©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Functional Consequences of Alterations to Amino Acids at the M5S5 Boundary of the Ca-ATPase of Sarcoplasmic Reticulum
MUTATION TYRGLY UNCOUPLES ATP HYDROLYSIS FROM Ca TRANSPORT (*)

(Received for publication, June 30, 1994; and in revised form, September 19, 1994)

Jens Peter Andersen (§)

From the Danish Biomembrane Research Centre, Institute of Physiology, University of Aarhus, DK-8000 Aarhus C, Denmark

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The roles of the hydrophobic side chains of residues Phe, Ile, Tyr, Leu, and Ile located at the M5S5 boundary of the Ca-ATPase of sarcoplasmic reticulum were analyzed by site-directed mutagenesis. Substitution of Tyr with glycine resulted in a new phenotypic variant of the Ca-ATPase that catalyzed a high rate of Ca-activated ATP hydrolysis without net accumulation of Ca in the microsomal vesicles. The ATPase activity of the Tyr Gly mutant displayed characteristics similar to the ATPase activity of the wild-type enzyme measured in the presence of calcium ionophore, and the mutant was able to form the ADP-insensitive phosphoenzyme intermediate. Mutants Phe Gly, Ile Gly, Leu Gly, and Ile Gly were able to accumulate Ca. In mutants Leu Gly and Ile Gly, the turnover rate was low due to inhibition of dephosphorylation of the ADP-insensitive phosphoenzyme intermediate. On the other hand, mutant Leu Lys dephosphorylated rapidly. Mutants Phe Gly and Leu Lys displayed apparent Ca affinities that were reduced two and three orders of magnitude, respectively, relative to that of the wild-type.


INTRODUCTION

The Ca-ATPase of sarcoplasmic reticulum belongs to the ion-translocating ATPases of P-type in which the hydrolysis of ATP is coupled to vectorial transport through formation of an acid-stable phosphoryl aspartyl enzyme intermediate that exists in two major conformational states E1P (^1)and E2P (De Meis, 1981; Andersen, 1989). Analysis of the amino acid sequence (MacLennan et al., 1985) along with extensive site-directed mutagenesis studies (MacLennan et al., 1992; Andersen and Vilsen, 1992a) and studies of two-dimensional membrane crystals (Taylor et al., 1986; Toyoshima et al., 1993) have concurred in a structural model for the Ca-ATPase in which the Ca binding region is located within a cluster of putative transmembrane alpha-helices, while the catalytic ATP-binding site is made up from residues in the cytoplasmic domains (Fig. 1). The presence in the Ca-ATPase of distinct structural entities associated with ATP binding and Ca binding, respectively, has recently been confirmed by studies of chimeric fusion proteins (Sumbilla et al., 1993; Nørregaard et al., 1993). Hence, the energy coupling between scalar and vectorial reactions seems to require long distance communication between the cytoplasmic and transmembrane parts of the pump through conformational changes (Andersen and Vilsen, 1992a). The mutational analysis of the Ca-ATPase has pinpointed the three transmembrane segments M4, M5, and M6 as central to events associated with Ca binding and occlusion (Clarke et al., 1989; Andersen and Vilsen, 1992b, 1994; Vilsen and Andersen, 1992d). In addition, this region may be involved in the countertransport of protons (Andersen and Vilsen 1992b, 1994; Vilsen and Andersen, 1992c). An important task is also to locate the amino acid residues responsible for maintenance of the normal physiological coupling of ATP hydrolysis to ion transport, but so far no site-directed mutagenesis data have revealed uncoupled mutants of the Ca-ATPase.


Figure 1: Structural model of the Ca-ATPase highlighting the residues at the M5S5 boundary. This model is based on the structure prediction by MacLennan et al.(1985) and Green(1989). S and M indicate stalk and transmembrane helices, respectively. Each circle corresponds to an amino acid residue indicated by the one-letter code inside the circle. The residues at the M5S5 boundary are shown enlarged in a box. Residues with oxygen-containing side chains that participate in Ca occlusion are indicated by filled circles (Vilsen and Andersen, 1992d; Andersen and Vilsen, 1994).



In the present study, the roles of the hydrophobic side chains of Phe, Ile, Tyr, Leu, and Ile located at the border between the fifth transmembrane segment M5 and the putative ``stalk'' helix S5 have been analyzed by substitution of these residues with glycine. In addition, Leu was replaced by lysine to examine the effect of the positive charge on Ca binding. These residues are totally conserved among the SERCA-type of Ca-ATPases. Other cation transporting ATPases of P-type contain motifs that are highly similar although not identical, and the aromatic nature of the residue found at the place of the tyrosine is well conserved (Green, 1989).

The results of the functional analysis of the mutants support the notion that these residues are located close to the Ca binding structure and demonstrate a role of Tyr in ensuring the coupling of ATP hydrolysis to Ca transport. Hence, the Tyr Gly mutant catalyzed ATP hydrolysis at a high rate in the absence of any net accumulation of Ca in the vesicles.


EXPERIMENTAL PROCEDURES

The construction and expression of mutant cDNAs as well as most of the methods employed in the analysis of enzyme function have previously been described in detail (Maruyama and MacLennan, 1988; Andersen et al., 1989; Andersen and Vilsen, 1992b; Vilsen et al., 1989, 1991; Vilsen and Andersen, 1992b). In brief, mutations were introduced into the rabbit fast twitch muscle Ca-ATPase cDNA using the site-specific mutagenesis method of Kunkel(1985). The presence of the correct mutation was confirmed by nucleotide sequencing according to Sanger et al.(1977). The entire Ca-ATPase cDNA containing the desired mutation was cloned into vector pMT2 (Kaufman et al., 1989) for expression in COS-1 cells (Gluzman, 1981). Microsomes were prepared from transfected cells, and the expression of exogenous Ca-ATPase protein was quantitated by a specific sandwich enzyme-linked immunosorbent assay (Andersen et al., 1989).

ATP-driven Ca uptake in the microsomes was measured by Millipore filtration (Vilsen et al., 1989) following incubation of 0.05-0.2 µg of Ca-ATPase protein at 37 °C for 1-30 min (depending on the specific transport rate) in medium containing 20 mM MOPS, pH 6.8, 100 mM KCl, 5 mM MgCl(2), 5 mM ATP, 0.5 mM EGTA, 5 mM potassium oxalate, 10^5 Bq Ca/ml, and various concentrations of CaCl(2) to set the concentrations of free Ca at the desired values according to calculations using published stability constants for Mg and Ca complexes of EGTA, ATP, and oxalate (Sillén and Martell, 1964; Fabiato and Fabiato, 1979; Dupont, 1982; Vilsen and Andersen, 1987). A Ca-selective electrode (Radiometer F2002) was used to adjust free Ca concentrations above 10 µM. All solutions were prepared freshly before use and kept at 37 °C to avoid precipitation of calcium oxalate. Following subtraction of the background Ca influx measured with microsomes harvested from cells transfected with vector without insert, the rate of Ca uptake was calculated from the linear part of the curves showing Ca uptake as a function of time (see inset in Fig. 2).


Figure 2: Ca dependence of the rate of ATP-driven Ca uptake in the microsomal vesicles. Ca uptake was measured as described under ``Experimental Procedures'' at 37 °C in the presence of oxalate, and the molecular turnover rate was calculated as the ratio between the rate of Ca uptake/mg of microsomal protein (µmol of Ca transported/min/mg of microsomal protein) and the phosphorylation capacity (nmol/mg of microsomal protein). The ordinate shows this number as percentage of that measured for the wild-type Ca-ATPase at 10 µM Ca. The inset shows examples of the time curves from which the rates were calculated, at pCa 3.8 for the wild-type and mutant Phe Gly and pCa 3.7 for mutant Tyr Gly. box, wild-type Ca-ATPase; , mutant Phe Gly; up triangle, mutant Ile Gly; down triangle, mutant Tyr Gly; , mutant Leu Gly; , mutant Ile Gly.



The rate of ATP hydrolysis was measured spectrophotometrically at 37 °C by the NADH-coupled assay described by Vilsen et al.(1991), in the presence of 0.15 mM NADH, 1 mM phosphoenolpyruvate, lactate dehydrogenase (approximately 10 IU/ml), and pyruvate kinase (approximately 10 IU/ml). Additional components included in the standard medium used in most experiments were 20 mM MOPS, pH 7.0, 100 mM KCl, 2 mM MgCl(2), 5 mM MgATP, 1 mM EGTA, 1 µM of the calcium ionophore A23187, and various concentrations of CaCl(2) to set the desired concentrations of free Ca. The ATPase activity referable to the Ca-ATPase was calculated following subtraction of the rate of ATP hydrolysis measured in the absence of free Ca (presence of 2 mM excess EGTA). MgATP concentration dependence was titrated as in Vilsen et al. (1991) keeping the free Mg concentration constant at 1 mM and the free Ca concentration at 100 µM. Inhibition by various concentrations of vanadate was tested in the presence of 100 µM Ca and 5 mM MgATP as described in Vilsen and Andersen (1992b).

The molecular turnover rates were calculated using the maximum amount of phosphoenzyme formed from ATP (see below) as a measure of the concentration of active enzyme sites present. In the figures these rates are presented as percentage of the maximum molecular turnover rate observed with the wild-type Ca-ATPase.

The phosphoenzyme intermediates formed in the presence of [-P]ATP or P(i) were quantitated as a function of Ca concentration following acid quenching at steady state. The phosphorylation from [-P]ATP was carried out at 0 °C for 15 s in the presence of 50 mM MOPS buffer, pH 7.0, 80 mM K, 5 mM Mg, 2 µM [-P]ATP, 1 mM EGTA, and CaCl(2) to set the indicated free Ca concentrations. To study the rate of dephosphorylation of the E1P phosphoenzyme intermediate in the absence of Ca accumulation in the vesicles, 1 µM of the calcium ionophore A23187 was added to the standard phosphorylation medium. Phosphorylation was terminated by the addition of excess EGTA (1 mM), with or without 1 mM ADP, followed by acid quenching at serial time intervals. Similar experiments were carried out following phosphorylation in a medium designed to accumulate the E2P phosphoenzyme intermediate (Andersen et al., 1989; Vilsen et al., 1989). This medium contained 100 mM TES/Tris buffer, pH 8.35, 10 mM Mg, 50 µM Ca, and 2 µM [-P]ATP. Phosphorylation from P(i) to form E2P in the ``backdoor'' reaction was carried out for 10 min at 25 °C in the presence of 100 µMP(i), 100 mM TES/Tris, pH 7.0, 5 mM Mg, 20% (v/v) dimethyl sulfoxide, and the indicated Ca concentrations set with EGTA. Dimethyl sulfoxide was added to increase the apparent affinity of the Ca-ATPase for P(i) (De Meis et al., 1980) so that the phosphorylation level was close to maximum in the absence of Ca. To study the rate of dephosphorylation of E2P formed from P(i), the phosphorylated sample was cooled to 0 °C. The phosphorylation was then terminated by a 20-fold dilution of the sample into ice-cold medium containing 50 mM MOPS, pH 7.0, 80 mM K, 5 mM Mg, 1 mM non-radioactive P(i), 2 mM EGTA, no dimethyl sulfoxide, followed by acid quenching at serial times.

The phosphorylated acid-precipitated microsomal protein was washed and subjected to SDS-polyacrylamide gel electrophoresis at pH 6.0, followed by autoradiography of the dried gels and quantitation by densitometric analysis, using an LKB 2202 Ultroscan Laser Densitometer, or by liquid scintillation counting of gel slices. The latter procedure was used to obtain the absolute site concentration needed for calculation of molecular turnover rate.

All experiments were repeated at least three times with consistent results. The data points shown in Fig. 2, Fig. 4, and Fig. 5are mean values of triplicate determinations. S.D. is indicated when larger than 5%.


Figure 4: Ca dependence of ATPase activity. The ATPase activity was measured in the presence of 1 µM calcium ionophore as described under ``Experimental Procedures'' and illustrated in Fig. 3. The molecular turnover rate was calculated as the ratio between the rate of ATP hydrolysis/mg of microsomal protein and the phosphorylation capacity/mg of microsomal protein. The ordinate shows this number as percentage of that measured for the wild-type Ca-ATPase at 50 µM Ca and 5 mM MgATP. The concentration of free Ca vas varied at a constant MgATP concentration of 5 mM. box, wild-type Ca-ATPase; , mutant Phe Gly; down triangle, mutant Tyr Gly; , mutant Leu Gly; , mutant Ile Gly.




Figure 5: Ca concentration dependence of phosphorylation from [-P]ATP or P(i). Phosphorylation of the microsomal fractions isolated from cells transfected with mutant or wild-type Ca-ATPase cDNA was carried out as described under ``Experimental Procedures.'' The acid-quenched samples containing equivalent amounts of expressed Ca-ATPase were subjected to SDS-polyacrylamide gel electrophoresis at pH 6.0. The autoradiograms of the dried gels were quantitated by densitometry. A, phosphorylation from ATP. B, phosphorylation from P(i). box, wild-type Ca-ATPase; , mutant Phe Gly; down triangle, mutant Tyr Gly; , mutant Leu Gly; , mutant Ile Gly; circle, mutant Leu Lys.




Figure 3: Ionophore sensitivity of Ca-activated ATPase activity. ATP hydrolysis was monitored spectrophotometrically by the NADH-coupled assay described under ``Experimental Procedures'' in the presence of 20 mM MOPS, pH 7.0, 100 mM KCl, 2 mM MgCl(2), 5 mM MgATP, and 100 µM Ca. The ordinate shows absorbance at 340 nm. The bar in the lower left corner indicates 0.1 absorbance unit. At 1, the microsomes were added. At 2, 1 µM of the calcium ionophore was added to relieve back-inhibition by accumulated Ca. At 3, EGTA was added to show the background ATPase activity not referable to Ca-ATPase. A, wild-type Ca-ATPase; B, mutant Tyr Gly; C, mutant Phe Gly; D, control microsomes harvested from cells transfected with vector without insert (note that more microsomal protein was inserted in this case).




RESULTS

Expression and Ca Uptake Activity of Mutants

The expression levels of mutants and wild-type Ca-ATPase were quantitated by relating the amount of expressed exogenous Ca-ATPase protein present in the microsomes (determined by enzyme-linked immunosorbent assay) to the total amount of microsomal protein. Mutants Phe Gly, Ile Gly, Leu Gly, and Ile Gly were expressed to levels ranging between 72 and 123% that of the wild-type Ca-ATPase in five transfections. The expression levels of mutants Tyr Gly and Leu Lys were significantly lower, ranging between 23 and 56% that of the wild-type in seven transfections. Only preparations corresponding to relative expression levels higher than 30% that of the wild-type were used for the functional characterization described below.

To be able to measure the rate of ATP-driven Ca transport into the microsomal vesicles containing expressed Ca-ATPase, in the presence of the background of passive diffusion of Ca into all vesicles in the preparation, oxalate is usually added to the reaction mixture. The precipitation of Ca oxalate at the high Ca concentrations generated inside the vesicles during active transport traps the calcium ions, thereby increasing the capacity for active Ca accumulation. Fig. 2shows the results of measurement of the specific Ca transport turnover rates of mutants Phe Gly, Ile Gly, Tyr Gly, Leu Gly, and Ile Gly, in the presence of oxalate. In mutant Ile Gly, the maximal transport rate measured at saturating Ca concentration and the apparent Ca affinity were rather similar to those of the wild-type Ca-ATPase. On the other hand, the maximal transport rates of mutants Leu Gly and Ile Gly were reduced below 15% that of the wild-type precluding an accurate determination of the Ca affinities of these mutants. Mutant Phe Gly was clearly able to transport Ca at a higher rate, but since this mutant displayed very low apparent Ca affinity (K(0.5) above 50 µM, see Fig. 2), it was not straightforward to determine its maximum Ca-uptake activity. Hence, at high medium Ca concentrations Ca oxalate crystals are formed not only inside the vesicles, but also outside, giving rise to a high filter background. For this reason, we have previously been unable to measure the oxalate-supported Ca uptake with accuracy at free Ca concentrations higher than 10-30 µM, but in the present study it was found that the high filter background can be avoided up to a free Ca concentration of about 200 µM (determined using the Ca selective electrode), provided the supersaturated Ca-oxalate solutions are prepared immediately before use and the Ca uptake measured at 37 °C instead of at 27 °C as previously. As seen in Fig. 2, these precautions permitted the measurement of ATP-driven Ca transport in mutant Phe Gly at Ca concentrations up to 200 µM Ca. At this Ca concentration, the turnover rate corresponded to about 50% that of the maximum turnover rate of the wild-type.

Fig. 2also shows that mutant Tyr Gly was unable to transport Ca at a measurable rate at all Ca concentrations up to the experimental limit of 200 µM. Even after 30 min of incubation at 200 µM Ca in the presence of 5 mM MgATP, it was not possible to measure any Ca uptake higher than the background corresponding to control microsomal preparations harvested from cells that had been mock-transfected with the vector without insert. In addition, the mutant with positively charged substituent, Leu Lys, was unable to transport Ca at Ca concentrations up to 200 µM (not shown).

Ca-activated ATPase Activity

Steady state Ca-ATPase activity was measured spectrophotometrically using the NADH-coupled assay system with ATP regeneration from phosphoenolpyruvate (Vilsen et al., 1991), in the absence and in the presence of the calcium ionophore A23187. Fig. 3shows examples of such measurements carried out at 100 µM Ca. The decrease of absorbance at 340 nm reflects hydrolysis of ATP. In these experiments there was no oxalate present to precipitate Ca inside the vesicles, and it is seen that in the wild-type enzyme as well as in mutant Phe Gly the ATPase activity increased significantly upon addition of the calcium ionophore. This is the result of relieving the back-inhibition exerted by accumulated Ca present at millimolar concentration inside the vesicles (De Meis, 1981). Surprisingly, a high Ca-activated ATPase activity was measured with mutant Tyr Gly, even though this mutant was found unable to accumulate Ca in the presence of oxalate as described above. Consistent with the lack of Ca accumulation in mutant Tyr Gly, there was no back-inhibition of ATP hydrolysis in this mutant, as evidenced by the absence of an activatory effect of the calcium ionophore (Fig. 3). Hence, in mutant Tyr Gly the ATPase activity appeared to be uncoupled from Ca transport. Fig. 4depicts the results of measurements of specific ATPase turnover rates in the presence of the calcium ionophore at various concentrations of Ca. The maximum ATPase turnover rate of mutant Tyr Gly was similar to that measured for the wild-type Ca-ATPase in the presence of ionophore. The apparent Ca affinity of mutant Tyr Gly was slightly reduced relative to that of the wild-type, saturation being reached at about 10 µM Ca. In mutant Phe Gly the apparent Ca affinity determined from Ca titration of ATPase activity was much more strongly reduced, consistent with the data for Ca activation of Ca transport shown in Fig. 2. Mutants Leu Gly and Ile Gly displayed rather low maximum ATPase activities. It is noteworthy, however, that the maximum ATPase activity of mutant Leu Gly was higher than that of mutant Ile Gly, while the corresponding maximum Ca uptake activity of mutant Leu Gly was lower than that of mutant Ile Gly (compare Fig. 2and Fig. 4). It also appears, therefore, as if mutant Leu Gly may have been uncoupled to some extent. Mutant Leu Lys was completely unable to hydrolyze ATP, even at a Ca concentration of 1 mM (not shown).

The ATPase activity was further characterized by measurement of the dependence on MgATP concentration at saturating Ca concentration (not shown). The activation curve for the Tyr Gly mutant was almost superposable on that of the wild-type, and like the wild-type Ca-ATPase, all the mutants displayed a biphasic activation pattern with a secondary rise in the millimolar concentration range indicative of a role for ATP as an allosteric modulator in addition to its role as substrate (Andersen, 1989). In addition, it was found that all mutants, including Tyr Gly, were sensitive to inhibition by vanadate.

Phosphorylation from ATP and P(i)

Like the wild-type Ca-ATPase, all the mutants discussed here were able to become phosphorylated from ATP provided the concentration of Ca was high enough and from P(i) in the absence of Ca. The ATP concentration dependence of phosphorylation from ATP of the mutants was indistinguishable from that of the wild-type, with K(0.5) for ATP below 1 µM (not shown). The Ca dependence of phosphorylation from ATP is shown in Fig. 5A. The uncoupled mutant Tyr Gly displayed an apparent Ca affinity only slightly lower than that of the wild-type. The apparent Ca affinities of mutants Leu Gly and Ile Gly were also quite similar to that of the wild-type. In accordance with the results of Ca titration of Ca transport and Ca-ATPase activity, the apparent Ca affinity of mutant Phe Gly determined in the phosphorylation assay with ATP was at least 100-fold lower than that of the wild-type. Mutant Leu Lys displayed even lower Ca affinity in the phosphorylation assay with a K(0.5) value above 10 mM, thus explaining the above described inability of this mutant to transport Ca and utilize ATP at submillimolar Ca concentrations.

Studies of the inhibition by Ca of the backdoor phosphorylation from P(i) may provide additional information on the function of the Ca sites (Andersen and Vilsen, 1992b, 1994). The results of such experiments are depicted in Fig. 5B. A comparison with Fig. 5A indicates that the apparent Ca affinities determined in the two types of phosphorylation assay were fairly consistent, except in the case of mutant Phe Gly. The apparent Ca affinity observed by titration of Ca inhibition of P(i) phosphorylation in this mutant was almost identical to that of the wild-type, in contrast to the low Ca affinity observed in the study of Ca activation of phosphorylation from ATP. Because the Ca-ATPase is thought to bind two calcium ions in a consecutive mechanism, and only the first binding site has to be occupied to inhibit phosphorylation from P(i), a possible explanation of the discrepancy between the apparent Ca affinities determined in the two types of phosphorylation assay with mutant Phe Gly is that Phe is directly or indirectly involved in the binding of the second calcium ion, but not in the binding of the first calcium ion in the sequence (Andersen and Vilsen, 1992b, 1994).

Dephosphorylation of the Phosphoenzyme Intermediate

Since the turnover rates of mutants Leu Gly and Ile Gly were found to be much reduced at concentrations of Ca and ATP that were sufficient to saturate the phosphorylation reaction, it seemed that a partial reaction following the phosphorylation must have been defective in these mutants. In the normal Ca-ATPase reaction cycle, the first phosphoenzyme intermediate formed is E1P, which is ADP sensitive, i.e. able to donate the phosphoryl group back to ADP forming ATP (De Meis, 1981). This intermediate traps the calcium ions in an occluded state with no direct access to the medium on either side of the membrane (Glynn and Karlish, 1990; McIntosh et al., 1991; Vilsen and Andersen, 1992a). The release of Ca at the luminal side of the membrane is believed to be coupled with the transition to a second phosphoenzyme intermediate, E2P, which is ADP insensitive. The latter intermediate appears to be identical to the phosphoenzyme intermediate formed in the backdoor reaction with P(i) (De Meis, 1981). The cycle is normally terminated by the hydrolysis of the acylphosphate bond in E2P and the release of P(i). Fig. 6, A and B, depict the results of experiments in which the stabilities of the E1P and E2P phosphoenzyme intermediates were examined. Phosphorylation was carried out either with ATP (Fig. 6A), under conditions where the E1P intermediate accumulates in the wild-type (Andersen et al., 1989; Vilsen et al., 1989), or with P(i) to obtain E2P (Fig. 6B). After the steady state had been reached during phosphorylation with ATP, the ADP sensitivity of the phosphoenzyme was tested (lane 2 in Fig. 6A), or the dephosphorylation in the forward direction was studied following addition of EGTA to terminate the phosphorylation by chelation of Ca (lanes 3 and 4 in Fig. 6A). In the wild-type Ca-ATPase, the phosphoenzyme disappears rapidly upon addition of either ADP or EGTA, as has previously been demonstrated (Andersen et al., 1989; Vilsen et al., 1989). As seen in Fig. 6A, a rapid dephosphorylation from E1P similar to that of the wild-type was observed with mutants Phe Gly, Ile Gly, and Tyr Gly, consistent with their abilities to hydrolyze ATP at a high rate. In mutant Leu Gly, the rate of disappearance of the phosphoenzyme in the presence of EGTA without ADP was reduced relative to that of the wild-type. The block of dephosphorylation was even more pronounced in mutant Ile Gly. The latter mutant was also partially insensitive to ADP. Under conditions where dephosphorylation in the forward direction of the reaction cycle is blocked, such as seen with mutant Ile Gly, the amount of phosphoenzyme remaining after the 5-s chase with ADP reflects the amount of E2P accumulated in steady state. The accumulation of a significant amount of E2P in mutant Ile Gly under conditions where the E1P intermediate prevails in the wild-type enzyme suggests that the high stability of the phosphoenzyme of mutant Ile Gly observed in the EGTA chase was due to a block of the dephosphorylation of E2P. This hypothesis was confirmed in the experiments shown in Fig. 6B, in which the dephosphorylation of E2P was followed directly after termination of the phosphorylation with P(i) by dilution of the phosphorylated samples in excess non-radioactive P(i). Again the mutants Phe Gly and Ile Gly dephosphorylated rapidly, while the dephosphorylation was strongly blocked in mutant Ile Gly. In addition, the dephosphorylation of E2P in mutant Leu Gly was partially blocked. The ability to dephosphorylate rapidly was, however, regained upon exchange of the glycine at position 764 with lysine. It is also seen that the rate of dephosphorylation of E2P was slightly reduced in the uncoupled mutant Tyr Gly.


Figure 6: Dephosphorylation of the phosphoenzyme intermediates formed from [-P]ATP or P(i). The acid-quenched P-phosphorylated samples were subjected to SDS-polyacrylamide gel electrophoresis at pH 6.0, and the autoradiograms of the dried gels are shown. A, phosphorylation with [-P]ATP was carried out at 0 °C in the presence of 50 mM MOPS buffer, pH 7.0, 80 mM K, 5 mM Mg, 0.1 mM Ca, 1 µM of the Ca ionophore A23187, and 2 µM [-P] ATP. Following 15-s reaction with [-P]ATP, the sample was acid-quenched directly (lane 1), or incubated with 1 mM ADP and 1 mM EGTA for 5 s (lane 2), or with 1 mM EGTA without ADP for 5 s (lane 3) or 10 s (lane 4), prior to acid-quenching. B, phosphorylation with P(i) was carried out at 25 °C for 10 min in the presence of 100 µMP(i), 100 mM TES/Tris pH 7.0, 5 mM Mg, 2 mM EGTA, and 20% (v/v) dimethyl sulfoxide. Following cooling to 0 °C, the sample was acid-quenched directly (0), or dephosphorylation was initiated by 20-fold dilution of an aliquot into ice-cold medium containing 50 mM MOPS, pH 7.0, 80 mM K, 5 mM Mg, 1 mM non-radioactive P(i), and 2 mM EGTA, and acid quenching was performed 5 or 10 s after the dilution. C, phosphorylation was carried out at 0 °C for 15 s in the presence of 100 mM TES/Tris, pH 8.35, 10 mM Mg, 50 µM Ca, and 2 µM [-P]ATP. The phosphorylated sample was either acid-quenched directly (lane 1), or incubated with 1 mM ADP and 1 mM EGTA for 5 s (lane 2), prior to acid-quenching. In lane 3, the phosphorylated sample was incubated with 1 mM EGTA without ADP for 20 s, prior to acid-quenching. In lane 4, the phosphorylated sample was incubated with 1 mM EGTA without ADP for 15 s followed by 1 mM ADP for 5 s, prior to acid-quenching.



Because the E2P phosphoenzyme intermediate is likely to represent the enzyme form that releases Ca at the luminal surface, a plausible explanation for the uncoupling of ATP hydrolysis from Ca transport in mutant Tyr Gly would be that E1P is hydrolyzed directly to produce P(i), thereby bypassing E2P. To test the ability of mutant Tyr Gly to form E2P in the forward direction from E1P, phosphorylation from ATP was in addition carried out at alkaline pH and in the absence of alkali metal ions. Under these conditions E2P accumulates as the major steady state intermediate in the wild-type Ca-ATPase, due to a reduced rate of dephosphorylation of E2P (Andersen et al., 1989; Vilsen et al., 1989). It is seen in Fig. 6C, lanes 1 and 2, that the E2P phosphoenzyme intermediate was accumulated in the Tyr Gly and Leu Gly mutants as well, as demonstrated by the fraction of phosphoenzyme being insensitive to ADP. Lanes 3 and 4 demonstrate that the ADP-insensitive fraction of the phosphoenzyme increased further in the Tyr Gly mutant during a 15-s incubation with EGTA.


DISCUSSION

The present study features mutant Tyr Gly as a new phenotypic variant of the Ca-ATPase which is uncoupled in the sense that it hydrolyzes ATP at a high rate in a Ca-activated reaction without any net accumulation of Ca in the microsomal vesicles. In this mutant, phosphorylation from ATP and the rate of hydrolysis of ATP were maximally stimulated by Ca at a Ca concentration as low as 10 µM, whereas no Ca accumulation was detected at Ca concentrations up to the experimental limit of 200 µM. The uncoupling of ATPase activity from Ca accumulation in the Tyr Gly mutant was furthermore documented by the lack of an activatory effect of ionophore on the ATP hydrolysis.

The uncoupled ATP hydrolysis catalyzed by the Tyr Gly mutant proceeded through formation of an acid-stable phosphoenzyme intermediate and displayed characteristics very similar to the ionophore-activated ATPase activity of the wild-type Ca-ATPase with respect to specific rate, MgATP concentration dependence, and vanadate inhibition. In the wild-type enzyme, dephosphorylation takes place through the partial reaction sequence E1P E2P E2 + P(i). It was directly demonstrated that the Tyr Gly mutant is able to form the E2P intermediate from E1P (Fig. 6C). Because E2P is the intermediate thought to release Ca at the luminal surface (De Meis, 1981; Andersen, 1989; McIntosh et al., 1991), it is possible that the machinery responsible for the translocation of Ca is intact in the Tyr Gly mutant, and that the lack of net accumulation of Ca by the mutant is due to an increased rate of efflux of transported Ca from the vesicles. Such an increased passive efflux of Ca would be reminiscent of the leak of Ca through the Ca-ATPase observed following binding of phenothiazines and local anesthetics to the pump (De Meis, 1991; Wolosker et al., 1992; De Meis and Inesi, 1992). It was suggested that this efflux could be mediated through stabilization of unphosphorylated reaction intermediates in the late part of the normal reaction cycle (De Meis, 1991).

Alternatively, the calcium ions are not translocated by the Tyr Gly mutant due to a defect in the early part of the reaction cycle. It is possible that the gate normally preventing the occluded calcium ions from dissociating to the cytoplasmic side upon phosphorylation is defective in the mutant so that an anomalous E1P intermediate with non-occluded Ca sites is created. The side chain of Tyr might play a critical role in the gating mechanism, not unlike the roles of the aromatic side chains in pore regions of voltage-gated ion channels (Heginbotham and MacKinnon, 1992; Miller, 1993). It is also possible that the removal of the bulky side chain of the tyrosine led to a less specific and more widespread disturbance of the transmembrane domain, for instance as a consequence of changes in helix packing.

The slight reduction of apparent Ca affinity of the uncoupled Tyr Gly mutant as well as the more pronounced changes in Ca affinity observed with mutants Phe Gly and Leu Lys are in accordance with the hypothesis (Fig. 1) that the M5S5 boundary is located in the vicinity of the entrance to the Ca-binding domain. The effect of the positively charged lysine side chain on Ca binding can be explained by electrostatic repulsion of the calcium ions.

The mutant Leu Lys was able to dephosphorylate at a normal rate, while the dephosphorylation of the E2P intermediate was blocked in mutants Leu Gly and Ile Gly, and more so in the latter mutant. The block of E2P dephosphorylation explains the low ATPase activity observed with mutants Leu Gly and Ile Gly. A similar block of dephosphorylation of E2P has been observed for other mutants with alterations to residues in transmembrane segments M4, M5, and M6, including the putative Ca liganding residues Glu, Glu, and Asn (Andersen and Vilsen, 1992b, 1994; Vilsen and Andersen, 1992c; Clarke et al., 1993). Since recent evidence suggests that protons are countertransported by the Ca-ATPase (Levy et al., 1990; Yu et al., 1993), very likely in a way similar to the K transport by Na,K-ATPase, we suggested that the role for these residues in the dephosphorylation of E2P might be associated with the donation of the side chain oxygen to form binding/occlusion site(s) for the H (possibly H(3)O) to be countertransported (Andersen and Vilsen, 1992b). It is possible that the bulky side chains of Leu and Ile are crucial to the dephosphorylation of E2P because they stabilize the proton binding structure or because they participate in the conformational change(s) mediating long range communication with the phosphorylation site. The recovery of the ability to dephosphorylate rapidly upon exchange of the glycine substituent with lysine might be ascribed solely to the bulk of the side chain of the lysine. It is, however, also possible that the presence of a positively charged side chain near the suggested proton-binding site mimicks a proton, thereby inducing dephosphorylation.

Finally, the ATPase activity of mutant Leu Gly was higher than that of mutant Ile Gly, while the corresponding Ca uptake was lower, indicating that also mutant Leu Gly may have been uncoupled to some extent.

In summary, the present study has pointed to the hydrophobic residues at the M5S5 boundary as important for the tight coupling between ATP hydrolysis and Ca transport, and for the partial reactions involved in Ca binding and E2P dephosphorylation. It will be important to try in the future to further resolve the reasons for the observed uncoupling.


FOOTNOTES

*
This research was supported by grants from the Danish Biomembrane Research Centre, the Danish Medical Research Council, and the NOVO Nordisk Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Institute of Physiology, University of Aarhus, Ole Worms Allé 160, Universitetsparken, DK-8000 Aarhus C, Denmark. Fax: 45-86-12-90-65.

(^1)
The abbreviations used are: E1P, ADP-sensitive phosphoenzyme intermediate; E1, conformation with cytoplasmically facing high affinity Ca-binding sites; E2, conformation with low Ca affinity; E2P, ADP-insensitive phosphoenzyme intermediate; M1-M10, putative transmembrane segments numbered from the NH(2)-terminal end of the peptide; S1-S5, stalk segments connecting M1-M5 with the cytoplasmic domains; TES, N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid.


ACKNOWLEDGEMENTS

I thank Dr. Bente Vilsen for discussion and constructive criticism of the manuscript, Jytte Jørgensen, Karin Kracht, and Lene Jacobsen for their expert and invaluable technical assistance, and Dr. R. J. Kaufman, Genetics Institute, Boston, for the gift of the expression vector pMT2.


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