Molecular Studies of Calcium Channels 
and the Dystrophin-Glycoprotein Complex 
Kevin p. Campbell, Ph.D. — Investigator 
Dr. Campbell is also Professor of Physiology and Biophysics at the University of Iowa, Iowa City. He 
received his B.S. degree in physics from Manhattan College, his master's degree from the University of 
Rochester School of Medicine and Dentistry, and his Ph.D. degree from the Department of Radiation 
Biology and Biophysics at the University of Rochester. He did postdoctoral studies in the laboratory of 
David MacLennan at the Banting and Best Department of Medical Research, University of Toronto, 
before coming to Iowa. 
CALCIUM ion (Ca^^) functions as a ubiquitous 
intracellular messenger regulating a wide va- 
riety of cellular responses, including contrac- 
tion, secretion, and cell proliferation. Two com- 
mon pathways by which intracellular Ca^"^ 
transients can be triggered have been identified. 
The first involves the influx of Ca^"^ into the cyto- 
plasm from the extracellular medium through 
specific plasma membrane channels, and the sec- 
ond involves the release of Ca^"^ into the cyto- 
plasm from intracellular stores through specific 
release channels. 
Over the past 10 years, we have investigated 
the structure and function of the membrane 
components involved in Ca^"*^ fluxes across mem- 
branes. In particular, we have focused on identi- 
fying, purifying, and characterizing the mem- 
brane proteins that function as surface Ca^^ 
channels and intracellular Ca^"^ release channels 
in excitable cells. 
Neuronal Calcium Release Channels 
A major area of research in my laboratory con- 
cerns the structure and function of intracellular 
Ca^^ release channels. In skeletal muscle, Ca^"^ 
release from the sarcoplasmic reticulum initi- 
ates contraction. We previously purified a high- 
affinity ryanodine receptor from skeletal muscle 
sarcoplasmic reticulum and showed it to be iden- 
tical to the sarcoplasmic reticulum Ca^^ release 
channel. 
In neurons, inositol 1 ,4,5-trisphosphate (IP3) 
is an important second messenger involved in 
Ca^"^ release. However, physiological and pharma- 
cological evidence has indicated the presence of 
non-IPj-gated Ca^^ pools in neurons. Recently 
my laboratory also identified and purified a neu- 
ronal ryanodine receptor. The purified receptor 
was shown to be a homotetramer composed of 
protein subunits of approximately 500 kDa. Re- 
constitution of the purified ryanodine receptor 
into lipid bilayers has demonstrated that it func- 
tions as a caffeine- and ryanodine-sensitive Ca^^ 
release channel that is distinct from the brain IP3 
receptor. Immunoblotting experiments indicate 
that the brain receptor is more like the cardiac 
receptor than the skeletal receptor. Thus we be- 
lieve that the brain ryanodine receptor may oper- 
ate as a Ca^^-induced release channel for intracel- 
lular Ca^^ pools in neurons. In the upcoming year 
further studies of this receptor should provide 
insights into its role in neuronal Ca^^ homeostasis 
and its distribution within the central nervous 
system. 
Voltage-gated Calcium Channels 
A second major area of research concerns volt- 
age-gated Ca^^ channels in excitable cells. We have 
studied in skeletal muscle the dihydropyridine- 
sensitive Ca^^ channel, which is essential to exci- 
tation-contraction coupling. The skeletal muscle 
dihydropyridine receptor has been purified and 
consists of four subunits (aj, and 7). 
In neurons, voltage-gated Ca^^ channels exist 
as several types (L, N, T, and P) with different 
kinetic and pharmacological properties. Dihy- 
dropyridines bind specifically to L-type Ca^"*" 
channels and alter their channel activity. For N- 
type channels, which are likely responsible for 
triggering neurotransmitter release at synapses, 
oj-conotoxin is largely specific. We are now using 
antibodies and cDNA probes to the various sub- 
units of the dihydropyridine-sensitive channels 
to study oj-conotoxin-sensitive Ca^^ channels. 
We have demonstrated that the brain 
conotoxin-sensitive Ca^^ channel contains a 
component homologous to the 18-subunit of the 
dihydropyridine-sensitive Ca'^'^ channel of skele- 
tal muscle. We have also isolated a cDNA clone 
from a brain cDNA library encoding a protein 
with high homology to the /8-subunit of the skele- 
tal muscle dihydropyridine-sensitive Ca^^ chan- 
nel. This brain /3-subunit cDNA encodes numer- 
ous consensus phosphorylation sites, suggesting 
a role in Ca^^ channel regulation. 
We are now purifying the w-conotoxin-sensi- 
tive Ca^^ channel, using affinity chromatography, 
in order to analyze its subunit composition and to 
demonstrate that it is identical to the N-type Ca^^ 
channel. Experiments are also in progress to 
coexpress the brain /3-subunit with the aj-subunit 
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