MOLECULAR PROPERTIES OF NEURONAL VOLTAGE-GATED CALCIUM CHANNELS 
Terry P. Snutch, Ph.D., International Research Scholar 
Dr. Snutch's laboratory investigates signal trans- 
duction in the mammalian central nervous system 
(CNS). The entry of calcium into neurons directly 
affects membrane potential and contributes to the 
electrical properties of cells. In addition, calcium 
acts as a second messenger and further affects cellu- 
lar events by regulating the activity of calcium- 
dependent enzymes and ion channels. The rapid 
entry of calcium into excitable cells is mediated by 
specific voltage-gated channels. The long-term 
goals of the research are to understand the molecu- 
lar nature of the diverse roles that these channels 
play in such processes as neuronal firing patterns, 
neurotransmitter release, neuromodulation, and 
gene regulation. 
Diversity of Neuronal Calcium Channels 
In both neurons and other cell types, various cal- 
cium channels can be distinguished by their electro- 
physiological and pharmacological properties (des- 
ignated T, L, N, and P types). A major concern is 
defining the molecular nature of calcium channel 
diversity. Dr. Snutch's laboratory has proposed that 
the expression of distinct isoforms of the pore- 
forming a J subunit of calcium channels is responsi- 
ble for a large amount of the observed functional 
heterogeneity. Recent molecular cloning studies in 
the laboratory have established that at least four dis- 
tinct subtypes of calcium channel subunits are 
expressed in the rat CNS (designated rbA-I, rbB-I, 
rbC-I, and rbD-I). The four cloned types of these 
subunits are between 240 and 262 kDa in predicted 
molecular mass, and each possesses four internal re- 
peated domains (I-IV) that are modeled to contain 
six to eight transmembrane regions. The overall 
transmembrane topologies and predicted secondary 
structures are similar to those proposed for voltage- 
gated sodium and potassium channels and indicate a 
common evolutionary origin for these molecules. 
Although the four calcium channel subunits of 
brain are highly similar in the four repeated do- 
mains, they diverge significantly from one another 
in both the segment-linking domains II and III and 
in their carboxyl regions. Both of these regions are 
modeled to be cytoplasmic. They contain many con- 
sensus sites for modulation by cAMP- and cGMP- 
dependent kinases, protein kinase C, and calmodu- 
lin kinase II, and may account for differences in the 
modulation of neuronal calcium currents. Examina- 
tion of the positively charged S4 segments shows 
that two distinct patterns occur in this putative volt- 
age-sensor region. In domains III and IV, the rbC-I 
and rbD-I channels have identical S4 segments, 
whereas the rbA-I and rbB-I channels have different 
patterns of positively charged residues in both do- 
mains. Structure-function studies will determine 
how these differences contribute to the diverse acti- 
vation properties observed for calcium channels. 
To determine the relationship between the 
cloned aj subunits and calcium channels defined in 
neurons, Dr. Snutch, in collaboration with Dr. Wil- 
liam Catterall (University of Washington), gener- 
ated polyclonal antisera specific for the four classes 
of brain calcium channels. Immunoprecipitation of 
rat brain membranes radiolabeled with calcium 
channel antagonists gave the unexpected result that 
both the rbC-I and rbD-I a, subunits encode distinct 
L-type calcium channels. Similar experiments show 
that the rbA-I a, subunit encodes neither L- nor N- 
type calcium channels. Together with the high lev- 
els of expression of rbA-I observed in the cerebellar 
Purkinje cells, these results suggest that rbA-I may 
encode a P-type calcium channel. These studies also 
demonstrate that rbB-I encodes an oj-conotoxin- 
sensitive N-type calcium channel. The molecular 
cloning of an N-type channel is significant in that 
these channels are thought to be concentrated at 
presynaptic nerve termini and that they play a major 
role in mediating chemical synaptic transmission. 
The availability of probes for an N-type channel 
will aid in studies examining the physiological roles 
of this channel in neuromodulation and in the 
small-cell lung carcinoma associated with Lambert- 
Eaton myasthenic syndrome. In agreement with stud- 
ies indicating that N-type calcium channels are a 
heteroligomeric complex, the expression of the 
rbB-I polypeptide alone does not result in func- 
tional N-type calcium channels. The goal of this proj- 
ect now is to express the cloned subunits to- 
gether with ancillary calcium channel subunits and 
to determine their electrophysiological and pharma- 
cological properties. 
Alternative Splicing of Calcium Channels 
Although initial studies demonstrated the exis- 
tence of the four main classes of brain calcium chan- 
nel a■^ subunit, further analysis shows that heteroge- 
neity also exists within each class. Molecular 
cloning and polymerase chain reaction studies dem- 
onstrate that the rat genome encodes single genes 
for the rbC and rbD a, subunits and that isoforms are 
generated by mutually exclusive alternative splic- 
INTERNATIONAL RESEARCH SCHOLARS 533 
