Biochemistry of Metabolism

Calcium Signals

Copyright © 1999-2007 by Joyce J. Diwan.

All rights reserved.
 Ca++ calmodulin

signal Ca++-release channel

++
endoplasmic
Ca reticulum

Modulation of Ca++-ATPase
cytosolic Ca++: ATP ++ ADP + Pi
Ca
Cytosolic [Ca++] Ca++ signal-
is usually 1 μM, ATP ADP + Pi activated
cytosol Ca++-ATPase channel
except during a
outside
Ca++ signal event. of cell Ca++

Ca++-ATPase pumps in plasma membranes & ER

membranes maintain this low concentration by transporting
Ca++ away from the cytosol, out of the cell or into the ER. 
 Ca++ calmodulin
 Extracellular signal Ca++-release channel
[Ca++] in
endoplasmic
mammals is in Ca ++
reticulum
the mM range.
Ca++-ATPase
Opening of ATP ++ ADP + Pi
plasma membrane Ca
Ca++ channels may Ca++ signal-
ADP + Pi activated
initiate or sustain cytosol
ATP
channel
Ca++-ATPase
a Ca++ signal.
outside
of cell Ca++

 [Ca++] is also relatively high in the lumen of the ER, which

serves as the major internal reservoir from which Ca++ is
released to the cytosol during Ca++ signaling.
 Mitochondria and lysosomes also serve as reservoirs for
Ca++ subject to release under certain conditions.
 Ca++-binding proteins in the ER lumen "buffer" free
[Ca++], and increase the capacity for Ca++ storage.
ER Ca++-binding proteins have 20-50 low-affinity Ca++-
binding sites per molecule, consisting of acidic residues.
Examples:
 Calsequestrin is in the lumen of the sarcoplasmic
reticulum (SR), a specialized ER of muscle.
 Calreticulin is in the lumen of the ER of non-muscle
cells. It also has a role in protein folding.
Ca++ concentration, within the cytosol or other cell
compartments, may be monitored using indicator dyes
or proteins that are either luminescent or change their
fluorescence when they bind Ca++.
Fluorescent indicators used with confocal fluorescence
microscopy can provide high-resolution imaging and
quantitation of Ca++ fluctuations within cells.
Ca++ regulates many cellular reactions and processes.
A transient Ca++ calmodulin
increase in signal Ca++-release channel
cytosolic Ca++
endoplasmic
may be localized Ca ++
reticulum
to the vicinity of
one or a few Ca++- Ca++-ATPase
release or Ca++- ATP
Ca ++ ADP + Pi

entry channels.
Ca++ signal-
Such a localized ATP ADP + Pi activated
cytosol Ca ++
-ATPase channel
Ca “puff” or
++
outside
“spark” may of cell Ca++
activate effectors
that induce additional Ca++ release, leading to a more
widespread increase in cytosolic Ca++.
A “wave” of higher Ca++ may spread to neighboring cells.
For example, see a website maintained by E. Niggli
showing recordings of Ca++ sparks and waves, using
fluorescent Ca++ indicators.
Ryanodine Receptor: A Ca++ Release Channel
A large Ca++ release channel in the membrane of
muscle sarcoplasmic reticulum (SR) is called the
ryanodine receptor, because of sensitivity to inhibition
by a plant alkaloid ryanodine.
Skeletal and cardiac muscle contraction is activated
when Ca++ is released from the SR lumen to the cytosol
via the ryanodine receptor.
 extracellular space
T tubules: invaginations of (T tubule lumen)
voltage-gated
muscle plasma membrane. Ca++ channel
Voltage-gated Ca++
channels in the T tubule
cytosol
membrane interact with
ryanodine receptors in ++
ryanodine Ca SR lumen
the closely apposed SR
receptor
membrane.
Activation of voltage-gated Ca++ channels, by an action
potential in the T tubule, leads to opening of ryanodine-
sensitive Ca++-release channels.
Ca++ moves from the SR lumen to the cytosol, passing
through the transmembrane part of the ryanodine receptor,
& then through the receptor's cytoplasmic assembly.
 extracellular space
voltage-gated (T tubule lumen)
Ca++ channel

cytosol

++
ryanodine Ca SR lumen
receptor

 The ryanodine receptor is itself activated by cytosolic

Ca++ at micromolar concentrations.
Thus a entry of a small amount of Ca++ into the cytosol
causes further Ca++ release.
 High (e.g., mM) cytosolic Ca++ inactivates the ryanodine
receptor channel, contributing to signal turn-off.
Three views of a 3D reconstruction of the structure of the
ryanodine-sensitive calcium channel at 30 Å resolution,
based on micrographs obtained by EM at varied tilt angles.
These images were provided by Terrence Wagenknecht of the
Wadsworth Center, NY State Dept. of Health.
Animation of conformational changes during channel
opening & closing.
A somewhat higher resolution structure of the
ryanodine receptor channel now available indicates the
presence of bent -helices adjacent to the lumen in the
transmembrane pore domain.
But an atomic resolution structure of the whole channel
has not yet been achieved.
For diagrams see article by Ludtke et al. (journal subscription
required).
 O PO 3 2  H
IP3 receptor Ca++ release channel
OH O PO 3 2 
H OH
In many mammalian cells, IP3 OH H
(inositol-1,4,5-trisphosphate) H H
triggers Ca++ release from the ER. H O PO 3 2 
IP 3
The 2nd messenger IP3 is inositol-1,3,4-trisphosphate
produced, e.g., in response
to hormonal signals, from Ca++ calmodulin
the membrane lipid
phosphatidylinositol. IP3 Ca++-release channel

++
endoplasmic
The IP3 receptor is a Ca reticulum
ligand-gated Ca++-release
channel embedded in ER Ca++-ATPase
membranes. ATP ++ ADP + Pi
Ca
 Ca++ calmodulin

IP3 Ca++-release channel

++
endoplasmic
Ca reticulum

Ca++-ATPase
ATP ++ ADP + Pi
Ca

 The IP3 receptor (IP3-activated Ca++-release channel)

is distinct from but partly homologous to the
ryanodine receptor channel.
 IP3 binds to a cytosolic domain of the receptor
promoting channel opening.
 IP3 may displace a regulatory phospho-protein IRBIT,
which binds at the same site. Diagram (RIKEN Inst)
Ca++ also binds to the ligand-
Ca++ calmodulin
binding domain of the IP3
receptor, & promotes IP3 Ca++-release channel
channel opening. ++
endoplasmic
Ca reticulum
However, high cytosolic
Ca++, which develops after Ca++-ATPase
channel opening, promotes ATP
Ca ++ ADP + Pi

channel closure.
Thus both IP3-activated & ryanodine-sensitive channels are
activated by low cytosolic Ca++ & inhibited by high Ca++.
The feedback inhibition of Ca++ release by high cytosolic
Ca++, along with activity of Ca++-ATPase pumps, contributes
to signal turnoff & makes possible observed oscillations in
Ca++ concentration.
 Ca++ calmodulin

IP3 Ca++-release channel

++
endoplasmic
Ca reticulum
View an animation of the
overall process of Ca++ Ca++-ATPase
cycling. ATP ++ ADP + Pi
Ca

Structures of cytosolic domains of the IP3 receptor,

including the IP3 binding site, have been solved, but the
pore structure of the receptor has not yet been determined
at atomic resolution. 
See website for a low-resolution structure of IP3 receptor.
(AIST, Japan, findings of C. Sato et al.)
 glutamate
PDB H
1CDM H3N+ C COO

CH2

CH2
Calmodulin, a Ca++- C
O
activated switch protein, O

mediates many of the Ca++ aspartate (Asp)

H
signal functions of Ca++.
H3N+ C COO
Calmodulin cooperatively helix-loop-helix CH2
binds 4 Ca++. motif in calmodulin COO

At each binding site, Ca++ interacts with O atoms, mainly of

Glu & Asp side-chain carboxyl groups, & of the protein
backbone, in a loop between 2 -helices at right angles.
This helix-loop-helix motif is called an EF hand.
There are 4 helix-loop-
helix motifs, 2 at each Calmodulin
end of calmodulin, which
is dumbbell shaped. Ca++ ( )

Ca++ binding promotes a

conformational change Target
that exposes hydrophobic peptide
residues along a concave
patch on each of the 2 lobes.
These are involved in protein-protein interactions.
Ca++-calmodulin then changes conformation again as it
wraps around the target domain of a protein.
 methionine (Met)
A typical calmodulin-binding target domain H
is a (+) charged, amphipathic -helix, with H3N+ C COO
polar & non-polar surfaces.
CH2
Terminal methyl groups of Met side-chains CH2
of calmodulin participate in binding to
S
hydrophobic residues in target domains of
CH3
some enzymes regulated by calmodulin.
However the interaction of Ca++-calmodulin with some
target proteins is different from what is described here.
Some proteins have bound calmodulin as part of their
quaternary structure, even in the absence of Ca++.
In either case, Ca++ binding to calmodulin may induce a
conformational change that alters target protein activity.
Many enzymes are regulated by Ca++-calmodulin. E.g.:
 Some protein kinases that transfer phosphate from
ATP to hydroxyl residues on other enzymes to be
regulated, are activated by Ca++-calmodulin.
These are referred to as CaM Kinases.
 The plasma membrane Ca++-ATPase that pumps Ca++
out of the cell is one of the target proteins activated
by Ca++-calmodulin.
Thus cytosolic Ca++ itself contributes further to
turning off Ca++ signals.

View an animation of Ca++-activated binding of

calmodulin to a target peptide.
 Ca++ calmodulin

signal Ca++-release channel

++
endoplasmic
Ca reticulum

Ca++-ATPase
ATP ++ ADP + Pi
Ca
Ca++ signal-
ATP ADP + Pi activated
cytosol Ca++-ATPase channel
outside
of cell Ca++

Defects in genes coding for Ca++ channel proteins, Ca+

+-ATPases, & intracellular Ca++ sensors are associated

with disease or death.