Molecular Studies on Neuronal Calcium Channels 
Terry p. Snutch, Ph.D. — International Research Scholar 
Dr. Snutch is Assistant Professor of Zoology and Neuroscience at the Biotechnology Laboratory, University 
of British Columbia, Vancouver. He obtained a B.Sc. degree in biochemistry from Simon Fraser University, 
Vancouver, and remained there to complete his Ph.D. degree for studies on the molecular genetics of the 
heat- shock response o/ Caenorhabditis elegans with David Baillie. After further research training in the 
laboratory of Norman Davidson at the California Institute of Technology, Dr. Snutch joined the newly 
formed Biotechnology Laboratory. He recently received a fellowship in neuroscience from the Alfred P. 
Sloan Research Foundation and the Killam Research Prize from the University of British Columbia. 
THE entry of calcium ions (Ca^^) into cells 
mediates a wide variety of cellular and physi- 
ological responses, including muscle contraction 
and hormone secretion. In the nervous system, an 
increase in intracellular Cd}^ concentration di- 
rectly affects the electrical properties of neurons. 
Ca^"^ entry has also been shown to have a role in 
regulating gene expression, modulating Ca^^- 
dependent enzymes, and mediating nerve grov^h 
and regeneration. Furthermore, an increase in 
C?}* concentration at the presynaptic nerve ter- 
minal triggers the release of neurotransmitter, in- 
hibiting or exciting postsynaptic neurons. 
The rapid entry of Ca^"*^ into neurons is me- 
diated by membrane proteins called Ca^"*^ chan- 
nels. These diverse molecules respond to voltage 
changes across the cell membrane by opening 
Ca'^"'^-selective transmembrane pores. Besides the 
wide variety of normal physiological effects that 
Ca^"^ channels mediate, they are implicated in a 
number of disorders, such as angina, hyperten- 
sion, migraine, and certain arrhythmias. The clin- 
ical treatment of these disorders is aided by Ca^"^ 
channel-blocking drugs. Our present studies uti- 
lize molecular cloning techniques to address ba- 
sic questions concerning the structure, function, 
and expression of Ca^"^ channels in the mamma- 
lian nervous system. 
Molecular Diversity of Neuronal 
Ca^* Channels 
Four types of Ca^"^ channel, called T, L, N, and 
P, have been identified, with differing electro- 
physiological and pharmacological properties. 
Among their subunits is the aj-subunit, which 
both responds to voltage changes and forms the 
ion-conducting pore. Although the differences 
among various Ca^^ channels may be due to a 
number of factors, we hypothesize that distinct 
a 1 -subunits account for most Ca^^ channel 
diversity. 
Utilizing molecular cloning techniques, we 
have isolated cDNAs encoding four distinct 
classes of Ca^^ channel from rat brain: classes 
A, B, C, and D. Their amino acid sequences show 
that they are large proteins (2,100-2,300 amino 
acids) and that they share a number of conserved 
features with cloned sodium and potassium 
channels. 
The class C channel found in brain is nearly 
identical to a Ca^^ channel previously found in 
heart and lung, while classes A, B, and D repre- 
sent novel forms. Hybridization of the clones to 
rat genomic DNA indicates that each of the classes 
of brain Ca^^ channel is encoded by a distinct 
gene. 
One interesting result of these studies is that 
within each class of aj-subunit cloned, several 
varieties have been identified, and the actual 
number of distinct a, -subunits is much larger 
than previously thought. Using molecular ge- 
netic techniques, we have demonstrated that 
these varieties, at least in the class C and D in- 
stances, are generated by alternative splicing. For 
class C Ca^"^ channels, two distinct isoforms are 
expressed at different levels in various regions of 
the rat brain and thus may make unique contribu- 
tions to distinct populations of neurons. 
Differential Localization of Ca^^ Channel 
Subtypes 
In an attempt to provide information concern- 
ing the physiological roles of the neuronal Ca^"*" 
channel subtypes, we are determining their cel- 
lular and subcellular localizations. Studies local- 
izing Ca^"^ channel gene expression by examina- 
tion of RNA levels show that the various classes of 
Ca^^ channel are differentially expressed in the 
brain. For example, in the cerebellum the class A 
channel is mostly localized to Purkinje cells, 
while class C channels are more widespread. In 
addition to their different spatial distributions in 
the brain, studies using antibodies generated 
against the cloned Ca^* channel aj -subunits show 
that the various Ca^^ channel proteins have dis- 
tinct subcellular localizations on individual neu- 
rons. For example, some Ca^* channel subtypes 
appear to be localized to dendritic regions, while 
others are located on both cell bodies and den- 
drites. We believe that the distinct cellular and 
529 
