Calcium Biochemistry
163
6.2.3
The Ca
2
+
Pump
The principle protein component of the
sarcoplasmic reticulum (SR) is the Ca
2
+
pump (SERCA) transporting Ca
2
+
from
the cytosol into the lumen of the retic-
ulum against the concentration gradient
across the membrane. This protein can
represent as much as 90% of the mem-
brane protein (in SR of skeletal muscles),
bu
tevenintheSRo
fhea
r
tce
l
ls
,i
tcan
still make up to 50%. Similar to the
plasma membrane Ca
2
+
pump, the pro-
tein consists of a single polypeptide chain
of about 100 kDa. The predicted secondary
structure and membrane topography as
suggested by MacLennan, Green et al. was
impressively conFrmed by the recently
solved structures of the Ca
2
+
-bound and
Ca
2
+
-free forms (the latter in the presence
of the SERCA-speciFc inhibitor thapsigar-
gin) by Toyoshima et al. (see ±ig. 5). The
properties of the SR Ca
2
+
pump can be
summarized as follows:
1. The hydrophobic portion is made up of
10 transmembrane helices as predicted.
2. Transmembrane helix M5 continues
into the cytosol (60
˚
At
o
t
a
ll
e
n
g
t
h
)
coupling the membrane portion to the
cytosolic domains.
3. The major protein mass protrudes into
the cytosol divided into the actuator
(A), phosphorylation (P), and the nu-
cleotide binding (N) domains, the latter
being inserted within the P-domain (see
±ig. 5). The P- and N-domains, con-
necting transmembrane helices 4 and
5, provide the catalytic center of the
enzyme, with the conserved aspartyl
residue (D351) being phosphorylated
during the reaction cycle.
4. Two
Ca
2
+
binding
sites
could
be
identiFed within the transmembrane
part of the SERCA pump comprising
polar residues of helices M4, M5, M6,
and M8. This is a necessary prerequisite
for the stoichiometry of two calcium
ions being pumped/ATP during one
reaction cycle, and conFrms strongly
those
residues participating
in the
transport of calcium as identiFed by
mutational experiments.
5. Large-scale movements of the A-, P-,
and N-domains occur during the reac-
tion cycle as suggested by comparing
the Ca
2
+
-free with the Ca
2
+
-bound
structure. These domain movements
are coupled to changes in tilt and posi-
tion of helices M1 to M6.
6. One of the signiFcant differences be-
tween the two Ca
2
+
pumps of plasma
and reticular membranes concerns the
C-terminal part. In contrast to the
plasma membrane Ca
2
+
pump, the
SR protein does
not
directly interact
with calmodulin; that is, it lacks the
corresponding regulatory domain, but
some isoforms can be regulated by a
CaM kinase through phosphorylation
of phospholamban (PLN) (see below).
7. The SR Ca
2
+
pump of cardiac or
smooth muscles, that is, of slow twitch
muscles,
is
regulated
by
a
highly
hydrophobic, phosphorylatable protein
called
phospholamban
(PLN), composed
of an N- and a C-terminal helix con-
nected by a short, flexible hinge region.
PLN interacts with the SERCA pump
within the transmembrane region as
well as with a small loop of the P-
domain. PLN can be phosphorylated by
different kinases, that is, by PKA and
by a CaM-dependent kinase, thereby
leading to a signiFcant stimulation (up
to Fvefold) of the SR Ca
2
+
pump.
Thus, the unphosphorylated form of
phospholamban can be viewed as an
endogenous inhibitor of the SR Ca
2
+
pump similar to the CaM binding do-
main of the Ca
2
+
pump of plasma
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