MOLECULAR MECHANISMS OF ION CHANNEL FUNCTION 
Gary Yellen, Ph.D., Assistant Investigator 
Ion channels are integral membrane proteins 
that determine the electrical properties of neurons 
and other excitable cells. Chemically gated chan- 
nels transduce neurotransmitter release at a syn- 
apse into an electrical signal; electrically gated 
channels modulate this electrical signal, propagate 
it over long distances, and convert this electrical 
signal (in the form of ion flux, particularly calcium 
influx) into a signal for neurotransmitter release at 
a second synapse or for cellular regulation. There 
are dozens of different types of ion channels, differ- 
ing in their mode of regulation and in which ions 
they permit to pass. 
Dr. Yellen's research is directed at the basic 
mechanisms of gating and permeation in ion chan- 
nels. How are these proteins engineered to permit 
the controlled opening and closing of an aqueous 
pore that is capable of selecting between small ions 
of similar size, and what structural features of these 
proteins are responsible for the differences be- 
tween different channels? Dr. Yellen's laboratory is 
using both molecular biological approaches and 
more traditional electrophysiological approaches to 
answer these questions. 
L Cloning of an Important Functional Variant of the 
Nicotinic Acetylcholine Receptor. 
Natural evolution of ion channel proteins can 
give significant clues to the functional organization 
of these proteins. Nature has provided an impor- 
tant functional variant of the "normal" nicotinic 
acetylcholine receptor (AChR) found in vertebrate 
muscle and brain. The normal nicotinic AChR con- 
tains an ion channel that allows Na^ and other cat- 
ions to carry current through the membrane. In the 
sea slug Aplysia and other molluscan species, there 
is a similar nicotinic receptor that contains an 
anion-selective channel. This channel, like its cat- 
ion-selective vertebrate counterpart, is inhibited by 
the snake toxin a-bungarotoxin. 
By using a combination of homology-based clon- 
ing and protein chemistry, Dr. Yellen and Dr. James 
McLaughlin are cloning the cDNAs that code for 
this AChR variant in Aplysia. 
II. Site-directed Mutagenesis of the Nicotinic AChR. 
Site-directed mutagenesis is one important tool 
for establishing links between specific structural 
features of the channel protein and specific func- 
tions. Dr. Yellen, Mark Jurman, and Dr. Gordon F. 
Tomaselli have been applying this method to the 
Torpedo nicotinic AChR. Oligonucleotide-directed 
mutations are produced in vitro, and the mutant 
channels are expressed by injection of mRNA into 
Xenopus oocytes. Findings from other laboratories 
show that changes in charged residues just outside 
one of the transmembrane hydrophobic regions of 
the protein produce a change in single-channel 
conductance. Dr. Yellen is extending these findings 
by altering other charged residues and determining 
the changes in single-channel conductance at very 
low ionic strength. At low ionic strength, the 
sphere of influence of each charged group is dra- 
matically increased. The effect of more distant 
charges can therefore be seen and their distances 
mapped by varying the ionic strength. This ap- 
proach should give additional information about 
channel topology, since the effects of charge on the 
intracellular and extracellular sides of the channel 
can be distinguished. 
Another set of site-directed mutations of the 
AChR is being prepared and tested in collaboration 
with Dr. Richard Huganir (HHMI, The Johns Hop- 
kins University). Dr. Huganir has found that specific 
amino acids in the AChR can be phosphorylated by 
protein kinases and that this phosphorylation ac- 
celerates receptor desensitization. Although it is dif- 
ficult to prepare completely unphosphorylated re- 
ceptor from tissue sources, it is possible to produce 
mutant receptors that cannot be phosphorylated. 
The goal of this experiment is to determine the 
features of desensitization in completely un- 
phosphorylated channels and then to see how the 
remaining desensitization is affected by charged 
amino acids (as opposed to phosphate groups). 
Mutations in several of the subunits have been pre- 
pared, and the mutant mRNAs are being tested in 
the oocyte expression system. It is now known that 
although phosphorylation can affect the rate of de- 
sensitization, it is not required for desensitization: 
desensitization still occurs when all of the poten- 
tial phosphorylation sites are removed from the 
protein. 
III. Selectable Expression Systems for Ion Channel 
Genes. 
Although site-directed mutagenesis of large pro- 
teins can be used to advantage, it requires a specific 
testable model. Specific biophysical theories (e.g., 
surface charge effects on permeation) or protein 
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