Proton Transport Pathway in the ClC Cl/H Antiporter

A fundamental question concerning the ClC Cl⁻/H⁺ antiporters is the nature of their proton transport (PT) pathway. We addressed this issue by using a novel computational methodology capable of describing the explicit PT dynamics in the ClC-ec1 protein. The main result is that the Glu²⁰³ residue delivers a proton from the intracellular solution to the core of ClC-ec1 via a rotation of its side chain and subsequent acid dissociation. After reorientation of the Glu²⁰³ side chain, a transient water-mediated PT pathway between Glu²⁰³ and Glu¹⁴⁸ is established that is able to receive and translocate the proton via Grotthuss shuttling after deprotonation of Glu²⁰³. A molecular-dynamics simulation of an explicit hydrated excess proton in this pathway suggests that a negatively charged Glu¹⁴⁸ and the central Cl⁻ ion act together to drive H⁺ to the extracellular side of the membrane. This finding is consistent with the experimental result that Cl⁻ binding to the central site facilitates the proton movement. A calculation of the PT free-energy barrier for the ClC-ec1 E203V mutant also supports the proposal that a dissociable residue is required at this position for efficient delivery of H⁺ to the protein interior, in agreement with recent experimental results.     

Multiscale Simulations Reveal Key Aspects of the Proton Transport Mechanism in the ClC-ec1 Antiporter

Multiscale reactive molecular dynamics simulations are used to study proton transport through the central region of ClC-ec1, a widely studied ClC transporter that enables the stoichiometric exchange of 2 Cl^– ions for 1 proton (H⁺). It has long been known that both Cl^– and proton transport occur through partially congruent pathways, and that their exchange is strictly coupled. However, the nature of this coupling and the mechanism of antiporting remain topics of debate. Here multiscale simulations have been used to characterize proton transport between E203 (Glu_in) and E148 (Glu_ex), the internal and external intermediate proton binding sites, respectively. Free energy profiles are presented, explicitly accounting for the binding of Cl^– along the central pathway, the dynamically coupled hydration changes of the central region, and conformational changes of Glu_in and Glu_ex. We find that proton transport between Glu_in and Glu_ex is possible in both the presence and absence of Cl^– in the central binding site, although it is facilitated by the anion presence. These results support the notion that the requisite coupling between Cl^– and proton transport occurs elsewhere (e.g., during proton uptake or release). In addition, proton transport is explored in the E203K mutant, which maintains proton permeation despite the substitution of a basic residue for Glu_in. This collection of calculations provides for the first time, to our knowledge, a detailed picture of the proton transport mechanism in the central region of ClC-ec1 at a molecular level.     

Intracellular Proton-Transfer Mutants in a CLC Cl?/H+ Exchanger

CLC-ec1, a bacterial homologue of the CLC family’s transporter subclass, catalyzes transmembrane exchange of Cl⁻ and H⁺. Mutational analysis based on the known structure reveals several key residues required for coupling H⁺ to the stoichiometric countermovement of Cl⁻. E148 (Glu_ex) transfers protons between extracellular water and the protein interior, and E203 (Glu_in) is thought to function analogously on the intracellular face of the protein. Mutation of either residue eliminates H⁺ transport while preserving Cl⁻ transport. We tested the role of Glu_in by examining structural and functional properties of mutants at this position. Certain dissociable side chains (E, D, H, K, R, but not C and Y) retain H⁺/Cl⁻ exchanger activity to varying degrees, while other mutations (V, I, or C) abolish H⁺ coupling and severely inhibit Cl⁻ flux. Transporters substituted with other nonprotonatable side chains (Q, S, and A) show highly impaired H⁺ transport with substantial Cl⁻ transport. Influence on H⁺ transport of side chain length and acidity was assessed using a single-cysteine mutant to introduce non-natural side chains. Crystal structures of both coupled (E203H) and uncoupled (E203V) mutants are similar to wild type. The results support the idea that Glu_in is the internal proton-transfer residue that delivers protons from intracellular solution to the protein interior, where they couple to Cl⁻ movements to bring about Cl⁻/H⁺ exchange.     

Intracellular Proton Access in a Cl?/H+ Antiporter

Chloride-transporting membrane proteins of the CLC family appear in two distinct mechanistic flavors: H⁺-gated Cl⁻ channels and Cl⁻/H⁺ antiporters. Transmembrane H⁺ movement is an essential feature of both types of CLC. X-ray crystal structures of CLC antiporters show the Cl⁻ ion pathway through these proteins, but the H⁺ pathway is known only inferentially by two conserved glutamate residues that act as way-stations for H⁺ in its path through the protein. The extracellular-facing H⁺ transfer glutamate becomes directly exposed to aqueous solution during the transport cycle, but the intracellular glutamate E203, Glu_in, is buried within the protein. Two regions, denoted “polar” and “interfacial,” at the intracellular surface of the bacterial antiporter CLC-ec1 are examined here as possible pathways by which intracellular aqueous protons gain access to Glu_in. Mutations at multiple residues of the polar region have little effect on antiport rates. In contrast, mutation of E202, a conserved glutamate at the protein–water boundary of the interfacial region, leads to severe slowing of the Cl⁻/H⁺ antiport rate. An X-ray crystal structure of E202Y, the most strongly inhibited of these substitutions, shows an aqueous portal leading to Glu_in physically blocked by cross-subunit interactions; moreover, this mutation has only minimal effect on a monomeric CLC variant, which necessarily lacks such interactions. The several lines of experiments presented argue that E202 acts as a water-organizer that creates a proton conduit connecting intracellular solvent with Glu_in.     

Antiport Mechanism for Cl/H in ClC-ec1 from Normal-Mode Analysis

ClC chloride channels and transporters play major roles in cellular excitability, epithelial salt transport, volume, pH, and blood pressure regulation. One family member, ClC-ec1 from Escherichia coli, has been structurally resolved crystallographically and subjected to intensive mutagenetic, crystallographic, and electrophysiological studies. It functions as a Cl⁻/H⁺ antiporter, not a Cl⁻ channel; however, the molecular mechanism for Cl⁻/H⁺ exchange is largely unknown. Using all-atom normal-mode analysis to explore possible mechanisms for this antiport, we propose that Cl⁻/H⁺ exchange involves a conformational cycle of alternating exposure of Cl⁻ and H⁺ binding sites of both ClC pores to the two sides of the membrane. Both pores switch simultaneously from facing outward to facing inward, reminiscent of the standard alternating-access mechanism, which may have direct implications for eukaryotic Cl⁻/H⁺ transporters and Cl⁻ channels.     

Surprises from an Unusual CLC Homolog

The chloride channel (CLC) family is distinctive in that some members are Cl⁻ ion channels and others are Cl⁻/H⁺ antiporters. The molecular mechanism that couples H⁺ and Cl⁻ transport in the antiporters remains unknown. Our characterization of a novel bacterial homolog from Citrobacter koseri, CLC-ck2, has yielded surprising discoveries about the requirements for both Cl⁻ and H⁺ transport in CLC proteins. First, even though CLC-ck2 lacks conserved amino acids near the Cl⁻-binding sites that are part of the CLC selectivity signature sequence, this protein catalyzes Cl⁻ transport, albeit slowly. Ion selectivity in CLC-ck2 is similar to that in CLC-ec1, except that SO₄²⁻ strongly competes with Cl⁻ uptake through CLC-ck2 but has no effect on CLC-ec1. Second, and even more surprisingly, CLC-ck2 is a Cl⁻/H⁺ antiporter, even though it contains an isoleucine at the Glu_in position that was previously thought to be a critical part of the H⁺ pathway. CLC-ck2 is the first known antiporter that contains a nonpolar residue at this position. Introduction of a glutamate at the Glu_in site in CLC-ck2 does not increase H⁺ flux. Like other CLC antiporters, mutation of the external glutamate gate (Glu_ex) in CLC-ck2 prevents H⁺ flux. Hence, Glu_ex, but not Glu_in, is critical for H⁺ permeation in CLC proteins.The chloride channel (CLC) family includes both Cl⁻ ion channels and Cl⁻/H⁺ antiporters (1). The ion channels allow Cl⁻ to diffuse passively down an electrochemical gradient, and antiporters couple the movement of chloride and protons in opposite directions across cellular membranes. So far, the only known CLC structures are those of antiporters (2–4). On the basis of sequence similarity and functional studies, it is thought that the basic structures of the ion channels and antiporters are similar, and that slight structural differences account for these diverse functions. Understanding how the CLC family has evolved to allow proteins of similar structure to carry out two distinct mechanisms remains a critical goal.In the Escherichia coli antiporter CLC-ec1, two glutamates, Glu_ex (E148) and Glu_in (E203), are absolutely required for H⁺ transport (5,6). Glu_ex is conserved in both CLC ion channels and antiporters. Glu_in is conserved only in antiporters and is instead a hydrophobic valine in all of the known ion channels. Hence, it was proposed that both Glu_in and Glu_ex are necessary to transfer protons through CLC antiporters (6). Studies of the CLC-4 and CLC-5 antiporters supported the notion that Glu_in and Glu_ex play critical roles in H⁺ transport (7,8). Surprisingly, however, recent experiments revealed that although the red algae homolog CmCLC contains a threonine at the Glu_in position, it is still Cl⁻/H⁺ antiporter (3). It is unknown whether this threonine has a shifted pKa that allows it to transfer protons or whether the H⁺ transport in CmCLC does not require a protonatable residue at this position. Further blurring the role of Glu_in, the CLC-0 ion channel, which contains a valine at the Glu_in position, requires slow transmembrane H⁺ transport for channel gating (9).To probe the molecular requirements for Cl⁻ and H⁺ transport in CLC proteins, we characterized a novel homolog from Citrobacter koseri called CLC-ck2. CLC-ck2 is 21% identical and 37% similar in amino acid sequence to CLC-ec1. CLC-ck2 contains an isoleucine at the Glu_in position, and hence we originally hypothesized that this protein would act as an ion channel. Additionally, CLC-ck2 lacks several amino acids that coordinate the central and internal Cl⁻-binding sites in CLC-ec1, most notably the GSGIP motif (Fig. S1 in the Supporting Material). With genomic information now revealing >1000 putative CLC homologs, we find that CLC-ck2 is not unique—several other uncharacterized homologs also lack these regions. To our knowledge, ours is the first study to characterize the function of a homolog missing these regions.Using Cl⁻ flux assays, we first sought to determine whether CLC-ck2 could catalyze Cl⁻ transport (10). With CLC-ck2-containing vesicles, slow but significant Cl⁻ efflux was observed upon addition of valinomycin (Vln; Fig. 1 A, blue trace). In control vesicles lacking CLC-ck2, no significant Cl⁻ flux was observed (Fig. 1 A, black). The CLC-ec1 inhibitor 4,4′-octanamidostilbene-2,2′-disulfonate (OADS) (11) completely inhibited Cl⁻ flux (Fig. 1 A, green). The Cl⁻ unitary turnover rate for wild-type CLC-ck2 was 31 ± 5 s⁻¹ (mean ± SE, n = 5). This rate is ∼2 orders of magnitude less than the Cl⁻ flux through the CLC-ec1 antiporter, and is much slower transport than expected for an ion channel. However, it is a similar to the rate catalyzed by the cyanobacterium antiporter CLC-sy1 (4).Open in a separate windowFigure 1(A) Representative Cl⁻ flux assays. Cl⁻ efflux was initiated by addition of Vln. Triton X-100 was added to disrupt liposomes and release all intracellular Cl⁻. The insert shows an expanded view of the efflux immediately after addition of Vln. (B) Representative H⁺ flux assays demonstrate Cl⁻-driven H⁺ influx. H⁺ flux was initiated by the addition of Vln. The H⁺ gradient was collapsed at the end by the addition of FCCP.To test whether CLC-ck2 is a Cl⁻ ion channel or Cl⁻/H⁺ antiporter, we performed H⁺ flux assays as previously described (10). If vesicles contain a Cl⁻/H⁺ antiporter, the efflux of Cl⁻ upon addition of Vln will drive the movement of protons into the vesicles against their concentration gradient. If vesicles contain a Cl⁻ ion channel, however, no movement of protons will be observed upon addition of Vln. We found that CLC-ck2 showed significant Cl⁻-driven H⁺ uptake. Fig. 1 B illustrates uphill movement of protons in the presence of a Cl⁻ gradient. H⁺ influx, like Cl⁻ efflux, was inhibited by the presence of OADS. These assays are not quantitative enough to determine Cl⁻/H⁺ stoichiometry. However, they qualitatively demonstrate that CLC-ck2 acts as a Cl⁻/H⁺ antiporter even though it lacks Glu_in.If Glu_in is important for maximizing H⁺ flux, we would expect that introducing a glutamate at the Glu_in position would increase the H⁺ flux observed through CLC-ck2. However, we found that the I175E mutation did not significantly alter H⁺ or Cl⁻ flux (Fig. 2). Hence, Glu_in does not enhance H⁺ transport through CLC-ck2.Open in a separate windowFigure 2Unitary turnover rates, calculated from initial velocities after addition of Vln in (A) Cl⁻ and (B) H⁺ flux assays. Reconstitutions contained 5–38 μg protein/mg lipid. Bars represent the mean ± SE for three to 17 assays.The external glutamate gate Glu_ex is conserved and required for H⁺ transport in all known CLC antiporters (5,7). To determine whether Glu_ex is also essential for H⁺ transport in CLC-ck2, we made the E122Q mutation. This mutant can still transport chloride but fails to move protons (Fig. 2). This mutant protein was not very stable in micelles, precipitating over the course of hours, and thus the unitary turnover rates shown in Fig. 2 represent lower limits. Nevertheless, this result is consistent with observations in other CLC antiporters and suggests that Glu_ex is important for H⁺ transport in all CLC antiporters.Because CLC-ck2 lacks amino acids that coordinate the Cl⁻ ions in the structure of CLC-ec1, we wondered whether the ion selectivity might differ. Indeed, the plant atCLC-a homolog has a single change in this region that makes it selective for NO₃⁻ over Cl⁻ (12). To determine the ion selectivity of CLC-ck2, we used radioactive uptake assays (11). In these assays, the amount of ³⁶Cl⁻ exchanged into CLC-ck2-containing vesicles loaded with cold Cl⁻ is measured as a function of time. Various anions were added to the extravesicular solution to test which ions were transported in preference to the ³⁶Cl⁻. A decrease in radioactive uptake indicates that the anion is permeant and/or blocks CLC-ck2. Fig. 3 A plots the amount of ³⁶Cl⁻ uptake with each of the various ions added; the ion selectivity (or block) was SO₄²⁻ ≫ Cl⁻ > NO₃⁻ > SCN⁻ >Br⁻ > F⁻ > P_i⁻ ≈ I⁻ ≫ isethionate⁻. This selectivity is similar to that of CLC-ec1 (13), with one noticeable exception: SO₄²⁻. SO₄²⁻ had no effect on CLC-ec1, but strongly competed with Cl⁻ uptake through CLC-ck2 (Fig. 3 B). Hence, the selectivity filter of CLC-ck2 is similar enough to other CLCs to transport Cl⁻, NO₃⁻, and Br⁻ as expected. However, further investigation is required to determine the structural differences that must underlie the distinct disparity in SO₄²⁻ permeability and/or block.Open in a separate windowFigure 3Ion selectivity of CLC-ck2. (A) Liposomes reconstituted with CLC-ck2 were screened for selectivity against various test ions in the presence of 1 mM ³⁶Cl⁻ at pH 4.5. All test ions were present at 10 mM, except for isethionate, which was present at 20 mM to confirm that it is inert. After 10 min, the radioactivity counts were measured to determine total ³⁶Cl⁻ uptake (for isethionate, uptake was stopped after 20 min). Counts were normalized with respect to liposome uptake in the absence of an external test ion. Bars represent the mean ± SE for three assays. (B) Comparison of effects of external sulfate on CLC-ec1 and CLC-ck2 on radioactive update assays, normalized as in part A.This study reveals that Glu_in is not essential for Cl⁻- coupled H⁺ transport in CLC-ck2, in direct contrast to the previous conclusion that the protonatable side chain of the glutamate is directly involved in the H⁺ transport pathway (14). Thus, our result brings into question the location of the H⁺ permeation pathway. The protons must be transferred via other protonatable residues or water molecules. The residue adjacent to Glu_in (E202 in CLC-ec1) is conserved in CLC-ck2. Unfortunately, mutation of this glutamate (E174F) in CLC-ck2 resulted in unstable protein that could not be characterized in functional studies. Using the structure of CLC-ec1 as a guide, we see no other obvious protonatable residues in CLC-ck2 available to transfer protons from the intracellular side to Glu_ex. One possibility is that H⁺ transport may require a water wire. The idea of a water wire is not new. In CLC-ec1, there is an ∼15 Å gap between Glu_in and Glu_ex, and it has never been clear exactly how protons cross this gap. Recent molecular-dynamics studies have supported the idea that the Glu_in in CLC-ec1 may help to position water molecules for a water wire to transfer protons to the extracellular glutamate (15). If indeed the role of Glu_in is simply to position water molecules properly to transfer protons, subtle changes in other parts of the structure could allow this water wire to exist in the absence of Glu_in. This could also explain how the eukaryotic CmCLC homolog, which has a threonine at the Glu_in position, is able to act as a coupled transporter as well. We have not yet been able to determine the structure of CLC-ck2 to understand how the lack of conserved amino acids near the Cl⁻-binding sites affects the structure. This study will inspire future work to investigate the molecular mechanism of CLC-ck2 and CLC-ck2 homologs in greater detail.     

Structure of the Membrane Domain of Human Erythrocyte Anion Exchanger 1 Revealed by Electron Crystallography

The membrane domain of human erythrocyte anion exchanger 1 (AE1) works as a Cl⁻/HCO₃⁻ antiporter. This exchange is a key step for CO₂/O₂ circulation in the blood. In spite of their importance, structural information about AE1 and the AE (anion exchanger) family are still very limited. We used electron microscopy to solve the three-dimensional structure of the AE1 membrane domain, fixed in an outward-open conformation by cross-linking, at 7.5-Å resolution. A dimer of AE1 membrane domains packed in two-dimensional array showed a projection map similar to that of the prokaryotic homolog of the ClC chloride channel, a Cl⁻/H⁺ antiporter. In a three-dimensional map, there are V-shaped densities near the center of the dimer and slightly narrower V-shaped clusters at a greater distance from the center of the dimer. These appear to be inserted into the membrane from opposite sides. The structural motifs, two homologous pairs of helices in internal repeats of the ClC transporter (helices B + C and J + K), are well fitted to those AE1 densities after simple domain movement.     

Anion- and proton-dependent gating of ClC-4 anion/proton transporter under uncoupling conditions

ClC-4 is a secondary active transporter that exchanges Cl⁻ ions and H⁺ with a 2:1 stoichiometry. In external SCN⁻, ClC-4 becomes uncoupled and transports anions with high unitary transport rate. Upon voltage steps, the number of active transporters varies in a time-dependent manner, resembling voltage-dependent gating of ion channels. We here investigated modification of the voltage dependence of uncoupled ClC-4 by protons and anions to quantify association of substrates with the transporter. External acidification shifts voltage dependence of ClC-4 transport to more positive potentials and leads to reduced transport currents. Internal pH changes had less pronounced effects. Uncoupled ClC-4 transport is facilitated by elevated external [SCN⁻] but impaired by internal Cl⁻ and I⁻. Block by internal anions indicates the existence of an internal anion-binding site with high affinity that is not present in ClC channels. The voltage dependence of ClC-4 coupled transport is modulated by external protons and internal Cl⁻ in a manner similar to what is observed under uncoupling conditions. Our data illustrate functional differences but also similarities between ClC channels and transporters.     

The Late Endosomal ClC-6 Mediates Proton/Chloride Countertransport in Heterologous Plasma Membrane Expression

Members of the CLC protein family of Cl⁻ channels and transporters display the remarkable ability to function as either chloride channels or Cl⁻/H⁺ antiporters. Due to the intracellular localization of ClC-6 and ClC-7, it has not yet been possible to study the biophysical properties of these members of the late endosomal/lysosomal CLC branch in heterologous expression. Whereas recent data suggest that ClC-7 functions as an antiporter, transport characteristics of ClC-6 have remained entirely unknown. Here, we report that fusing the green fluorescent protein (GFP) to the N terminus of ClC-6 increased its cell surface expression, allowing us to functionally characterize ClC-6. Compatible with ClC-6 mediating Cl⁻/H⁺ exchange, Xenopus oocytes expressing GFP-tagged ClC-6 alkalinized upon depolarization. This alkalinization was dependent on the presence of extracellular anions and could occur against an electrochemical proton gradient. As observed in other CLC exchangers, ClC-6-mediated H⁺ transport was abolished by mutations in either the “gating” or “proton” glutamate. Overexpression of GFP-tagged ClC-6 in CHO cells elicited small, outwardly rectifying currents with a Cl⁻ > I⁻ conductance sequence. Mutating the gating glutamate of ClC-6 yielded an ohmic anion conductance that was increased by additionally mutating the “anion-coordinating” tyrosine. Additionally changing the chloride-coordinating serine 157 to proline increased the NO₃⁻ conductance of this mutant. Taken together, these data demonstrate for the first time that ClC-6 is a Cl⁻/H⁺ antiporter.     

A model of lysosomal pH regulation

Lysosomes must maintain an acidic luminal pH to activate hydrolytic enzymes and degrade internalized macromolecules. Acidification requires the vacuolar-type H⁺-ATPase to pump protons into the lumen and a counterion flux to neutralize the membrane potential created by proton accumulation. Early experiments suggested that the counterion was chloride, and more recently a pathway consistent with the ClC-7 Cl^–/H⁺ antiporter was identified. However, reports that the steady-state luminal pH is unaffected in ClC-7 knockout mice raise questions regarding the identity of the carrier and the counterion. Here, we measure the current–voltage characteristics of a mammalian ClC-7 antiporter, and we use its transport properties, together with other key ion regulating elements, to construct a mathematical model of lysosomal pH regulation. We show that results of in vitro lysosome experiments can only be explained by the presence of ClC-7, and that ClC-7 promotes greater acidification than Cl^–, K⁺, or Na⁺ channels. Our models predict strikingly different lysosomal K⁺ dynamics depending on the major counterion pathways. However, given the lack of experimental data concerning acidification in vivo, the model cannot definitively rule out any given mechanism, but the model does provide concrete predictions for additional experiments that would clarify the identity of the counterion and its carrier.     

A solid-supported membrane electrophysiology assay for efficient characterization of ion-coupled transport

Transport stoichiometry determination can provide great insight into the mechanism and function of ion-coupled transporters. Traditional reversal potential assays are a reliable, general method for determining the transport stoichiometry of ion-coupled transporters, but the time and material costs of this technique hinder investigations of transporter behavior under multiple experimental conditions. Solid-supported membrane electrophysiology (SSME) allows multiple recordings of liposomal or membrane samples adsorbed onto a sensor and is sensitive enough to detect transport currents from moderate-flux transporters that are inaccessible to traditional electrophysiology techniques. Here, we use SSME to develop a new method for measuring transport stoichiometry with greatly improved throughput. Using this technique, we were able to verify the recent report of a fixed 2:1 stoichiometry for the proton:guanidinium antiporter Gdx, reproduce the 1H⁺:2Cl⁻ antiport stoichiometry of CLC-ec1, and confirm loose proton:nitrate coupling for CLC-ec1. Furthermore, we were able to demonstrate quantitative exchange of internal contents of liposomes adsorbed onto SSME sensors to allow multiple experimental conditions to be tested on a single sample. Our SSME method provides a fast, easy, general method for measuring transport stoichiometry, which will facilitate future mechanistic and functional studies of ion-coupled transporters.     

A Mathematical Model of Action Potential in Cells of Vascular Plants

A mathematical model of action potential (AP) in vascular plants cells has been worked out. The model takes into account actions of plasmalemma ion transport systems (K⁺, Cl^? and Ca²⁺ channels; H⁺- and Ca²⁺-ATPases; 2H⁺/Cl^? symporter; and H⁺/K⁺ antiporter), changes of ion concentrations in the cell and in the extracellular space, cytoplasmic and apoplastic buffer capacities and the temperature dependence of active transport systems. The model of AP simulates a stationary level of the membrane potential and ion concentrations, generation of AP induced by electrical stimulation and gradual cooling and the impact of external Ca²⁺ for AP development. The model supports a hypothesis about participation of H⁺-ATPase in AP generation.     

Protonation of Glu135 Facilitates the Outward-to-Inward Structural Transition of Fucose Transporter

Major facilitator superfamily (MFS) transporters typically need to alternatingly sample the outward-facing and inward-facing conformations, in order to transport the substrate across membrane. To understand the mechanism, in this work, we focused on one MFS member, the L-fucose/H⁺ symporter (FucP), whose crystal structure exhibits an outward-open conformation. Previous experiments imply several residues critical to the substrate/proton binding and structural transition of FucP, among which Glu¹³⁵, located in the periplasm-accessible vestibule, is supposed as being involved in both proton translocation and conformational change of the protein. Here, the structural transition of FucP in presence of substrate was investigated using molecular-dynamics simulations. By combining the equilibrium and accelerated simulations as well as thermodynamic calculations, not only was the large-scale conformational change from the outward-facing to inward-facing state directly observed, but also the free energy change during the structural transition was calculated. The simulations confirm the critical role of Glu¹³⁵, whose protonation facilitates the outward-to-inward structural transition both by energetically favoring the inward-facing conformation in thermodynamics and by reducing the free energy barrier along the reaction pathway in kinetics. Our results may help the mechanistic studies of both FucP and other MFS transporters.     

Asp and Thr are critical for cation exchange mediated by NhaD, Na/H antiporter of Vibrio cholerae

The Vc-NhaD is an Na⁺/H⁺ antiporter from Vibrio cholerae belonging to a new family of bacterial Na⁺/H⁺ antiporters, the NhaD family. In the present work we mutagenized five conserved Asp and Glu residues and one conserved Thr residue to Ala in order to identify amino acids that are critical for the antiport activity. All mutations fall into two distinct groups: (i) four variants, Glu¹⁰⁰Ala, Glu²⁵¹Ala, Glu³⁴²Ala, and Asp³⁹³Ala, did not abolish antiport activity but shifted the pH optimum to more alkaline pH, and (ii) variants Asp³⁴⁴Ala, Asp³⁴⁴Asn, and Thr³⁴⁵Ala caused a complete loss of both Na⁺/H⁺ and Li⁺/H⁺ antiport activity whereas the Asp³⁴⁴Glu variant exhibited reduced Na⁺/H⁺ and Li⁺/H⁺ antiport activity. This is the first mutational analysis of the antiporter of NhaD type and the first demonstration of Thr residue being indispensable for Na⁺/H⁺ antiport. We discuss the possible role of Asp³⁴⁴ and Thr³⁴⁵ in the functioning of Vc-NhaD.     

Plasma membrane K+/H+-ATPase From leishmania donovani

Leishmania donovani has an active ^K+/H⁺ exchange system on the surface membrane. Modulation of external K⁺ concentration resulted in a corresponding change in internal pH (pH_i) suggesting a link between proton and potassium transport. Although a Na⁺/H⁺ antiporter is present on the plasma membrane, its sensitivity to amiloride suggests that it operates independent of K⁺/H⁺ exchange. Reduction of cellular ATP with NaN₃ and KCN inhibits K⁺/H⁺ exchange showing thereby that the process is energy dependent. The K⁺/H⁺ exchange is sensitive to inhibitors of the gastric K⁺/H⁺-ATPase. It is concluded that the H⁺-ATPase previously reported on the plasma membrane of L. donovani is in fact a K⁺/H⁺-ATPase. © 1994 wiley-Liss, Inc.     

Comparative analysis of cation/proton antiporter superfamily in plants

The cation/proton antiporter superfamily is associated with the transport of monovalent cations across membranes. This superfamily was annotated in the Arabidopsis genome and some members were functionally characterized. In the present study, a systematic analysis of the cation/proton antiporter genes in diverse plant species was reported. We identified 240 cation/proton antiporters in alga, moss, and angiosperm. A phylogenetic tree was constructed showing these 240 members are separated into three families, i.e., Na⁺/H⁺ exchangers, K⁺ efflux antiporters, and cation/H⁺ exchangers. Our analysis revealed that tandem and/or segmental duplications contribute to the expansion of cation/H⁺ exchangers in the examined angiosperm species. Sliding window analysis of the nonsynonymous/synonymous substitution ratios showed some differences in the evolutionary fate of cation/proton antiporter paralogs. Furthermore, we identified over-represented motifs among these 240 proteins and found most motifs are family specific, demonstrating diverse evolution of the cation/proton antiporters among three families. In addition, we investigated the co-expressed genes of the cation/proton antiporters in Arabidopsis thaliana. The results showed some biological processes are enriched in the co-expressed genes, suggesting the cation/proton antiporters may be involved in these biological processes. Taken together, this study furthers our knowledge on cation/proton antiporters in plants.     

Mechanisms of passive potassium influx in corn mitochondria

Corn mitochondria in 100 millimolar KCl show accelerated passive swelling upon addition of uncoupler. This unusual response has been compared with swelling produced by valinomycin, tripropyltin, and nigericin. It is concluded that the driving force for swelling lies with the chloride gradient and a high P_Cl:P_K ratio, the chloride influx creating a negative membrane potential. The action of uncoupler is to facilitate K⁺ influx via the endogenous H⁺/K⁺ antiporter. The antiporter is active over the pH range 6 to 8, is not sensitive to Mg²⁺ concentration, and is not inactivated by aging. It is not clear why corn mitochondria show this exceptional activity of the H⁺/K⁺ antiporter in K⁺ influx. It is speculated that during isolation the antiporter may be exposed or activated, and that it contributes to cyclic K⁺ transport and high State 4 respiration rates.     

Enhanced V-ATPase activity contributes to the improved salt tolerance of transgenic tobacco plants overexpressing vacuolar Na(+)/H (+) antiporter AtNHX1

AtNHX1, a vacuolar Na⁺/H⁺ antiporter gene from Arabidopsis thaliana, was introduced into tobacco genome via Agrobacterium tumefaciens-mediated transformation to evaluate the role of vacuolar energy providers in plants salt stress response. Compared to the wild-type plants, over-expression of AtNHX1 increased salt tolerance in the transgenic tobacco plants, allowing higher germination rates of seeds and successful seedling establishment in the presence of toxic concentrations of NaCl. More importantly, the induced Na⁺/H⁺ exchange activity in the transgenic plants was closely correlated to the enhanced activity of vacuolar H⁺-ATPase (V-ATPase) when exposed to 200 mM NaCl. In addition, inhibition of V-ATPase activity led to the malfunction of Na⁺/H⁺ exchange activity, placing V-ATPase as the dominant energy provider for the vacuolar Na⁺/H⁺ antiporter AtNHX1. V-ATPase and vacuolar Na⁺/H⁺ antiporter thus function in an additive or synergistic way. Simultaneous overexpression of V-ATPase and vacuolar Na⁺/H⁺ antiporter might be appropriate for producing plants with a higher salt tolerance ability.