transport. The alternating barrel system may comprise as little as 1/5 the entire permease. In one configuration the barrel forms a pocket with hydrogen bonding residues oriented to the outside of the cell. In the other configuration the barrel forms an analogous pocket oriented towards the inside. Six particular amino acids participate in the substrate hydrogen bonding schemes of both forms, providing a mechanism to shuttle lactose from the outside to the inside or vice versa. A trigger for change of forms which could couple the beta-barrel to H(+)-transport is easily devised, and it involves the apparently critical His322-Glu325 charge relay system. The Na+/H+ antiporter can be organized similarly with an interchanging beta-barrel-beta-clamshell structure attached to 7-transmembrane helices. Charged amino acid sidechains form the basis of an ionic shuttle which is analogous to the lactose shuttle. In this case, too, coupling of Na+ transport to H+ transport may be accomplished by a histidine-glutamate charge relay system.     

An antiport mechanism for a member of the cation diffusion facilitator family: divalent cations efflux in exchange for K+ and H+

Members of the cation diffusion facilitator (CDF) family of membrane transport proteins are found in eukaryotes and prokaryotes. The family encompasses transporters of zinc ions, with cobalt, cadmium and lead ions being additional substrates for some prokaryotic examples. No transport mechanism has previously been established for any CDF protein. It is shown here that the CzcD protein of Bacillus subtilis, a CDF protein, uses an antiporter mechanism, catalysing active efflux of Zn2+ in exchange for K+ and H+. The exchange is probably electroneutral, energized by the transmembrane pH gradient and oppositely oriented gradients of the other cation substrates. The data suggest that Co2+ and Cd2+ are additional cytoplasmic substrates for CzcD. A second product of the same operon that encodes czcD has sequence similarity to oxidoreductases and is here designated CzcO. CzcO modestly enhances the activity of CzcD but is not predicted to be an integral membrane protein and has no antiport activity of its own.