bodies. These results suggest the presence of a ryan- 
odine-sensitive Ca^^ release channel within the sea 
urchin egg cortex. In the upcoming year studies of 
this receptor should provide insights into its role in 
Ca^"^ homeostasis in excitable and nonexcitable 
cells and its distribution within the central nervous 
system. 
Voltage-Sensitive Ca^^ Channels in Neurons 
A second major area of research concerns voltage- 
sensitive Ca^"*^ channels in excitable cells. The dihy- 
dropyridine receptor (DHPR), previously purified 
in Dr. Campbell's laboratory from rabbit skeletal 
muscle, consists of four subunits (a,, |8, and 7). 
In neurons, voltage-sensitive Ca^^ channels exist as 
several types (L, N, T, and P) with different kinetic 
and pharmacological properties. Dihydropyridines 
bind specifically to L-type Ca^"^ channels and alter 
their channel activity. For N-type channels, which 
are likely responsible for triggering neurotransmit- 
ter release at synapses, oj-conotoxin is largely 
specific. 
In the past year, Dr. Campbell's laboratory has 
used antibodies and cDNA probes to the various sub- 
units of the dihydropyridine-sensitive channels of 
skeletal muscle to study N-type Ca^^ channels (co- 
conotoxin sensitive) in neurons. Antibodies against 
the subunits of the dihydropyridine-sensitive L-type 
Ca^^ channel were tested for their ability to immu- 
noprecipitate the high-afifinity (K^ = 0.13 nM) *^^I- 
co-conotoxin GVIA receptor from rabbit brain mem- 
branes. Monoclonal antibody VD2j against the |8 
subunit of the DHPR specifically immunoprecipi- 
tated up to 86% of the '^^I-co-conotoxin receptor, 
whereas specific antibodies against the aj, and 7 
subunits did not precipitate the brain receptor. 
The co-conotoxin receptor immunoprecipitated by 
monoclonal antibody VD2i showed high-affinity 
'^'l-oj-conotoxin binding, which was inhibited by 
unlabeled co-conotoxin and by CaCl2 but not by 
various dihydropyridines. These results suggest that 
the brain co-conotoxin-sensitive Ca^^ channel con- 
tains a component homologous to the /3 subunit of 
the dihydropyridine-sensitive Ca^^ channel of skele- 
tal muscle and brain. Work is now in progress to 
purify the co-conotoxin-sensitive Ca^^ channel, us- 
ing affinity chromatography to analyze its subunit 
composition and to demonstrate that it is the N-type 
Ca^^ channel. 
A cDNA clone encoding a protein with high ho- 
mology to the (8 subunit of the rabbit skeletal muscle 
dihydropyridine-sensitive Ca^^ channel was also 
isolated. This rat brain ;S-subunit cDNA hybridizes to 
a 3 .4-kb message that is expressed in high levels in 
the cerebral hemispheres and hippocampus but is 
significantly reduced in cerebellum. The open read- 
ing frame encodes 597 amino acids with a predicted 
mass of 65,679 Da and is 82% homologous with the 
skeletal muscle /3 subunit. The brain cDNA encodes 
a unique 153-amino acid carboxyl terminus and 
predicts the absence of a muscle-specific 50-amino 
acid internal segment. It also encodes numerous 
consensus phosphorylation sites, suggesting a role 
in Ca^^ channel regulation. Thus the encoded brain 
18 subunit, which has a primary structure highly simi- 
lar to its isoform in skeletal muscle, may have a com- 
parable role as an integral regulatory component of 
a neuronal Ca^^ channel. A grant from the National 
Institutes of Health provided support for the project 
described above. 
Dystrophin-Glycoprotein Complex 
Dystrophin, the high-molecular-weight protein 
product of the Duchenne muscular dystrophy 
(DMD) gene, is localized to the sarcolemma of nor- 
mal skeletal muscle but is absent from the skeletal 
muscle of DMD patients and mdx mice. The pre- 
dicted amino acid sequence of dystrophin suggests 
that dystrophin is a membrane cytoskeletal protein 
involved in the anchoring of sarcolemmal proteins 
to the underlying cytoskeleton. Dr. Campbell and 
his colleagues discovered the dystrophin-glycopro- 
tein complex, a large oligomeric complex contain- 
ing dystrophin, a 59-kDa protein triplet, and four 
sarcolemma glycoproteins of 156, 50, 43, and 35 
kDa. In the past year the membrane organization of 
this complex, including the stoichiometry, cellular 
location, glycosylation, and hydrophobic properties 
of the components, has been determined. The 1 56-, 
59-, 50-, 43-, and 35-kDadystrophin-associated pro- 
teins (DAPs) each possess unique antigenic determi- 
nants, enrich quantitatively with dystrophin, and 
were localized to the skeletal muscle sarcolemma. 
The 156-, 50-, 43-, and 35-kDa DAPs contain Asn- 
linked oligosaccharides. The 156-kDa dystrophin- 
associated glycoprotein (DAG) contained termi- 
nally sialylated Ser/Thr-linked oligosaccharides. 
Dystrophin, the 156-kDa, and the 59-kDa DAPs are 
peripheral membrane proteins; the 50-, 43-, and 
35-kDa DAGs and the 2 5 -kDa DAP are integral mem- 
brane proteins. Thus dystrophin and its 59- 
kDa associated protein are cytoskeletal elements 
that are tightly linked to a 156-kDa extracellular 
glycoprotein by a complex of transmembrane 
proteins. The membrane organization of the 
dystrophin-glycoprotein complex and the high den- 
sity of dystrophin in the sarcolemma membrane sug- 
gest that this complex could have an important 
structural role in skeletal muscle. 
In the past year Dr. Campbell's group also exam- 
CELL BIOLOGY AND REGULATION 
31 
