Molecular Studies on Neuronal Calcium Channels 
subcellular distributions of the Ca channel sub- 
types reflect their unique contributions to neuro- 
nal physiology. A long-term goal of this research 
is to define how the individual subtypes uniquely 
contribute to neuronal functioning. 
Functional Expression of Neuronal 
Ca^^-Channels 
We are taking two approaches to determining 
the functional properties of the cloned Ca^"^ 
channel aj-subunits. First, full-length clones for 
the four main classes of rat brain aj-subunit are 
being introduced into mammalian cell lines that 
normally do not express any Ca^"^ channels. The 
electrical and pharmacological properties of the 
Cd}^ channels will then be determined with the 
patch-clamp technique. Second, we are using the 
DNA sequences derived from the cloned Ca^^ 
channels to generate small synthetic pieces of 
DNA (oligonucleotides) that will hybridize to 
mRNAs encoding these molecules. 
Microinjection of oligonucleotides into cells 
that already express defined Ca^^ channels re- 
sults in the oligonucleotide hybridizing to Ca^"*" 
channel mRNA and inhibiting expression. By in- 
hibiting the expression of a single type of Ca^"*" 
channel in cells that normally express several 
types, we hope both to identify the specific type 
of Ca^"^ channel blocked by the oligonucleotide 
and also to obtain some insight into the physiolog- 
ical roles that individual Ca^"^ channel types con- 
tribute to neurons. Using this technique we have 
found that the rat brain class C aj-subunit en- 
codes a dihydropyridine-sensitive (L-type) Ca^"^ 
channel. Similar studies with the other classes of 
brain Ca^"^ channel are under way. 
Detection of single- copy sequences in fluores- 
cence micrographs of metaphase chromo- 
somes, interphase nuclei, and free chromatin, 
a ) Metaphase chromosomes from a human dip- 
loid lymphocyte culture, hybridized with four 
cosmid probes (cM58-3-6, CF14, cf21, and 
cWlO-20) together, spanning 341 kb. b) Hy- 
bridization of the same probes with interphase 
nucleus. Arrows indicate two sets of hybridiza- 
tion signals, c-f) Results of hybridization with 
different combinations of cosmids in the 7q31 
region, c) cNH24 and cf21; d) cM58-3-6 and 
cf21; e) CM58-3.6, CF14 and cf21 (note: two 
sets of hybridization signals); f) cM58-3-6, 
CF14, cf21, and cW 10-20. 
Research and photograph by Henry Heng in 
the laboratory of Lap-Chee Tsui. 
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