Uncoupling of a CLC Cl-/H+ exchange transporter by polyatomic anions

CLC-ec1 is a bacterial archetype of CLC transporters, a ubiquitous class of proteins that catalyze transmembrane exchange of Cl- and H+ necessary for pH regulation of numerous physiological processes. Despite a profusion of high-resolution structures, the molecular mechanism of exchange remains unknown. Here, we rigorously demonstrate strict exchange stoichiometry of 2 Cl-/1 H+. In addition to Cl- and Br-, two non-halide ions, NO3- and SCN-, are shown to be transported by CLC-ec1, but with reduced H+ counter-transport. The loss of proton coupling to these anions is accompanied by an absence of bound anions in the central and external Cl- binding sites in the protein's anion selectivity region, as revealed by crystallographic comparison of Br- and SeCN- bound to this region.     

Determinants of coupled transport and uncoupled current by the electrogenic SLC26 transporters

Members of the SLC26 family of anion transporters mediate the transport of diverse molecules ranging from halides to carboxylic acids and can function as coupled transporters or as channels. A unique feature of the two members of the family, Slc26a3 and Slc26a6, is that they can function as both obligate coupled and mediate an uncoupled current, in a channel-like mode, depending on the transported anion. To identify potential features that control the two modes of transport, we performed in silico modeling of Slc26a6, which suggested that the closest potential fold similarity of the Slc26a6 transmembrane domains is to the CLC transporters, despite their minimal sequence identity. Examining the predicted Slc26a6 fold identified a highly conserved glutamate (Glu(-); Slc26a6(E357)) with the predicted spatial orientation similar to that of the CLC-ec1 E148, which determines coupled or uncoupled transport by CLC-ec1. This raised the question of whether the conserved Glu(-) in Slc26a6(E357) and Slc26a3(E367) have a role in the unique transport modes by these transporters. Reversing the Glu(-) charge in Slc26a3 and Slc26a6 resulted in the inhibition of all modes of transport. However, most notably, neutralizing the charge in Slc26a6(E357A) eliminated all forms of coupled transport without affecting the uncoupled current. The Slc26a3(E367A) mutation markedly reduced the coupled transport and converted the stoichiometry of the residual exchange from 2Cl(-)/1HCO(3)(-) to 1Cl(-)/1HCO(3)(-), while completely sparing the current. These findings suggest the possibility that similar structural motif may determine multiple functional modes of these transporters.     

Conversion of the 2 Cl−/1 H+ antiporter ClC‐5 in a NO3−/H+ antiporter by a single point mutation

Several members of the CLC family are secondary active anion/proton exchangers, and not passive chloride channels. Among the exchangers, the endosomal ClC-5 protein that is mutated in Dent''s disease shows an extreme outward rectification that precludes a precise determination of its transport stoichiometry from measurements of the reversal potential. We developed a novel imaging method to determine the absolute proton flux in Xenopus oocytes from the extracellular proton gradient. We determined a transport stoichiometry of 2 Cl⁻/1 H⁺. Nitrate uncoupled proton transport but mutating the highly conserved serine 168 to proline, as found in the plant NO₃⁻/H⁺ antiporter atClCa, led to coupled NO₃⁻/H⁺ exchange. Among several amino acids tested at position 168, S168P was unique in mediating highly coupled NO₃⁻/H⁺ exchange. We further found that ClC-5 is strongly stimulated by intracellular protons in an allosteric manner with an apparent pK of ∼7.2. A 2:1 stoichiometry appears to be a general property of CLC anion/proton exchangers. Serine 168 has an important function in determining anionic specificity of the exchange mechanism.     

Synergism between halide binding and proton transport in a CLC-type exchanger

The Cl-/H+ exchange-transporter CLC-ec1 mediates stoichiometric transmembrane exchange of two Cl- ions for one proton. A conserved tyrosine residue, Y445, coordinates one of the bound Cl- ions visible in the structure of this protein and is located near the intersection of the Cl- and H+ pathways. Mutants of this tyrosine were scrutinized for effects on the coupled transport of Cl- and H+ determined electrophysiologically and on protein structure determined crystallographically. Despite the strong conservation of Y445 in the CLC family, substitution of F or W at this position preserves wild-type transport behavior. Substitution by A, E, or H, however, produces uncoupled proteins with robust Cl- transport but greatly impaired movement of H+. The obligatory 2 Cl-/1 H+ stoichiometry is thus lost in these mutants. The structures of all the mutants are essentially identical to wild-type, but apparent anion occupancy in the Cl- binding region correlates with functional H+ coupling. In particular, as determined by anomalous diffraction in crystals grown in Br-, an electrophysiologically competent Cl- analogue, the well-coupled transporters show strong Br- electron density at the "inner" and "central" Cl- binding sites. However, in the uncoupled mutants, Br- density is absent at the central site, while still present at the inner site. An additional mutant, Y445L, is intermediate in both functional and structural features. This mutant clearly exchanges H+ for Cl-, but at a reduced H+-to-Cl- ratio; likewise, both the central and inner sites are occupied by Br-, but the central site shows lower Br- density than in wild-type (or in Y445F,W). The correlation between proton coupling and central-site occupancy argues that halide binding to the central transport site somehow facilitates movement of H+, a synergism that is not readily understood in terms of alternating-site antiport schemes.     

Mechanism of formate–nitrite transporters by dielectric shift of substrate acidity

Bacterial formate–nitrite transporters (FNTs) regulate the metabolic flow of small, weak mono-acids. Recently, the eukaryotic PfFNT was identified as the malaria parasite's lactate transporter and novel drug target. Despite crystal data, central mechanisms of FNT gating and transport remained unclear. Here, we show elucidation of the FNT transport mechanism by single-step substrate protonation involving an invariant lysine in the periplasmic vestibule. Opposing earlier gating hypotheses and electrophysiology reports, quantification of total uptake by radiolabeled substrate indicates a permanently open conformation of the bacterial formate transporter, FocA, irrespective of the pH. Site-directed mutagenesis, heavy water effects, mathematical modeling, and simulations of solvation imply a general, proton motive force-driven FNT transport mechanism: Electrostatic attraction of the acid anion into a hydrophobic vestibule decreases substrate acidity and facilitates protonation by the bulk solvent. We define substrate neutralization by proton transfer for transport via a hydrophobic transport path as a general theme of the Amt/Mep/Rh ammonium and formate–nitrite transporters.     

Functional characterization of two novel mammalian electrogenic proton-dependent amino acid cotransporters

We cloned two cDNAs encoding proton/amino acid cotransporters, designated as mPAT1 and mPAT2, from murine tissues. They were identified by sequence similarity to the amino acid/auxin permease family member of lower eukaryotes. We functionally characterized both transporters by flux studies and electrophysiology after expression in Xenopus laevis oocytes. Both mPAT1 and mPAT2 induced a pH-dependent electrogenic transport activity for small amino acids (glycine, alanine, and proline) that is altered by membrane potential. Direct evidence for amino acid/H(+)-symport was shown by intracellular acidification, and a flux coupling stoichiometry for proline/H(+)-symport of 1:1 was determined for both transporters. Besides small apolar L-amino acids, the transporters also recognize their D-enantiomers and selected amino acid derivatives such as gamma-aminobutyric acid. The mPAT1 transporter, the murine orthologue of the recently cloned rat LYAAT-1 transporter, can be considered as a low affinity system when compared with mPAT2. The mRNA of mPAT1 is highly expressed in small intestine, colon, kidney, and brain; the mPAT2-mRNA is mainly found in heart and lung. Phenotypically, the PAT1 transporter possesses the same functional characteristics as the previously described proton-dependent amino acid transport process in apical membranes of intestinal and renal epithelial cells.     

H+/ATP stoichiometry for the gastric (K++H+)-ATPase

Summary The initial rate of ATP-dependent proton uptake by hog gastric vesicles was measured at pH's between 6.1 and 6.9 by measuring the loss of protons from the external space with a glass electrode. The apparent rates of proton loss were corrected for scalar proton production due to ATP hydrolysis. For vesicles in 150mm KCl and pH 6.1, corrected rates of proton uptake and ATP hydrolysis were 639±84 and 619±65 nmol/min×mg protein, respectively, giving an H⁺/ATP ratio of 1.03±0.7. Furthermore, at all pH's tested the ratio of the rate of proton uptake to the rate of ATP hydrolysis was not significantly different than 1.0. No proton uptake (<10 nmol/min×mg protein) was exhibited by vesicles in 150mm NaCl at pH 6.1 despite ATP hydrolysis of 187±46 nmol/min×mg (nonproductive hydrolysis). Comparison of the rates of proton transport and ATP hydrolysis in various mixture of KCl and NaCl showed that the H⁺/ATP stoichiometries were not significantly different than 1.0 at all concentrations of K⁺ greater than 10mm. This fact suggests that the nonproductive rate is vanishingly small at these concentrations, implying that the measured H⁺/ATP stoichiometry is equal to the enzymatic stoichiometry. This result shows that the isolated gastric (K⁺+H⁺)-ATPase is thermodynamically capable of forming the observed proton gradient of the stomach.     

Evidence for an intestinal Na+: Sugar transport coupling stoichiometry of 2.0

Membrane potentials maintained by normally-energized intestinal epithelium interfere with an accurate determination of the Na⁺: sugar coupling stoichiometry associated with Na⁺-dependent transport systems. The interference is due to the fact that basal Na⁺ influx is itself a potential-dependent event, and sugar transport induces a membrane depolarization which therefore modifies basal Na⁺ entry. New information obtained under circumstances in which the membrane potential is maintained near 0 indicates that the true coupling stoichiometry is 2:1 rather than the commonly-accepted value of 1:1. A 2:1 stoichiometry means that cellular electrochemical Na⁺ gradients are adequate to account for recently observed 70-fold sugar gradients maintained by these cells under certain conditions.     

Characterizing unexpected interactions of a glutamine transporter inhibitor with members of the SLC1A transporter family

The solute carrier 1A family comprises a group of membrane proteins that act as dual-function amino acid transporters and chloride (Cl⁻) channels and includes the alanine serine cysteine transporters (ASCTs) as well as the excitatory amino acid transporters. ASCT2 is regarded as a promising target for cancer therapy, as it can transport glutamine and other neutral amino acids into cells and is upregulated in a range of solid tumors. The compound L-γ-glutamyl-p-nitroanilide (GPNA) is widely used in studies probing the role of ASCT2 in cancer biology; however, the mechanism by which GPNA inhibits ASCT2 is not entirely clear. Here, we used electrophysiology and radiolabelled flux assays to demonstrate that GPNA activates the Cl⁻ conductance of ASCT2 to the same extent as a transported substrate, whilst not undergoing the full transport cycle. This is a previously unreported phenomenon for inhibitors of the solute carrier 1A family but corroborates a body of literature suggesting that the structural requirements for transport are distinct from those for Cl⁻ channel formation. We also show that in addition to its currently known targets, GPNA inhibits several of the excitatory amino acid transporters. Together, these findings raise questions about the true mechanisms of its anticancer effects.     

Altering CLC stoichiometry by reducing non-polar side-chains at the dimerization interface

CLC-ec1 is a Cl⁻/H⁺ antiporter that forms stable homodimers in lipid bilayers, with a free energy of −10.9 kcal/mol in 2:1 POPE/POPG lipid bilayers. The dimerization interface is formed by four transmembrane helices: H, I, P and Q, that are lined by non-polar side-chains that come in close contact, yet it is unclear as to whether their interactions drive dimerization. To investigate whether non-polar side-chains are required for dimer assembly, we designed a series of constructs where side-chain packing in the dimer state is significantly reduced by making 4–5 alanine substitutions along each helix (H-ala, I-ala, P-ala, Q-ala). All constructs are functional and three purify as stable dimers in detergent micelles despite the removal of significant side-chain interactions. On the other hand, H-ala shows the unique behavior of purifying as a mixture of monomers and dimers, followed by a rapid and complete conversion to monomers. In lipid bilayers, all four constructs are monomeric as examined by single-molecule photobleaching analysis. Further study of the H-helix shows that the single mutation L194A is sufficient to yield monomeric CLC-ec1 in detergent micelles and lipid bilayers. X-ray crystal structures of L194A reveal the protein re-assembles to form dimers, with a structure that is identical to wild-type. Altogether, these results demonstrate that non-polar membrane embedded side-chains play an important role in defining dimer stability, but the stoichiometry is highly contextual to the solvent environment. Furthermore, we discovered that L194 is a molecular hot-spot for defining dimerization of CLC-ec1.     

Mammalian Complex I Pumps 4 Protons per 2 Electrons at High and Physiological Proton Motive Force in Living Cells

Mitochondrial complex I couples electron transfer between matrix NADH and inner-membrane ubiquinone to the pumping of protons against a proton motive force. The accepted proton pumping stoichiometry was 4 protons per 2 electrons transferred (4H⁺/2e⁻) but it has been suggested that stoichiometry may be 3H⁺/2e⁻ based on the identification of only 3 proton pumping units in the crystal structure and a revision of the previous experimental data. Measurement of proton pumping stoichiometry is challenging because, even in isolated mitochondria, it is difficult to measure the proton motive force while simultaneously measuring the redox potentials of the NADH/NAD⁺ and ubiquinol/ubiquinone pools. Here we employ a new method to quantify the proton motive force in living cells from the redox poise of the bc₁ complex measured using multiwavelength cell spectroscopy and show that the correct stoichiometry for complex I is 4H⁺/2e⁻ in mouse and human cells at high and physiological proton motive force.     

The mutation F227I increases the coupling of metal ion transport in DCT1

Metal ion transport by DCT1, a member of the natural resistance-associated macrophage protein family, is driven by protons. The stoichiometry of the proton to metal ion is variable, and under optimal transport conditions, more than 10 protons are co-transported with a single metal ion. To understand this phenomenon better, we used site-directed mutagenesis of DCT1 and analyzed the mutants by complementation of yeast suppressor of mitochondria import function-null mutants and electrophysiology with Xenopus oocytes. The mutation F227I resulted in an increase of up to 14-fold in the ratio between metal ions to protons transported. This observation suggests that low metal ion to proton transport of DCT1 resulting from a proton slippage is not a necessity of the transport mechanism in which positively charged protons are driving two positive charges of the metal ion in the same direction. It supports the idea that the proton slippage has a physiological advantage, and the proton slip was positively selected during the evolution of DCT1.     

pH effects on light dependence of the stoichiometry of proton influx and efflux to electron transport in spinach chloroplasts

Initial and steady state rates of proton transport at low light intensity have been measured and compared with steady state rates of electron transport in the pH range of 6.0–7.6 in envelope-free spinach chloroplasts. At pH 6–7, the H⁺/e^- values computed using the initial rate of proton transport varied with light intensity, from a value of 2 at low light to almost 5 at high light. In contrast, the H⁺/e^- values computed using the steady state rate of proton transport did not show a dependence on light intensity, having a constant value of 1.7±0.2. Likewise, at pH 7.6, the H⁺/e^- ratio, computed using either the initial or steady state rates of proton transport did not vary with light intensity but was constant at H⁺/e^-=1.7±0.1. Analysis of the light dependence of electron and proton transport allowed determination of (a) the quantam requirements of transport, (b) the rates of transport at light saturation, and (c) H⁺/e^- ratios for initial and steady state proton transport. Extrapolating the initial proton transport to zero light, we found that both H⁺/photon and H⁺/e^- values were not strongly dependent on pH, approaching a near constant value of 2.0. Using the initial rate of proton transport extrapolated to saturating light intensity we found the H⁺/e^- ratio to be strongly pH-dependent. We suggest that internal pH controls electron transport at high light intensities. The true stoichiometry is reflected only in measurements taken at low light using the initial proton transport data. Our findings and interpretation reconcile some conflicting data in the literature regarding the pH-dependence of the H⁺/e^- ratio and support the concept that internal pH controls noncyclic electron transport.Abbreviations Bicine N, N-bis [2-hydroxyethyl] glycine - HEPES N-2-hydroxy-ethylpiperazine-N-2-ethansulfonic acid - MES 2-(N-morpholino) ethanesulfonic acid     

The Thr505 and Ser557 residues of the AGT1-encoded alpha-glucoside transporter are critical for maltotriose transport in Saccharomyces cerevisiae

Aims:  The main objective of this study was to identify amino acid residues in the AGT1‐encoded α‐glucoside transporter (Agt1p) that are critical for efficient transport of maltotriose in the yeast Saccharomyces cerevisiae. Methods and Results:  The sequences of two AGT1‐encoded α‐glucoside transporters with different efficiencies of maltotriose transport in two Saccharomyces strains (WH310 and WH314) were compared. The sequence variations and discrepancies between these two proteins (Agt1p_WH310 and Agt1p_WH314) were investigated for potential effects on the functionality and maltotriose transport efficiency of these two AGT1‐encoded α‐glucoside transporters. A 23‐amino‐acid C‐terminal truncation proved not to be critical for maltotriose affinity. The identification of three amino acid differences, which potentially could have been instrumental in the transportation of maltotriose, were further investigated. Single mutations were created to restore the point mutations I505T, V549A and T557S one by one. The single site mutant V549A showed a decrease in maltotriose transport ability, and the I505T and T557S mutants showed complete reduction in maltotriose transport. Conclusions:  The amino acids Thr⁵⁰⁵ and Ser⁵⁵⁷, which are respectively located in the transmembrane (TM) segment TM¹¹ and on the intracellular segment after TM¹² of the AGT1‐encoded α‐glucoside transporters, are critical for efficient transport of maltotriose in S. cerevisiae. Significance and Impact of the Study:  Improved fermentation of starch and its dextrin products, such as maltotriose and maltose, would benefit the brewing and whisky industries. This study could facilitate the development of engineered maltotriose transporters adapted to starch‐efficient fermentation systems, and offers prospects for the development of yeast strains with improved maltose and maltotriose uptake capabilities that, in turn, could increase the overall fermentation efficiencies in the beer and whisky industries.     

The static head method for determining the charge stoichiometry of coupled transport systems Applications to the sodium-coupled d-glucose transporters of the renal proximal tubule

The static head method for determining the charge stoichiometry (the number of moles of charge translocated per mole of substrate) of a coupled transport system is presented. The method involves establishing experimental conditions under which a membrane potential exactly balances the thermodynamic driving force of a known substrate gradient. The charge stoichiometry can then be calculated from thermodynamic principles. In contrast to the usual steady-state method for determining charge stoichiometry in cell suspensions and vesicle preparations, the static head method is applicable to systems which are not capable of maintaining a constant membrane potential over time. The charge stoichiometries of two renal sodium coupled d-glucose transporters previously identified in brush-border membrane vesicle preparations from the outer cortex (early proximal tubule) and outer medulla (late proximal tubule) are determined. The charge stoichiometries of these transporters are in good agreement with their sodium/glucose coupling ratios arguing against the possibility that glucose transport is coupled to ions other than sodium in these membranes.     

Molecular Mechanism of Nramp-Family Transition Metal Transport

The Natural resistance-associated macrophage protein (Nramp) family of transition metal transporters enables uptake and trafficking of essential micronutrients that all organisms must acquire to survive. Two decades after Nramps were identified as proton-driven, voltage-dependent secondary transporters, multiple Nramp crystal structures have begun to illustrate the fine details of the transport process and provide a new framework for understanding a wealth of preexisting biochemical data. Here we review the relevant literature pertaining to Nramps’ biological roles and especially their conserved molecular mechanism, including our updated understanding of conformational change, metal binding and transport, substrate selectivity, proton transport, proton-metal coupling, and voltage dependence. We ultimately describe how the Nramp family has adapted the LeuT fold common to many secondary transporters to provide selective transition-metal transport with a mechanism that deviates from the canonical model of symport.     

Liposomes in Reconstitution of Ion-Pumps. Electrogenic Properties of the Na+,K+-Atpase and the Sarcoplasmic Ca2+-Atpase

Abstract

Any electrogenic ion-pump carrying a net-current during turnover is an electromotive device creating a transmembrane potential in tight vesicles, which can be detected by the potential sensitive fluorochrome oxonol VI. For the Na⁺,K⁺-ATPase the coupling ratio Na⁺:K⁺:ATP during physiological Na⁺:K⁺-exchange is 3:2:1, giving one positive net-charge translocated per ATP split. The same stoichiometry is found for the electrogenic Na⁺:Na⁺-exchange, whereas during uncoupled Na⁺-efflux this net-charge stoichiometry changes to three, in accordance with a transport stoichiometry 3:0:1. By inducing internal electrostatic potentials in the proteoliposome bilayer using the hydrophobic ions TPB or TPP⁺ it could be shown that the backreaction which normally translocates K⁺ changes from electroneutral to electrogenic during the uncoupled Na⁺-efflux where no ions are returned.

For Ca²⁺-transport a stoichiometry of close to, but lower than 2 Ca²⁺-ions per ATP split is found. Recent findings indicate that protons may be exchanged during this transport, but it was uncertain if this proton transport took place primarily on the Ca²⁺-pump, or was a secondary consequence of the established membrane pump-potential. Using the pH-sensitive fluorescent probe pyranine we have investigated these questions by measurements of generated proton gradients associated with Ca -pump turnover during conditions where the pump potential is short-circuited. From this it can be concluded that protons are countertransported during Ca²⁺-transport, but the stoichiometry apparently varies.     

Investigation of sugar binding kinetics of the E. coli sugar/H+ symporter XylE using solid-supported membrane-based electrophysiology

Bacterial transporters are difficult to study using conventional electrophysiology because of their low transport rates and the small size of bacterial cells. Here, we applied solid-supported membrane–based electrophysiology to derive kinetic parameters of sugar translocation by the Escherichia coli xylose permease (XylE), including functionally relevant mutants. Many aspects of the fucose permease (FucP) and lactose permease (LacY) have also been investigated, which allow for more comprehensive conclusions regarding the mechanism of sugar translocation by transporters of the major facilitator superfamily. In all three of these symporters, we observed sugar binding and transport in real time to determine K_M, V_max, K_D, and k_obs values for different sugar substrates. K_D and k_obs values were attainable because of a conserved sugar-induced electrogenic conformational transition within these transporters. We also analyzed interactions between the residues in the available X-ray sugar/H⁺ symporter structures obtained with different bound sugars. We found that different sugars induce different conformational states, possibly correlating with different charge displacements in the electrophysiological assay upon sugar binding. Finally, we found that mutations in XylE altered the kinetics of glucose binding and transport, as Q175 and L297 are necessary for uncoupling H⁺ and d-glucose translocation. Based on the rates for the electrogenic conformational transition upon sugar binding (>300 s⁻¹) and for sugar translocation (2 s⁻¹ − 30 s⁻¹ for different substrates), we propose a multiple-step mechanism and postulate an energy profile for sugar translocation. We also suggest a mechanism by which d-glucose can act as an inhibitor for XylE.     

Electrogenicity, pH-Dependence, and Stoichiometry of the Proton-Sucrose Symport

The electrogenicity, pH-dependence, and stoichiometry of the proton-sucrose symport were examined in plasma membrane vesicles isolated from sugar beet (Beta vulgaris L. cv Great Western) leaves. Symport mediated sucrose transport was electrogenic as demonstrated by the effect of membrane potential on ΔpH-dependent flux. In the absence of significant charge compensation, a low rate of sucrose transport was observed. When membrane potential was clamped at zero with symmetric potassium concentrations and valinomycin, the rate of sucrose flux was stimulated fourfold. In the presence of a negative membrane potential, transport increased six-fold. These results are consistent with electrogenic sucrose transport which results in a net flux of positive charge into the vesicles. The effect of membrane potential on the kinetics of sucrose transport was on V_max only with no apparent change in K_m. Sucrose transport rates driven by membrane potential only, i.e. in the absence of ΔpH, were comparable to ΔpH-driven flux. Both membrane potential and ΔpH-driven sucrose transport were used to examine proton binding to the symport and the apparent K_m for H⁺ was 0.7 micromolar. The kinetics of sucrose transport as a function of proton concentration exhibited a simple hyperbolic relationship. This observation is consistent with kinetic models of ion-cotransport systems when the stoichiometry of the system, ion:substrate, is 1:1. Quantitative measurements of proton and sucrose fluxes through the symport support a 1:1 stoichiometry. The biochemical details of protoncoupled sucrose transport reported here provide further evidence in support of the chemiosmotic hypothesis of nutrient transport across the plant cell plasma membrane.