Photochemical study of a cyanobacterial chloride-ion pumping rhodopsin

Mastigocladopsis repens halorhodopsin (MrHR) is a Cl^?-pumping rhodopsin that belongs to a distinct cluster far from other Cl^? pumps. We investigated its pumping function by analyzing its photocycle and the effect of amino acid replacements. MrHR can bind I^? similar to Cl^? but cannot transport it. I^?-bound MrHR undergoes a photocycle but lacks the intermediates after L, suggesting that, in the Cl^?-pumping photocycle, Cl^? moves to the cytoplasmic (CP) channel during L decay. A photocycle similar to that of the I^?-bound form was also observed for a mutant of the Asp200 residue, which is superconserved and assumed to be deprotonated in most microbial rhodopsins. This residue is probably close to the Cl^?-binding site and the protonated Schiff base, in which a chromophore retinal binds to a specific Lys residue. However, the D200N mutation affected neither the Cl^?-binding affinity nor the absorption spectrum, but completely eliminated the Cl^?-pumping function. Thus, the Asp200 residue probably protonates in the dark state but deprotonates during the photocycle. Indeed, a H⁺ release was detected for photolyzed MrHR by using an indium?tin oxide electrode, which acts as a good time-resolved pH sensor. This H⁺ release disappeared in the I^?-bound form of the wild-type and Cl^?-bound form of the D200N mutant. Thus, Asp200 residue probably deprotonates during L decay and then drives the Cl^? movement to the CP channel.     

Anion-Sensitive, H-Pumping ATPase of Oat Roots : Direct Effects of Cl, NO(3), and a Disulfonic Stilbene

To understand the mechanism and molecular properties of the tonoplast-type H⁺-translocating ATPase, we have studied the effect of Cl⁻, NO₃⁻, and 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid (DIDS) on the activity of the electrogenic H⁺-ATPase associated with low-density microsomal vesicles from oat roots (Avena sativa cv Lang). The H⁺-pumping ATPase generates a membrane potential (Δψ) and a pH gradient (ΔpH) that make up two interconvertible components of the proton electrochemical gradient (μh⁺). A permeant anion (e.g. Cl⁻), unlike an impermeant anion (e.g. iminodiacetate), dissipated the membrane potential ([¹⁴C]thiocyanate distribution) and stimulated formation of a pH gradient ([¹⁴C]methylamine distribution). However, Cl⁻-stimulated ATPase activity was about 75% caused by a direct stimulation of the ATPase by Cl⁻ independent of the proton electrochemical gradient. Unlike the plasma membrane H⁺-ATPase, the Cl⁻-stimulated ATPase was inhibited by NO₃⁻ (a permeant anion) and by DIDS. In the absence of Cl⁻, NO₃⁻ decreased membrane potential formation and did not stimulate pH gradient formation. The inhibition by NO₃⁻ of Cl⁻-stimulated pH gradient formation and Cl⁻-stimulated ATPase activity was noncompetitive. In the absence of Cl⁻, DIDS inhibited the basal Mg,ATPase activity and membrane potential formation. DIDS also inhibited the Cl⁻-stimulated ATPase activity and pH gradient formation. Direct inhibition of the electrogenic H⁺-ATPase by NO₃⁻ or DIDS suggest that the vanadate-insensitive H⁺-pumping ATPase has anion-sensitive site(s) that regulate the catalytic and vectorial activity. Whether the anion-sensitive H⁺-ATPase has channels that conduct anions is yet to be established.     

Potential-dependent anion transport in tonoplast vesicles from oat roots

Potential-dependent anion movement into tonoplast vesicles from oat roots (Avena sativa L. var Lang) was monitored as dissipation of membrane potentials (Δψ) using the fluorescence probe Oxonol V. The potentials (positive inside) were generated with the H⁺-pumping pyrophosphatase, which is K⁺ stimulated and anion insensitive. The relative rate of ΔΨ dissipation by anions was used to estimate the relative permeabilities of the anions. In decreasing order they were: SCN⁻ (100) > NO₃⁻ (72) = Cl⁻ (70) > Br⁻ (62) > SO₄²⁻ (5) = H₂PO₄⁻ (5) > malate (3) = acetate (3) > iminodiacetate (2). Kinetic studies showed that the rate of Δψ dissipation by Cl⁻ and NO₃⁻, but not by SCN⁻, was saturable. The K_m values for Cl⁻ and NO₃⁻ uptake were about 2.3 and 5 millimolar, respectively, suggesting these anions move into the vacuole through proteinaceous porters. In contrast to a H⁺-coupled Cl⁻ transporter on the same vesicles, the potential-dependent Cl⁻ transport was insensitive to 4,4′-diisothiocyano-2,2′-stilbene disulfonate. These results suggest the existence of at least two different mechanisms for Cl⁻ transport in these vesicles. The potentials generated by the H⁺-translocating ATPase and H⁺-pyrophosphatase were nonadditive, giving support to the model that both pumps are on tonoplast vesicles. No evidence for a putative Cl⁻ conductance on the anion-sensitive H⁺-ATPase was found.     

Decrease of pH Gradients in Tonoplast Vesicles by NO(3) and Cl: Evidence for H-Coupled Anion Transport

Chloride or nitrate decreased a pH gradient (measured as [¹⁴C]methylamine accumulation) in tonoplast-enriched vesicles. The ΔpH decrease was dependent on the anion concentration. These effects are independent of the anion-sensitive H⁺-ATPase of the tonoplast, since the pH gradient (acid inside) was imposed artificially using a pH jump or a K⁺ gradient and nigericin. 4,4′-Diisothiocyano-2,2′-stilbene disulfonic acid partially prevented the decrease in pH gradient induced by Cl⁻. Two possible models to account for this anion-dependent decrease of ΔpH are: (a) H⁺ loss is accompanied by Cl⁻ or NO₃⁻ efflux from the vesicles via H⁺/anion symport systems on the tonoplast and (b) H⁺ loss is accompanied by Cl⁻ or NO₃⁻ uptake into the vesicles via H⁺/anion antiport systems. Depending on the requirements and conditions of the cell, these two systems would serve to either mobilize Cl⁻ and NO₃⁻ stored in the vacuole for use in the cytoplasm or to drive anions into the vacuole. Chloride or nitrate also decreased a pH gradient in fractions containing plasma membrane and Golgi, implying that these membranes may have similar H⁺-coupled anion transport systems.     

An Acid-loading Chloride Transport Pathway in the Intraerythrocytic Malaria Parasite,Plasmodium falciparum

The intraerythrocytic malaria parasite exerts tight control over its ionic composition. In this study, a combination of fluorescent ion indicators and ³⁶Cl⁻ flux measurements was used to investigate the transport of Cl⁻ and the Cl⁻-dependent transport of “H⁺-equivalents” in mature (trophozoite stage) parasites, isolated from their host erythrocytes. Removal of extracellular Cl⁻, resulting in an outward [Cl⁻] gradient, gave rise to a cytosolic alkalinization (i.e. a net efflux of H⁺-equivalents). This was reversed on restoration of extracellular Cl⁻. The flux of H⁺-equivalents was inhibited by 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid and, when measured in ATP-depleted parasites, showed a pronounced dependence on the pH of the parasite cytosol; the flux was low at cytosolic pH values < 7.2 but increased steeply with cytosolic pH at values > 7.2. ³⁶Cl⁻ influx measurements revealed the presence of a Cl⁻ uptake mechanism with characteristics similar to those of the Cl⁻-dependent H⁺-equivalent flux. The intracellular concentration of Cl⁻ in the parasite was estimated to be ∼48 mm in situ. The data are consistent with the intraerythrocytic parasite having in its plasma membrane a 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid-sensitive transporter that, under physiological conditions, imports Cl⁻ together with H⁺-equivalents, resulting in an intracellular Cl⁻ concentration well above that which would occur if Cl⁻ ions were distributed passively in accordance with the parasite''s large, inwardly negative membrane potential.     

Light-Dependent Changes of the Cytoplasmic H and Cl Activity in the Green Alga Eremosphaera viridis

Ion-sensitive microelectrodes were used to measure Cl⁻ and H⁺ activities in the cytoplasm of the unicellular green alga Eremosphaera viridis de Bary. In the light, cytoplasmic Cl⁻ activity was 2.2 millimolar at most and cytoplasmic H⁺ activity was about 5.4·10⁻⁸ molar (pH 7.3). Darkening resulted in a permanent increase of the Cl⁻ activity to 3.2 millimolar and in a transient acidification, which was compensated within 3 to 5 minutes. Switching light on again decreased the Cl⁻ activity to the light level (2.2 millimolar). Simultaneously, a transient alkalization of the cytoplasm was observed. The transient character of the light-dependent pH changes was probably caused by pH-stat mechanisms, whereas the light-dependent Cl⁻ activity changes were compensated to a much smaller degree. Studies with different inhibitors (3-(3,4-dichlorophenyl)-1, 1-dimethylurea, piretanide, venturicidin) indicated a direct relation between the light-driven H⁺ flow across the thylakoid membrane and the observed light-dependent Cl⁻ and H⁺ activity changes in the cytoplasm. It is suggested that light-driven H⁺ flux across the thylakoid membrane was in part electrically compensated by a parallel Cl⁻ flux. The resulting Cl⁻ and H⁺ activity changes in the stroma were compensated by Cl⁻ and H⁺ fluxes across the chloroplast envelope giving rise to the observed Cl⁻ and H⁺ activity changes in the cytoplasm.     

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.     

Proton Pump Activity of Mitochondria-rich Cells : The Interpretation of External Proton-concentration Gradients

We have hypothesized that a major role of the apical H⁺-pump in mitochondria-rich (MR) cells of amphibian skin is to energize active uptake of Cl⁻ via an apical Cl⁻/HCO₃ ⁻-exchanger. The activity of the H⁺ pump was studied by monitoring mucosal [H⁺]-profiles with a pH-sensitive microelectrode. With gluconate as mucosal anion, pH adjacent to the cornified cell layer was 0.98 ± 0.07 (mean ± SEM) pH-units below that of the lightly buffered bulk solution (pH = 7.40). The average distance at which the pH-gradient is dissipated was 382 ± 18 μm, corresponding to an estimated “unstirred layer” thickness of 329 ± 29 μm. Mucosal acidification was dependent on serosal pCO₂, and abolished after depression of cellular energy metabolism, confirming that mucosal acidification results from active transport of H⁺. The [H⁺] was practically similar adjacent to all cells and independent of whether the microelectrode tip was positioned near an MR-cell or a principal cell. To evaluate [H⁺]-profiles created by a multitude of MR-cells, a mathematical model is proposed which assumes that the H⁺ distribution is governed by steady diffusion from a number of point sources defining a set of particular solutions to Laplace''s equation. Model calculations predicted that with a physiological density of MR cells, the [H⁺] profile would be governed by so many sources that their individual contributions could not be experimentally resolved. The flux equation was integrated to provide a general mathematical expression for an external standing [H⁺]–gradient in the unstirred layer. This case was treated as free diffusion of protons and proton-loaded buffer molecules carrying away the protons extruded by the pump into the unstirred layer; the expression derived was used for estimating stationary proton-fluxes. The external [H⁺]-gradient depended on the mucosal anion such as to indicate that base (HCO₃ ⁻) is excreted in exchange not only for Cl ⁻, but also for Br⁻ and I⁻, indicating that the active fluxes of these anions can be attributed to mitochondria-rich cells.     

Evidence for a highly specific k/h antiporter in membrane vesicles from oil-seed rape hypocotyls

We present evidence strongly suggesting that a proton gradient (acid inside) is used to drive an electroneutral, substrate-specific, K⁺/H⁺ antiport in both tonoplast and plasma membrane-enriched vesicles obtained from oilseed rape (Brassica napus) hypocotyls. Proton fluxes into and out of the vesicles were monitored both by following the quenching and restoration of quinacrine fluorescence (indicating a transmembrane pH gradient) and of oxonol V fluorescence (indicating membrane potential.) Supply of K⁺ (with Cl⁻ or SCN⁻) after a pH gradient had been established across the vesicle membrane by provision of ATP to the H⁺-ATPase dissipated the transmembrane pH gradient but did not depolarize the positive membrane potential. Evidence that the K⁺/H⁺ exchange thus indicated could not be accounted for by mere electric coupling included the findings that, first, no positive potential was generated when KSCN or KCl was supplied, even in the absence of 100 millimolar Cl⁻ and, second, efflux of K⁺ from K⁺-loaded vesicles drives intravesicular accumulation of H⁺ against the electrochemical potential gradient. Neither was the exchange due to competition between K⁺ and quinacrine for membrane sites, nor to inhibition of the H⁺-ATPase. Thus, it is likely that it was effected by a membrane component. The exchanger utilized primarily K⁺ (at micromolar concentrations); Na⁺/H⁺ antiport was detected only at concentrations two orders of magnitude higher. Rb⁺, Li⁺, or Cs⁺ were ineffective. Dependence of tonoplast K⁺/H⁺ antiport on K⁺ concentration was complex, showing saturation at 10 millimolar K⁺ and inhibition by concentrations higher than 25 millimolar. Antiport activity was associated both with tonoplast-enriched membrane vesicles (where the proton pump was inhibited by more than 80% by 50 millimolar NO₃⁻ and showed no sensitivity to vanadate or oligomycin) and with plasma membrane-enriched fractions prepared by phase separation followed by separation on a sucrose gradient (where the proton pump was vanadate and diethylstilbestrol-sensitive but showed no sensitivity to NO₃⁻ or oligomycin). The possible physiological role of such a K⁺/H⁺ exchange mechanism is discussed.     

Potassium Transport in Corn Roots : IV. Characterization of the Linear Component

A detailed examination was conducted on the linear, or first-order kinetic component for K⁺(⁸⁶Rb⁺) influx into root segments of both low- and high-salt grown corn seedlings (Zea mays [A632 × Oh 43]). In tissue from both low- and high-salt grown roots, replacement of Cl⁻ in the uptake solution by either SO₄²⁻, H₂PO₄⁻, or NO₃⁻ caused a significant (50-60%) and specific inhibition of the linear component of K⁺ influx. The anion transport inhibitor, 4,4′-diisothiocyano-2,2′-disulfonic acid, was found to abolish saturable Cl⁻ influx in corn roots while causing a significant (50-60%) and specific inhibition of the linear K⁺ uptake system; this inhibition was identical to that observed when Cl⁻ was replaced by other anions in the K⁺ uptake solution. Additionally, the quaternary ammonium cation, tetraethylammonium, which has been shown to block K⁺ channels in nerve axons, also caused a dramatic (70%) and specific inhibition of the linear component of K⁺ influx, but this was obtained only in high-salt roots. The reasons for this difference are discussed with respect to the differing abilities of low- and high-salt roots to absorb tetraethylammonium.

Our present results indicate that the linear component of K⁺ influx may occur by a passive process involving transmembrane K⁺ channels. Fluxes through these K⁺ channels may be partly coupled to a saturating Cl⁻ influx mechanism.

     

Solute Accumulation in Tobacco Cells Adapted to NaCl

Cells of Nicotiana tabacum L. var Wisconsin 38 adapted to NaCl (up to 428 millimolar) which have undergone extensive osmotic adjustment accumulated Na⁺ and Cl⁻ as principal solutes for this adjustment. Although the intracellular concentrations of Na⁺ and Cl⁻ correlated well with the level of adaptation, these ions apparently did not contribute to the osmotic adjustment which occurred during a culture growth cycle, because the concentrations of Na⁺ and Cl⁻ did not increase during the period of most active osmotic adjustment. The average intracellular concentrations of soluble sugars and total free amino acids increased as a function of the level of adaptation; however, the levels of these solutes did not approach those observed for Na⁺ and Cl⁻. The concentration of proline was positively correlated with cell osmotic potential, accumulating to an average concentration of 129 millimolar in cells adapted to 428 millimolar NaCl and representing about 80% of the total free amino acid pool as compared to an average of 0.29 millimolar and about 4% of the pool in unadapted cells. These results indicate that although Na⁺ and Cl⁻ are principal components of osmotic adjustment, organic solutes also may make significant contributions.     

Ion Transport in Isolated Protoplasts from Tobacco Suspension Cells: II. Selectivity and Kinetics

Protoplasts were enzymically isolated from suspension cultured cells of Nicotiana glutinosa L. and aspects of transport selectivity and kinetics were studied. In the presence of Ca²⁺, transport was selective for K⁺ (⁸⁶Rb) over Na⁺. ³⁶Cl⁻ transport was inhibited by Br⁻ or I⁻ but not by H₂PO₄⁻. The kinetic data for short term (30 minutes) K⁺ influx over the range of 0.05 to 100 millimolar KCl were complex but similar to those observed in other plant tissues. In contrast, the kinetic data for Cl⁻ and H₂³²PO₄⁻ over the same concentration range were different from those observed for K⁺, and could be accounted for by a single isotherm in the range of 0.05 to 4 millimolar and by an almost linear increase in influx rate above 4 millimolar. The kinetic data for Cl⁻ transport into intact cultured cells were identical in character to those observed for isolated protoplasts. The results support the view that enzymic removal of the cell wall produced no significant alteration in the transport properties of the protoplast.     

Anion-sensitive, h-pumping ATPase in membrane vesicles from oat roots

H⁺-pumping ATPases were detected in microsomal vesicles of oat (Avena sativa L. var Lang) roots using [¹⁴C]methylamine distribution or quinacrine fluorescent quenching. Methylamine (MeA) accumulation into vesicles and quinacrine quench were specifically dependent on Mg,ATP. Both activities reflected formation of a proton gradient (ΔpH) (acid inside) as carbonyl cyanide m-chlorophenylhydrazone, nigericin (in the presence of K⁺), or gramicidin decreased MeA uptake or increased quinacrine fluorescence. The properties of H⁺ pumping as measured by MeA uptake were characterized. The K_mapp for ATP was about 0.1 millimolar. Mg,GTP and Mg, pyrophosphate were 19% and 30% as effective as Mg,ATP. MeA uptake was inhibited by N,N′-dicyclohexylcarbodiimide and was mostly insensitive to oligomycin, vanadate, or copper. ATP-dependent MeA was stimulated by anions with decreasing order of potency of Cl⁻ > Br⁻ > NO₃⁻ > SO₄²⁻, iminodiacetate, benzene sulfonate. Anion stimulation of H⁺ pumping was caused in part by the ability of permeant anions to dissipate the electrical potential and in part by a specific requirement of Cl⁻ by a H⁺ -pumping ATPase. A pH gradient, probably caused by a Donnan potential, could be dissipated by K⁺ in the presence or absence of ATP. MeA uptake was enriched in vesicles of relatively low density and showed a parallel distribution with vanadate-insensitive ATPase activity on a continuous dextran gradient. ΔpH as measured by quinacrine quench was partially vanadate-sensitive. These results show that plant membranes have at least two types of H⁺ -pumping ATPases. One is vanadate-sensitive and probably enriched in the plasma membrane. One is vanadate-resistant, anion-sensitive and has many properties characteristic of a vacuolar ATPase. These results are consistent with the presence of electrogenic H⁺ pumps at the plasma membrane and tonoplast of higher plant cells.     

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.     

Characterization of in vitro proton pumping by microsomal vesicles isolated from corn coleoptiles

Corn (Zea mays L. cv Golden Cross Bantam) coleoptile microsomal vesicles have been isolated which are capable of ATP-driven H⁺-transport as measured by [¹⁴C]methylamine accumulation and quinacrine fluorescence quenching. Formation of the pH gradient in vitro shows a high specificity for ATP·Mg, is temperature-sensitive, exhibits a pH optimum at 7.5, and is inhibited by carbonyl cyanide-m-chlorophenylhydrazone. Of the divalent cations tested, Mn²⁺ is almost as effective as Mg²⁺, while Ca²⁺ is ineffective. Excess divalent cations, particularly Ca²⁺, reduces the pH gradient. H⁺ transport is strongly promoted by anions, especially chloride, while potassium does not affect pump activity. Studies with ³⁶Cl⁻ indicate that ATP-driven H⁺ transport into the vesicles is associated with chloride uptake. Both carbonyl cyanide-m-chlorophenylhydrazone and the anion transport inhibitor, 4,4′-diisothiocyano-2,2′-disulfonic acid stilbene, inhibit methylamine accumulation and ³⁶Cl⁻ uptake. Proton pumping is also blocked by diethyl stilbestrol and N,N′-dicyclohexylcarbodiimide, but is insensitive to oligomycin and vanadate. These properties of the pump are inconsistent with either a mitochondrial or plasma membrane origin.     

Ion transport in isolated protoplasts from tobacco suspension cells: I. General characteristics

An investigation was conducted into the feasibility of using enzymically isolated protoplasts from suspension-cultured cells of Nicotiana glutinosa L. to study ion transport. Transport of K⁺ (⁸⁶Rb), ³⁶Cl⁻, H₂³²PO₄⁻ and ⁴⁵Ca²⁺ from 1 millimolar salt solutions was determined after separation of intact protoplasts from nonabsorbed tracers by centrifugation through a Ficoll step gradient. Influx of K⁺, Cl⁻, and H₂PO₄⁻ measured over a 30-minute period was reduced (up to 99%) by respiratory inhibitors such as 5 micrograms per milliliter oligomycin, 0.1 millimolar dinitrophenol, 0.1 millimolar cyanide, or N₂ gas. In contrast, Ca²⁺ influx was not tightly coupled to respiratory energy production. The influx of K⁺ was highest between pH 6.5 and 7.5 whereas the influx of H₂PO₄⁻ and Cl⁻ was greatest between pH 4.5 and 5.5. Influx of K⁺ and Cl⁻ was maximal at 35 and 45 C, respectively, and was almost completely inhibited below 10 C. Fusicoccin (0.01 millimolar) stimulated K⁺ influx by more than 200% but had no effect on the influx of either Cl⁻ or H₂PO₄⁻. Apparent H⁺ efflux, as measured by decrease in solution pH, was enhanced by K⁺, stimulated further by 0.01 millimolar fusicoccin, and inhibited by 0.1 millimolar dinitrophenol or 5 micrograms per milliliter oligomycin. The measured ionic fluxes into protoplasts were similar to those obtained with intact cultured cells. The results indicate that enzymic removal of the cell wall produced no significant alteration in the transport properties of the protoplast, and that it is feasible to use isolated protoplasts for studies on ion transport.     

Extracellular Chloride Modulates the Desensitization Kinetics of Acid-sensing Ion Channel 1a (ASIC1a)

Acid-sensing ion channels (ASICs) are sodium channels gated by extracellular protons. The recent crystallization of ASIC1a identified potential binding sites for Cl⁻ in the extracellular domain that are highly conserved between ASIC isoforms. However, the significance of Cl⁻ binding is unknown. We investigated the effect of Cl⁻ substitution on heterologously expressed ASIC1a current and H⁺-gated currents from hippocampal neurons recorded by whole-cell patch clamp. Replacement of extracellular Cl⁻ with the impermeable and inert anion methanesulfonate (MeSO₃⁻) caused ASIC1a currents to desensitize at a faster rate and attenuated tachyphylaxis. However, peak current amplitude, pH sensitivity, and selectivity were unchanged. Other anions, including Br⁻, I⁻, and thiocyanate, also altered the kinetics of desensitization and tachyphylaxis. Mutation of the residues that form the Cl⁻-binding site in ASIC1a abolished the modulatory effects of anions. The results of anion substitution on native ASIC channels in hippocampal neurons mirrored those in heterologously expressed ASIC1a and altered acid-induced neuronal death. Anion modulation of ASICs provides new insight into channel gating and may prove important in pathological brain conditions associated with changes in pH and Cl⁻.     

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.     

Ae4 (Slc4a9) Anion Exchanger Drives Cl? Uptake-dependent Fluid Secretion by Mouse Submandibular Gland Acinar Cells

Transcellular Cl⁻ movement across acinar cells is the rate-limiting step for salivary gland fluid secretion. Basolateral Nkcc1 Na⁺-K⁺-2Cl⁻ cotransporters play a critical role in fluid secretion by promoting the intracellular accumulation of Cl⁻ above its equilibrium potential. However, salivation is only partially abolished in the absence of Nkcc1 cotransporter activity, suggesting that another Cl⁻ uptake pathway concentrates Cl⁻ ions in acinar cells. To identify alternative molecular mechanisms, we studied mice lacking Ae2 and Ae4 Cl⁻/HCO₃⁻ exchangers. We found that salivation stimulated by muscarinic and β-adrenergic receptor agonists was normal in the submandibular glands of Ae2^−/− mice. In contrast, saliva secretion was reduced by 35% in Ae4^−/− mice. The decrease in salivation was not related to loss of Na⁺-K⁺-2Cl⁻ cotransporter or Na⁺/H⁺ exchanger activity in Ae4^−/− mice but correlated with reduced Cl⁻ uptake during β-adrenergic receptor activation of cAMP signaling. Direct measurements of Cl⁻/HCO₃⁻ exchanger activity revealed that HCO₃⁻-dependent Cl⁻ uptake was reduced in the acinar cells of Ae2^−/− and Ae4^−/− mice. Moreover, Cl⁻/HCO₃⁻ exchanger activity was nearly abolished in double Ae4/Ae2 knock-out mice, suggesting that most of the Cl⁻/HCO₃⁻ exchanger activity in submandibular acinar cells depends on Ae2 and Ae4 expression. In conclusion, both Ae2 and Ae4 anion exchangers are functionally expressed in submandibular acinar cells; however, only Ae4 expression appears to be important for cAMP-dependent regulation of fluid secretion.     

Separation of two types of electrogenic h-pumping ATPases from oat roots

Microsomal vesicles of oat roots (Avena sativa var Lang) were separated with a linear dextran (0.5-10%, w/w) or sucrose (25-45%, w/w) gradient to determine the types and membrane identity of proton-pumping ATPases associated with plant membranes. ATPase activity stimulated by the H⁺/K⁺ exchange ionophore nigericin exhibited two peaks of activity on a linear dextran gradient. ATPase activities or ATP-generated membrane potential (inside positive), monitored by SCN⁻ distribution, included a vanadate-insensitive and a vanadate-sensitive component. In a previous communication, we reported that ATP-dependent pH gradient formation (acid inside), monitored by quinacrine fluorescence quenching, was also partially inhibited by vanadate (Churchill and Sze 1983 Plant Physiol 71: 610-617). Here we show that the vanadate-insensitive, electrogenic ATPase activity was enriched in the low density vesicles (1-4% dextran or 25-32% sucrose) while the vanadate-sensitive activity was enriched at 4% to 7% dextran or 32% to 37% sucrose. The low-density ATPase was stimulated by Cl⁻ and inhibited by NO⁻₃ or 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid (DIDS). The distribution of Cl⁻-stimulated ATPase activity in a linear dextran gradient correlated with the distribution of H⁺ pumping into vesicles as monitored by [¹⁴C]methylamine accumulation. The vanadate-inhibited ATPase was mostly insensitive to anions or DIDS and stimulated by K⁺. These results show that microsomal vesicles of plant tissues have at least two types of electrogenic, proton-pumping ATPases. The vanadate-insensitive and Cl⁻-stimulated, H⁺-pumping ATPase may be enriched in vacuolar-type membranes; the H⁺-pumping ATPase that is stimulated by K⁺ and inhibited by vanadate is most likely associated with plasma membrane-type vesicles.