Identification of the transmembrane metal binding site in Cu+-transporting PIB-type ATPases

P(IB)-type ATPases have an essential role maintaining copper homeostasis. Metal transport by these membrane proteins requires the presence of a transmembrane metal occlusion/binding site. Previous studies showed that Cys residues in the H6 transmembrane segment are required for metal transport. In this study, the participation in metal binding of conserved residues located in transmembrane segments H7 and H8 was tested using CopA, a model Cu(+)-ATPase from Archaeoglobus fulgidus. Four invariant amino acids in the central portion of H7 (Tyr(682) and Asn(683)) and H8 (Met(711) and Ser(715)) were identified as required for Cu(+) binding. Replacement of these residues abolished enzyme activity. These proteins did not undergo Cu(+)-dependent phosphorylation by ATP but were phosphorylated by P(i) in the absence of Cu(+). Moreover, the presence of Cu(+) could not prevent the enzyme phosphorylation by P(i). Other conserved residues in the H7-H8 region were not required for metal binding. Mutation of two invariant Pro residues had little effect on enzyme function. Replacement of residues located close to the cytoplasmic end of H7-H8 led to inactive enzymes. However, these were able to interact with Cu(+) and undergo phosphorylation. This suggests that the integrity of this region is necessary for conformational transitions but not for ligand binding. These data support the presence of a unique transmembrane Cu(+) binding/translocation site constituted by Tyr-Asn in H7, Met and Ser in H8, and two Cys in H6 of Cu(+)-ATPases. The likely Cu(+) coordination during transport appears distinct from that observed in Cu(+) chaperone proteins or catalytic/redox metal binding sites.     

Importance for absorption of Na+ from freshwater of lysine, valine and serine substitutions in the alpha1a-isoform of Na,K-ATPase in the gills of rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar)

In the gills of rainbow trout and Atlantic salmon, the alpha1a- and alpha1b-isoforms of Na,K-ATPase are expressed reciprocally during salt acclimation. The alpha1a-isoform is important for Na(+) uptake in freshwater, but the molecular basis for the functional differences between the two isoforms is not known. Here, three amino acid substitutions are identified in transmembrane segment 5 (TM5), TM8 and TM9 of the alpha1a-isoform compared to the alpha1b-isoform, and the functional consequences are examined by mutagenesis and molecular modeling on the crystal structures of Ca-ATPase or porcine kidney Na,K-ATPase. In TM5 of the alpha1a-isoform, a lysine substitution, Asn783 --> Lys, inserts the epsilon-amino group in cation site 1 in the E(1) form to reduce the Na(+)/ATP ratio. In the E(2) form the epsilon-amino group approaches cation site 2 to force ejection of Na(+) to the blood phase and to interfere with binding of K(+). In TM8, a Asp933 --> Val substitution further reduces K(+) binding, while a Glu961 --> Ser substitution in TM9 can prevent interaction of FXYD peptides with TM9 and alter Na(+) or K(+) affinities. Together, the three substitutions in the alpha1a-isoform of Na,K-ATPase act to promote binding of Na(+) over K(+) from the cytoplasm, to reduce the Na(+)/ATP ratio and the work done in one Na,K pump cycle of active Na(+) transport against the steep gradient from freshwater (10-100 microM: Na(+)) to blood (160 mM: Na(+)) and to inhibit binding of K(+) to allow Na(+)/H(+) rather than Na(+)/K(+) exchange.     

KtrB, a member of the superfamily of K+ transporters

KtrB is the K(+)-translocating subunit of the K(+)-uptake system KtrAB from bacteria. It is a member of the superfamily of K(+)transporters (SKT proteins) with other sub-families occurring in archaea, bacteria, fungi, plants and trypanosomes. SKT proteins may have originated from small K(+) channels by at least two gene duplication and two gene fusion events. They contain four covalently linked M(1)PM(2) domains, in which M(1) and M(2) stand for transmembrane stretches, and P for a P-loop, which folds back from the external medium into the membrane. SKT proteins distinguish themselves in two important aspects from K(+) channels: first, with just one conserved glycine residue in their P-loops they contain a much simpler K(+)-selectivity filter sequence than K(+) channels with their conserved Thr-Val-Gly-Tyr-Gly sequence. Secondly, the middle part M(2C2) from the long transmembrane stretch M(2C) of KtrB from the bacterium Vibrio alginolyticus forms a gate inside the membrane, which prevents K(+) permeation to the cytoplasm. Beside the mechanism of K(+) transport via KtrB and other SKT proteins existing hypotheses of how the KtrA protein regulates the K(+)-transport activity of KtrB are discussed.     

Activation of Archaeoglobus fulgidus Cu(+)-ATPase CopA by cysteine

CopA, a thermophilic ATPase from Archaeoglobus fulgidus, drives the outward movement of Cu(+) across the cell membrane. Millimolar concentration of Cys dramatically increases ( congruent with 800%) the activity of CopA and other P(IB)-type ATPases (Escherichia coli ZntA and Arabidopsis thaliana HMA2). The high affinity of CopA for metal ( congruent with 1 microM) together with the low Cu(+)-Cys K(D) (<10(-10)M) suggested a multifaceted interaction of Cys with CopA, perhaps acting as a substitute for the Cu(+) chaperone protein present in vivo. To explain the activation by the amino acid and further understand the mechanism of metal delivery to transport ATPases, Cys effects on the turnover and partial reactions of CopA were studied. 2-20 mM Cys accelerates enzyme turnover with little effect on CopA affinity for Cu(+), suggesting a metal independent activation. Furthermore, Cys activates the p-nitrophenyl phosphatase activity of CopA, even though this activity is metal independent. Cys accelerates enzyme phosphorylation and the forward dephosphorylation rates yielding higher steady state phosphoenzyme levels. The faster dephosphorylation would explain the higher enzyme turnover in the presence of Cys. The amino acid has no significant effect on low affinity ATP K(m) suggesting no changes in the E(1)<-->E(2) equilibrium. Characterization of Cu(+) transport into sealed vesicles indicates that Cys acts on the cytoplasmic side of the enzyme. However, the Cys activation of truncated CopA lacking the N-terminal metal binding domain (N-MBD) indicates that activation by Cys is independent of the regulatory N-MBD. These results suggest that Cys is a non-essential activator of CopA, interacting with the cytoplasmic side of the enzyme while this is in an E1 form. Interestingly, these effects also point out that Cu(+) can reach the cytoplasmic opening of the access path into the transmembrane transport sites either as a free metal or a Cu(+)-Cys complex.     

Residues within transmembrane domains 4 and 6 of the Na,K-ATPase alpha subunit are important for Na+ selectivity

The Na,K- and H,K-ATPases are plasma membrane enzymes responsible for the active exchange of extracellular K(+) for cytoplasmic Na(+) or H(+), respectively. At present, the structural determinants for the specific function of these ATPases remain poorly understood. To investigate the cation selectivity of these ATPases, we constructed a series of Na,K-ATPase mutants in which residues in the membrane spanning segments of the alpha subunit were changed to the corresponding residues common to gastric H,K-ATPases. Thus, mutants were created with substitutions in transmembrane domains TM1, TM4, TM5, TM6, TM7, and TM8 independently or together (designated TMAll). The function of each mutant was assessed after coexpression with the beta subunit in Sf-9 cells using baculoviruses. The enzymatic properties of TM1, TM7, and TM8 mutants were similar to the wild-type Na,K-ATPase, and while TM5 showed modest changes in apparent affinity for Na(+), TM4, TM6, and TMAll displayed an abnormal activity. This resulted in a Na(+)-independent hydrolysis of ATP, a 2-fold higher K(0.5) for Na(+) activation, and the ability to function at low pH. These results suggest a loss of discrimination for Na(+) over H(+) for the enzymes. In addition, TM4, TM6, and TMAll mutants exhibited a 1.5-fold lower affinity for K(+) and a 4-5-fold decreased sensitivity to vanadate. Altogether, these results provide evidence that residues in transmembrane domains 4 and 6 of the alpha subunit of the Na,K-ATPase play an important role in determining the specific cation selectivity of the enzyme and also its E1/E2 conformational equilibrium.     

Proximity of transmembrane segments M3 and M1 of the alpha subunit of Na+,K+-ATPase revealed by specific oxidative cleavage mediated by a complex of Cu2+ ions and 4,7-diphenyl-1,10-phenanthroline

This paper describes a novel approach to specific oxidative cleavage of Na(+),K(+)-ATPase, mediated by Cu(2+) ions and a hydrophobic phenanthroline, 4,7-diphenyl-1,10-phenanthroline (DPP), in the presence of ascorbate and H(2)O(2). The cleavage produces two major fragments of the alpha subunit, with apparent molecular masses of 96.5 and 76 kDa, and N-termini near the cytoplasmic entrance of transmembrane segments M1 and M3, respectively, The kinetics indicate that both cleavages are mediated by a single Cu(2+)-DPP complex. We infer that M3 and M1 are in proximity near the cytoplasmic surface. The yields of 96.5 and 76 kDa fragments are not significantly affected by ligands that stabilize different E(1) and E(2) conformations. In E(2)(K) and E(2)P conformations, a minor 5.5 kDa fragment with its N-terminus in M10 is also observed. The 96.5 and 76 kDa fragments are indistinguishable from two fragments near M3 and M1 produced by Fe(2+)-catalyzed cleavage described previously [Goldshleger, R., and Karlish, S. J. D. (1999) J. Biol. Chem. 274, 16213-16221], whereas other Fe(2+)-catalyzed cleavage fragments in the cytoplasmic P and A domains are not observed with the Cu(2+)-DPP complex. These findings provide experimental support for the concept of two separate Fe(2+) sites. A homology model, with Na(+),K(+)-ATPase residues within transmembrane segments and connecting loops substituted into the crystal structure of Ca(2+)-ATPase, shows the proximity between the sequences HFIH in M3 and EVWK in M1, near the cytoplasmic surface. Thus, the model strongly supports the conclusions based on cleavages mediated by the Cu(2+)-DPP complex (or Fe(2+) at site 2). As a corollary, the cleavages provide evidence for similar packing of M1 and M3 of Na(+),K(+)-ATPase and Ca(2+)-ATPase.     

Access of extracellular cations to their binding sites in Na,K-ATPase: role of the second extracellular loop of the alpha subunit

Na,K-ATPase, the main active transport system for monovalent cations in animal cells, is responsible for maintaining Na(+) and K(+) gradients across the plasma membrane. During its transport cycle it binds three cytoplasmic Na(+) ions and releases them on the extracellular side of the membrane, and then binds two extracellular K(+) ions and releases them into the cytoplasm. The fourth, fifth, and sixth transmembrane helices of the alpha subunit of Na,K-ATPase are known to be involved in Na(+) and K(+) binding sites, but the gating mechanisms that control the access of these ions to their binding sites are not yet fully understood. We have focused on the second extracellular loop linking transmembrane segments 3 and 4 and attempted to determine its role in gating. We replaced 13 residues of this loop in the rat alpha1 subunit, from E314 to G326, by cysteine, and then studied the function of these mutants using electrophysiological techniques. We analyzed the results using a structural model obtained by homology with SERCA, and ab initio calculations for the second extracellular loop. Four mutants were markedly modified by the sulfhydryl reagent MTSET, and we investigated them in detail. The substituted cysteines were more readily accessible to MTSET in the E1 conformation for the Y315C, W317C, and I322C mutants. Mutations or derivatization of the substituted cysteines in the second extracellular loop resulted in major increases in the apparent affinity for extracellular K(+), and this was associated with a reduction in the maximum activity. The changes produced by the E314C mutation were reversed by MTSET treatment. In the W317C and I322C mutants, MTSET also induced a moderate shift of the E1/E2 equilibrium towards the E1(Na) conformation under Na/Na exchange conditions. These findings indicate that the second extracellular loop must be functionally linked to the gating mechanism that controls the access of K(+) to its binding site.     

Cysteine-scanning mutagenesis of transmembrane segments 4 and 5 of the Tn10-encoded metal-tetracycline/H+ antiporter reveals a permeability barrier in the middle of a transmembrane water-filled channel

Cysteine-scanning mutants as to putative transmembrane segments 4 and 5 and the flanking regions of Tn10-encoded metal-tetracycline/H(+) antiporter (TetA(B)) were constructed. All mutants were normally expressed. Among the 57 mutants (L99C to I155C), nine conserved arginine-, aspartate-, and glycine-replaced ones exhibited greatly reduced tetracycline resistance and almost no transport activity, and five conserved glycine- and proline-replaced mutants exhibited greatly reduced tetracycline transport activity in inverted membrane vesicles despite their high or moderate drug resistance. All other cysteine-scanning mutants retained normal drug resistance and normal tetracycline transport activity except for the L142C and I143C mutants. The transmembrane (TM) regions TM4 and TM5 were determined to comprise 20 amino acid residues, Leu-99 to Ile-118, and 17 amino acid residues, Ala-136 to Ala-152, respectively, on the basis of N-[(14)C]ethylmaleimide ([(14)C]NEM) reactivity. The NEM reactivity patterns of the TM4 and TM5 mutants were quite different from each other. TM4 could be divided into two halves, that is, a NEM nonreactive periplasmic half and a periodically reactive cytoplasmic half, indicating that TM4 is tilted toward a water-filled transmembrane channel and that only its cytoplasmic half faces the channel. On the other hand, NEM-reactive mutations were observed periodically (every two residues) along the whole length of TM5. A permeability barrier for a membrane-impermeable sulfhydryl reagent, 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, was present in the middle of TM5 between Leu-142 and Gly-145, whereas all the NEM-reactive mutants as to TM4 were not accessible to 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, indicating that the channel-facing side of TM4 is located inside the permeability barrier. Tetracycline protected the G141C mutant from the NEM binding, whereas the other mutants in TM4 and TM5 were not protected by tetracycline.     

Functional comparison of the K+-Cl- cotransporters KCC1 and KCC4

The K(+)-Cl(-) cotransporters (KCCs) are members of the cation-chloride cotransporter gene family and fall into two phylogenetic subgroups: KCC2 paired with KCC4 and KCC1 paired with KCC3. We report a functional comparison in Xenopus oocytes of KCC1 and KCC4, widely expressed representatives of these two subgroups. KCC1 and KCC4 exhibit differential sensitivity to transport inhibitors, such that KCC4 is much less sensitive to bumetanide and furosemide. The efficacy of these anion inhibitors is critically dependent on the concentration of extracellular K(+), with much higher inhibition in 50 mm K(+) versus 2 mm K(+). KCC4 is also uniquely sensitive to 10 mm barium and to 2 mm trichlormethiazide. Kinetic characterization reveals divergent affinities for K(+) (K(m) values of approximately 25.5 and 17.5 mm for KCC1 and KCC4, respectively), probably due to variation within the second transmembrane segment. Although the two isoforms have equivalent affinities for Cl(-), they differ in the anion selectivity of K(+) transport (Cl(-) > SCN(-) = Br(-) > PO(4)(-3) > I(-) for KCC1 and Cl(-) > Br(-) > PO(4)(-3) = I(-) > SCN(-) for KCC4). Both KCCs express minimal K(+)-Cl(-) cotransport under isotonic conditions, with significant activation by cell swelling under hypotonic conditions. The cysteine-alkylating agent N-ethylmaleimide activates K(+)-Cl(-) cotransport in isotonic conditions but abrogates hypotonic activation, an unexpected dissociation of N-ethylmaleimide sensitivity and volume sensitivity. Although KCC4 is consistently more volume-sensitive, the hypotonic activation of both isoforms is critically dependent on protein phosphatase 1. Overall, the functional comparison of these cloned K(+)-Cl(-) cotransporters reveals important functional, pharmacological, and kinetic differences with both physiological and mechanistic implications.     

Cysteine-scanning mutagenesis and thiol modification of the Rickettsia prowazekii ATP/ADP translocase: evidence that transmembrane regions I and II, but not III, are structural components of the aqueous translocation channel

The contribution of transmembrane regions I, II, and III of the Rickettsia prowazekii ATP/ADP translocase to the structure of the putative water-filled ATP translocation channel was evaluated from the accessibility of hydrophilic, thiol-reactive, methanethiosulfonate reagents to a library of 68 independent cysteine-substitution mutants heterologously expressed in Escherichia coli. The MTS reagents used were MTSES (negatively charged) and MTSET and MTSEA (both positively charged). Mutants F036C, Y042C, and R046C (TM I), K066C and P072C (TM II), and F101C, F105C, F108C, Y113C, and P114C (TM III) had no assayable transport activity, indicating that cysteine substitution at these positions may not be tolerated. All three MTS reagents inhibit the transport of ATP in mutants of TM I (L039C, S043C, S047C, I048C) and TM II (S061C, S063C, T067C, I069C, V070C, A074C). Further, these residues appear to cluster along a single face of the transmembrane domain. Preexposure of MTS-reactive mutants S047C (TM I) and T067C (TM II) to high levels of ATP resulted in protection from MTS-mediated inhibition. This indicated that both TM I and TM II make major contributions to the structure of an aqueous ATP translocation pathway. Finally, on the basis of the lack of accessibility of charged MTS reagents to the thiol groups in mutants of TM III, it appears that TM III is not exposed to the ATP translocation channel. Cysteine substitution of residues constituting a highly conserved "phenylalanine face" in TM III resulted in ablation of ATP transport activity. Further, substituting these phenylalanine residues for either isoleucine or tyrosine also resulted in much lower transport activity, indicating that some property of phenylalanine at these positions that is not shared by cysteine, isoleucine, or tyrosine is critical to translocase activity.     

Role of copper in folding and stability of cupredoxin-like copper-carrier protein CopC

CopC is a periplasmic copper carrier that, in contrast to cytoplasmic copper chaperones, has a beta-barrel fold and two metal-binding sites distinct for Cu(II) and Cu(I). The copper sites are located in each end of the molecule: the Cu(I) site involves His and Met coordination whereas the Cu(II) site consists of charged residues. To reveal biophysical properties of this protein, we have explored the effects of the cofactors on CopC unfolding in vitro. We demonstrate that Cu(II) coordination affects both protein stability and unfolding pathway, whereas Cu(I) has only a small effect on stability. Apo-CopC unfolds in a two-state reaction between pH 4 and 7.5 with maximal stability at pH 6. In contrast, Cu(II)-CopC unfolds in a three-state reaction at pH6 that involves a partly folded intermediate that retains Cu(II). This intermediate exhibits high thermal and chemical stability. Unique energetic and structural properties of different metalated CopC forms may help facilitate metal transport to many partners in vivo.     

Definition of membrane topology and identification of residues important for transport in subunit a of the vacuolar ATPase

Subunit a of the vacuolar H(+)-ATPases plays an important role in proton transport. This membrane-integral 100-kDa subunit is thought to form or contribute to proton-conducting hemichannels that allow protons to gain access to and leave buried carboxyl groups on the proteolipid subunits (c, c', and c″) during proton translocation. We previously demonstrated that subunit a contains a large N-terminal cytoplasmic domain followed by a C-terminal domain containing eight transmembrane (TM) helices. TM7 contains a buried arginine residue (Arg-735) that is essential for proton transport and is located on a helical face that interacts with the proteolipid ring. To further define the topology of the C-terminal domain, the accessibility of 30 unique cysteine residues to the membrane-permeant reagent N-ethylmaleimide and the membrane-impermeant reagent polyethyleneglycol maleimide was determined. The results further define the borders of transmembrane segments in subunit a. To identify additional buried polar and charged residues important in proton transport, 25 sites were individually mutated to hydrophobic amino acids, and the effect on proton transport was determined. These and previous results identify a set of residues important for proton transport located on the cytoplasmic half of TM7 and TM8 and the lumenal half of TM3, TM4, and TM7. Based upon these data, we propose a tentative model in which the cytoplasmic hemichannel is located at the interface of TM7 and TM8 of subunit a and the proteolipid ring, whereas the lumenal hemichannel is located within subunit a at the interface of TM3, TM4, and TM7.     

Visualizing the mapped ion pathway through the Na,K-ATPase pump

The Na+,K+-ATPase pump achieves thermodynamically uphill exchange of cytoplasmic Na+ ions for extracellular K+ ions by using ATP-mediated phosphorylation, followed by autodephosphorylation, to power conformational changes that allow ion access to the pump's binding sites from only one side of the membrane at a time. Formally, the pump behaves like an ion channel with two tightly coupled gates that are constrained to open and close alternately. The marine agent palytoxin disrupts this coupling, allowing both gates to sometimes be open, so temporarily transforming a pump into an ion channel. We made a cysteine scan of Na+,K+-ATPase transmembrane (TM) segments TM1 to TM6, and used recordings of Na+ current flow through palytoxin-bound pump-channels to monitor accessibility of introduced cysteine residues via their reaction with hydrophilic methanethiosulfonate (MTS) reagents. To visualize the open-channel pathway, the reactive positions were mapped onto a homology model of Na+,K+-ATPase based on the structure of the related sarcoplasmic- and endoplasmic-reticulum (SERCA) Ca2+-ATPase in a BeF3--trapped state1,2, in which the extra-cytoplasmic gate is wide open (although the cytoplasmic access pathway is firmly shut). The results revealed a single unbroken chain of reactive positions that traverses the pump from the extracellular surface to the cytoplasm, comprises residues from TM1, TM2, TM4, and TM6, and passes through the equivalent of cation binding site II in SERCA, but not through site I. Cavity search analysis of the homology model validated its use for mapping the data by yielding a calculated extra-cytoplasmic pathway surrounded by MTS-reactive residues. As predicted by previous experimental results, that calculated extra-cytoplasmic pathway abruptly broadens above residue T806, at the outermost end of TM6 which forms the floor of the extracellular-facing vestibule. These findings provide a structural basis for further understanding cation translocation by the Na+,K+-ATPase and by other P-type pumps like the Ca2+- and H+,K+-ATPases.     

K+-Phosphatase activity of gill (Na+, K+)-ATPase from the blue crab, Callinectes danae: low-salinity acclimation and expression of the alpha-subunit

The kinetic properties of a microsomal gill (Na(+), K(+)) ATPase from the blue crab, Callinectes danae, acclimated to 15 per thousand salinity for 10 days, were analyzed using the substrate p-nitrophenylphosphate. The (Na(+), K(+))-ATPase hydrolyzed the substrate obeying Michaelian kinetics at a rate of V=102.9+/-4.3 U.mg(-1) with K(0.5)=1.7+/-0.1 mmol.L(-1), while stimulation by magnesium (V=93.7+/-2.3 U.mg(-1); K(0.5)=1.40+/-0.03 mmol.L(-1)) and potassium ions (V=94.9+/-3.5 U.mg(-1); K(0.5)=2.9+/-0.1 mmol.L(-1)) was cooperative. K(+)-phosphatase activity was also stimulated by ammonium ions to a rate of V=106.2+/-2.2 U. mg(-1) with K(0.5)=9.8+/-0.2 mmol.L(-1), following cooperative kinetics (n(H)=2.9). However, K(+)-phosphatase activity was not stimulated further by K(+) plus NH(4) (+) ions. Sodium ions (K(I)=22.7+/-1.7 mmol.L(-1)), and orthovanadate (K(I)=28.1+/-1.4 nmol.L(-1)) completely inhibited PNPPase activity while ouabain inhibition reached almost 75% (K(I)=142.0+/-7.1 micromol.L(-1)). Western blotting analysis revealed increased expression of the (Na(+), K(+))-ATPase alpha-subunit in crabs acclimated to 15 per thousand salinity compared to those acclimated to 33 per thousand salinity. The increase in (Na(+), K(+))-ATPase activity in C. danae gill tissue in response to low-salinity acclimation apparently derives from the increased expression of the (Na(+), K( (+) ))-ATPase alpha-subunit; phosphate-hydrolyzing enzymes other than (Na(+), K(+))-ATPase are also expressed. These findings allow a better understanding of the kinetic behavior of the enzymes that underlie the osmoregulatory mechanisms of euryhaline crustaceans.     

Na<Superscript>+</Superscript> gradient-dependent transport of hypoxanthine by calf intestinal brush border membrane vesicles

The properties of hypoxanthine transport were investigated in purified brush border membrane vesicles isolated from calf proximal and distal jejunum. Hypoxanthine uptake in the vesicles was stimulated by a transmembrane Na(+) gradient and an inside negative potential resulting in a transient accumulation of intravesicular hypoxanthine, especially in the proximal jejunum. Na(+)-dependent hypoxanthine uptake at this site seemed to occur by two saturable transport systems, a high affinity (K(m)=0.33 micromol/l) and a low affinity (K(m)=165 micromol/l) transporter. Guanine, hypoxanthine, thymine and uracil inhibited intravesicular hypoxanthine uptake, whereas adenine and the nucleosides inosine and thymidine were without effect. These findings represent the first demonstration of active Na(+) gradient-dependent nucleobase transport in intestinal brush border membrane vesicles.     

Molecular basis of the cooperative binding of Cu(I) and Cu(II) to the CopK protein from Cupriavidus metallidurans CH34

The bacterium Cupriavidus metallidurans CH34 is resistant to high environmental concentrations of many metal ions. Upon copper challenge, it upregulates the periplasmic protein CopK (8.3 kDa). The function of CopK in the copper resistance response is ill-defined, but CopK demonstrates an intriguing cooperativity: occupation of a high-affinity Cu(I) binding site generates a high-affinity Cu(II) binding site, and the high-affinity Cu(II) binding enhances Cu(I) binding. Native CopK and targeted variants were examined by chromatographic, spectroscopic, and X-ray crystallographic probes. Structures of two distinct forms of Cu(I)Cu(II)-CopK were defined, and structural changes associated with occupation of the Cu(II) site were demonstrated. In solution, monomeric Cu(I)Cu(II)-CopK features the previously elucidated Cu(I) site in Cu(I)-CopK, formed from four S(δ) atoms of Met28, -38, -44, and -54 (site 4S). Binding of Cu(I) to apo-CopK induces a conformational change that releases the C-terminal β-strand from the β-sandwich structure. In turn, this allows His70 and N-terminal residues to form a large loop that includes the Cu(II) binding site. In crystals, a polymeric form of Cu(I)Cu(II)-CopK displays a Cu(I) site defined by the S(δ) atoms of Met26, -38, and -54 (site 3S) and an exogenous ligand (modeled as H(2)O) and a Cu(II) site that bridges dimeric CopK molecules. The 3S Cu(I) binding mode observed in crystals was demonstrated in solution in protein variant M44L where site 4S is disabled. The intriguing copper binding chemistry of CopK provides molecular insight into Cu(I) transfer processes. The adaptable nature of the Cu(I) coordination sphere in methionine-rich clusters allows copper to be relayed between clusters during transport across membranes in molecular pumps such as CusA and Ctr1.     

Thallium ions can replace both sodium and potassium ions in the glutamate transporter excitatory amino acid carrier 1

The excitatory amino acid carrier EAAC1 belongs to a family of glutamate transporters that use the electrochemical transmembrane gradients of sodium and potassium to mediate uphill transport of glutamate into the cell. While the sites of cation interaction with EAAC1 are unknown, two cation binding sites were observed in the crystal structure of the bacterial glutamate transporter homologue GltPh. Although occupied by Tl(+) in the crystal structure, these sites were proposed to be Na(+) binding sites. Therefore, we tested whether Tl(+) has the ability to replace Na(+) also in the mammalian transporters. Our data demonstrate that Tl(+) can bind to EAAC1 with high affinity and mediate a host of different functions. Tl(+) can functionally replace potassium when applied to the cytoplasm and can support glutamate transport current. When applied extracellularly, Tl(+) induces some behavior that mimics that of the Na(+)-bound transporter, such as activation of the cation-induced anion conductance and creation of a substrate binding site, but it cannot replace Na(+) in supporting glutamate transport current. Moreover, our data show a differential effect of mutations to two acidic amino acids potentially involved in cation binding (D367 and D454) on Na(+) and Tl(+) affinity. Overall, our results demonstrate that the ability of the glutamate transporters to interact with Tl(+) is conserved between GltPh and a mammalian member of the transporter family. However, in contrast to GltPh, which does not bind K(+), Tl(+) is more efficient in mimicking K(+) than Na(+) when interacting with the mammalian protein.     

A kinetic study of the gill (Na+, K+)-ATPase, and its role in ammonia excretion in the intertidal hermit crab, Clibanarius vittatus

To better comprehend the role of gill ion regulatory mechanisms, the modulation by Na(+), K(+), NH(4)(+) and ATP of (Na(+), K(+))-ATPase activity was examined in a posterior gill microsomal fraction from the hermit crab, Clibanarius vittatus. Under saturating Mg(2+), Na(+) and K(+) concentrations, two well-defined ATP hydrolyzing sites were revealed. ATP was hydrolyzed at the high-affinity sites at a maximum rate of V=19.1+/-0.8 U mg(-1) and K(0.5)=63.8+/-2.9 nmol L(-1), obeying cooperative kinetics (n(H)=1.9); at the low-affinity sites, hydrolysis obeyed Michaelis-Menten kinetics with K(M)=44.1+/-2.6 mumol L(-1) and V=123.5+/-6.1 U mg(-1). Stimulation by Na(+) (V=149.0+/-7.4 U mg(-1); K(M)=7.4+/-0.4 mmol L(-1)), Mg(2+) (V=132.0+/-5.3 U mg(-1); K(0.5)=0.36+/-0.02 mmol L(-1)), NH(4)(+) (V=245.6+/-9.8 U mg(-1); K(M)=4.5+/-0.2 mmol L(-1)) and K(+) (V=140.0+/-4.9 U mg(-1); K(M)=1.5+/-0.1 mmol L(-1)) followed a single saturation curve and, except for Mg(2+), obeyed Michaelis-Menten kinetics. Under optimal ionic conditions, but in the absence of NH(4)(+), ouabain (K(I)=117.3+/-3.5 mumol L(-1)) and orthovanadate inhibited up to 67% of the ATPase activity. The inhibition studies performed suggest the presence of F(0)F(1), V- and P-ATPases, but not Na(+)-, K(+)- or Ca(2+)-ATPases as contaminants in the gill microsomal preparation. (Na(+), K(+))-ATPase activity was synergistically modulated by NH(4)(+) and K(+). At 20 mmol L(-1) K(+), a maximum rate of V=290.8+/-14.5 U mg(-1) was seen as NH(4)(+) concentration was increased up to 50 mmol L(-1). However, at fixed NH(4)(+) concentrations, no additional stimulation was found for increasing K(+) concentrations (V=135.2+/-4.1 U mg(-1) and V=236.6+/-9.5 U mg(-1) and for 10 and 30 mmol L(-1) NH(4)(+), respectively). This is the first report to detail ionic modulation of gill (Na(+), K(+))-ATPase in C. vittatus, revealing an asymmetrical, synergistic stimulation of the enzyme by K(+) and NH(4)(+), as yet undescribed for other (Na(+), K(+))-ATPases, and should provide a better understanding of NH(4)(+) excretion in pagurid crabs.     

Ultracytochemical location of Na(+)/K(+)-atpase activity and effect of high salinity acclimation in gill and renal epithelia of the freshwater shrimp Macrobrachium olfersii (Crustacea, Decapoda).

Accumulation sites of lead phosphate reaction product consequent to Na(+)/K(+)-ATPase activity in gill and renal epithelia of the freshwater shrimp Macrobrachium olfersii were located ultracytochemically by para-nitrophenyl-phosphate hydrolysis and lead precipitation, and quantified per unit membrane area and cytoplasmic volume. In shrimps in freshwater (<0.5 per thousand S, 20 mOsm/kg H(2)O, 0.7 mEq Na(+)/liter), numerous sites of electron-dense, Na(+)/K(+)-ATPase reaction product accumulation were demonstrated in the membrane invaginations of the mitochondria-rich, intralamellar septal cells (12.5 +/- 1.7 sites/microm(2) membrane, 179 +/- 22 sites/microm(3) cytoplasm, mean+/- SEM, N 相似文献   

Alanine-scanning mutagenesis of the sixth transmembrane segment of gastric H+,K+-ATPase alpha-subunit

The sixth transmembrane (M6) segment of the catalytic subunit plays an important role in the ion recognition and transport in the type II P-type ATPase families. In this study, we singly mutated all amino acid residues in the M6 segment of gastric H(+),K(+)-ATPase alpha-subunit with alanine, expressed the mutants in HEK-293 cells, and studied the effects of the mutation on the functions of H(+),K(+)-ATPase; overall K(+)-stimulated ATPase, phosphorylation, and dephosphorylation. Four mutants, L819A, D826A, I827A, and L833A, completely lost the K(+)-ATPase activity. Mutant L819A was phosphorylated but hardly dephosphorylated in the presence of K(+), whereas mutants D826A, I827A, and L833A were not phosphorylated from ATP. We found that almost all of these amino acid residues, which are important for the function, are located on the same side of the alpha-helix of the M6 segment. In addition, we found that amino acids involved in the phosphorylation are located exclusively in the cytoplasmic half of the M6 segment and those involved in the K(+)-dependent dephosphorylation are in the luminal half. Several mutants such as I821A, L823A, T825A, and P829A partly retained the K(+)-ATPase activity accompanying the decrease in the rate of phosphorylation.