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 Bi- 
ology 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 
ion fluxes giving rise to the generation, propaga- 
tion, and integration of electrical signals in neu- 
rons, muscle, and other electrically active cells. 
By forming aqueous pores right through the heart 
of the channel protein (and hence across the 
membrane that the protein spans), channels act 
as "leakage" pathways for ions down their prees- 
tablished thermodynamic gradients. These pro- 
teins are intelligent leaks. Channels can discrimi- 
nate fiercely among the different species of 
inorganic ions present in the aqueous solutions 
bathing the cell membrane. They can also open 
and close their conduction pores rapidly in re- 
sponse to external signals, such as binding of neu- 
rotransmitters or changes in the transmembrane 
electric field. 
Work here is directed toward questions of basic 
molecular mechanisms of ion channel operation 
and of the underlying protein structures in- 
volved. Since no high-resolution structures have 
been obtained for this class of proteins (and since 
none is coming over the horizon) , one must draw 
structural inferences from close examination of 
ion channel function. This can be done because 
ion channels, unique among all classes of pro- 
teins, can be studied at the single-molecule level. 
In this laboratory, heavy use is made of the tech- 
nique of "single-channel reconstitution," in 
which individual ion channel molecules are in- 
serted into an artificial membrane under simple, 
chemically controllable conditions. This ap- 
proach 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 distance of the pore 
entryway from the lipid bilayer surface, and the 
number of ions inside the channel during the 
conduction process. We are currently comple- 
menting these purely functional and mechanistic 
studies with recent advances in methods of mem- 
brane 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 up the channel's externally 
facing "mouth," we are now utilizing the pep- 
tide as a probe of this important region of the 
channel. Employing site-directed mutagenesis 
with the Drosophila Shaker channel, we have 
identified residues that locally and specifically 
alter the binding of CTX. These residues are evi- 
dently located near the ion entryway, so we are 
homing in on regions of the protein that form the 
transmembrane pore. Two developments of the 
past year have placed us in position to use this 
toxin as a structural probe of the K"^ channel's 
outer mouth. First, using two-dimensional nu- 
clear magnetic resonance, we determined the 
solution structure of the toxin. Second, we con- 
structed a synthetic gene for CTX and success- 
fully overexpressed the fully functional peptide 
in Escherichia coli. Using high-level expression, 
routine structure determination of the toxin, and 
site-specific mutagenesis of both toxin and chan- 
nel, we are now attempting to map the locations 
in the K"*" channel mouth of residues that make 
direct contact with residues on CTX. 
Purification and Reconstitution 
of Cl" Channels 
The electric ray Torpedo californica carries in 
its electric organ a C 1 "-specific channel with an 
unusual structural characteristic. The channel is 
built as a dimeric, or "double-barreled," com- 
plex, with two identical Cl~ diffusion pathways 
in a single molecular unit. We have developed a 
functional assay for this channel protein in a solu- 
bilized state and are presently using it to perform 
conventional purification studies. We intend to 
study the purified channel protein as a supple- 
ment to structure-function work at the cDNA 
level on this recently cloned anion channel. 
Structure-Function Relations in a Minimal 
K+ Channel 
We are beginning a structure-function analysis 
on a K^-specific channel that was first cloned 
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