Fundamental Mechanisms of Ion Channel Proteins 
Christopher Miller, Ph.D. — Investigator 
Dr. Miller is also Professor of Biochemistry at Brandeis University and Adjunct Professor of Molecular 
Biology at Massachusetts General Hospital, Boston. He received his B.A. degree in physics from 
Swarthmore College and his Ph.D. degree in molecular biology from the University of Pennsylvania. He 
carried out postdoctoral work in membrane biochemistry with Efraim Racker at Cornell University for 
two years and then joined the Graduate Department of Biochemistry at Brandeis. 
ION channels are the most fundamental ele- 
ments of molecular hardware in the nervous 
system. They are the membrane-spanning pro- 
teins that directly mediate the transmembrane 
ionic fluxes by which electrical signals are gener- 
ated, propagated, and integrated in neurons, 
muscle, and other electrically active cells. By 
forming aqueous pores through the heart of the 
channel protein (and hence through the mem- 
brane that the protein spans), channels act as 
"leakage" pathways for ions down their preestab- 
lished thermodynamic gradients. These proteins 
make intelligent leaks. Channels can discrimi- 
nate fiercely among the different species of inor- 
ganic ions present in the aqueous solutions bath- 
ing the cell membrane. They can also rapidly 
open and close their conduction pores in re- 
sponse to external signals, such as binding of neu- 
rotransmitters or changing of the transmembrane 
electric field. 
Work here is directed toward questions of basic 
molecular mechanisms of ion channel operation 
and of the underlying protein structures. Since no 
high-resolution structures have been obtained 
(or are soon likely to be) for this class of proteins, 
it is necessary to draw structural inferences from 
close examination of ion channel function. This 
can be done because ion channels, unique among 
proteins, can be studied quantitatively at the 
single-molecule level. In this laboratory, heavy 
use is made of the technique of "single-channel 
reconstitution," in which individual ion channel 
molecules are inserted into an artificial mem- 
brane under simple, chemically controllable 
conditions. 
This approach has allowed us to develop crude 
physical pictures of several ion channels in 
which crucial dimensions have been deduced: 
the conduction pore's width and length, the dis- 
tance of its entryway from the lipid bilayer sur- 
face, and the number of ions inside the channel 
during the conduction process. We are currently 
complementing these purely functional and 
mechanistic studies with recently developed 
methods of membrane protein biochemistry and 
manipulation of ion channels at the genetic level. 
Use of Peptide Neurotoxins as Probes 
of Channel Structure 
Charybdotoxin (CTX) is a scorpion venom- 
derived peptide that blocks a small family of 
K^-specific channels. Having shown that it acts by 
physically plugging the channel's externally fac- 
ing mouth, we are now utilizing CTX as a probe 
of this important region. We are using two differ- 
ent channels for these efforts: the high- 
conductance Ca^^-activated channel from skel- 
etal muscle reconstituted into planar lipid 
bilayers, and the Shaker channel expressed in 
Xenopus oocytes. Each of these channels has a 
particular advantage, the former lending itself to 
close mechanistic study and the latter to molecu- 
lar genetic manipulation. 
Using a synthetic gene for CTX expressed in 
Escherichia colt, in combination with the known 
structure of the peptide, we have mapped the in- 
teraction surface of the CTX, using both types of 
channels as CTX receptors. As a result, we 
know that the peptide makes intimate contact 
with the channel on a well-defined surface built 
from 7-8 residues. One of these residues inter- 
acts electrostatically with a ion residing in the 
channel's conduction pore. 
In parallel with mapping the toxin, we have 
used site-directed mutagenesis with the Shaker 
channel to identify channel residues making 
up the CTX receptor located in the channel's ex- 
ternal mouth. With this information in hand, we 
are currently trying to identify channel-toxin in- 
teraction partners by constructing complemen- 
tary mutants of peptide and receptor. This will 
allow us to deduce physical distances between 
residues lining the mouth of the K"^ channel. 
Purification and Reconstitution 
of CI" Channels 
The electric ray Torpedo californica carries in 
its electric organ a CP-specific channel with an 
unusual structural characteristic. The channel is 
built as a dimeric, or "double-barreled" com- 
plex, with two identical CI" diffusion pathways 
in a single molecular unit. We have developed a 
functional assay for this channel protein in a solu- 
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