10.1 Calcium Regulation of the Actomyosin System
333
then effects an increase in the affinity of the Ca2+ATPase of the sarcoplasmic reticulum. Phospholamban is present in skeletal and heart muscle as
a pentamer of 52-amino-acid subunits; the
sequence of this protein varies only slightly
between different mammals, and these differ by
only 22-29 % from the chicken phospholamban
[195,285]. Calsequestrin in the reticulum membrane binds Ca 2 + in large amounts, although with
a low affinity, and it thus maintains the free calcium concentration of the reticulum lumen at
0.5-1.0 mmolll; this restricts the concentration
gradient between the sarcoplasma and the reticulum, against which the calcium pump must work
[294]. The sarcoplasmic calcium concentration is
not reduced quickly enough by uptake into the
reticulum to ensure rapid muscle relaxation.
Instead, much of the Ca 2 + is bound temporarily to
parvalbumin, which functions as a soluble relaxation factor [238].
There are three main mechanisms by which
calcium ions regulate the actomyosin ATPase;
one affects the thin actin filaments (A regulation), and the other two affect the thick myosin
filaments (M regulation). Vertebrate skeletal and
heart muscle, for example, are regulated actin
specifically. In this case, ATPase is inhibited at
low calcium concentrations by the troponintropomyosin complex of the thin filaments; binding of Ca 2 + to troponin C relieves this inhibition.
Until recently, the inhibitory effect of the
troponin-tropomyosin complex in vertebrate
muscle was thought to be due solely to steric
hindrance of the actin-myosin interaction; this
was supported by the observation that tropomyosin changes its position on the actin filaments at
increased calcium concentrations. However, it
has now been shown that actin-myosin binding
may be stable in the absence of calcium ions,
even though both ATPase activity and contraction
are inhibited. Apparently, the absence of Ca2+
inhibits a step in ATP hydrolysis which occurs
after the binding of myosin to actin. In preparations of various mollusc muscles, e.g. the striped
adductor muscle of the scallop Aequipecten irradians or the mantle muscle of the cephalopod
Loligo pealii, myosin-actin binding also persists
in the presence of ATP and the absence of calcium, although it may be weaker. In contrast, the
ATPase activity declines by 90 %, in particular by
reduction of the V max. Ca 2 + thus influences the
kinetics of ATP hydrolysis as well as of actinmyosin binding [238].
A myosin-specific regulation mechanism is active in various mollusc muscles and in vertebrate
smooth muscle. The contraction of mollusc muscles is triggered by direct binding of Ca 2 + to myosin. The Ca 2 + -binding regulatory domain includes
the regulatory and the essential light chains (LCs)
and a part of the heavy chain (HC) in the neck
region of the myosin head. Ca 2 + apparently binds
specifically to the essential LC, which in mollusc
myosins, in contrast to those not directly regulated by Ca 2 +, carries a functional Ca2+ -binding
site [153]. Ca2+ regulation of vertebrate smooth
muscle is also myosin specific; here, however, the
inhibitory effect of the regulatory LC is prevented
through phosphorylation by a specific Ca 2 +calmodulin-dependent myosin light-chain kinase
(MLCK). Phosphorylation of the regulatory LC
at a further site by the Ca2+ -phospholipiddependent protein kinase C has a deactivating
effect; this does not occur in embryonal smooth
muscle [158]. The regulatory LC in striated skeletal muscle can also be phosphorylated by
calmodulin-dependent MLCK; phosphorylation
of at least part of the LC is important for both the
strength and the efficiency of muscle contraction
[238]. A mutant, mdf, of Drosophila melanogaster has been isolated and shown to have threefold
lower myosin ATPase activity due to a lack of
phosphorylation [278].
In addition to myosin phosphorylation, caldesmon appears to play a role in the regulation of
vertebrate smooth-muscle (chicken stomach,
bovine aorta and uterus) and non-muscle (blood
platelets) cells; this is a calcium-regulated dimeric
protein with the ability to bind in a flip-flop manner to F-actin and calmodulin. In relaxed muscle
at low calcium concentration, caldesmon is bound
to actin and ATPase is inhibited; at higher calcium levels, caldesmon binds to calmodulin via an
interaction with tropomyosin and the inhibition is
relieved. Regulation in vertebrate smooth muscle
probably depends upon cooperation between
both the mechanisms described. Through its
effect on phosphorylation of the regulatory chain,
MLCK brings about the correct conditions for
actin-myosin interaction, which is then switched
on or off by the caldesmon flip-flop mechanism
[238, 250]. Caldesmon occurs in two tissuespecific isoforms: h-caldesmon (h for heavy) is
typical of smooth muscle, and l-caldesmon (1 for
light) is found in the brain and other non-muscle
cells; caldesmon is absent from skeletal and heart
muscle. The heavy form is composed of 771
amino acids, and l-caldesmon differs only by the
deletion of 232 amino acids from the repetitively
organized central region [252]. A caldesmon-like
protein has also been isolated from striated
333
then effects an increase in the affinity of the Ca2+ATPase of the sarcoplasmic reticulum. Phospholamban is present in skeletal and heart muscle as
a pentamer of 52-amino-acid subunits; the
sequence of this protein varies only slightly
between different mammals, and these differ by
only 22-29 % from the chicken phospholamban
[195,285]. Calsequestrin in the reticulum membrane binds Ca 2 + in large amounts, although with
a low affinity, and it thus maintains the free calcium concentration of the reticulum lumen at
0.5-1.0 mmolll; this restricts the concentration
gradient between the sarcoplasma and the reticulum, against which the calcium pump must work
[294]. The sarcoplasmic calcium concentration is
not reduced quickly enough by uptake into the
reticulum to ensure rapid muscle relaxation.
Instead, much of the Ca 2 + is bound temporarily to
parvalbumin, which functions as a soluble relaxation factor [238].
There are three main mechanisms by which
calcium ions regulate the actomyosin ATPase;
one affects the thin actin filaments (A regulation), and the other two affect the thick myosin
filaments (M regulation). Vertebrate skeletal and
heart muscle, for example, are regulated actin
specifically. In this case, ATPase is inhibited at
low calcium concentrations by the troponintropomyosin complex of the thin filaments; binding of Ca 2 + to troponin C relieves this inhibition.
Until recently, the inhibitory effect of the
troponin-tropomyosin complex in vertebrate
muscle was thought to be due solely to steric
hindrance of the actin-myosin interaction; this
was supported by the observation that tropomyosin changes its position on the actin filaments at
increased calcium concentrations. However, it
has now been shown that actin-myosin binding
may be stable in the absence of calcium ions,
even though both ATPase activity and contraction
are inhibited. Apparently, the absence of Ca2+
inhibits a step in ATP hydrolysis which occurs
after the binding of myosin to actin. In preparations of various mollusc muscles, e.g. the striped
adductor muscle of the scallop Aequipecten irradians or the mantle muscle of the cephalopod
Loligo pealii, myosin-actin binding also persists
in the presence of ATP and the absence of calcium, although it may be weaker. In contrast, the
ATPase activity declines by 90 %, in particular by
reduction of the V max. Ca 2 + thus influences the
kinetics of ATP hydrolysis as well as of actinmyosin binding [238].
A myosin-specific regulation mechanism is active in various mollusc muscles and in vertebrate
smooth muscle. The contraction of mollusc muscles is triggered by direct binding of Ca 2 + to myosin. The Ca 2 + -binding regulatory domain includes
the regulatory and the essential light chains (LCs)
and a part of the heavy chain (HC) in the neck
region of the myosin head. Ca 2 + apparently binds
specifically to the essential LC, which in mollusc
myosins, in contrast to those not directly regulated by Ca 2 +, carries a functional Ca2+ -binding
site [153]. Ca2+ regulation of vertebrate smooth
muscle is also myosin specific; here, however, the
inhibitory effect of the regulatory LC is prevented
through phosphorylation by a specific Ca 2 +calmodulin-dependent myosin light-chain kinase
(MLCK). Phosphorylation of the regulatory LC
at a further site by the Ca2+ -phospholipiddependent protein kinase C has a deactivating
effect; this does not occur in embryonal smooth
muscle [158]. The regulatory LC in striated skeletal muscle can also be phosphorylated by
calmodulin-dependent MLCK; phosphorylation
of at least part of the LC is important for both the
strength and the efficiency of muscle contraction
[238]. A mutant, mdf, of Drosophila melanogaster has been isolated and shown to have threefold
lower myosin ATPase activity due to a lack of
phosphorylation [278].
In addition to myosin phosphorylation, caldesmon appears to play a role in the regulation of
vertebrate smooth-muscle (chicken stomach,
bovine aorta and uterus) and non-muscle (blood
platelets) cells; this is a calcium-regulated dimeric
protein with the ability to bind in a flip-flop manner to F-actin and calmodulin. In relaxed muscle
at low calcium concentration, caldesmon is bound
to actin and ATPase is inhibited; at higher calcium levels, caldesmon binds to calmodulin via an
interaction with tropomyosin and the inhibition is
relieved. Regulation in vertebrate smooth muscle
probably depends upon cooperation between
both the mechanisms described. Through its
effect on phosphorylation of the regulatory chain,
MLCK brings about the correct conditions for
actin-myosin interaction, which is then switched
on or off by the caldesmon flip-flop mechanism
[238, 250]. Caldesmon occurs in two tissuespecific isoforms: h-caldesmon (h for heavy) is
typical of smooth muscle, and l-caldesmon (1 for
light) is found in the brain and other non-muscle
cells; caldesmon is absent from skeletal and heart
muscle. The heavy form is composed of 771
amino acids, and l-caldesmon differs only by the
deletion of 232 amino acids from the repetitively
organized central region [252]. A caldesmon-like
protein has also been isolated from striated