Substrate Cotransport Research Articles

Mechanism of Transport Modulation by an Extracellular Loop in an Archaeal Excitatory Amino Acid Transporter (EAAT) Homolog

Secondary transporters in the excitatory amino acid transporter family terminate glutamatergic synaptic transmission by catalyzing Na(+)-dependent removal of glutamate from the synaptic cleft. Recent structural studies of the aspartate-specific archaeal homolog, Glt(Ph), suggest that transport is achieved by a rigid body, piston-like movement of the transport domain, which houses the substrate-binding site, between the extracellular and cytoplasmic sides of the membrane. This transport domain is connected to an immobile scaffold by three loops, one of which, the 3-4 loop (3L4), undergoes substrate-sensitive conformational change. Proteolytic cleavage of the 3L4 was found to abolish transport activity indicating an essential function for this loop in the transport mechanism. Here, we demonstrate that despite the presence of fully cleaved 3L4, Glt(Ph) is still able to sample conformations relevant for transport. Optimized reconstitution conditions reveal that fully cleaved Glt(Ph) retains some transport activity. Analysis of the kinetics and temperature dependence of transport accompanied by direct measurements of substrate binding reveal that this decreased transport activity is not due to alteration of the substrate binding characteristics but is caused by the significantly reduced turnover rate. By measuring solute counterflow activity and cross-link formation rates, we demonstrate that cleaving 3L4 severely and specifically compromises one or more steps contributing to the movement of the substrate-loaded transport domain between the outward- and inward-facing conformational states, sparing the equivalent step(s) during the movement of the empty transport domain. These results reveal a hitherto unknown role for the 3L4 in modulating an essential step in the transport process.

Conserved Tyrosine in the First Transmembrane Segment of Solute:Sodium Symporters Is Involved in Na+-coupled Substrate Co-transport

Solute:sodium symporters (SSSs) transport vital molecules across the plasma membrane of all living organisms. vSGLT, the Na(+)/galactose transporter of Vibrio parahemeolyticus, is the only SSS for which high resolution structural information is available, revealing a LeuT-like fold and a Na(+)-binding site analogous to the Na2 site of LeuT. Whereas the core transmembrane segments (TMs) of SSSs share high structural similarity with other transporters of LeuT-like fold, TM1 does not correspond to any TM in those structural homologs and was only resolved for the backbone atoms in the initial vSGLT structure (Protein Data Bank code 3DH4). To assess the role of TM1 in Na(+)-coupled substrate symport by the SSSs, here we have studied the role of a conserved residue in TM1 by computational modeling in conjunction with radiotracer transport and binding studies. Based on our sequence alignment and much topological data for homologous PutP, the Na(+)/proline transporter, we have simulated a series of vSGLT models with shifted TM1 residue assignments. We show that in two converged vSGLT models that retained the original TM1 backbone conformation, a conserved residue, Tyr-19, is associated with the Na(+) binding interaction network. In silico and in vitro mutagenesis of homologous Tyr-14 in PutP revealed the involvement of this conserved residue in Na(+)-dependent substrate binding and transport. Thus, our combined computational and experimental data provide the first clues about the importance of a conserved residue in TM1, a unique TM in the proteins with LeuT-like fold, in the Na(+)-coupled symport mechanism of SSSs.

Variable NMR visibility of intracellular sodium induced by Na+‐substrate cotransport in dog cortical tubules

The intracellular sodium concentration [( Na+]i) of dog kidney cortical tubules was monitored by flame photometry and 23Na NMR using dysprosium tripolyphosphate as shift reagent. Upon addition of substrates cotransported with sodium, flame photometry showed an increase in [Na+]i while no change (glutamine, glucose) or even a decrease (lactate) in the Na+i NMR signal was observed. This discrepancy could not be explained by a lack of ATP prior to the addition of substrates or by a decrease of NMR visibility of Nai+ induced by binding of substrate to membrane transporters (and pump). We propose that a variation of the "apparent visibility" of Nai+ may occur, arising from either a compartmentation of Nai+ in dog cortical tubules or an inhomogeneous extracellular distribution of the shift reagent.

Renal Fanconi syndrome: developmental basis for a new animal model with relevance to human disease

Using succinylacetone (SA), a metabolite of tyrosine excreted in excess by infants and children with hereditary tyrosinemia and the renal Fanconi syndrome (FS), we have investigated developmentally-related membrane transport events leading to emergence of the generalized renal tubular dysfunction seen in human FS. SA was found to impair sugar and amino acid uptake by both newborn renal tubules and 7-day renal brush-border membrane vesicles (BBMV). This impairment by SA was due in part to a slowing of substrate cotransport rate of 22Na+-entry into BBMV. Concentration-dependent uptake studies indicated SA inhibited the newborn high-affinity transport systems for sugars and amino acids. SA also caused an increase in membrane fluidity and a shift in the thermotropic transition temperature. The demonstrated dual nature of SA's effect on membrane fluidity and O2 consumption, together with the relative contribution of each component to SA-induced transport impairment helps to provide a basis for an understanding of the age-related increases in glucosuria, aminoaciduria and natriuria seen in infants with FS.

Na+(Li+)/Branched-chain amino acid cotransport inPseudomonas aeruginosa

A transport system for branched-chain amino acids (designated as LIV-II system) in Pseudomonas aeruginosa requires Na+ for its operation. Coupling cation for this system was identified by measuring cation movement during substrate entry using cation-selective electrodes. Uptakes of Na+ and Li+ were induced by the imposition of an inwardly-directed concentration gradient of leucine, isoleucine, or valine. No uptake of H+ was found, however, under the same conditions. In addition, effects of Na+ and Li+ on the kinetic property of the system were examined. At chloride salt concentration of 2.5 mM, values of apparent Km and Vmax for leucine uptake were larger in the presence of Na+ than Li+. These results indicate that the LIV-II transport system is a Na+(Li+)/substrate cotransport system, although effects of Na+ and Li+ on kinetics of the system are different.

Effects of membrane potential on Na cotransports in eel intestinal brush-border membrane vesicles: Studies with a fluorescent dye

The Na-dependent transport of a number of organic molecules (d-glucose,l-proline,l-alanine,l-phenylalanine) in brush-border membrane vesicles isolated from the intestine of the eel (Anguilla anguilla) was monitored by recording the fluorescence quenching of the voltage-sensitive cyanine dye 3,3′-diethylthiacarbocyanine iodide (DiS-C2(5)). The experimental approach consisted of: a) generating an inside-negative membrane potential mimicking “in vivo” conditions: b) measuring the rate of membrane potential decay (i.e., the rate of fluorescence quenching decay) due to Na-neutral substrate cotransport. Rates of membrane potential decay showed saturation on substrate concentration andKapp values (the substrate concentration giving 50% of the maximal rate) were estimated for Na-dependent transport ofd-glucose (0,099mm),l-alanine (0.516mm),l-proline (0.118mm) andl-phenylalanine (2.04mm). The influence of an inside-negative membrane potential on the affinity of the transporter for glucose and for sodium is discussed.

Isolation and properties of a mutant of Escherichia coli possessing defective Na+/H+ antiporter.

A mutant of Escherichia coli with defective Na+/H+ antiporter was isolated. The rationale for its isolation was that cells possessing defective Na+/H+ antiporter, which is essential for establishment of a Na+ gradient, could not grow with a carbon source that was taken up with Na+. The mutant had no appreciable Na+/H+ antiporter activity, but its K+/H+ antiporter and Ca2+/H+ antiporter activities were normal. Judging from the reversion frequency, the defect seems to be due to a single mutation. The mutant could not grow at alkaline pH. Therefore, the Na+/H+ antiporter, but not the K+/H+ antiporter or the Ca2+/H+ antiporter, seems to be responsible for pH regulation in alkaline medium. This mutant will be useful for cloning the Na+/H+ antiporter gene and for detection of Na+-substrate cotransport systems.

細菌細胞膜のＨ｀＋´‐有機物質共輸送系

Progresses on H+ -solute cotransport systems are reviewed. We focus mainly on the lactose transport system of Escherichia coli, which is the best characterized H+ -substrate cotransport system. Structure-function relationship in the transport carrier, which has been studied from biochemical and genetic aspect, is discussed.

Active transport of sugars coupled to Na+ in Escherichia coli

Cation-nutrient cotransport is ubiquitous in nature. For cotransport, coupling between H+ and substrate is common in microbial cells, and that between Na+ and substrate is common in animal cells. We have reported that the melibiose transport system of Escherichia coli could utilize Na+ as a coupling cation. This is the first example of Na+-substrate cotransport found in microbial cells. Versatility in the combination between cation and substrate was revealed. This system seems to be a suitable model system for the analysis of cation-substrate cotransport systems. Physiological, biochemical and genetical properties of this system are summarized.

Altered cation coupling to melibiose transport in mutants of Escherichia coli.

The alpha-galactoside transport system of Escherichia coli utilizes either H+ of Na+ as a coupling cation for melibiose transport. Mutants were isolated which showed altered cation recognition for melibiose transport. The transport carrier of the mutants has lost the ability to accept H+ but can utilize either Na/ or Li+ for co-transport with melibiose. Such mutants might be predicted if the melibiose carrier were viewed as a descendant of an evolutionary transition between the H+-substrate co-transport systems of primitive cells and the Na+-substrate systems of more complex organisms. A second mutation was found in these mutants which involves the Na+ (Li+)-H+ exchange carrier. The maximal rate of Li+-H+ exchange in one of the mutants was 11 times higher than the parent and the Km for Li+ by this carrier was one-sixth that of the parent.

Metabolic control of lactose entry in Escherichia coli

A general method has been developed for determining the rate of entry of lactose into cells of Escherichia coli that contain β-galactosidase. Lactose entry is measured by either the glucose or galactose released after lactose hydrolysis. Since lactose is hydrolyzed by β-galactosidase as soon as it enters the cell, this assay measures the activity of the lactose transport system with respect to the translocation step. Using assays of glucose release, lactose entry was studied in strain GN2, which does not phosphorylate glucose. Lactose entry was stimulated 3-fold when cells were also presented with readily metabolizable substrates. Entry of o- nitrophenyl-β- d-galactopyranoside (ONPG) was only slightly elevated (1.5-fold) under the same conditions. The effects of arsenate treatment and anaerobiosis suggest that lactose entry may be limited by the need for reextrusion of protons which enter during H +/sugar cotransport. Entry of o- nitrophenyl-β- d-galactopyranoside is less dependent on the need for proton reextrusion, probably because the stoichiometry of H +/substrate cotransport is greater for lactose than for ONPG.

The uptake and release of calcium by heart mitochondria

The kinetics of uptake of Ca(2+) by rat heart mitochondria were studied by a spectrophotometric method with Arsenazo III indicator. The exponential rate coefficients measured with or without added phosphate increase with the amount of Ca(2+) added up to about 24mum. Evidence is given that the effect is attributable to a combination of formation of chelates at low concentrations to act as Ca(2+) buffers, with co-transport of substrate to provide more respiratory fuel. The inhibitory effect of Mg(2+) depends on the Ca(2+) concentration, so with a constant [Mg(2+)] the low concentrations of Ca(2+) are most inhibited, and the rate coefficients are still more Ca(2+)-dependent. Ca(2+) uptake is slowed by local anaesthetics such as butacaine and dibucaine, and also by propranolol and palmitoyl-CoA. After an uptake, the release of Ca(2+) was investigated. The spontaneous release involves an initially slow and small appearance of free Ca(2+) and is followed by an auto-accelerated phase. The release is accompanied by a gradual decrease in internal ATP; it is initiated by palmitoyl-CoA (reversed by carnitine), by lysophosphatidylcholine, by Na(+) salts (reversed by oligomycin) and by K(+) salts added to a K(+)-free medium containing valinomycin. The process is probably a response to an increased energy load imposed on the mitochondria by the various conditions, which include the spontaneous action of phospholipase activated by traces of Ca(2+). The problem of how much mitochondrial activity is participating in normal heart Ca(2+) turnover is discussed, and experiments showing only 7-14% exchange of the mitochondrial Ca(2+) occurring in vivo in 10 or 20min are reported.

Electrical evidence for rhythmic changes in the cotransport of sucrose and hydrogen ions in Samanea pulvini

Measurements with microelectrodes implanted into Samanea saman (Jacq.) Merrill leaf pulvini showed that membrane potentials were rhythmically sensitive to the application of sucrose. The magnitude of the electrical depolarizations induced by sucrose were dependent on the concentration of H(+) in the medium, yet changes in [H(+)] alone did not greatly affect the potential. During sucrose-induced electrical depolarization, there was a slight increase in the pH of the bathing medium; both effects were abolished by high levels of K(+), Na(+) or Ca(2+) in the medium. These observations indicate that H(+) enter the cells by some cooperative action with sucrose. A model of H(+)-substrate cotransport is proposed in which a sugar carrier in the membrane is made more permeable by the attachment of a proton. The rhythmic nature of this proposed cotransport may be related to circadian leaf-movements in this plant.

Models of lon and substrate cotransport and the effect of the membrane potential

The implications of a carrier model of ion and substrate cotransport are worked out. Each carrier is assumed to have one ion and one substrate binding site. The model includes features that have not been included in previously published models. These features are the effect of the membrane potential and of the assumption that all carrier forms, with or without bound substrate and with or without various bound ions, can cross the membrane. The model is of a two-state (gate-type) carrier with transition rate constants. In one state the carrier interacts with outer bulk phase; in the other state it interacts with the inner bulk phase. Equilibrium in the reactions between ion, substrate, and carrier is assumed at each surface.