The Molecular Physiology of Calcium 
D. Martin Watterson, Ph.D. — Investigator 
Dr. Watterson is also Professor of Pharmacology at Vanderbilt University School of Medicine. He received 
his Ph.D. degree in biochemistry from Emory University. He was then an NIH postdoctoral fellow at Duke 
University Medical Center. Before joining the faculty at Vanderbilt, Dr. Watterson was Assistant Professor 
(Andrew Mellon Fellow) and Associate Professor of Cell Biology at the Rockefeller University. 
THE maintenance of a viable basal state in eu- 
karyotic cells (homeostasis), and the re- 
sponses of cells to environmental stimuli, involve 
a fine-tuned regulatory network that is modulated 
by transient changes in the intracellular levels of 
ionized calcium. The molecular mechanisms by 
which quantitative changes in calcium concen- 
tration are transduced into qualitative changes in 
cell behavior (altered cell motility, proliferation, 
receptor capping, cell morphology, etc.) involve 
the reversible interaction of calcium with a class 
of protein receptors that bind it selectively in the 
presence of higher concentrations of other 
ions, such as magnesium. A prototypical member 
of this class of calcium-binding proteins is 
calmodulin. 
Calmodulin is ubiquitous among the plant and 
animal kingdoms and has multiple biological 
roles. It fulfills these roles through its presence as 
an integral subunit of a diverse array of enzymes, 
cytoskeletal structures, and membrane-asso- 
ciated transport systems. Insight into the funda- 
mentals of this regulatory network would provide 
a framework from which to interpret biological 
phenomena and guide clinical treatments. It is 
the goal of our research to elucidate the calmod- 
ulin-mediated signal transduction system and 
thereby gain a more complete understanding of 
how calcium regulates cell processes and how 
this homeostatic network might be altered in cer- 
tain disease states or susceptibilities. 
Calmodulin-regulated Protein Kinases 
One class of enzymes that have calmodulin as 
an integral calcium-binding regulatory subunit 
are the protein kinases. Protein kinases catalyze 
the transfer of phosphate from an ATP molecule 
to a specific site on certain proteins {phosphory- 
lation), resulting in a rapid alteration of the 
phosphorylated protein's biological functioning. 
In the basal or "resting" state of the cell, the 
active site of the protein kinase is inhibited by 
another region of the kinase molecule {autoinhi- 
bition). When calcium binds to calmodulin, 
which is loosely bound to the kinase, the cal- 
modulin's three-dimensional structure is subtly 
changed. This in turn results in a change in the 
three-dimensional arrangement of the protein ki- 
nase molecule that relieves the inhibition of the 
kinase active site. The kinase can now phosphor- 
ylate a specific set of proteins in the cell and 
thereby set off a cascade of events. 
The basal state is reestablished by at least two 
closely coupled reactions: the removal of phos- 
phate from the phosphorylated protein by an en- 
zyme called a phosphatase, and the dissociation 
of calcium from calmodulin with a resultant res- 
toration of the protein kinase to its autoinhibited 
state. During this past year, we elucidated the pri- 
mary structure of one of these calmodulin- 
regulated protein kinases — called nonmuscle 
myosin light-chain kinase (nmMLCK) — that is 
found in all nonmuscle tissues examined. 
We have also determined, through use of mo- 
lecular genetic and protein chemistry technolo- 
gies, some fundamental features of both the pro- 
tein kinase and calmodulin that are required for 
these two proteins to recognize each other in a 
milieu of macromolecules, with the resultant for- 
mation of a fully functional calcium signal-trans- 
duction complex. 
By serendipity, we also discovered a novel ge- 
netic relationship among this protein kinase and 
some other proteins produced from the same 
gene. As a result, we have extended our investiga- 
tions to include an analysis of how the various 
proteins with different biological functions are 
made from this single gene and a more detailed 
localization of the gene in the human genome. 
Calmodulin-regulated Ion Channels 
Ion channels are cell membrane proteins that 
regulate ion transport across the membrane. They 
do not appear to be enzymes like the protein ki- 
nases, but represent another class of cellular pro- 
teins whose functions can be regulated by cal- 
modulin. Our previous studies of mutant 
organisms with ion channel defects demonstrated 
that it is possible to have inherited mutations of 
calmodulin that can selectively alter one cal- 
modulin-regulated pathway. (Previously it was 
thought that any inherited mutation of calmodu- 
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