Specific Sites in the Cytoplasmic N Terminus Modulate Conformational Transitions of the Na,K-ATPase (original) (raw)
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The Na,K-ATPase is a major ion-motive ATPase of the P-type family responsible for many aspects of cellular homeostasis. To determine the structure of the pathway for cations across the transmembrane portion of the Na,K-ATPase, we mutated 24 residues of the fourth transmembrane segment into cysteine and studied their function and accessibility by exposure to the sulfhydryl reagent 2-aminoethyl-methanethiosulfonate. Accessibility was also examined after treatment with palytoxin, which transforms the Na,K-pump into a cation channel. Of the 24 tested cysteine mutants, seven had no or a much reduced transport function. In particular cysteine mutants of the highly conserved "PEG" motif had a strongly reduced activity. However, most of the non-functional mutants could still be transformed by palytoxin as well as all of the functional mutants. Accessibility, determined as a 2-aminoethyl-methanethiosulfonate-induced reduction of the transport activity or as inhibition of the membrane conductance after palytoxin treatment, was observed for the following positions: Phe 323 , Ile 322 , Gly 326 , Ala 330 , Pro 333 , Glu 334 , and Gly 335 . In accordance with a structural model of the Na,K-ATPase obtained by homology modeling with the two published structures of sarcoplasmic and endoplasmic reticulum calcium ATPase (Protein Data Bank codes 1EUL and 1IWO), the results suggest the presence of a cation pathway along the side of the fourth transmembrane segment that faces the space between transmembrane segments 5 and 6. The phenylalanine residue in position 323 has a critical position at the outer mouth of the cation pathway. The residues thought to form the cation binding site II ( 333 PEGL) are also part of the accessible wall of the cation pathway opened by palytoxin through the Na,K-pump.
Biochemistry, 2004
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 R 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 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 R 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.
Journal of Biological Chemistry, 1996
The ␣2 isoform of the Na,K-ATPase exhibits kinetic behavior distinct from that of the ␣1 isoform. The distinctive behavior is apparent when the reaction is carried out under conditions (micromolar ATP concentration) in which the K ؉ deocclusion pathway of the reaction cycle is rate-limiting; the ␣1 activity is inhibited by K ؉ , whereas ␣2 is stimulated. When 32 NH 2terminal amino acid residues are removed from ␣1, the kinetic behavior of the mutant enzyme (␣1M32) is similar to that of ␣2 (
Journal of Biological Chemistry, 2003
The -subunit of the Na,K-ATPase is required to deliver functional ␣؊heterodimers to the plasma membrane (PM) of baculovirus-infected insect cells. We have investigated the molecular determinants in the -subunit for the assembly and delivery processes. Trafficking of both subunits was analyzed by Western blots of fractionated membranes enriched in endoplasmic reticulum (ER), Golgi, and PM. Heterodimer assembly was evaluated by co-immunoprecipitation, and enzymatic activity was measured by ATPase assay. Elimination of enzymatic activity by D369A point mutation of the ␣-subunit had no effect on the compartmental distribution of the Na,K-ATPase, demonstrating that enzymatic functioning is not a prerequisite for PM delivery. Replacement of all three N-glycosylation site asparagines with glutamines produced no effect on the delivery to the PM or the activity of the enzyme, but increased susceptibility to degradation was observed. Analysis of -subunits in which the disulfide bonds were removed through substitution reveals that the bridges are important for PM targeting but not for assembly of the heterodimer. Assembly is supported by -subunits with greatly truncated extracellular domains. The presence of the amino-terminal domain and transmembrane segment is sufficient for assembly and PM delivery. Intermediate length truncated -subunits and some disulfide bridge substitution mutants assemble with the ␣-subunit but are not able to exit the ER. We conclude that there are different and separable requirements for the assembly of Na,K-ATPase heterodimer complexes, exit of the dimer from the ER, delivery to the PM, and catalytic activity of the dimer.
Journal of Biological Chemistry, 2008
The -subunits of Na,K-ATPase and H,K-ATPase have important functions in maturation and plasma membrane targeting of the catalytic ␣-subunit but also modulate the transport activity of the holoenzymes. In this study, we show that tryptophan replacement of two highly conserved tyrosines in the transmembrane domain of both Na,K-and gastric H,K-ATPase -subunits resulted in considerable shifts of the voltagedependent E 1 P/E 2 P distributions toward the E 1 P state as inferred from presteady-state current and voltage clamp fluorometric measurements of tetramethylrhodamine-6-maleimidelabeled ATPases. The shifts in conformational equilibria were accompanied by significant decreases in the apparent affinities for extracellular K ؉ that were moderate for the Na,K-ATPase -(Y39W,Y43W) mutation but much more pronounced for the corresponding H,K-ATPase -(Y44W,Y48W) variant. Moreover in the Na,K-ATPase -(Y39W,Y43W) mutant, the apparent rate constant for reverse binding of extracellular Na ؉ and the subsequent E 2 P-E 1 P conversion, as determined from transient current kinetics, was significantly accelerated, resulting in enhanced Na ؉ competition for extracellular K ؉ binding especially at extremely negative potentials. Analogously the reverse binding of extracellular protons and subsequent E 2 P-E 1 P conversion was accelerated by the H,K-ATPase -(Y44W,Y48W) mutation, and H ؉ secretion was strongly impaired. Remarkably tryptophan replacements of residues in the M7 segment of Na,K-and H,K-ATPase ␣-subunits, which are at interacting distance to the -tyrosines, resulted in similar E 1 shifts, indicating their participation in stabilization of E 2. Thus, interactions between selected residues within the transmembrane regions of ␣and -subunits of P 2C-type ATPases exert an E 2-stabilizing effect, which is of particular importance for efficient H ؉ pumping by H,K-ATPase under in vivo conditions. * This work was supported by the Max-Planck-Gesellschaft zur Fö rderung der Wissenschaften e.V. and the Deutsche Forschungsgemeinschaft (Grants SFB 472 and SFB 740 and Cluster of Excellence "Unifying Concepts in Catalysis").
ATP hydrolysis by Na ؉ /K ؉ -ATPase proceeds via the interaction of simultaneously existing and cooperating high (E 1 ATP) and low (E 2 ATP) substrate binding sites. It is unclear whether both ATP sites reside on the same or on different catalytic ␣-subunits. To answer this question, we looked for a fluorescent label for the E 2 ATP site that would be suitable for distance measurements by Fö rster energy transfer after affinity labeling of the E 1 ATP site by fluorescein 5-isothiocyanate (FITC). Erythrosin 5-isothiocyanate (ErITC) inactivated, in an E 1 ATP site-blocked enzyme (by FITC), the residual activity of the E 2 ATP site, namely K ؉ -activated p-nitrophenylphosphatase in a concentration-dependent way that was ATP-protectable. The molar ratios of FITC/␣-subunit of 0.6 and of ErITC/␣-subunit of 0.48 indicate 2 ATP sites per (␣) 2 diprotomer. Measurements of Fö rster energy transfer between the FITC-labeled E 1 ATP and the ErITC-labeled or Co(NH 3 ) 4 ATP-inactivated E 2 ATP sites gave a distance of 6.45 ؎ 0.64 nm. This distance excludes 2 ATP sites per ␣-subunit since the diameter of ␣ is 4 -5 nm. Fö rster energy transfer between cardiac glycoside binding sites labeled with anthroylouabain and fluoresceinylethylenediamino ouabain gave a distance of 4.9 ؎ 0.5 nm. Hence all data are consistent with the hypothesis that Na ؉ /K ؉ -ATPase in cellular membranes is an (␣) 2 diprotomer and works as a functional dimer (Thoenges, D., and Schoner, W. (1997) J. Biol. Chem. 272, 16315-16321).
Journal of Biological Chemistry, 1998
Mutations comprising either deletion of 32 amino acids from the NH 2 terminus (␣1M32) or a Glu 233 3 Lys substitution in the first M2-M3 cytoplasmic loop (E233K) of the ␣1-subunit of the Na,K-ATPase result in a shift in the steady-state E 1 7 E 2 conformational equilibrium toward E 1 form(s). In the present study, the functional consequences of both NH 2-terminal deletion and Glu 233 substitution provide evidence for mutual interactions of these cytoplasmic regions. Following transfection and selection of HeLa cells expressing the ouabain-resistant ␣1M32E233K double mutant, growth was markedly reduced unless the K ؉ concentration in the culture medium was increased to at least 10 mM. Marked changes effected by this double mutation included 1) a 15-fold reduction in catalytic turnover (V max /EP max), 2) a 70-fold increase in apparent affinity for ATP, 3) a marked decrease in vanadate sensitivity, and 4) marked (Ϸ10-fold) K ؉ activation of the Na-ATPase activity measured at micromolar ATP under which condition the E 2 (K) 3 3 E 1 pathway is normally (␣1) rate-limiting and K ؉ is inhibitory. The decrease in catalytic turnover was associated with a 5-fold decrease in V max and a compensatory Ϸ3-fold increase in expressed ␣1M32E233K protein. In contrast to the behavior of either ␣1M32 or E233K, ␣1M32E233K also showed alterations in apparent cation affinities. K Na was decreased Ϸ2-fold and K K was increased Ϸ2-fold. The importance of the charge at residue 233 is underscored by the consequences of single and double mutations comprising either a conservative change (E233D) or neutral substitution (E233Q). Thus, whereas mutation to a positively charged residue (E233K) causes a drastic change in enzymatic behavior, a conservative change causes only a minor change and the neutral substitution, an intermediate effect. Overall, the combined effects of the NH 2-terminal deletion and the Glu 233 substitutions are synergistic rather than additive, consistent with an interaction between the NH 2terminal region, the first cytoplasmic loop, and possibly the large M4-M5 cytoplasmic loop bearing the nucleotide binding and phosphorylation sites.
Interactions between Na,K-ATPase alpha -Subunit ATP-binding Domains
Journal of Biological Chemistry, 2003
The reaction mechanism of the Na,K-ATPase is thought to involve a number of ligand-induced conformational changes. The specific amino acid residues responsible for binding many of the important ligands have been identified; however, details of the specific conformational changes produced by ligand binding are largely undescribed. The experiments described in this paper begin to identify interactions between domains of the Na,K-ATPase ␣-subunit that depend on the presence of particular ligands. The major cytoplasmic loop (between TM4 and TM5), which we have previously shown contains the ATP-binding domain, was overexpressed in bacteria either with a His 6 tag or as a fusion protein with glutathione S-transferase. We have observed that these polypeptides associate in the presence of MgATP. Incubation with [␥-32 P]ATP under conditions that result in phosphorylation of the full-length Na,K-ATPase did not result in 32 P incorporation into either the His 6 tag or glutathione S-transferase fusion proteins. The MgATPinduced association was strongly inhibited by prior modification of the fusion proteins with fluorescein isothiocyanate or by simultaneous incubation with 10 M eosin, indicating that the effect of MgATP is due to interactions within the nucleotide-binding domain. These data are consistent with Na,K-ATPase associating within cells via interactions in the nucleotide-binding domains. Although any functional significance of these associations for ion transport remains unresolved, they may play a role in cell function and in modulating interactions between the Na,K-ATPase and other proteins.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1993
The Na+/K+-ATPase couples the hydrolysis of ATP to the transport of Na + and K ÷ via a phosphorylated intermediate and conformational changes. In order to identify these conformational changes, we have probed the sequence of steps from EP{3Na~} to EP + 3Na+out with three fluorescent probes (IAF: 5-iodoacetamidofluorescein; BIPM: N-[p-(2-benzimidazolyl) phenyl]maleimide; and RH421) and the sensitivity of their fluorescence changes to oligomycin and divalent cations (Ca 2÷ and Mn2+). The magnitude (%AF) and rate constant (kob s) of ATP-induced fluorescence changes were measured on a fluorescence stopped-flow apparatus, and yielded the following results. (a) With RH421, kobs and %AF varied with [Na ÷] (maximal kob s = 100 S-1, KI/2 = 6 mM; %AFmax = 6%, K1/2-1 mM); these values are comparable to those previously reported using IAF-labeled enzyme (