Calcium Ion Is a Ubiquitous Cytosolic Messenger

Figure 15.17. Metabolism of Diacylglycerol. Diacylglycerol may be (1) phosphorylated to phosphatidate or (2)
hydrolyzed to glycerol and fatty acids.
II. Transducing and Storing Energy
15. Signal-Transduction Pathways: An Introduction to Information Metabolism
15.3. Calcium Ion Is a Ubiquitous Cytosolic Messenger
We have already seen that Ca2+ is an important component of one signal-transduction circuit, the phosphoinositide
cascade. Indeed, Ca2+ is itself an intracellular messenger in many eukaryotic signal-transducing pathways. Why is this
ion commonly found to mediate so many signaling processes? First, an apparent drawback is in fact an advantage:
calcium complexes of phosphorylated and carboxylated compounds are often insoluble, but such compounds are
fundamental to many biochemical processes in the cell. Consequently, the intracellular levels of Ca2+ must be kept low
to prevent precipitation of these compounds. These low levels are maintained by transport systems for the extrusion of
Ca2+. In eukaryotic cells, two in particular the Ca2+ ATPase (Section 13.2.1) and the sodium-calcium exchanger are
especially important. Because of their actions, the cytosolic level of Ca2+ in unexcited cells is typically 100 nM, several
orders of magnitude lower than the concentration in the blood, which is approximately 5 mM. This steep concentration
2+
concentration can be abruptly raised for
gradient presents cells with a matchless opportunity: the cytosolic Ca
signaling purposes by transiently opening calcium channels in the plasma membrane or in an intracellular membrane.
A second property of Ca2+ that makes it a highly suitable intracellular messenger is that it can bind tightly to proteins
(Figure 15.18). Negatively charged oxygen atoms (from the side chains of glutamate and aspartate) and uncharged
oxygen atoms (main-chain carbonyl groups and side-chain oxygen atoms from glutamine and asparagine) bind well to
2+
to be coordinated to multiple ligands from six to eight oxygen atoms
Ca2+. The capacity of Ca
link different segments of a protein and induce significant conformational changes.
enables it to cross-
15.3.1. Ionophores Allow the Visualization of Changes in Calcium Concentration
Our understanding of the role of calcium in cellular processes has been greatly enhanced by the use of calcium-specific
reagents. Ionophores such as A23187 and ionomycin can traverse a lipid bilayer because they have a hydrophobic
periphery.
They can be used to introduce Ca2+ into cells and organelles. Many physiological responses that are normally triggered
by the binding of hormones to cell-surface receptors can also be elicited by using calcium ionophores to raise the
cytosolic calcium level. Conversely, the concentration of unbound calcium in a cell can be made very low (nanomolar or
less) by introducing a calcium-specific chelator such as EGTA. The concentration of free Ca2+ in intact cells can be
monitored by using polycyclic chelators such as Fura-2.
The fluorescence properties of this and related indicators change markedly when Ca2+ is bound (Figure 15.19). These
compounds can be introduced into cells to allow measurement of the available Ca2+ concentration in real time through
the use of fluorescence microscopy. Such methods allow the direct detection of calcium fluxes and diffusion within
living cells in response to the activation of specific signal-transduction pathways (Figure 15.20).
15.3.2. Calcium Activates the Regulatory Protein Calmodulin, Which Stimulates Many
Enzymes and Transporters
Calmodulin (CaM), a 17-kd protein with four calcium-binding sites, serves as a calcium sensor in nearly all eukaryotic
2+
when the cytosolic calcium level is raised above about 500 nM.
cells. Calmodulin is activated by the binding of Ca
Calmodulin is a member of the EF-hand protein family. The EF hand is a Ca2+-binding motif that consists of a helix, a
loop, and a second helix. This motif, originally discovered in the protein parvalbumin, was named the EF hand because
helices designated E and F in parvalbumin form the calcium-binding motif and because the two helices are positioned
like the forefinger and thumb of the right hand (Figure 15.21). Seven oxygen atoms are coordinated to each Ca2+: six
from the protein and one from a bound water molecule.
Let us consider how calmodulin changes conformation in response to Ca2+ binding and how these conformational
changes enable the calmodulin to interact with other proteins (Figure 15.22). In the absence of bound Ca2+, calmodulin
consists of two domains, each consisting of a pair of EF-hand motifs joined by a flexible helix. Many of the residues that
typically participate in calcium binding are on the surface of these domains, oriented in a manner inappropriate for Ca2+
binding. On the binding of one Ca2+ to each EF hand, these units change conformation: the Ca2+-binding sites turn
inward to bind the Ca2+, moving hydrophobic residues from the inside to the outside of the domains. These
conformational changes generate hydrophobic patches on the surface of each domain that are suitable for interacting
with other proteins.
2+
The Ca
-calmodulin complex stimulates a wide array of enzymes, pumps, and other target proteins. Two targets are
especially noteworthy: one that propagates the signal and another that abrogates it. Calmodulin-dependent protein
kinases (CaM kinases) phosphorylate many different proteins. These enzymes regulate fuel metabolism, ionic
permeability, neurotransmitter synthesis, and neurotransmitter release. The binding of Ca2+-calmodulin to CaM kinase
activates the enzyme and enables it to phosphorylate target proteins. In addition, the activated enzyme phosphorylates
itself and is thus partly active even after Ca2+concentration falls and calmodulin is released from the kinase. In essence,
2+
-ATPase
autophosphorylation of CaM kinase is the memory of a previous calcium pulse. The plasma membrane Ca
pump is another important target of Ca2+-calmodulin. Stimulation of the pump by Ca2+-calmodulin drives the calcium
level down to restore the lowcalcium basal state, thus helping to terminate the signal.
Are there structural features common to all calmodulin target proteins? Comparisons of the amino acid sequences of
calmodulin-binding domains of target proteins suggests that calmodulin recognizes positively charged, amphipathic α
helices. The results of structural studies fully support this conclusion and reveal the details of calmodulin-target
interactions (Figure 15.23). The two domains of calmodulin surround the amphipathic helix and are linked to it by
extensive hydrophobic and ionic interactions. The α helix linking the two EF hands folds back onto itself to facilitate
binding to the target helix. How does this binding of calmodulin to its target lead to enzyme activation? In regard to CaM
kinase I, calmodulin targets a peptide present at the carboxyl terminus of the kinase. This region interacts with a loop
that is crucial for ATP binding and holds it in a conformation inappropriate for ATP binding. Calmodulin surrounds this
peptide and extracts it, freeing the kinase active site to bind ATP and phosphorylate appropriate substrates.
II. Transducing and Storing Energy
15. Signal-Transduction Pathways: An Introduction to Information Metabolism
15.3. Calcium Ion Is a Ubiquitous Cytosolic Messenger
Figure 15.18. Mode of Binding of CA2+ to Calmodulin. Calcium is coordinated to six oxygen atoms from the protein
and one (top) of water.
II. Transducing and Storing Energy
15. Signal-Transduction Pathways: An Introduction to Information Metabolism
15.3. Calcium Ion Is a Ubiquitous Cytosolic Messenger
Figure 15.19. Calcium Indicator. The fluorescence spectra of the calcium-binding dye Fura-2 can be used to measure
available calcium ion concentrations in solution and in cells. [After S. J. Lippard and J. M. Berg, Principles of
Bioinorganic Chemistry. (University Science Books, 1994), p. 193.]
II. Transducing and Storing Energy
15. Signal-Transduction Pathways: An Introduction to Information Metabolism
15.3. Calcium Ion Is a Ubiquitous Cytosolic Messenger
Figure 15.20. Calcium Imaging. A series of images shows Ca2+ spreading across a cell. These images were obtained
through the use of a fluorescent calcium-binding dye. The images are false colored: red represents high Ca2+
concentrations and blue low Ca2+ concentrations. [Courtesy of Dr. Masashi Isshiki, Dept. of Nephrology, University of
Tokyo, and Dr. G. W. Anderson, Dept. of Cell Biology, University of Texas Southwestern Medical School.]
II. Transducing and Storing Energy
15. Signal-Transduction Pathways: An Introduction to Information Metabolism
15.3. Calcium Ion Is a Ubiquitous Cytosolic Messenger
Figure 15.21. EF Hand. Formed by a helix-loop-helix unit, an EF hand is a binding site for calcium in many calcium
sensing proteins. Here, the E helix is yellow, the F helix is blue, and calcium is represented by the green sphere.
II. Transducing and Storing Energy
15. Signal-Transduction Pathways: An Introduction to Information Metabolism
15.3. Calcium Ion Is a Ubiquitous Cytosolic Messenger
Figure 15.22. Conformational Changes in Calmodulin on Calcium Binding. In the absence of calcium (top), the EF
hands have hydrophobic cores. On binding of a calcium ion (green sphere) to each EF hand, structural changes
expose hydrophobic patches on the calmodulin surface. These patches serve as docking regions for target proteins.
Acidic residues are shown in red, basic residues in blue, and hydrophobic residues in black. The central helix in
calmodulin remains somewhat flexible, even in the calcium-bound state.
II. Transducing and Storing Energy
15. Signal-Transduction Pathways: An Introduction to Information Metabolism
15.3. Calcium Ion Is a Ubiquitous Cytosolic Messenger
Figure 15.23. Calmodulin Binds to Amphipathic α Helices. (A) An amphipatic α helix (purple) in CaM kinase I is a