Genetic suppression of agrin reduces mania-like behavior in Na+ , K+ -ATPase α3 mutant mice.

Myshkin mice heterozygous for an inactivating mutation in the neuron-specific Na(+) ,K(+) -ATPase α3 isoform show behavior analogous to mania, including an abnormal endogenous circadian period. Agrin is a proteoglycan implicated as a regulator of synapses that has been proposed to inhibit activity of Na(+) ,K(+) -ATPase α3. We examined whether the mania-related behavior of Myshkin mice could be rescued by a reduction in the expression of agrin through genetic knockout. The suppression of agrin reduced hyperambulation and holeboard exploration, restored anxiety-like behavior (or reduced risk-taking behavior), improved prepulse inhibition and shortened the circadian period. Hence, agrin is important for regulating mania-like behavior and circadian rhythms. In Myshkin mice, the suppression of agrin increased brain Na(+) ,K(+) -ATPase activity by 11 ± 4%, whereas no effect on Na(+) ,K(+) -ATPase activity was detected when agrin was suppressed in mice without the Myshkin mutation. These results introduce agrin as a potential therapeutic target for the treatment of mania and other neurological disorders associated with reduced Na(+) ,K(+) -ATPase activity and neuronal hyperexcitability.

Decreased neuronal Na+, K+ -ATPase activity in Atp1a3 heterozygous mice increases susceptibility to depression-like endophenotypes by chronic variable stress.

Unipolar depression and bipolar depression are prevalent and debilitating diseases in need of effective novel treatments. It is becoming increasingly evident that depressive disorders manifest from a combination of inherited susceptibility genes and environmental stress. Genetic mutations resulting in decreased neuronal Na(+) ,K(+) -ATPase (sodium-potassium adenosine triphosphatase) activity may put individuals at risk for depression given that decreased Na(+) ,K(+) -ATPase activity is observed in depressive disorders and animal models of depression. Here, we show that Na(+) ,K(+) -ATPase α3 heterozygous mice (Atp1a3(+/-) ), with 15% reduced neuronal Na(+) ,K(+) -ATPase activity, are vulnerable to develop increased depression-like endophenotypes in a chronic variable stress (CVS) paradigm compared to wild-type littermates (Atp1a3(+/+) ). In Atp1a3(+/+) mice CVS did not decrease Na(+) ,K(+) -ATPase activity, however led to despair-like behavior in the tail suspension test (TST), anhedonia in a sucrose preference test and a minimal decrease in sociability, whereas in Atp1a3(+/-) mice CVS decreased neuronal Na(+) ,K(+) -ATPase activity to 33% of wild-type levels, induced despair-like behavior in the TST, anhedonia in a sucrose preference test, anxiety in the elevated plus maze, a memory deficit in a novel object recognition task and sociability deficits in a social interaction test. We found that a mutation that decreases neuronal Na(+) ,K(+) -ATPase activity interacts with stress to exacerbate depression. Furthermore, we observed an interesting correlation between Na(+) ,K(+) -ATPase activity and mood that may relate to both unipolar depression and bipolar disorder. Pharmaceuticals that increase Na(+) ,K(+) -ATPase activity or block endogenous Na(+) , K(+) -ATPase inhibition may provide effective treatment for depressive disorders and preclude depression in susceptible individuals.

ATP binding residues of sarcoplasmic reticulum Ca2+-ATPase.

ATP-binding residues in the N and P domains of sarcoplasmic reticulum Ca-ATPase have been investigated using mutagenesis in combination with a binding assay based on the photolabeling of Lys(492) with [g-(32)P] 2',3'-O-(2,4,6 trinitrophenyl)-8-azido-ATP and competition with nucleotide. In the N domain, mutations to several residues in conserved motifs, (438)GEATE, (487)FSRDRK, (515)KGAPE, and (560)RCLALA produce nucleotide-binding defects. Key residues include Thr(441), Glu(442), Phe(487), Arg(489), Lys(492), Lys(515), Arg(560), and Leu(562). In the absence of Mg(2+), Arg(489), Lys(492), and Arg(560) are most important, whereas in its presence Thr(441) and Glu(442) also play a crucial role. In the P domain, Asp(351) is striking for its strong electrostatic repulsion of the gamma-phosphate, especially in the presence of Mg(2+). Lys(352) is a key residue, and Asp(627) and Lys(684) must come close to the nucleotide. Thr(353), Asn(359), Asp(601), and Asp(703) interact only in the presence of Mg(2+). Asn(706) and Asp(707) are unimportant for nucleotide binding. The results identify several ATP binding residues in the N and P domains and suggest that Mg(2+) changes the nucleotide/protein interaction in both. Models of bound ATP and MgATP are presented.

Mutational effects on conformational changes of the dephospho- and phospho-forms of the Na+,K+-ATPase.

Gly263 of the rat kidney Na(+),K(+)-ATPase is highly conserved within the family of P-type ATPases. Mutants in which Gly263 or the juxtaposed Arg264 had been replaced by alanine were expressed at high levels in COS-1 cells and characterized functionally. Titrations of Na(+),K(+), ATP, and vanadate dependencies of Na(+),K(+)-ATPase activity showed changes in the apparent affinities relative to wild-type compatible with a displacement of the E(1)-E(2) conformational equilibrium in favor of E(1). The level of the K(+)-occluded form was reduced in the Gly263-->Ala and Arg264-->Ala mutants, and the rate constant characterizing deocclusion of K(+) or Rb(+) was increased as much as 20-fold in the Gly263-->Ala mutant. Studies of the sensitivity of the phosphoenzyme to K(+) and ADP showed a displacement of the E(1)P-E(2)P equilibrium of the phosphoenzyme in favor of E(1)P, and dephosphorylation experiments carried out at 25 degrees C on a millisecond time scale using a quenched-flow technique demonstrated a reduction of the E(1)P to E(2)P conversion rate in the mutants. Hence, the mutations displaced the conformational equilibria of dephosphoenzyme and phosphoenzyme in parallel in favor of the E(1) and E(1)P forms. The observed effects were more pronounced in the Gly263-->Ala mutant compared with the Arg264-->Ala mutant. Leu332 mutations that likewise displaced the conformational equilibria in favor of E(1) and E(1)P were also studied. Unlike the Gly263-->Ala mutant the Leu332 mutants displayed a wild-type like rate of K(+) deocclusion. Thus, the effect of the Gly263 mutation on the E(1)-E(2) conformational equilibrium seems to be caused mainly by an acceleration of the K(+)-deoccluding step, whereas in the Leu332 mutants the rate of the reverse reaction seems to be reduced.

Importance of transmembrane segment M3 of the sarcoplasmic reticulum Ca2+-ATPase for control of the gateway to the Ca2+ sites.

The specific functional roles of various parts of the third transmembrane segment (M3) of the sarcoplasmic reticulum Ca(2+)-ATPase were examined by functionally characterizing a series of mutants with multiple or single substitutions of M3 residues. Steady-state and transient kinetic measurements, assisted by computer simulation of the time and Ca(2+) dependences of the phosphorylation level, were used to study the partial reaction steps of the enzyme cycle, including the binding and dissociation of Ca(2+) at the high affinity cytoplasmically facing sites. The mutation Lys-Leu-Asp-Glu(255) --> Glu-Ile-Glu-His resulted in a conspicuous increase in the rate of Ca(2+) dissociation as well as a displacement of the major conformational equilibria of the phosphoenzyme and dephosphoenzyme forms. The point mutant Phe(256) --> Ala also showed an increased rate of Ca(2+) dissociation, whereas a conspicuous decrease both in the rate of Ca(2+) dissociation and in the rate of Ca(2+) binding was found for the mutant Gly-Glu-Gln-Leu(260) --> Ile-His-Leu-Ile. These findings suggest that the NH(2)-terminal half of M3 is involved in control of the gateway to the Ca(2+) sites. The main effect of two mutations to the COOH-terminal half of M3, Ser-Lys-Val-Ile-Ser(265) --> Thr-Gly-Val-Ala-Val and Leu-Ile-Cys-Val-Ala-Val-Trp-Leu-Ile(274) --> Phe-Leu-Gly-Val-Ser-Phe-Phe-Ile-Leu, was a block of the dephosphorylation.

Fast kinetic analysis of conformational changes in mutants of the Ca(2+)-ATPase of sarcoplasmic reticulum.

Rapid quench experiments at 25 degrees C were carried out on selected mutants of the sarco(endo)plasmic reticulum Ca(2+)-ATPase to assess the kinetics of the conformational changes of the dephosphoenzyme associated with ATP binding/phosphoryl transfer and the binding and dissociation of Ca(2+) at the cytoplasmically facing transport sites. The mutants Gly(233) --> Glu, Gly(233) --> Val, Pro(312) --> Ala, Leu(319) --> Arg, and Lys(684) --> Arg differed conspicuously with respect to the behavior of the dephosphoenzyme, although they were previously shown to display a common block of the transformation of the phosphoenzyme from an ADP-sensitive to an ADP-insensitive form. The maximum rate of the ATP binding/phosphoryl transfer reaction was reduced 3.6-fold in mutant Gly(233) --> Glu and more than 50-fold in mutant Lys(684) --> Arg, relative to wild type. In mutant Leu(319) --> Arg, the rate of the Ca(2+)-binding transition was reduced as much as 10-30-fold depending on the presence of ATP. In mutants Gly(233) --> Glu, Gly(233) --> Val, and Pro(312) --> Ala, the rate of the Ca(2+)-binding transition was increased at least 2-3-fold at acid pH but not significantly at neutral pH, suggesting a destabilization of the protonated form. The rate of Ca(2+) dissociation was reduced 12-fold in mutant Pro(312) --> Ala and 3.5-fold in Leu(319) --> Arg, and increased at least 4-fold in a mutant in which the putative Ca(2+) liganding residue Glu(309) was replaced by aspartate. The data support a model in which Pro(312) and Leu(319) are closely associated with the cation binding pocket, Gly(233) is part of a long-range signal transmission pathway between the ion-binding sites and the catalytic site, and Lys(684) is an essential catalytic residue that may function in the same way as its counterpart in the soluble hydrolases belonging to the haloacid dehalogenase superfamily.

Mutant Phe788 --> Leu of the Na+,K+-ATPase is inhibited by micromolar concentrations of potassium and exhibits high Na+-ATPase activity at low sodium concentrations.

Mutant Phe788 --> Leu of the rat kidney Na+,K(+)-ATPase was expressed in COS cells to active-site concentrations between 40 and 60 pmol/mg of membrane protein. Analysis of the functional properties showed that the discrimination between Na+ and K+ on the two sides of the system is severely impaired in the mutant. Micromolar concentrations of K+ inhibited ATP hydrolysis (K(0.5) for inhibition 107 microM for the mutant versus 76 mM for the wild-type at 20 mM Na+), and at 20 mM K+, the molecular turnover number for Na+,K(+)-ATPase activity was reduced to 11% that of the wild-type. This inhibition was counteracted by Na+ in high concentrations, and in the total absence of K+, the mutant catalyzed Na(+)-activated ATP hydrolysis ("Na(+)-ATPase activity") at an extraordinary high rate corresponding to 86% of the maximal Na+,K(+)-ATPase activity. The high Na(+)-ATPase activity was accounted for by an increased rate of K(+)-independent dephosphorylation. Already at 2 mM Na+, the dephosphorylation rate of the mutant was 8-fold higher than that of the wild-type, and the maximal rate of Na(+)-induced dephosphorylation amounted to 61% of the rate of K(+)-induced dephosphorylation. The cause of the inhibitory effect of K+ on ATP hydrolysis in the mutant was an unusual stability of the K(+)-occluded E2(K2) form. Hence, when E2(K2) was formed by K+ binding to unphosphorylated enzyme, the K(0.5) for K+ occlusion was close to 1 microM in the mutant versus 100 microM in the wild-type. In the presence of 100 mM Na+ to compete with K+ binding, the K(0.5) for K+ occlusion was still 100-fold lower in the mutant than in the wild-type. Moreover, relative to the wild-type, the mutant exhibited a 6-7-fold reduced rate of release of occluded K+, a 3-4-fold increased apparent K+ affinity in activation of the pNPPase reaction, a 10-11-fold lower apparent ATP affinity in the Na+,K(+)-ATPase assay with 250 microM K+ present (increased K(+)-ATP antagonism), and an 8-fold reduced apparent ouabain affinity (increased K(+)-ouabain antagonism).

Interaction of nucleotides with Asp(351) and the conserved phosphorylation loop of sarcoplasmic reticulum Ca(2+)-ATPase.

The nucleotide binding properties of mutants with alterations to Asp(351) and four of the other residues in the conserved phosphorylation loop, (351)DKTGTLT(357), of sarcoplasmic reticulum Ca(2+)-ATPase were investigated using an assay based on the 2', 3'-O-(2,4,6-trinitrophenyl)-8-azidoadenosine triphosphate (TNP-8N(3)-ATP) photolabeling of Lys(492) and competition with ATP. In selected cases where the competition assay showed extremely high affinity, ATP binding was also measured by a direct filtration assay. At pH 8.5 in the absence of Ca(2+), mutations removing the negative charge of Asp(351) (D351N, D351A, and D351T) produced pumps that bound MgTNP-8N(3)-ATP and MgATP with affinities 20-156-fold higher than wild type (K(D) as low as 0.006 microM), whereas the affinity of mutant D351E was comparable with wild type. Mutations K352R, K352Q, T355A, and T357A lowered the affinity for MgATP and MgTNP-8N(3)-ATP 2-1000- and 1-6-fold, respectively, and mutation L356T completely prevented photolabeling of Lys(492). In the absence of Ca(2+), mutants D351N and D351A exhibited the highest nucleotide affinities in the presence of Mg(2+) and at alkaline pH (E1 state). The affinity of mutant D351A for MgATP was extraordinarily high in the presence of Ca(2+) (K(D) = 0.001 microM), suggesting a transition state like configuration at the active site under these conditions. The mutants with reduced ATP affinity, as well as mutants D351N and D351A, exhibited reduced or zero CrATP-induced Ca(2+) occlusion due to defective CrATP binding.

Structure-function relationships of the calcium binding sites of the sarcoplasmic reticulum Ca(2+)-ATPase.

Site-directed mutagenesis studies of the structure and function of the Ca2+ binding sites of the sarcoplasmic reticulum Ca(2+)-ATPase are reviewed. The Ca2+ binding properties of six mutants with alterations to amino acid residues with oxygen-containing side chains in the membrane segments M4, M5, M6, and M8 were investigated. The mutations to Glu309 in M4, Glu771 in M5, Asn796, Thr799, and Asp800 in M6 all disrupted Ca2+ occlusion, suggesting that the side chains of these residues donate oxygen ligands to Ca2+ binding at the high-affinity sites and/or are involved in conformational changes that occlude the sites. Alanine substitution of Glu908 in transmembrane segment M8 did not prevent Ca2+ occlusion, thereby excluding this residue from playing a central role in Ca2+ coordination. Titrations of Ca2+ activation of phosphorylation from ATP and of inhibition by Ca2+ of phosphorylation from Pi allowed us to assign Ca2+ liganding residues separately to the two high-affinity Ca2+ sites. Hence, residues Glu771 and Thr799 are associated with the site binding the first calcium ion in the sequential mechanism ("site 1"), whereas Glu309 and Asn796 are associated with the site binding the second calcium ion ("site 2"), and Asp800 donates Ca2+ ligands to both sites. On this basis we discuss two possible structural models for the Ca2+ sites.

Mutation to the glutamate in the fourth membrane segment of Na+,K+-ATPase and Ca2+-ATPase affects cation binding from both sides of the membrane and destabilizes the occluded enzyme forms.

The functional consequences of mutations Glu329 --> Gln in the Na+,K+-ATPase and Glu309 --> Asp in the sarco(endo)plasmic reticulum Ca2+-ATPase were analyzed and compared. Relative to the wild-type Na+,K+-ATPase, the Glu329 --> Gln mutant exhibited a 20-fold reduction in the apparent K+ affinity determined by titration of the rate of ATP hydrolysis at 50 microM ATP, and the rate of release of occluded K+ or Rb+ to the cytoplasmic side of the membrane was up to 30-fold enhanced by the mutation, as measured in kinetic studies of the phosphorylation by ATP of enzyme equilibrated with K+ or Rb+. The apparent affinity for extracellular K+ was 12-fold reduced by the Glu329 --> Gln mutation, as determined by K+ titration of the dephosphorylation. The maximum rate of phosphorylation by ATP of the Na+ form of the enzyme was reduced more than 2-fold by the mutation, but this effect could be counteracted by stabilizing Na+ occlusion with oligomycin. Similar studies on the Glu309 --> Asp mutant of the Ca2+-ATPase showed that the maximum rate of phosphorylation of the Ca2+ form was 8-9-fold reduced relative to that of the wild-type Ca2+-ATPase, and no Ca2+ occlusion could be detected in the mutant. Dephosphorylation of the phosphoenzyme intermediate formed with Pi was blocked in the Ca2+-ATPase mutant. The sensitivity to inhibition by thapsigargin, which binds selectively to the putative proton-occluded form of the Ca2+-ATPase, was reduced almost 300-fold in the mutant at neutral pH, but only 3-4-fold at pH 6.0. These data indicate that the mutations destabilize the occluded enzyme forms and interfere with cation binding from the extracytoplasmic side as well as with the gating process at the cytoplasmic entrance to the cation occlusion pocket.

Mutagenesis of Sarcoplasmic Reticulum Ca(2+)-ATPase.

Ca(2+)-ATPases of sarco(endo)plasmic reticulum utilize energy derived from hydrolysis of ATP to drive active uptake of calcium ions. Site-directed mutagenesis analysis of Ca(2+)-ATPase structure-function relationships has identified protein domains and single amino acid residues involved in the binding and occlusion of the calcium ions during translocation, as well as amino acid residues crucial to the energy-transducing intramolecular signaling that links events in the catalytic ATP hydrolysis site with rearrangements in the calcium sites. The interaction between the cardiac muscle sarcoplasmic reticulum Ca(2+)-ATPase and the regulatory protein phospholamban, which mediates the activation of Ca(2+) transport by ß-agonists, has also been elucidated by mutagenesis.

Mutation Lys758 --> Ile of the sarcoplasmic reticulum Ca2+-ATPase enhances dephosphorylation of E2P and inhibits the E2 to E1Ca2 transition.

The highly conserved lysine residue Lys758 in the fifth stalk segment of the sarcoplasmic reticulum Ca2+-ATPase was substituted with either isoleucine or arginine by site-directed mutagenesis. The substitution with arginine was without significant effects on Ca2+-ATPase function, whereas multiple changes of functional characteristics were observed with the Lys758 --> Ile mutant. These included insensitivity of ATPase activity to the calcium ionophore A23187, an alkaline shift of the pH dependence of ATPase activity, reduced maximum molecular turnover rate and steady-state phosphorylation level, reduced apparent affinities for Ca2+ and inorganic phosphate, as well as increased sensitivity to inhibition by vanadate. Analysis of the partial reaction steps of the enzyme cycle traced these changes to two steps. The rate of dephosphorylation of the ADP-insensitive phosphoenzyme intermediate (E2P) was increased, irrespective of variations of pH, K+, Ca2+, and dimethyl sulfoxide concentration. In addition, the rate of conversion of the dephosphoenzyme with low Ca2+ affinity (E2) to the Ca2+-bound form activated for phosphorylation (E1Ca2) was reduced in the mutant, and the ATP-induced rate enhancement of this step required higher ATP concentrations in the mutant compared with the wild type.

Leucine 332 at the boundary between the fourth transmembrane segment and the cytoplasmic domain of Na+,K+-ATPase plays a pivotal role in the ion translocating conformational changes.

Mutants Gly330-->Ala, Leu332-->Ala, Leu332-->Pro, and Pro780-->Ala of the alpha1-isoform of rat kidney Na+,K+-ATPase were expressed in COS-1 cells to active site concentrations between 20 and 70 pmol per mg of membrane protein. The functional properties of the mutants were characterized by titrations of Na+-, K+-, and ATP-dependencies of Na+,K+-ATPase activity as well as by a series of assays measuring the K+-dependence of the steady-state phosphoenzyme level, the kinetics of dephosphorylation under a variety of conditions, and the ADP-ATP exchange activity. In mutants Gly330-->Ala, Leu332-->Ala, and Leu332-->Pro, the molecular turnover number was reduced relative to that of the wild-type Na+,K+-ATPase, and the steady-state phosphoenzyme level was high even in the presence of several millimolar K+. At a low Na+ concentration in the absence of K+, mutants Leu332-->Pro and Gly330-->Ala displayed high ADP-ATP exchange activity and formed a high level of the ADP-sensitive phosphoenzyme (E1P). The phosphoenzyme decayed slowly following a jump in salt concentration and chase with ATP and K+. Hence, the conversion of E1P to the K+-sensitive phosphoenzyme (E2P) was inhibited in mutants Leu332-->Pro and Gly330-->Ala. In the Leu332-->Ala mutant, a high level of E2P was accumulated in the absence of K+, and the ADP-ATP exchange activity was low at low Na+ concentration in the absence of K+, but rose sharply on addition of K+. Dephosphorylation experiments indicated that in the Leu332-->Ala mutant K+ induced reversal of the phosphoenzyme interconversion, forming E1P from E2P. Leu332 is therefore a pivotal residue in the conformational change. Mutants Gly330-->Ala and Pro780-->Ala displayed reduced K+ affinities relative to the wild type, determined in three independent assays.

Mutagenesis of segment 487Phe-Ser-Arg-Asp-Arg-Lys492 of sarcoplasmic reticulum Ca2+-ATPase produces pumps defective in ATP binding.

The lysine residue Lys492 located in the large cytoplasmic domain of sarcoplasmic reticulum Ca2+-ATPase is implicated in nucleotide binding through affinity labeling. The contribution of segment 487Phe-Ser-Arg-Asp-Arg-Lys492 to ATP binding and pump function has been investigated through the introduction of 11 site-directed amino acid mutations. ATP binding was measured through competitive inhibition of [gamma-32P]2',3'-O-(2,4, 6-trinitrophenyl)-8-azido-adenosine triphosphate photolabeling of Lys492 or its substitute. Mutations F487S and positional swap F487S/S488F produced pumps that were severely defective in ATP binding (KD > 1 mM), and mutant F487S, together with F487E, exhibited low ATPase activity and low ATP-supported calcium transport and phosphorylation and failed to show CrATP-dependent Ca2+ occlusion. Mutations F487L, R489L, and K492Y were less inhibitory to ATP binding (KD = 8-49 microM) and, together with K492L and R489D/D490R, produced correspondingly smaller changes in ATP-mediated activities. The ATP dependence of ATPase activity of these five mutants showed deviations from the wild-type profile in the low, intermediate, and high concentration ranges, suggesting defects in ATP-dependent conformational changes. Mutations S488A and D490A had no effect on ATP binding (KD = 0.4 microM) or ATP-mediated activities. None of the mutations significantly affected phosphorylation from Pi or acetyl phosphate-supported Ca2+ transport. Mutations F487L and F487S, and not those at residue 492, increased the K0.5 for Ca2+ activation of transport 2- and 8-fold, respectively. The results implicate Phe487, Arg489, and Lys492 in binding ATP in both a catalytic and a regulatory mode.

Functional consequences of mutation Asn326-->Leu in the 4th transmembrane segment of the alpha-subunit of the rat kidney Na+, K(+)-ATPase.

Site-specific mutagenesis was used to replace Asn326 in transmembrane segment M4 of the ouabain-insensitive alpha 1-isoform of rat kidney Na+, K(+)-ATPase. Mutant Asn326-->Leu was functional as demonstrated by the ability of COS cells expressing the mutant enzyme to grow in the presence of ouabain. In three independent assays encompassing Na+ titrations of Na+,K(+)-ATPase activity, Na(+)-ATPase activity, and phosphorylation from ATP, the Asn326-->Leu mutant displayed a reduced apparent affinity for Na+. By contrast, this mutant exhibited a slightly increased apparent affinity for K+ relative to the wild-type enzyme. In the presence of Na+ without K+, the Asn326-->Leu mutant hydrolyzed ATP at a high rate corresponding to 32% of the maximal Na+,K(+)-ATPase activity, and the rate of dephosphorylation of the phosphoenzyme intermediate was enhanced in the mutant relative to that of the wild-type enzyme. Oligomycin, known to stabilize the Na(+)-occluded phosphoenzyme intermediate, reduced the dephosphorylation rate of the mutant and increased the steady-state phosphoenzyme level formed by the mutant at least 3-fold, whereas an increase in the steady-state phosphoenzyme level of only 10-15% was determined for the wild-type enzyme. The molecular turnover number for the Na+,K(+)-ATPase reaction, calculated when the steady-state phosphoenzyme level obtained in the presence of oligomycin was taken as a measure of the concentration of active sites, was slightly reduced relative to that of the wild-type enzyme. The data are discussed in terms of a role for Asn326 in binding of cytoplasmic Na+ and in mediation of inhibition of dephosphorylation.

Structure-function relationships of cation translocation by Ca(2+)- and Na+, K(+)-ATPases studied by site-directed mutagenesis.

Site-directed mutagenesis studies of the sarcoplasmic reticulum Ca(2+)-ATPase have pinpointed five amino acid residues that are essential to Ca2+ occlusion, and these residues have been assigned to different parts of a Ca2+ binding pocket with channel-like structure. Three of the homologous Na+, K(+)-ATPase residues have been shown to be important for binding of cytoplasmic Na+ at transport sites. In addition, three of the above mentioned Ca(2+)-ATPase residues appear to participate in the countertransport of H+, and two of the Na+, K(+)-ATPase residues to participate in the countertransport of K+. Residues involved in energy transducing conformational changes have also been identified by mutagenesis. In the Ca(2+)-ATPase, ATP hydrolysis is uncoupled from Ca2+ transport following mutation of a tyrosine residue located at the top of transmembrane segment M5. This tyrosine, present also in the Na+, K(+)-ATPase, may play a critical role in closing the gate to a transmembrane channel.

Mutant Glu781-->Ala of the rat kidney Na+,K(+)-ATPase displays low cation affinity and catalyzes ATP hydrolysis at a high rate in the absence of potassium ions.

Site-specific mutagenesis was used to replace Glu329, Glu781, Asp806, Thr809, and Asp810 in the transmembrane domain of the ouabain-insensitive alpha 1-isoform of rat kidney Na+,K(+)-ATPase. cDNAs encoding any of the mutants Glu329-->Ala, Glu781-->Ala, Asp806-->Asn, Thr809-->Ala, and Asp810-->Asn were transfected into COS cells, and transfectants were grown in the presence of ouabain to inhibit the endogenous COS cell Na+,K(+)-ATPase. Mutants Glu781-->Ala and Thr809-->Ala were functional as evidenced by their ability to confer ouabain resistance to the cells, whereas mutants Glu329-->Ala, Asp806-->Asn, and Asp810-->Asn were inactive. The apparent Na+ affinities determined by titrations of Na+,K(+)-ATPase activity, Na(+)-ATPase activity, and phosphorylation from ATP in mutants Glu781-->Ala and Thr809-->Ala were strongly reduced relative to the affinity of the wild type (6-8-fold increase in K0.5 for Na+ in the phosphorylation assay for both mutants). The Glu781-->Ala mutant displayed a 3-4-fold reduction in the apparent affinity for K+ and was able to hydrolyze ATP at a high rate in the absence of K+ (Vmax for Na(+)-ATPase activity 5-fold higher than that of the wild-type enzyme). The steady-state phosphoenzyme level formed by the Glu781-->Ala mutant was increased 3-fold by addition of oligomycin, whereas only a slight effect of oligomycin was observed for mutant Thr809-->Ala and the wild type.(ABSTRACT TRUNCATED AT 250 WORDS)