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Originally published In Press as doi:10.1074/jbc.M604781200 on October 20, 2006

J. Biol. Chem., Vol. 281, Issue 50, 38285-38292, December 15, 2006
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Potassium as an Intrinsic Uncoupler of the Plasma Membrane H+-ATPase*Formula

Morten J. Buch-Pedersen1, Elena L. Rudashevskaya, Torben S. Berner, Kees Venema, and Michael G. Palmgren

From the Department of Plant Biology, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark

Received for publication, May 18, 2006 , and in revised form, September 18, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The plant plasma membrane proton pump (H+-ATPase) is stimulated by potassium, but it has remained unclear whether potassium is actually transported by the pump or whether it serves other roles. We now show that K+ is bound to the proton pump at a site involving Asp617 in the cytoplasmic phosphorylation domain, from where it is unlikely to be transported. Binding of K+ to this site can induce dephosphorylation of the phosphorylated E1P reaction cycle intermediate by a mechanism involving Glu184 in the conserved TGES motif of the pump actuator domain. Our data identify K+ as an intrinsic uncoupler of the proton pump and suggest a mechanism for control of the H+/ATP coupling ratio. K+-induced dephosphorylation of E1P may serve regulatory purposes and play a role in negative regulation of the transmembrane electrochemical gradient under cellular conditions where E1P is accumulating.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
P-type ATPases constitute a large ubiquitous family of cation pumps characterized by forming a phosphorylated intermediate as part of their catalytic cycle. In most organisms, except plants (1–3), members of this family are involved in active transport of K+ (4, 5). Examples of P-type K+ pumps are Kdp ATPases from bacterial systems (6), K+-ATPases in fungi (7), and animal Na+/K+- and H+/K+-ATPases (8, 9), which exchange K+ with either Na+ or H+. The main primary transporter in the plant plasma membrane is the P-type plasma membrane H+-ATPase, an electrogenic proton pump that generates the plasma membrane electrochemical gradient of H+ and the ATP hydrolytic activity of which is stimulated by K+ (10, 11). It has been speculated that the plasma membrane proton pump is an H+/K+ counterion ATPase mechanistically similar to the animal Na+/K+-, H+/K+-, and Ca2+/H+-ATPases (12, 13), but decisive proof for active K+ transport has not been obtained. It therefore seems plausible that K+ could serve other role(s) in the plasma membrane H+-ATPase.

During turnover, P-type ATPases alternate between different conformations that have very different structures. A simplified scheme operates with two basal conformations, Enzyme 1 (E1) and Enzyme 2(E2) (14, 15) (Fig. 1A). Phosphorylation from ATP of E1, in which an ion binding site in the membranous region is facing the cytoplasm, results in formation of a phosphorylated pump intermediate, E1P, that is spontaneously converted to E2P, in which the ion is now exposed to the extracellular medium. E2P will become dephosphorylated to produce E2 that spontaneously reverts to E1. According to this model, binding of H+ to plasma membrane H+-ATPase triggers phosphorylation of E1 to form E1P, whereas, in case it was a counterion, binding of K+ to E2P would be expected to trigger dephosphorylation to E2 (16). In Na+/K+- and H+/K+-ATPases, uptake of K+ is associated with the E2 to E1 conformational step, which is preceded by dephosphorylation of E2P (17, 18).

The D684N mutant of plasma membrane H+-ATPase is H+ transport-incompetent but can hydrolyze ATP and, as it is defective in the E1P–E2P conformational step, E1P will start to accumulate (19, 20). The E1P intermediate slowly dephosphorylates and returns to E1, which results in Pi release not associated with proton transport (20). As no inhibitors of E1P–E2P partial reactions are known for plasma membrane H+-ATPases, the D684N mutant serves as a unique tool to investigate partial reactions involving E1P. Unexpectedly, we found that D684N H+-ATPase is hypersensitive to K+. The D684N mutant proved effective for identifying new mutations that are completely insensitive to K+ but otherwise with normal catalytic properties. The study of these mutants has allowed for a detailed characterization of the role of K+ in the ATP hydrolytic mechanism of the proton pump. We demonstrate that K+ has the capacity to induce rapid dephosphorylation of the E1P phosphoform. As transport takes place between the E1 and E2 conformational states, K+-induced dephosphorylation of E1P serves as a pump uncoupling mechanism.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Site-directed Mutagenesis—The construction of the wild-type H+-ATPase vector for heterologous expression in the yeast Saccharomyces cerevisiae, as well as the construction of the D684N mutant, has been described (19). Other mutants were constructed by site-directed mutagenesis using an overlap extension polymerase chain reaction (21). All mutated sequences were verified by DNA sequencing. All proteins were expressed without the COOH-terminal 73-amino-acid residues but with a COOH-terminal Met-Arg-Gly-Ser-His6 (MRGSH6) tag. The COOH-terminal deletion transforms the enzyme into a constitutively activated state, simplifying functional studies. The addition of the His6 tag allows affinity purification of recombinant H+-ATPase (22).


Figure 1
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  		FIGURE 1.
A, proposed reaction cycle of the plasma membrane H+-ATPase. E1, pump conformation with H+ (alternatively H3O+) binding site(s) facing the cytoplasm; E1P, ADP-sensitive phosphoenzyme intermediate with bound H+; E2, pump conformation with very low affinity for H+; E2P, ADP-insensitive phosphoenzyme intermediate with exit pathway for H+ toward the extracellular side. B, schematic presentation of the plasma membrane H+-ATPase AHA2. A, actuator (phosphatase) domain); P, phosphorylation domain; N, nucleotide binding domain; M1–10, transmembrane segments 1–10; boxed P, the position of the phosphorylated Asp329; highlighted circles, substituted residues analyzed in this study. The aha2{Delta}73 mutant (A876MRGSH6STOP) was used as the basis for studies in this work (and thus termed "wild type"). In this mutant, 73 residues from the autoinhibitory COOH-terminal domain are removed to generate a constitutively active enzyme.

 
Expression in Yeast and Protein Affinity Purification—The S. cerevisiae strain RS-72 (23) was transformed and cultured essentially as described previously (24). Affinity purification of heterologously expressed wild-type and D684N mutant H+-ATPases was performed as described (20) except that purified proteins after purification were dialyzed against GMED20 (20% (w/v) glycerol, 100 mM MES2 (pH 6.5 with N-methyl-D-glucamine), 1 mM EDTA, and 1 mM dithiothreitol).

ATPase Assay—ATPase activity at 3 mM ATP was determined as described (24). The assay was carried out at 30 °C in a buffer (300 µl) containing 20 mM MOPS, 4 mM MgSO4, and 3 mM Tris-ATP. The pH of the buffer was adjusted to pH 6.5 with N-methyl-D-glucamine. The reaction was initiated by the addition of purified membrane protein in reactivation buffer (GMED20 with 10 µg/µl L-phosphatidylcholine and 0.7% (w/v) N-dodecyl-beta-D-maltoside).

Measurement of ATPase activity at 1 µM ATP was performed at 0 °C in a buffer (300 µl) containing 20 mM MOPS (pH adjusted to 6.5 with N-methyl-D-glucamine), 1 mM MgSO4, and purified protein in reactivation buffer and various cations as indicated. The addition of [{gamma}-32P]ATP to a final concentration of 1 µM initiated the ATP hydrolysis reaction. ATP hydrolysis was stopped by the addition of 6% trichloroacetic acid and 10% (w/v) charcoal. After centrifugation, the amount of liberated inorganic phosphate was determined by scintillation counting.

Stability Measurements—Purified proteins in reactivation buffer were incubated 46 °C for 20 min in 20 mM MOPS (pH adjusted to 6.5 with N-methyl-D-glucamine) and either in the absence or in the presence of 50 mM monovalent cation (LiCl, NaCl, or KCl). After heat treatment, proteins were diluted 50 times into buffer containing Tris-ATP (3 mM), 1 mM MgSO4, and 50 mM KCl. The ATP hydrolysis reaction was followed as described above. The activity remaining after 20 min at 46 °C were related to proteins incubated 20 min on ice (100% activity).

Reconstitution of H+-ATPases into Artificial Liposomes—To determine the sidedness of the K+ effect, protein was reconstituted at a lipid to protein ratio of 200:1 as described (22) in 10 mM MES (adjusted to pH 6.5 with N-methyl-D-glucamine), 50 mM Na2SO4 or 50 mM K2SO4, and 20% (v/v) glycerol. Reconstituting wild-type protein with Na+ substituting for K+ consistently resulted in vesicles that were less transport-competent (data not shown). Immediately prior to use, the outside medium of K2SO4 containing vesicles was exchanged by spinning a 100-µl sample (12 µg of protein) through a 2-ml column of Sephadex G50 equilibrated in 10 mM MES (adjusted to pH 6.5 with N-methyl-D-glucamine), 50 mM Na2SO4, and 20% (w/v) glycerol.

Phosphorylation and Dephosphorylation of the Phosphorylated Intermediate—Phosphorylation by [{gamma}-32P]ATP and dephosphorylation initiated with 10 mM EDTA, 10 mM EDTA supplemented 2 mM ADP or various cations were performed as described (19).

Protein Determination—Protein concentrations were determined by the method of Bradford (25) employing bovine serum albumin as a standard.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The ATP Hydrolytic Activity of the D684N Mutant Is Stimulated to High Values by K+—The only conserved acidic residue in the proton coordination center of plasma membrane H+-ATPase is Asp684 in transmembrane segment 6 (16)(Fig. 1B). If this residue is coordinating K+ as a counter-transported ligand, a D684N substituted pump would be expected to have reduced K+ sensitivity. The ATP hydrolytic activities of affinity-purified wild-type and mutant D684N plasma membrane H+-ATPase proteins were measured in the presence of various concentrations of KCl (Fig. 2A). Although physiological concentrations of KCl (50 mM) stimulated ATP hydrolysis by the wild-type protein less than 2-fold, KCl stimulated the Asn684 substituted enzyme more than 4-fold when measured at standard conditions (pH = 6.5, 30 °C) (Fig. 2A). K+ stimulated ATPase activity of both the wild-type and the D684N substituted protein with the same apparent Km (1 mM at pH 6.5). The effect of KCl on the D684N substituted enzyme was specific for K+ as other physiologically relevant monovalent cations did not stimulate ATP hydrolysis to the same degree as KCl (Fig. 2B). The effect of monovalent cations on ATP hydrolytic activity of D684N was as the same order (K+ > Rb+ > Na+ > Li+) as seen for wild-type proteins (Fig. 2B) (26).


Figure 2
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  		FIGURE 2.
The K+ stimulatory effect is potentiated in the D684N substituted plasma membrane H+-ATPase. A, KCl stimulation of ATP hydrolysis by wild-type (•, {blacksquare}) and D684N substituted ({circ}, {square}) pumps at 30 °C (•, {circ}; 3 mM ATP) or 0 °C ({blacksquare}, {square}; 1 µM ATP as in phosphorylation assays). The ATP hydrolytic activities in the absence of monovalent cations were taken as 0% and are indicated in Table 1. B, the effect of monovalent cations (25 mM) on ATP hydrolytic activity. Gray bars, LiCl; white bars, NaCl; black bars, KCl; light gray bars, RbCl. WT, wild type. C, insensitivity toward vanadate of the D684N substituted enzyme ({circ}, •) when compared with wild-type ({square}, {blacksquare}) and D617A ({triangleup}, {blacktriangleup}) substituted enzymes indicates that K+ does not induce E1–E2 transitions. KCl was either excluded from the ATPase assay ({square}, {triangleup}, {circ}) or added at 50 mM ({blacksquare}, {blacktriangleup}, •). In A–C, constant ionic strength (125 mM) was obtained by adding Tris-HCl. Error bars represent the standard deviation of at least three independent experiments.

 
K+ Does Not Induce E1–E2 Transitions in the D684N Mutant—One explanation for the above finding could be that K+ accelerates transition of E1P into the E2P conformation that in turn is dephosphorylated. Vanadate is a transition state analogue of inorganic phosphate in the E2 conformation of P-type ATPases, and for this reason, vanadate can be used as a conformational probe of P-type ATPases (4). The vanadate sensitivity of ATP hydrolysis was measured for the wild-type and D684N mutant proteins in the absence and in the presence of 50 mM KCl (Fig. 2C). K+ increased the vanadate sensitivity of the wild-type protein (Fig. 2C; Table 1; Ki = 50 µM in the absence of KCl, Ki = 10 µM at 50 mM KCl). However, K+ did not affect the vanadate insensitivity of the D684N mutant protein. Thus we conclude that the stimulatory effect of K+ is not due to an induced E1–E2 transition in the D684N mutant protein.


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  		TABLE 1
Kinetic properties of plasma membrane H+-ATPase mutant proteins

 
K+ Induces Dephosphorylation of the E1P Phosphoform of the D684N Protein—In P-type ATPases, the E1P phosphoform, but not E2P, is capable of ATP synthesis from ADP and bound phosphate and dephosphorylates to E1 in the presence of ADP (4). As expected for the E1P phosphoform, all of the phosphorylated intermediate accumulating in the D684N mutant of plant H+-ATPase was sensitive toward the application of ADP (Fig. 3A). In the absence of ADP, application of 50 mM KCl triggered dephosphorylation of a large fraction ({approx}40%) of the E1P phosphoform (Fig. 3A). The K+ effect upon the ATP hydrolytic reaction was intact at the conditions used for investigating the phosphorylated intermediate (i.e. 0 °C, 1 µM ATP) (Fig. 2A). Likewise, monovalent cations induced E1P dephosphorylation to the same degree as they stimulated ATP hydrolysis (K+ > Rb+ > Na+ > Li+; Fig. 3B). Since K+ does not induce an E1–E2 transition in this mutant we conclude that the E1P phosphoform of the D684N mutant is K+-sensitive.

An increase in the KCl concentration from 50 to 1000 mM only resulted in a slightly increased degree of E1P dephosphorylation (Fig. 3C), which indicates that K+-induced breakdown of E1P in the D684N mutant is saturable. The remaining EP was still sensitive toward application of ADP, which suggests that it belongs to the E1P pool (Fig. 3A). This indicates the presence of two E1P reaction cycle intermediate populations: one that is K+- and ADP-sensitive and another that is K+-insensitive but ADP-sensitive.


Figure 3
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  		FIGURE 3.
K+ promotes EP dephosphorylation of the E1P accumulating D684N mutant protein. A, the K+-sensitive EP form is ADP-sensitive. Dephosphorylation of D684N in the absence ({circ}) or in the presence of either KCl (50 mM; •) or ADP (2 mM; {triangleup}) is shown. In addition, ADP was added as t = 5s(arrow) to a replicate sample initially dephosphorylated in the presence of 50 mM KCl, and the amount of EP was measured after additional1 s ({blacktriangleup}). B, dephosphorylation (dephos.) of D684N in the presence of various cations (50 mM). C, dephosphorylation of D684N by KCl (various concentrations). In both B and C, the amount of EP was detected after 5 s of dephosphorylation, and EPat t = 0 was set to 100%. D, monovalent cation effect upon Asp684 substituted enzymes. ATP hydrolysis was measured in the absence or in the presence of 50 mM LiCl (white bars) or 50 mM KCl (black bars) for affinity-purified Asp684 substituted enzymes and the wild-type (WT) protein. Constant ionic strength was maintained with Tris-HCl (125 mM). Values are given relative to the measured ATP hydrolytic activity in the absence of added cation. Gray bars represent the steady state level of EP measured and given relative to the wild-type EP level (100%). In A–D, error bars represent the standard deviation of at least three independent experiments.

 
The Effect of K+ on Other Asp684 Substituted Enzymes—Like D684N, other substitutions at this site (D684A, D684V, and D684R but not D684E) are blocked in the E1–E2 transition, albeit the phosphorylated intermediate accumulates to various levels and can be purified in the E1 conformation (20). The degree by which K+ stimulated the ATP hydrolysis reaction was in accordance with the degree of E1P intermediate accumulation (Fig. 3D). This correlation indicates that K+ stimulation of ATP hydrolysis is not a general characteristic of E1 accumulating mutants but rather could be related to the degree of accumulation of the E1P conformational state.

K+ Exerts Its Effect from the Cytoplasmic Face of the Enzyme—K+ has been suggested to stimulate ATP hydrolysis from the cytoplasmic side of plasma membrane H+-ATPase (27). To test the sidedness of the K+ effect, detergent-solubilized and purified D684N protein was reconstituted into artificial proteoliposomes. As no ATP is included in the reconstitution buffer, only ATPase reconstituted with its cytoplasmic ATP binding site facing the outside of the vesicles will split ATP. D684N protein was reconstituted into proteoliposomes in the absence of K+, and the phosphorylation reaction was initiated by application of [{gamma}-32P]ATP to the extravesicular medium. K+ efficiently promoted EP breakdown under these conditions (Fig. 4A), suggesting that binding of K+ occurs to the same part of the ATPase as does ATP, i.e. to the cytoplasmic side of the pump. Reconstitution in the presence of K+ with subsequent removal of K+ from the extravesicular medium resulted in relatively stable EP intermediate that could be triggered to dephosphorylate by the addition of K+ to the extravesicular medium (Fig. 4A).

Monovalent Cation Stimulation Is Lost in D617A/D684N Double Substituted Enzymes—The cytoplasmic part of P-type ATPases is subdivided into the A (actuator), N (nucleotide binding), and P (phosphorylation) domains (28, 29) (Fig. 1B). We hypothesized that K+ facilitates water attack in the P domain either by directly changing the geometry of the covalently bound phosphate or, alternatively, by indirectly influencing the phosphate geometry trough induction of a conformational change.

In a homology model of AHA2 plasma membrane H+-ATPase (30), the P domain is comprised of residues 308–337 and 491–622. Negatively charged amino acid moieties are often found in context with monovalent cation binding sites (31), and to investigate whether residues in the phosphorylation domain of the H+-ATPase mediate the K+ response, 8 conserved H+-ATPase acidic residues (Glu318, Asp492, Glu520, Glu556, Asp559, Asp600, Asp610, and Asp617) were selected for mutagenic analysis (Fig. 1B). Conserved residues that in related P-type pumps are essential for the catalytic mechanism had already been excluded. All mutations were introduced into the D684N mutant H+-ATPase background, and the corresponding proteins were expressed in yeast and affinity-purified (data not shown).

Monovalent cations (K+ > Na+ > Li+) stimulated release of Pi in the ATPase assay from the E318A/D684N, D492A/D684N, E520A/D684N, E556A/D684N, D559A/D684N, D600A/D684N, and D610/D684N double mutants (Fig. 4B). However, in the D617A/D684N mutant, both the stimulation effect of K+ on ATP hydrolytic activity (Fig. 4B) and the K+-induced E1P dephosphorylation (Fig. 4C) were completely lost. The D617A/D684N double mutant resembled the D684N single point substituted enzyme with respect to pH optimum for ATP hydrolysis and specific activity in the absence of K+ (Table 1). Furthermore, the lost K+ effect was apparent at all pH values investigated (pH 5.5, 6.5, and 7.2; Fig. 5), ruling out an effect of H+ competing out K+.


Figure 4
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  		FIGURE 4.
Identification of a cytoplasmically located K+ binding site involving Asp617. A, K+ affects D684N substituted pump from the cytoplasmic side. In reconstituted proteoliposomes harboring purified D684N, phosphorylation was initiated with [{gamma}-32P]ATP, and dephosphorylation (dephos.) of the phosphorylated intermediate was initiated after 8 s with EDTA only (–Addition) or EDTA including either KCl (50 mM) or ADP (2 mM). After either 0 s or 5 s of dephosphorylation, the EP remaining was determined (expressed as the percentage of the amount present at the t = 0) by acid quenching. B, mutating P-domain-associated acidic residues in the D684N background reveals that the monovalent cation effect is dependent on Asp617. ATP hydrolysis was measured in the absence or in the presence of 50 mM LiCl (gray bars), 50 mM NaCl (white bars), or 50 mM KCl (black bars). Constant ionic strength was maintained with Tris-HCl (125 mM). Values are given relative to the measured ATP hydrolytic activity in the absence of added cation. C, EPof the D617A/D684N mutant is insensitive toward K+. Purified D617A/D684N protein was phosphorylated, and the amount of EP was detected after 5 s of dephosphorylation initiated in the absence and in the presence of KCl (50 mM), NaCl (50 mM) or ADP (2 mM). The amount of EPat t = 0 is set to 100%. In A–C, error bars represent the standard deviation of at least three independent experiments.

 
To test the hypothesis that Asp617 is essential for mediating the K+ effect, a single D617A substituted protein was produced. As expected, K+ stimulation of ATP hydrolytic activity was lost in the D617A mutant at all pH values determined (Fig. 5), and the pH optimum for ATP hydrolysis resembled that of the wild-type protein (Table 1). In contrast to the wild-type protein, K+ did not influence the vanadate sensitivity of the D617A substituted enzyme (Fig. 2C). The specific activity of the D617A single point substituted pump was found higher than the unmodified wild-type protein (Table 1).


Figure 5
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  		FIGURE 5.
D617A single substituted mutant protein is insensitive to KCl. ATP hydrolysis of wild-type (•), D684N ({circ}), D617A ({blacksquare}), and D617A/D684N ({square}) proteins was measured in the presence of various concentrations of KCl and at pH = 5.5, pH = 6.5, and pH = 7.2. Error bars represent the standard deviation of at least three independent experiments.

 
Glu184 in the A domain Is Necessary for Uncoupled Dephosphorylation of E1P—Biochemical and structural data have demonstrated that the A domain of P-type ATPases undergoes substantial domain movements during catalysis (17, 32, 33). In the A domain of sarcoplasmic reticulum Ca2+-ATPase (SERCA1a), Glu183 in the conserved P-type ATPase signature motif TGESis essential for dephosphorylation of EP (34–36). In the plasma membrane H+-ATPase, the corresponding residue is Glu184 (Fig. 1B). We generated E184A mutations in the wild-type and D684N mutant backgrounds to investigate whether the phosphatase function of the A domain is required in the plasma membrane H+-ATPase for dephosphorylation of E1P. Neither E184A nor E184A/D684N substituted enzymes exhibited any detectable Pi release in the ATPase assay (Fig. 6A), and both mutant proteins accumulated to a high degree in the EP phosphorylated state (Fig. 6A). This indicates the buildup of stable EP forms. A major fraction ({approx}65%) of the EP pool of the E184A single substituted protein was insensitive to ADP, suggesting accumulation of the E2P phosphointermediate (Fig. 6B). Accumulation of E2P in response to mutations in the TGES motif is in accordance with results obtained in other pumps (35). In contrast herewith, the phosphoprotein that formed in the E184A/D684N double substituted enzyme was highly sensitive toward ADP (Fig. 6B), which indicates that the E1P conformational state accumulates in this mutant. K+ had no effect on breakdown on EP in either E184A or E184A/D684N substituted proteins, which is strong evidence that the phosphatase activity of the A domain is required for K+-induced dephosphorylation of the E1P phosphointermediate.


Figure 6
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  		FIGURE 6.
Glu184 in the TGES phosphatase motif of the actuator domain is required for dephosphorylation of E1P. A, ATPase activity (measured as Pi release) (black bars) and steady state phosphorylation (white bars) of wild-type (WT), E184A, D684N, and E184A/D684N substituted enzymes. The Pi release activity and phosphoenzyme level is given relative to the wild-type protein (100%). B, dephosphorylation of the phosphorylated intermediate measured after 1 s with dephosphorylation initiated in the absence (gray bars) or in the presence of ADP (dark gray) and in the presence of KCl (50 mM; white bars). The amount of EP remaining is given relative to at t = 0(black bars). In A and B, error bars represent the standard deviation of at least three independent experiments.

 
K+ Binding to Asp617 Provides Protection to the H+-ATPase against Thermal Inactivation—If a role of K+ is to promote domain interactions in the pump protein, e.g. between the P domain and the A domain, it could be expected that K+ has a positive effect on enzyme stability. Indeed, it was found that monovalent cations protected wild-type and D684N H+-ATPase against thermal inactivation (Fig. 7). The substitution of Asp617, to abolish the K+ binding site, lead to a loss of K+ protection against thermal inactivation (Fig. 7). This was seen for both D617A and D617A/D684N mutant proteins, indicating that K+ binding to Asp617 not only influences enzyme turnover reactions but also affects overall protein stability. Na+ also influenced wild-type and D684N protein stability positively (Fig. 7), indicative that the protective effect of K+ against thermal inactivation is less specific than its effect on the ATP hydrolytic reaction (Fig. 2B).


Figure 7
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  		FIGURE 7.
K+ binding to Asp617 provides protection to the H+-ATPase against thermal inactivation. Affinity-purified wild-type (WT), D617A, and D684N single substituted enzymes and D617A/D684N double substituted enzyme were incubated in the presence of 50 mM cation for 20 min at 46 °C. Gray bars, Li+; white bars, Na+; black bars, K+. Residual ATP hydrolytic activity was measured in the presence of excess potassium. Error bars represent the standard deviation of at least three independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
K+ Stimulation of Plasma Membrane H+-ATPase Involves Asp617 Situated in a Cytoplasmic Domain—In this study, we show that in a E1P–E2P conformationally blocked plasma membrane H+-ATPase, K+ stimulates dephosphorylation of the E1P phosphointermediate. Following the substitution of Asp617 in the cytoplasmic phosphorylation domain, K+ stimulation of Pi release and K+-induced E1P dephosphorylation were completely lost. Asp617 is not situated in transmembrane segments that facilitate ion transfer, but is predicted to be close to the aspartyl phosphate group at Asp329 (30) (Fig. 1B).

The fact that the K+ effect is completely lost when substituting a single negatively charged residue, without having other measurable effects on H+-ATPase kinetics, strongly suggests a stoichiometry of 1 K+ bound per pump molecule. In a typical plant cell, the cytoplasmic concentration of K+ exceeds 50 mM, which indicates that the K+ binding site (apparent Km = 1mM) will be occupied under normal conditions.

Asp617 Is Part of a Conserved P-type ATPase K+ Binding Site—The activities of Ca2+- and Na+/K+-ATPases are both stimulated by cytoplasmic monovalent cations (37–39). Further, using fast kinetic analysis, K+ has been demonstrated to affect the EP conformations of the plasma membrane Ca2+-ATPase (40) and the plasma membrane H+-ATPase (41). Although the mechanism is not clarified, it appears that cytoplasmic K+ promotes dephosphorylation of P-type Ca2+-, Na+/K+, and H+-ATPase phosphoenzymes. This demonstrates that a K+ effect on pump dephosphorylation is a general phenomenon in P-type ATPases.

In sequence alignments (the P-type ATPase data base), Asp617 in the plant plasma membrane H+-ATPase AHA2 corresponds to Glu732 in sarcoplasmic reticulum Ca2+-ATPase SERCA1a. A K+ binding site that involves Glu732 (SERCA1a) has recently been identified in crystal structures of sarcoplasmic reticulum Ca2+-ATPase (42). K+ coordinating groups at this site involves oxygen atoms derived from the negatively charged Glu732 (SERCA1a) and three oxygen atoms in backbone carbonyl residues (Leu711, Lys712, and Ala714 in SERCA1a). Mutation of Glu732 in the sarcoplasmic reticulum Ca2+-ATPase reduces the rate of phosphoenzyme dephosphorylation in the presence of K+. A negative charge is present at an equivalent position in many P-type ATPases, which indicates the presence of a conserved K+ binding site in the P domain of P-type ATPases. It is an attractive hypothesis that binding of K+ to this site promotes pump dephosphorylation in the same P-type pumps.

Mutational Evidence for Potassium-sensitive and Potassium-insensitive E1P Conformational States—We found that the complete E1P pool is ADP-sensitive, whereas only part of the E1P pool is K+-sensitive. This is compatible with the presence of two different E1P phosphoforms, in only one of which does K+ induce a rapid discharge of the covalently bound phosphate. In E1–E2 conformationally blocked Na+/K+-ATPase, an ADP- and K+-sensitive E1P intermediate (termed E1P*) has been described (39, 43). The E1P* of this enzyme is sensitive toward application of cytoplasmic K+ and is a conversion product between E1P (ADP-sensitive) and E2P (ADP-insensitive, K+-sensitive) (39, 43). The E1P* intermediate of the Na+/K+-ATPase could resemble the K+-sensitive form of E1Pinthe plasma membrane H+-ATPase.

Mechanism of the K+ Effect on Pump Dephosphorylation—Our finding that K+ positively influences the stability of purified H+-ATPase supports a model in which K+ induces a compact conformation of the protein. Crystal structures of sarcoplasmic reticulum Ca2+-ATPase have demonstrated that the A domain undergoes substantial movement and rotates during the E1–E2 conformational step (32, 33). Linker regions between the membranous region and the A domain direct this rotation, the purpose of which is to bring the TGES motif in the A domain in close proximity to the aspartyl phosphate group in the P domain. It has been proposed that K+ bound to the P domain assists in attracting the linker regions connected to the A domain (42).

In support of this model, the effect of K+ bound to the P domain at Asp617 was completely lost in mutants carrying substitutions at Glu184 (in the sequence TGES) in the A domain. The negative charge of the conserved TGES motif in the A domain has been suggested to interact with the water molecule that attacks the phosphoryl group (32, 35). The simplest hypothesis in accordance with our data is that K+ accelerates docking of the A domain to the P domain, and in this way, promotes A domain-catalyzed decay of the phosphoryl group situated in the P domain.

According to this model, K+ is not an uncoupler of the H+-ATPase per se, which would require that it specifically dephosphorylates E1P, but rather, it has the capacity to stimulate dephosphorylation of both the E1P and the E2P phosphoenzymes. What determines whether K+ acts as an uncoupler or not is the time the enzyme spends in the E1P conformation before it proceeds to the E2P conformation. If the E1P–E2P transition is very fast, and faster than A domain movements, E2P will be dephosphorylated by the A domain, and the K+ effect will be seen as stimulation of coupled pump turnover. If the conformational transition is slow, the A domain moves in before the E1P–E2P transition, and K+ will act as a pump uncoupler. This could be the case if the membrane potential is close to the reversal potential of the pump. In such a situation, K+-induced dephosphorylation of E1P would act as a safety valve.

Regulation of P-type ATPases by Cytoplasmic K+—An important question that arises is why a mechanism has developed that potentially allows K+ to dephosphorylate E1Pina way that uncouples ATP hydrolysis from transport. Uncoupling has been described in several P-type ATPases and appears to be an inherent property of these pumps (44). Several lines of evidence suggest that proton transport and ATP hydrolysis in plasma membrane H+-ATPase are partially uncoupled in the resting state (45–47). Yeast and plant plasma membrane H+-ATPases are controlled by an autoinhibitory COOH-terminal regulatory domain (11) (Fig. 1B). Following relief of this constraint, e.g. by post-translational regulatory processes, proton transport is typically stimulated to a much higher degree than ATP hydrolysis (45–47). This would suggest that post-translational activation involving the regulatory domain is linked to the onset of strict coupling. The found connection between the K+ binding site and the phosphorylated aspartic acid could represent a mechanism controlling the coupling ratio depending on the activation state of the pump molecule. K+-induced dephosphorylation of E1P may thus well serve regulatory purposes and play a role in negative regulation of the transmembrane electrochemical gradient under cellular conditions where E1P is accumulating. As a shift in conformational equilibrium between E1 and E2 is likely to determine the extent of the K+ effect, any effector that influences this equilibrium would in turn control the coupling state of the pump.


   FOOTNOTES
 
* This work was supported by the European Union's Framework 6 Program, a fellowship from the Danish Carlsberg Foundation (to M. B. P.), and a fellowship from the Danish Ministry of Education (to E. L. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains a supplemental figure. Back

1 To whom correspondence should be addressed. Tel.: 45 3528 2595; Fax: 45 3528 3365; E-mail: mob{at}kvl.dk.

2 The abbreviations used are: MES, 2-morpholinoethansulfonic acid; MOPS, 3-(N-morpholino)propanesulfonic acid. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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