©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Two Residues That May Ligate Ca in Transmembrane Domain Six of the Plasma Membrane Ca-ATPase (*)

(Received for publication, August 22, 1995)

Aderonke O. Adebayo Agnes Enyedi (§) Anil K. Verma Adelaida G. Filoteo John T. Penniston (¶)

From the Department of Biochemistry and Molecular Biology, Mayo Clinic/Foundation, Rochester, Minnesota 55905

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

In order to identify Ca ligands in the putative transmembrane domain 6 of the plasma membrane Ca pump, amino acids Asn, Met, Asp, and Ser were singly altered. Asn, Met, and Asp were chosen because the corresponding amino acids have been proposed as Ca ligands in the sarcoplasmic reticulum Ca pump (Clarke, D. M., Loo, T. W., and MacLennan, D. H.(1990) J. Biol. Chem. 265, 6262-6267). For the alterations, a fully active truncated version of the pump was used, because the interaction of Ca with the pump could be studied without interference from calmodulin binding. The mutants at Asn and Asp did not carry out ATP-supported Ca uptake and formed no acylphosphate from [-P]ATP, suggesting that, like the corresponding amino acids in the sarcoplasmic reticulum Ca pump, these two are Ca ligands. However, all the mutants at the position of Met showed some activity. Indeed, the Met Ile mutant was fully active at a saturating Ca concentration and only the K for Ca activation was shifted slightly upward. Converting the Met to Thr (which is the corresponding residue in the sarcoplasmic reticulum Ca pump) reduced the activity to 20% of the wild type, further emphasizing the differences between the two Ca pumps. The mutant Ser Ala was expressed in greater amounts than, and had a specific activity about 50% higher than, the wild type, indicating that this serine also could not be a Ca ligand and could not replace the missing Thr at position Met


INTRODUCTION

There are two ATP-energized Ca pumps in mammalian cells, both of which have the role of removing Ca from the cytosol to a metabolically inactive compartment. The sarcoplasmic reticulum Ca pump (SERCA) (^1)moves calcium to the lumen of the sarcoplasmic or endoplasmic reticulum, while the plasma membrane Ca pump (PMCA) moves calcium to the outside of the cell. These pumps are both P-type ATPases, which means that they form an acylphosphate from ATP and the side chain carboxylate of an aspartic acid as a part of their transport mechanism. This acylphosphate is broken down as Ca is released to the metabolically inactive compartment. As P-type ATPases, these pumps share certain crucial regions of primary structure, but large stretches of the primary sequence do not show any strong similarity between the two Ca pumps. SERCA1a and hPMCA4b (the most commonly studied isoforms) share only a 32% sequence identity overall. The transmembrane regions show an interesting type of homology: the distribution and number of putative transmembrane domains is the same in both of these pumps, but in general only the hydrophobicity of these domains is conserved. The sequence of these domains in most parts of the enzyme shows no conservation. Putative transmembrane domain 6 (Brandl et al., 1986) is an exception to this generalization; it shows a significant degree of similarity between these two pumps.

M6 is a particularly interesting region, because site-directed mutagenesis studies (Clarke et al., 1989a) in SERCA have shown that Ca transporting activity is unusually sensitive to mutations in this region. Extensive study of mutants (Clarke et al., 1990) has identified three residues in this region as proposed Ca ligands, which are said to be involved in binding Ca during its transit through the membrane. Site-directed mutagenesis studies on SERCA1a showed that Asn, Thr, and Asp had kinetic properties consistent with their being the ligands. Fig. 1shows the sequence of this region in SERCA1a. Also shown in this figure is the corresponding region of the human plasma membrane Ca pump hPMCA4. The similarities between these two Ca pumps in region M6 is evident from Fig. 1, and these similarities have recently been found to extend to other putative Ca pumps. Fig. 2illustrates this by comparing the M6 region of known or putative Ca pumps (top seven lines), with a few examples of other P-type ATPases (bottom five lines). The first two lines are the same pumps as were listed in Fig. 1; both of these have been extensively documented as Ca pumps. The next two lines show sequence from two probable Ca pumps from yeast. Genetic evidence has shown that null mutants in these genes have differences in Ca sensitivity consistent with the identification of the gene products as Ca pumps. The fifth through seventh lines are sequences from molecules which have been identified as Ca pumps only because of their sequence similarity to the known Ca pumps, when they are compared over the entire length of the molecule (usually 900-1000 amino acid residues). The conservation of the M6 region among the Ca pumps and the greater divergence among pumps which move other kinds of ions is evident. This relationship suggests that the M6 region is important for the ionic specificity of P-type ATPases.


Figure 1: Point mutations in the M6 transmembrane region of human plasma membrane Ca-ATPase (hPMCA). Stars indicate the position of the mutation in hPMCA4b(ct120). Capital L indicates putative Ca ligands.




Figure 2: The alignment of the M6 regions of known P-type ATPases. The italicized capital L indicates the putative Ca ligands in SERCA. The consensus was based on agreement of seven of the sequences, and residues agreeing with the consensus are capitalized.



In order to investigate the potential Ca ligands in hPMCA, we made point mutations in the three residues which corresponded to the putative Ca ligands proposed by MacLennan and co-workers (Clarke et al., 1990) and in Ser. Instead of making the mutants in the full-length hPMCA4b, the truncated form called ct120 was used. This form is already fully activated and allows the study of the interaction of the pump with Ca without interference from the Ca-calmodulin interaction (Enyedi et al., 1993). Of the three putative ligands, only the Thr of SERCA is not conserved in hPMCA4b, where the corresponding residue is Met. This alteration already suggested that it was unlikely that this Met would be a ligand, since the sulfur ether side chain is not expected to have a strong affinity for Ca. However, because of its two unshared electron pairs the possibility that it ligated with Ca could not be ruled out, except by experiment. The results reported below are consistent with the notion that Asn and Asp are Ca ligands. They also show that Met and Ser cannot be ligands.


MATERIALS AND METHODS

Oligonucleotide-directed Mutagenesis

The construction of the hPMCA4b (ct120)-truncated mutant has been described in detail previously (Enyedi et al., 1993). Mutations were performed using the altered site mutagenesis kit, which uses the pAlter 1 vector (Promega Corp., Madison, WI). Specific oligonucleotides containing the desired changes were synthesized by the Mayo Molecular Biology Core Facility and hybridized with the single strand hPMCA4b(ct120) cDNA contained in the pAlter1 vector. After transformation and ampicillin selection, the clones containing the mutations were sequenced using the Applied Biosystems Automatic Sequencer, using dideoxy chain termination technology. The mutant hPMCA4b(ct120) was cut out of the pAlter 1 vector with restriction nucleases SalI and KpnI and cloned into a smaller vector pSP72 (Promega) that does not have the unique restriction sites BspEI and NsiI. The 868-base pair BspEI-NsiI fragment containing the mutations was sequenced again and then cloned into the pSP72 hPMCA4b(ct120) from which the original BspEI-NsiI fragment had been removed. Subsequently, the hPMCA4b(ct120) mutants were then removed from pSP72 with the restriction nucleases SalI and KpnI and transferred into the pMM2 vector for expression in COS-1 cells (Gluzman, 1981).

Transfection

The M6 isolates of COS-1 cells obtained from Genetics Institute (Cambridge, MA) were transfected with the mutant DNA preparations as described in a previous publication (Verma et al., 1994), using 20 µg DNA/150-cm^2 flask. After transfection (3 h) and posttransfection (48 h), the cells were harvested for the preparation of microsomes.

Isolation of Microsomes from COS-1 Cells

The crude microsomal membrane fractions were prepared by a modified version of the procedure of Clarke et al. (1989b) as described by Enyedi et al.(1993).

Ca Transport Assay

Calcium influx into microsomal vesicles was measured at 37 °C by rapid filtration through Millipore membrane filters (0.45 µM pore size, type HA) as described in a previous paper (Enyedi et al., 1993). The transport medium contained 100 mM KCl, 25 mM TES-triethanolamine (pH 7.2), 40 mM KH(2)PO(4)/K(2)HPO(4) (pH 7.2), 200 nM thapsigargin, 5 mM NaN(3), 4 µg/ml oligomycin, 0.5 mM ouabain, 7 mM MgCl(2), 100 µM CaCl(2) (labeled with Ca), appropriate concentrations of vanadate where called for, and sufficient EGTA to obtain the desired free Ca concentration. Microsomes at 15-20 µg/ml were preincubated for 3 min at 37 °C before initiating calcium uptake by the addition of 6 mM ATP. When ct120 was expressed, the amount of calcium uptake observed was about 20 times higher than that when the empty plasmid pMM2 was expressed.

Phosphorylation of the Calcium Pump Protein

10 µg of microsomal protein was added to an ice-cold solution containing 25 mM TES-triethanolamine (pH 7.2), 400 nM thapsigargin, 50 µM CaCl and (when needed) 50 µM LaCl(3) in a reaction volume of 0.25 ml. The reaction was initiated by the addition of 1 µM [-P]ATP and stopped after 30 s with 0.015 ml of 100% trichloroacetic acid followed immediately by 50 µl of 1 mg/ml bovine serum albumin. After addition of 2 ml of a washing solution containing 6% (w/v) trichloroacetic acid, 1 mM cold ATP, and 10 mM inorganic phosphate, the denatured proteins were collected by centrifugation at 5,000 rpm for 20 min, washed three times with the washing solution and once with distilled water. The trichloroacetic acid-precipitated samples were dissolved in solubilizing solution containing 62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 5 mM EDTA, 125 mg/ml urea, 100 mM dithiothreitol. An aliquot containing 2-5 µg of the phosphorylated membrane protein was applied to a slightly acidic 7.5% SDS acrylamide gel according to Sarkadi et al.(1986). The gel samples were dried for 15 min using a gel drier (Fisher Biotech). Autoradiography of the dried gel was performed with 24-72 h of exposure at -70 °C using X-Omat x-ray films.

Phosphorylation from P(i), as was done in studies of the Ca ligands of SERCA, was not feasible because of the inefficiency of this process in PMCA. Only in purified enzyme has phosphorylation from P(i) been demonstrated in PMCA, and this demonstration required a very large amount of enzyme and high levels of P(i) and dimethyl sulfoxide to achieve phosphorylation of only about 12% of the available molecules (Chiesi et al., 1984). Because of the low amount of PMCA protein in actual membranes, and the presence of large amounts of other proteins which bind P, it is not possible to carry out phosphorylation from P(i) in the system used here.

Quantitation of Ca Pump in COS Cell Membrane Preparations

Different amounts of microsomes were dissolved in solubilizing solution as described above. Using a Bio-Rad Mini-PROTEAN System, the samples were electrophoresed on a 7.5% SDS acrylamide gel according to Laemmli(1970). The samples were subsequently transblotted to polyvinylidene difluoride membrane using 25 mM Tris, 0.7 M glycine as transfer solution. The transfer was run for 35 min at a constant voltage of 60 volts. A block of ice was used to prevent overheating during transfer. The blots were immunostained using monoclonal antibody 5F10. The amount of Ca pump in the mutants, as compared with the wild type, was determined by scanning the immunoblots digitally using MacIntosh Image 1.44.

Protein Concentrations

These were measured by the method of Lowry et al.(1951).


RESULTS

Table 1shows a list of the point mutants which were successfully expressed in COS cells. Of these mutants, those involving Asn and Met were expressed in amounts approximately equal to that of the wild type ct120, while mutants involving Asp and Ser were expressed to a much higher degree. This latter group was expressed at approximately a 3-fold higher level than the wild type, based on the intensity of staining of immunoblots with antibody 5F10. The extent of expression in each individual preparation of COS cell membranes was used to calculate the V(max) for Ca transport. In such cases, the immunoblots were done at three different membrane concentrations and the amount of expression used as the divisor in calculating V(max). As is evident from the table, the mutants involving Asn and Asp were inactive, consistent with their proposed role as Ca ligands. However, the mutations to Met and Ser all showed activity, with Met Ile having an activity equal to that of the wild type and Ser Ala showing an activity even higher than the wild type.



The dependence of the Ca uptake on Ca concentration is shown in Fig. 3and in the third column of Table 1. The Ser Ala mutant showed no significant change in K for calcium when compared with the wild type, while the mutants involving Met showed a somewhat lower apparent affinity for Ca.


Figure 3: Ca concentration dependence of Ca transport by the wild type ct120 and mutant pumps. Ca uptake by vesicles made from cells transfected by vector alone have been subtracted from all data points. The maximum Ca uptake of the wild type ct120 was used to calculate the relative percent activities. Inverted triangles, ct120; diamonds, Met Ala; circles, Met Thr; open diamonds, Met Ile; open triangles, Ser Ala. The lines represent the best fit of the data given by the Hill equation. The V(max) and K values are given in Table 1.



The vanadate sensitivity of the enzymes was also tested and is reported in Fig. 4. The wild type and the mutants Met Ile and Ser Ala all showed the same vanadate sensitivity with a half-maximal inhibition at about 4-5 µM vanadate, and the Met Ala mutant required approximately three times as much vanadate for half inhibition. The most notable result of this experiment was that Met Thr showed a very low sensitivity to vanadate, with 80% of the original activity remaining even at 50 µM vanadate. Since the E(2) state of the pump is believed to be the one which interacts with vanadate, these data indicate that in the Met Ala and Met Thr mutants, the proportion of E(2) formed during the reaction cycle is lower than in the wild type. This would also explain the lowered activity of these mutants.


Figure 4: The effect of vanadate on the Met and Ser Ala mutants as compared with hPMCA4b(ct120). Symbols are the same as in Fig. 3. The activities of each mutant are expressed as a percent of the values of the same samples measured in the absence of vanadate.



Studies on the phosphorylated intermediate: the ability of all the mutants to form an acylphosphate from ATP in the presence of Ca was assessed both in the presence and the absence of lanthanum. Lanthanum is known to block the dephosphorylation step, thereby increasing the level of phosphoenzyme. As Fig. 5shows, in the absence of lanthanum small amounts of phosphoenzyme were present in the wild type, but it was not possible to demonstrate the presence of phosphoenzyme in any of the mutants except for Ser Ala. Surprisingly, this mutant showed a much higher level of phosphoenzyme than did the wild type. In the presence of lanthanum, the phosphoenzyme level in the wild type responded in the expected way, with a substantial increase in intensity. This increase due to lanthanum was much less in the Ser Ala mutant, leaving the intensities of the bands in these two constructs nearly equal. The other two mutants having substantial activity (Met Ala and Met Ile) showed smaller amounts of phosphoenzyme. Insignificant amounts of phosphoenzyme were observed in the other mutants, indicating that the inhibition was at or before the phosphorylation step. It is worth mentioning that none of the interactions increased the sensitivity of the pump to thapsigargin which was present in all assays in order to inhibit SERCA (data not shown).


Figure 5: Phosphorylation of the Ca-ATPase in microsomal fractions from COS1 cells transfected with wild type ct120 and mutant cDNA. Phosphoenzyme formation with ATP was carried out in the absence (A) and or presence (B) of lanthanum as described under ``Materials and Methods.'' Samples containing an equivalent amount of expressed ATPase were separated by electrophoresis by the method of Sarkadi et al.(1986), using 7.5% acrylamide gels. Radioactivity was detected by autoradiography.




DISCUSSION

The assignment of three possible ligands involved in transport of Ca across the membrane in SERCA (Clarke et al., 1989a, 1990) has demonstrated the importance of this region to the enzyme's function. When the corresponding regions of other P type ATPases are aligned, it becomes apparent, as shown in Fig. 2, that the Ca pumps and putative Ca pumps share a high degree of conservation of sequence in this region, while the pumps of other ions diverge substantially from this sequence. This suggests that this region is involved in the selectivity for Ca over other ions and that all P-type Ca pumps use a similar general strategy for binding Ca.

In order to assess the role of some of these residues in the plasma membrane calcium pump, the three residues proposed as Ca ligands were altered. The amino acid substitutions that were made in the positions of Asn and Asp all caused complete inactivation of the enzyme, even when a minimal change was made by substituting Asn for Asp. None of the mutants in these positions formed a phosphoenzyme from ATP. These results are consistent with the concept that these two residues are Ca ligands as in the sarcoplasmic reticulum Ca pump.

The results with substitutions at Met were quite different. All of the substitutions made here retained some activity and the changes in K for Ca transport, while significant, were only by a factor of about two. Thus, it appeared that changes to this residue were causing significant changes in the enzyme but not inactivating it. The mutant Met Ile was particularly interesting, because it gave a fully active product. This substitution is highly conservative, since the size of isoleucine is similar to that of methionine, and studies of related proteins from different species frequently show substitution of one of these for the other (Kyte, 1995). The full activity of this construct demonstrates that this methionine cannot be a Ca ligand. Nonetheless this construct had somewhat different properties from the wild type; less acylphosphate was formed in this construct either in the presence or in the absence of lanthanum, and its K for Ca was also somewhat lower.

Conversion of methionine 882 to alanine decreased the activity of the enzyme somewhat while converting this methionine to the threonine found in SERCA caused the lowest activity found among the substitutions at this position. Both mutations decreased the sensitivity of the pump to vanadate inhibition. This indicates that the inhibition results in a lower fraction of E(2) in the steady state. These results showed that this region of M6 is very sensitive to changes even in residues which do not bind Ca. The fact that converting this methionine to the threonine found in SERCA substantially decreased the enzyme's activity demonstrated that significant differences remain in the nature of the Ca binding region of these two enzymes even though they have substantial fundamental similarities. These differences may also be reflected in the different stoichiometries reported for PMCA and SERCA. The latter transports two Ca per ATP hydrolyzed (Inesi et al., 1990), while PMCA appears to transport only one Ca per ATP (Niggli et al., 1981, Hao et al., 1994).

The results of the Ser Ala mutant were also interesting. They show not only that Ser cannot be a ligand, but also that it is possible to make alterations in this region which cause an increase in V(max). Not only was the activity of this construct higher, but the amount of acylphosphate (when measured in the absence of lanthanum) was substantially higher than that seen in the wild type. This indicates that the higher activity might be due to a faster EP formation from ATP in the presence of Ca.

Fig. 6is a diagram of the M6 region showing the arrangement which would exist looking down the axis of an alpha helix. It shows the Asn and Asp to be near one another on the same side of the helix, favorably situated for ligation of a metal ion, while Met and Ser flank them. If M6 is in a helical conformation, Met or the corresponding threonine in SERCA would be poorly situated to chelate an ion which was chelated by Asn and Asp. The effects of all the mutants studied here are consistent with the notion that Asn and Asp are intimately involved in Ca binding, while Met and Ser are in nearby positions where they can influence, but not prevent, Ca transport. They also support that SERCA and PMCA, while generally similar in their handling of Ca, have substantial differences which may result in an altered stoichiometry.


Figure 6: Helical wheel plot of transmembrane domain six. The amino acid residues Asn and Asp are on the same side of the helical wheel, while Met and Ser occupy flanking positions.




FOOTNOTES

*
This work was supported in part by the National Institutes of Health Grant GM 28835. 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.

§
Present address: National Institute of Hematology, Blood Transfusion and Immunology, 1113 Budapest, Daroczi Ut 24, Hungary.

To whom correspondence should be addressed. Tel.: 507-284-2295; Fax: 507-284-9759.

(^1)
The abbreviations used are: SERCA, sarco/endoplasmic reticulum Ca pump; PMCA, plasma membrane Ca pump; hPMCA4b, human plasma membrane Ca pump, isoform 4b; M6, transmembrane domain 6; ct120, hPMCA4b with the downstream 120 amino acids removed; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.