Cotransport Mechanism Research Articles

Co-transport of uranyl carbonate and silica colloids in saturated quartz sand under different hydrochemical conditions

Uranyl carbonate (UC) and silica colloids (cSiO2) are widely distributed in carbonate-rich subsurface environments associated with uranium pollution. Mobile colloids such as cSiO2 can affect uranium's transport efficiency in the groundwater environment. Therefore, elucidating the mechanism of UC and cSiO2 co-transport in a saturated porous medium with different ionic strength (IS), pH, and UC concentration is essential for the prevention and control of groundwater radioactive pollution. At low UC concentrations (<2.1 × 10−5 M), cSiO2 is more prone to be deposited on the surfaces of quartz sand (QS) than UC, resulting in cSiO2 preventing UC transport. Compared to pH 7 and 9, at pH 5 the adsorption of uranium [in the form of 81.5% UO2CO3(aq), 8.6% UO22+, and 5.2% UO2OH+] on cSiO2 renders cSiO2 more prone to aggregate, causing smaller amounts of cSiO2 (86.6%) and UC (55.8%) to be recovered. Mechanisms responsible for the evolution of the pH and zeta potential in effluents have been proposed. Chemical reactions (ligand-exchange reactions and deprotonation) that occur in the QS column between UC and cSiO2/QS cause the pH of the suspension to varying, which in turn causes changes in the zeta potential and particle size of cSiO2. Eventually, the recovery rates of cSiO2 and UC are changed, depending upon the colloid particle size. Changes in ionic strength can seriously affect the stability of cSiO2 particles, and that effect is more significant when UC is present. Moreover, colloidal filtration theory, a non-equilibrium two-site model, and the Derjaguin–Landau–Verwey–Overbeek theory successfully describe the individual-transport and co-transport of cSiO2 and UC in the column. This study provides a strong basis for investigating UC pollution control in porous media.

Co-transport behavior of ammonium and colloids in saturated porous media under different hydrochemical conditions.

To investigate co-transport behavior of ammonium and colloids in saturated porous media under different hydrochemical conditions, NH4+ was selected as the target contaminant, and silicon and humic acid (HA) were selected as typical organic and inorganic colloids in groundwater. Column experiments were then conducted to investigate the transport of NH4+ colloids under various hydrochemical conditions. The results showed that because of the different properties of colloidal silicon and HA after combining with NH4+, the co-transport mechanism became significantly different. During transport by the NH4+-colloid system, colloidal silicon occupied the adsorption sites on the medium surface to promote the transport of NH4+, while humic acid (HA) increased the number of adsorption sites of the medium to hinder the transport of NH4+. The co-transport of NH4+ and colloids is closely related to hydrochemical conditions. In the presence of HA, competitive adsorption and morphological changes of HA caused NH4+ to be more likely to be transported at a higher ionic strength (IS = 0.05m, CaCl2) and alkalinity (pH = 9.3). In the presence of colloidal silicon, blocking action caused the facilitated transport to be dependent on higher ionic strength and acidity (pH = 4.5), causing the recovery of NH4+ to improve by 7.99%, 222.25% (stage 1), and 8.63%, respectively. Moreover, transport increases with the colloidal silicon concentrations of 20mg/L then declines at 40mg/L, demonstrating that increased concentrations will lead to blocking and particle aggregation, resulting in delayed release in the leaching stage. Graphical abstract.

Principles and Practice of Oral Rehydration.

An understanding of fluid and electrolyte losses from diarrhea and mechanisms of solute cotransport led to development of oral rehydration solution (ORS), representing a watershed in efforts to reduce diarrheal disease morbidity and mortality. This report reviews the scientific rationale and modifications of ORS and barriers to universal application. Solutions with osmolality and electrolyte composition different from original ORS for routine and unique pathophysiology such as in malnutrition have met with varying success. Following the conceptual rationale of sodium-glucose cotransportation to facilitate water absorption, other cotransporters and formulations have been explored with the aim to improve ORS efficacy and acceptance. ORS remains the anchor of acute watery diarrhea and dehydration management worldwide. Despite development of different formulations, the current standard solution is the mainstay of treatment for nearly all situations. Efforts to improve oral hydration solution and to increase acceptance and usage are ongoing.

Cryo-EM structures of DrNKCC1 and hKCC1: a new milestone in the physiology of cation-chloride cotransporters.

New milestones have been reached in the field of cation-Cl- cotransporters with the recently released cryo-electron microscopy (EM) structures of the Danio rerio (zebrafish) Na+-K+-2Cl- cotransporter (DrNKCC1) and the human K+-Cl- cotransporter (hKCC1). In this review we provide a brief timeline that identifies the multiple breakthroughs in the field of solute carrier 12 transporters that led to the structure resolution of two of its key members. While cation-Cl- cotransporters share the overall architecture of carriers belonging to the amino acid-polyamine-organocation (APC) superfamily and some of their substrate binding sites, several new insights are gained from the two individual structures. A first major feature relates to the largest extracellular domain between transmembrane domain (TMD) 5 and TMD6 of KCC1, which stabilizes the dimer and forms a cap that likely participates in extracellular gating. A second feature is the conservation of the K+ and Cl- binding sites in both structures and evidence of an unexpected second Cl- coordination site in the KCC1 structure. Structural data are discussed in the context of previously published studies that examined the basic and kinetics properties of these cotransport mechanisms. A third characteristic is the evidence of an extracellular gate formed by conserved salt bridges between charged residues located toward the end of TMD3 and TMD4 in both transporters and the existence of an additional neighboring bridge in the hKCC1 structure. A fourth feature of these newly solved structures relates to the multiple points of contacts between the monomer forming the cotransporter homodimer units. These involve the TMDs, the COOH-terminal domains, and the large extracellular loop for hKCC1.

Physiological roles and molecular mechanisms of K+ -Cl- cotransport in the mammalian kidney and cardiovascular system: where are we?

In the early 80s, renal microperfusion studies led to the identification of a basolateral K+ -Cl- cotransport mechanism in the proximal tubule, thick ascending limb of Henle and collecting duct. More than ten years later, this mechanism was found to be accounted for by three different K+ -Cl- cotransporters (KCC1, KCC3 and KCC4) that are differentially distributed along the renal epithelium. Two of these isoforms (KCC1 and KCC3) were also found to be expressed in arterial walls, the myocardium and a variety of neurons. Subsequently, valuable insights have been gained into the molecular and physiological properties of the KCCs in both the mammalian kidney and cardiovascular system. There is now robust evidence indicating that KCC4 sustains distal renal acidification and that KCC3 regulates myogenic tone in resistance vessels. However, progress in understanding the functional significance of these transporters has been slow, probably because each of the KCC isoforms is not identically distributed among species and some of them share common subcellular localizations with other KCC isoforms or sizeable conductive Cl- pathways. In addition, the mechanisms underlying the process of K+ -Cl- cotransport are still ill defined. The present review focuses on the knowledge gained regarding the roles and properties of KCCs in renal and cardiovascular tissues.

Intensification of zirconium and hafnium separation through the hollow fiber renewal liquid membrane technique using synergistic mixture of TBP and Cyanex-272 as extractant

Abstract The novel synergistic mixture of TBP and Cyanex-272 is used as the extractant in the hollow fiber renewal liquid membrane (HFRLM) technique for Zr/Hf separation. The effects of the chemical and operational parameters such as HNO3 concentration in the donor phase, NH4F concentration in the acceptor phase, Cyanex-272 and TBP concentration in the liquid membrane phase, the lumen and shell side flow rates, and aqueous/organic volume ratio on the mass transfer and separation performance of HFRLM method were investigated. The obtained results reveal the intensification potential of proposed HFRLM technique for selective extraction of Zr over Hf with separation factor higher than 100. The HFRLM method provides two times higher mass transfer flux in comparison with hollow fiber supported liquid membrane (HFSLM). Also, the HFRLM method shows satisfactory stability for 700 min of continuous operation. The Zr ion transport through the LM phase follows the coupled co-transport mechanism and the diffusion in the renewal layer is recognized as the rate-controlling step in the HFRLM process. Moreover, the Zr mass transfer coefficient and molar flux in the HFRLM method are calculated in the range of 1 × 10−8 to 8.4 × 10−7 m·s−1 and 4.9 × 10−6 to 20.1 × 10−6 mol·m−2·s−1, respectively.

Modeling and Simulation of hGAT1: A Mechanistic Investigation of the GABA Transport Process

Human γ-Aminobutyric acid transporter 1 (hGAT1) is a Na+/Cl− dependent co-transporter that plays a key role in the inhibitory neurotransmission of GABA in the brain. Due to the lack of structural data, the exact co-transport mechanism of GABA reuptake by hGAT1 remains unclear. To examine the roles of the co-transport ions and the nature of their interactions with GABA, homology modeling and molecular dynamics simulations of the hGAT1 in the open-to-out conformation were carried out. Our study focused on the sequential preloading of Na+ and Cl− ions, followed by GABA binding. Our simulations showed pre-loading of ions maintains the transport ready state of hGAT1 in the open-to-out conformation essential for GABA binding. Of the four putative preloaded states, GABA binding to the fully loaded state is most favored. Binding of Na+ ion to the Na1 site helps to maintain the open-to-out conformation for GABA binding as compared to the Na2 site. GABA binding to the mono-sodium or the di-sodium loaded states leads to destabilization of Na+ ions within their binding sites. The two most prominent interactions required for GABA binding include interaction between carboxylate group of GABA with the bound Na+ ion in Na1 binding site and the hydroxyl group of Y140. Overall our results support the fully loaded state as the predominate state for GABA binding. Our study further illustrates that Na+ ion within the Na1 site is crucial for GABA recognition. Therefore, a revised mechanism is proposed for the initial step of hGAT1 translocation cycle.

Natural colloid mobilization and leaching in wettable and water repellent soil under saturated condition

AbstractThe coupled transport of pollutants that are adsorbed to colloidal particles has always been a major topic for environmental sciences due to many unfavorable effects on soils and groundwater. This laboratory column study was conducted under saturated moisture conditions to compare the hydrophobic character of the suspended and mobilized colloids in the percolates released from a wettable subsoil and a water repellent topsoil. Both soils with different organic matter content were analyzed for wettability changes before and after leaching using sessile drop contact angles as well as water and ethanol sorptivity curves, summarized as repellency index. Hydrophobicity of the effluent suspensions was assessed using the C18 adsorption method. Water repellency level of the repellent soil decreased after leaching but remained on a lower level of water repellency, while, the wettable soil remained wettable. The leached colloids from the repellent soil were predominantly hydrophilic and the percentage of the hydrophobic colloid fraction in the effluent did not systematically changed with time. Total colloid release depended on soil carbon stock but not on soil wettability. Our results suggest that due to the respective character of transported colloids a similar co-transport mechanism for pollutants may occur which does not depend explicitly on soil wettability of the releasing horizon, but could be more affected by total SOM content. Further studies with a wider range of soils are necessary to determine if the dominant hydrophilic character of leached colloids is typical. Due to the mostly hydrophilic colloid character we conclude also that changes in wettability status, i.e. of wettable subsoil horizons due to the leachate, may not necessarily occur very fast, even when the overlaying topsoil is a repellent soil horizon with a high organic matter content.

Reassessment of the Transport Mechanism of the Human Zinc Transporter SLC39A2.

The human zinc transporter SLC39A2, also known as ZIP2, was shown to mediate zinc transport that could be inhibited at pH <7.0 and stimulated by HCO3-, suggesting a Zn2+/HCO3- cotransport mechanism [Gaither, L. A., and Eide, D. J. (2000) J. Biol. Chem. 275, 5560-5564]. In contrast, recent experiments in our laboratory indicated that the functional activity of ZIP2 increases at acidic pH [Franz, M. C., et al. (2014) J. Biomol. Screening 19, 909-916]. The study presented here was therefore designed to reexamine the findings about the pH dependence and to extend the functional characterization of ZIP2. Our current results show that ZIP2-mediated transport is modulated by extracellular pH but independent of the H+ driving force. Also, in our experiments, ZIP2-mediated transport is not modulated by extracellular HCO3-. Moreover, a high extracellular [K+], which induces depolarization, inhibited ZIP2-mediated transport, indicating that the transport mechanism is voltage-dependent. We also show that ZIP2 mediates the uptake of Cd2+ ( Km ∼ 1.57 μM) in a pH-dependent manner ( KH+ ∼ 66 nM). Cd2+ transport is inhibited by extracellular [Zn2+] (IC50 ∼ 0.32 μM), [Cu2+] (IC50 ∼ 1.81 μM), and to a lesser extent [Co2+], but not by [Mn2+] or [Ba2+]. Fe2+ is not transported by ZIP2. Accordingly, the substrate selectivity of ZIP2 decreases in the following order: Zn2+ > Cd2+ ≥ Cu2+ > Co2+. Altogether, we propose that ZIP2 is a facilitated divalent metal ion transporter that can be modulated by extracellular pH and membrane potential. Given that ZIP2 expression has been reported in acidic environments [Desouki, M. M., et al. (2007) Mol. Cancer 6, 37; Inoue, Y., et al. (2014) J. Biol. Chem. 289, 21451-21462; Tao, Y. T., et al. (2013) Mol. Biol. Rep. 40, 4979-4984], we suggest that the herein described H+-mediated regulatory mechanism might be important for determining the velocity and direction of the transport process.

Mechanism of Water and Solute Cotransport by the Sodium Glucose Cotransporter SGLT1

Mechanism of Water and Solute Cotransport by the Sodium Glucose Cotransporter SGLT1

Distinguishable co-transport mechanisms of phenanthrene and oxytetracycline with oxidized-multiwalled carbon nanotubes through saturated soil and sediment columns: vehicle and competition effects

To date mechanisms underlying co-transports of engineered nanomaterials (ENMs) with contaminants have not been adequately explored, which involve complex interactions among ENMs, contaminants, and soils. This study investigated co-transport behaviors of 3 oxidized-multiwalled carbon nanotubes (o-MWCNTs) with phenanthrene (PHE) and oxytetracycline (OTC) in soil and sediment columns. Sorptions and desorptions of PHE and OTC by the o-MWCNTs were examined to facilitate the discussion of co-transport mechanisms. The results showed that mobilities of PHE and OTC in the columns were significantly enhanced by the presences of o-MWCNTs in the influents; the eluted o-MWCNTs were positively correlated to the eluted total PHE but negatively correlated to the eluted total OTC; the eluted PHE was mainly in the o-MWCNTs-associated form, while it was mainly the dissolved OTC breaking through the columns. It was thus concluded that the o-MWCNTs acted as vehicles facilitating the PHE transport, while besides the vehicle effect the o-MWCNTs also competed for the adsorption sites on soil particles with OTC and thereby enhancing the OTC mobility. These findings provide new insight into the mechanisms regulating co-transports of ENMs and contaminants in porous media.

Identification of phlorizin binding domains in sodium-glucose cotransporter family: SGLT1 as a unique model system.

The sodium glucose cotransporter SGLT1 expressed mainly in the intestine and kidney has been explored extensively for understanding the mechanism of sugar cotransport and its inhibition by a classical competitive inhibitor, phlorizin (Pz). It has been shown that inhibition of SGLT1 by Pz involves its interaction followed by major conformational changes in the Pz binding domain (PBD) in C-terminal loop 13. However, the mechanism of Pz inhibition and its interaction with other members of SGLT is not known. In this hypothesis, we performed molecular modeling of SGLT1-loop 13 with Pz and carried out primary sequence analyses and secondary structure predictions to determine qualitatively similar PBDs in C-termini of human SGLT2-4, except for vSGLT, which contains an unstructured short C-terminus. The ranking of predictions of Pz interaction strongly agrees with the following ranking of previously reported Pz inhibition: SGLT2>SGLT1>SGLT4>SGLT3>>vSGLT. In addition, the sugar binding residues were found to be quite conserved among all SGLT members investigated here. Based on these preliminary analyses, we propose that other Pz-sensitive SGLTs are also inhibited via mechanism similar to SGLT1 where an aglucone of Pz, phloretin, interacts with PBD and glucoside moiety with sugar binding residues. Our hypothesis sets the stage for future analyses on investigation of Pz interaction with SGLT family and further suggests that Pz modeling may be explored to design novel inhibitors targeting several SGLT members.

The significance of water co-transport for sustaining transpirational water flow in plants: a quantitative approach.

In a recent Opinion paper, Wegner (Journal of Experimental Botany 65, 381-392, 2014) adapts a concept developed for water flow in animal tissues to propose a model, which can explain the loading of water into the root xylem against a difference in water potential (Ψ) between the xylem parenchyma cell (more negative Ψ) and the xylem vessel (less negative Ψ). In this model, the transport of water is energized through the co-transport of ions such as K(+) and Cl(-) through plasma membrane-located transporters. The emphasis of the model is on the thermodynamic feasibility of the co-transport mechanism per se. However, what is lacking is a quantitative evaluation of the energy input required at the organismal level to sustain such a co-transport mechanism in the face of considerable net (transpirational) flows of water through the system. Here, we use a ratio of 500 water molecules being co-transported for every pair of K(+) and Cl(-) ions, as proposed for the animal system, to calculate the energy required to sustain daytime and night-time transpirational water flow in barley plants through a water co-transport mechanism. We compare this energy with the total daily net input of energy through photosynthetic carbon assimilation. Water co-transport can facilitate the filling of xylem against a difference in Ψ of 1.0MPa and puts a minor drain on the energy budget of the plant. Based on these findings it cannot be excluded that water co-transport in plants contributes significantly to xylem filling during night-time and possibly also daytime transpiration.

P-glycoprotein induction in Caco-2 cells by newly synthetized thioxanthones prevents paraquat cytotoxicity

The induction of P-glycoprotein (P-gp), an ATP-dependent efflux pump, has been proposed as a strategy against the toxicity induced by P-gp substrates such as the herbicide paraquat (PQ). The aim of this study was to screen five newly synthetized thioxanthonic derivatives, a group known to interact with P-gp, as potential inducers of the pump's expression and/or activity and to evaluate whether they would afford protection against PQ-induced toxicity in Caco-2 cells. All five thioxanthones (20µM) caused a significant increase in both P-gp expression and activity as evaluated by flow cytometry using the UIC2 antibody and rhodamine 123, respectively. Additionally, it was demonstrated that the tested compounds, when present only during the efflux of rhodamine 123, rapidly induced an activation of P-gp. The tested compounds also increased P-gp ATPase activity in MDR1-Sf9 membrane vesicles, indicating that all derivatives acted as P-gp substrates. PQ cytotoxicity was significantly reduced in the presence of four thioxanthone derivatives, and this protective effect was reversed upon incubation with a specific P-gp inhibitor. In silico studies showed that all the tested thioxanthones fitted onto a previously described three-feature P-gp induction pharmacophore. Moreover, in silico interactions between thioxanthones and P-gp in the presence of PQ suggested that a co-transport mechanism may be operating. Based on the in vitro activation results, a pharmacophore model for P-gp activation was built, which will be of further use in the screening for new P-gp activators. In conclusion, the study demonstrated the potential of the tested thioxanthonic compounds in protecting against toxic effects induced by P-gp substrates through P-gp induction and activation.

An Insight into the Skin Penetration Enhancement Mechanism of N-Methylpyrrolidone

This work aims to elucidate the mechanism by which N-methylpyrrolidone (NMP) enhances the skin permeation of a compound by combining experimental data with molecular dynamic (MD) simulations. The addition of 10% NMP significantly increased the propranolol (PR) permeation through the human epidermis (∼ 15 μg/cm(2) vs ∼ 30 μg/cm(2)) while resulting inefficacious on hydrocortisone (HC) diffusion. No significant alterations in the stratum corneum structure were found after the in vitro treatment of human epidermis with NMP dispersed in mineral oil or water by attenuated total reflectance Fourier transform infrared (ATR-FTIR) analyses. MD simulations revealed the formation of a complex by H-bonds and the π-π stacking interactions between the NMP's amido group and the drug's aromatic systems. The size of the depicted NMP/PR clusters was in line with the hydrodynamic radius derived by dynamic light scattering analyses (∼ 2.00 nm). Conversely, no interaction, and consequently cluster formation, between NMP and HC occurred. These results suggest that NMP is effective in enhancing the drug permeation through human epidermis by a cotransport mechanism when NMP/drug interaction occurs.

Induction and activation of P-glycoprotein by dihydroxylated xanthones protect against the cytotoxicity of the P-glycoprotein substrate paraquat

Xanthones are a family of compounds with several known biological activities and therapeutic potential for which information on their interaction with membrane transporters is lacking. Knowing that P-glycoprotein (P-gp) acts as a cellular defense mechanism by effluxing its toxic substrates, the aim of this study was to investigate the potential of five dihydroxylated xanthones as inducers of P-gp expression and/or activity and to evaluate whether they could protect Caco-2 cells against the cytotoxicity induced by the toxic P-gp substrate paraquat (PQ). After 24h of incubation, all tested xanthones caused a significant increase in both P-gp expression and activity, as evaluated by flow cytometry using the UIC2 antibody and rhodamine 123, respectively. Additionally, after a short 45-min incubation, all the tested xanthones induced a rapid increase in P-gp activity, indicating direct pump activation without increased P-gp protein expression. The tested compounds also increased P-gp ATPase activity in MDR1-Sf9 membrane vesicles, demonstrating to be P-gp substrates. Moreover, when simultaneously incubated with PQ, all xanthones significantly reduced the cytotoxicity of the herbicide, and these protective effects were completely reversed upon incubation with a specific P-gp inhibitor. In silico studies evaluating the interactions between xanthones and P-gp in the presence of PQ suggested that a co-transport mechanism may be operating. A quantitative structure-activity relationship model was developed and validated, and the maximal partial charge for an oxygen atom was the descriptor predicted as being implicated in P-gp activation by the dihydroxylated xanthones. These results disclose new perspectives in preventing PQ- and other P-gp substrates-induced poisonings.

Computational Investigation of Charge Transfer Mechanisms in Ammonium Transporters

The mechanism by which proteins of the Amt/MEP/Rh family transport ammonium remains largely unknown. No convincing mechanistic picture has emerged yet as to whether it is an electroneutral NH3 transport or an electrogenic NH3/H+ co-transport. using free energy calculations with polarizable force fields and with hybrid QM/MM descriptions, we have investigated the binding of ammonium and its transport across the pore of Escherichia coli's AmtB protein. The simulations reveal a novel cotransport mechanism in which the NH4+ substrate binds deeply into the pore and translocates as separate NH3 and H+ fragments. Critical to the cotransport mechanism is a pair of highly-conserved histidine residues, that enhance NH4+ binding, form a “proton wire” for charge transfer, and provide a scaffold for a water chain in the pore lumen.

Structural Insights into Genetic Variants of Na+/Glucose Cotransporter SGLT1 Causing Glucose–Galactose Malabsorption: vSGLT as a Model Structure

Current advances in structural biology provide valuable insights into structure-function relationship of membrane transporters by solving crystal structures of bacterial homologs of human transporters. Therefore, scientists consider bacterial transporters as useful structural models for designing of drugs targeted in human diseases. The functional homology between Vibrio parahaemolyticus Na(+)/galactose transporter (vSGLT) and Na(+)/glucose cotransporter SGLT1 has been well established a decade ago. Now the crystal structure of vSGLT is considered quite valuable in explaining not only the cotransport mechanisms, but it also acts as a representative protein in understanding the protein stability and amino acid interactions within the core structure. We investigated the molecular mechanisms of genetic variations in SGLT1 that cause glucose-galactose malabsorption (GGM) defects using the crystal structure of vSGLT as a model sugar transporter. Our in silico mutagenesis and modeling analysis suggest that the GGM genetic variations lead to conformational changes either by structure destabilization or by formation of unnecessary interaction within the core structure of SGLT1 thereby explaining the genetic defects in Na(+) dependent sugar translocation across the cell membrane.

Swelling-Activated Anion Channels Are Essential for Volume Regulation of Mouse Thymocytes

Channel-mediated trans-membrane chloride movement is a key process in the active cell volume regulation under osmotic stress in most cells. However, thymocytes were hypothesized to regulate their volume by activating a coupled K-Cl cotransport mechanism. Under the patch-clamp, we found that osmotic swelling activates two types of macroscopic anion conductance with different voltage-dependence and pharmacology. At the single-channel level, we identified two types of events: one corresponded to the maxi-anion channel, and the other one had characteristics of the volume-sensitive outwardly rectifying (VSOR) chloride channel of intermediate conductance. A VSOR inhibitor, phloretin, significantly suppressed both macroscopic VSOR-type conductance and single-channel activity of intermediate amplitude. The maxi-anion channel activity was largely suppressed by Gd3+ ions but not by phloretin. Surprisingly, [(dihydroindenyl)oxy] alkanoic acid (DIOA), a known antagonist of K-Cl cotransporter, was found to significantly suppress the activity of the VSOR-type single-channel events with no effect on the maxi-anion channels at 10 μM. The regulatory volume decrease (RVD) phase of cellular response to hypotonicity was mildly suppressed by Gd3+ ions and was completely abolished by phloretin suggesting a major impact of the VSOR chloride channel and modulatory role of the maxi-anion channel. The inhibitory effect of DIOA was also strong, and, most likely, it occurred via blocking the VSOR Cl− channels.

Substrate-modulated gating dynamics in a Na+-coupled neurotransmitter transporter homologue

Neurotransmitter/Na(+) symporters (NSSs) terminate neuronal signalling by recapturing neurotransmitter released into the synapse in a co-transport (symport) mechanism driven by the Na(+) electrochemical gradient. NSSs for dopamine, noradrenaline and serotonin are targeted by the psychostimulants cocaine and amphetamine, as well as by antidepressants. The crystal structure of LeuT, a prokaryotic NSS homologue, revealed an occluded conformation in which a leucine (Leu) and two Na(+) are bound deep within the protein. This structure has been the basis for extensive structural and computational exploration of the functional mechanisms of proteins with a LeuT-like fold. Subsequently, an 'outward-open' conformation was determined in the presence of the inhibitor tryptophan, and the Na(+)-dependent formation of a dynamic outward-facing intermediate was identified using electron paramagnetic resonance spectroscopy. In addition, single-molecule fluorescence resonance energy transfer imaging has been used to reveal reversible transitions to an inward-open LeuT conformation, which involve the movement of transmembrane helix TM1a away from the transmembrane helical bundle. We investigated how substrate binding is coupled to structural transitions in LeuT during Na(+)-coupled transport. Here we report a process whereby substrate binding from the extracellular side of LeuT facilitates intracellular gate opening and substrate release at the intracellular face of the protein. In the presence of alanine, a substrate that is transported ∼10-fold faster than leucine, we observed alanine-induced dynamics in the intracellular gate region of LeuT that directly correlate with transport efficiency. Collectively, our data reveal functionally relevant and previously hidden aspects of the NSS transport mechanism that emphasize the functional importance of a second substrate (S2) binding site within the extracellular vestibule. Substrate binding in this S2 site appears to act cooperatively with the primary substrate (S1) binding site to control intracellular gating more than 30 Å away, in a manner that allows the Na(+) gradient to power the transport mechanism.