Transcript Biochemistry of Metabolism Calcium Signals Copyright © 1999-2007 by Joyce J. Diwan. All rights reserved.

Biochemistry of Metabolism
Calcium Signals
Copyright © 1999-2007 by Joyce J. Diwan.
All rights reserved.
Ca++
calmodulin
Ca++-release channel
signal
endoplasmic
reticulum
++
Ca
Modulation of
cytosolic Ca++:
Cytosolic [Ca++]
is usually <1 μM,
except during a
Ca++ signal event.
ATP
Ca++-ATPase
++ ADP + Pi
Ca
Ca++
cytosol
outside
of cell
ATP
ADP + Pi
Ca++-ATPase
signalactivated
channel
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++
 Extracellular
[Ca++] in
mammals is in
the mM range.
Opening of
plasma membrane
Ca++ channels
may initiate or
sustain a Ca++
signal.
calmodulin
Ca++-release channel
signal
endoplasmic
reticulum
++
Ca
ATP
Ca++-ATPase
++ ADP + Pi
Ca
Ca++
cytosol
outside
of cell
ATP
ADP + Pi
Ca++-ATPase
signalactivated
channel
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
increase in
cytosolic Ca++
may be localized
to the vicinity of
one or a few Ca++release or Ca++entry channels.
Ca++
calmodulin
Ca++-release channel
signal
endoplasmic
reticulum
++
Ca
ATP
Ca++-ATPase
++ ADP + Pi
Ca
Ca++
signalactivated
channel
ADP + Pi
Such a localized
ATP
cytosol
++
++
Ca
-ATPase
Ca “puff” or
outside
“spark” may
Ca++
of cell
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
membrane interact with
ryanodine receptors in
the closely apposed SR
membrane.
cytosol
++
Ca
ryanodine
SR lumen
receptor
Activation of voltage-gated Ca++ channels, by an action
potential in the T tubule, leads to opening of ryanodinesensitive 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
(T tubule lumen)
voltage-gated
Ca++ channel
cytosol
++
Ca
ryanodine
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 a-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).
IP3 receptor
Ca++
OPO32 H
release channel
In many mammalian cells, IP3
(inositol-1,4,5-trisphosphate)
triggers Ca++ release from the ER.
The 2nd messenger IP3 is
produced, e.g., in response
to hormonal signals, from
the membrane lipid
phosphatidylinositol.
The IP3 receptor is a
ligand-gated Ca++-release
channel embedded in ER
membranes.
OPO32
OH
H
OH
OH
H
H
H
H
IP3
OPO32
inositol-1,3,4-trisphosphate
Ca++
Ca++-release channel
IP3
Ca
ATP
calmodulin
Ca
++
endoplasmic
reticulum
Ca++-ATPase
++ ADP + Pi
Ca++
Ca++-release channel
IP3
Ca
ATP
calmodulin
Ca
++
endoplasmic
reticulum
Ca++-ATPase
++ ADP + Pi
 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-binding domain of
the IP3 receptor, & promotes
channel opening.
Ca++
Ca++-release channel
IP3
Ca
However, high cytosolic
Ca++, which develops after
channel opening, promotes
channel closure.
ATP
calmodulin
Ca
++
endoplasmic
reticulum
Ca++-ATPase
++ ADP + Pi
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++
Ca++-release channel
IP3
Ca
View an animation of the
overall process of Ca++
cycling.
ATP
calmodulin
Ca
++
endoplasmic
reticulum
Ca++-ATPase
++ ADP + Pi
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.)
PDB
1CDM
glutamate
H
H3N+
COO
C
CH2
Calmodulin, a Ca++activated switch protein,
mediates many of the
signal functions of Ca++.
Calmodulin cooperatively
binds 4 Ca++.
CH2
C
O
++
Ca
O
aspartate (Asp)
H
H3N+
helix-loop-helix
motif in calmodulin
C
COO
CH2
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 a-helices at right angles.
This helix-loop-helix motif is called an EF hand.
There are 4 helix-loophelix motifs, 2 at each
end of calmodulin, which
is dumbbell shaped.
Calmodulin
Ca++ ( )
Ca++ binding promotes a
Target
conformational change
peptide
that exposes hydrophobic
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.
A typical calmodulin-binding target domain
is a (+) charged, amphipathic a-helix, with
polar & non-polar surfaces.
Terminal methyl groups of Met side-chains
of calmodulin participate in binding to
hydrophobic residues in target domains of
some enzymes regulated by calmodulin.
methionine (Met)
H
H3N+
C
COO
CH2
CH2
S
CH3
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
Ca++-release channel
signal
endoplasmic
reticulum
++
Ca
ATP
Ca++-ATPase
++ ADP + Pi
Ca
Ca++
cytosol
outside
of cell
ATP
ADP + Pi
Ca++-ATPase
signalactivated
channel
Ca++
Defects in genes coding for Ca++ channel proteins,
Ca++-ATPases, & intracellular Ca++ sensors are
associated with disease or death.