(Received for publication, August 22, 1995)
From the
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
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) (
)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.
Phosphorylation from P, 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
been demonstrated in PMCA, and this
demonstration required a very large amount of enzyme and high levels of
P
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
in
the system used here.
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
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
. 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
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
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
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.
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 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
. 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 E
P 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
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.