analogues at position 27 will serve as sensitive, 
well-calibrated probes of ionic interactions inside 
the channel pore. 
Purification and Reconstitution 
of Voltage-Dependent Cl"^ Channels 
The electric ray Torpedo californica carries in its 
electric organ a Cr-specific channel with an un- 
usual structural characteristic: the channel is built 
as a dimeric, or double-barreled complex, with two 
identical Cl~ diffusion pathways in a single molecu- 
lar unit. Dr. Miller and Dr. Richard Middleton, in a 
project partially supported by the National Insti- 
tutes of Health, are using a functional assay for this 
channel protein in a solubilized state for protein- 
level purification and active-site labeling studies. 
At this point the protein has been purified at ana- 
lytical scale, using a monospecific immunoafiBnity 
column, and the subunit structure of the complex is 
being investigated. The longer-range goal is to scale 
up the purification to exploit the huge advantage of 
the electroplax: the high level at which the protein 
is expressed in this organ. In principle, it should be 
possible to use this source for routine purification 
of tens of milligrams of voltage-dependent CI" chan- 
nel protein. 
High-Level Expression of Ion Channel Proteins 
Over the past few years, ion channel genes have 
been cloned at a rapidly accelerating pace, and 
they can be expressed in systems that allow high- 
sensitivity electrophysiological assays. But it would 
be desirable to have a high-level expression system 
available for producing milligram-scale protein for 
cloned channel genes. This is a fundamental stum- 
bling block for future molecular attacks on ion 
channels at the level of protein biochemistry. For 
this reason, Dr. Miller is using the Shaker K"^ channel 
as a test for high-level expression in several heterolo- 
gous systems, including baculovirus-infected insect 
cells, mammalian cells, yeast, and transgenic mice 
(and eventually goats) with the channel targeted to 
the mammary epithelium. The rationale here is that 
although this line of work is highly empirical, the 
channel under test, which contains an engineered 
high-afifinity CTX-binding site, is well suited for the 
goals. Specifically, it lends itself to straightforward 
immunoaffinity purification, and the toxin-binding 
site allows a clear distinction between total ex- 
pressed protein and assembled tetrameric channel. 
Ion Conduction in Ca^^-activated Channels 
Dr. Miller, with Qiang Lu, a graduate student, has 
recently begun to apply single-channel reconstitu- 
tion methods, developed earlier, to a genetically 
manipulable Ca^^-activated K"^ channel, the "slow- 
poke" channel of Drosophila. This project is now in 
its infancy, but the directions are clear: to identify, 
through mutagenesis and single-channel analysis, 
the tight binding sites for K"^ and Ba^^ ions residing 
within the conduction pore. These experiments will 
initially require the development of routine tech- 
niques to transfer expressed channels to the planar 
lipid bilayer system, where the necessary Ba^^-K^ 
interactions can be measured. 
Dr. Miller is also Professor of Biochemistry at 
Brandeis University, Waltham, and Adjunct Pro- 
fessor of Molecular Biology at Massachusetts Gen- 
eral Hospital, Boston. 
Articles 
Goldberg, A.F.X., and Miller, C. 1991. Solubiliza- 
tion and functional reconstitution of a chloride 
channel from Torpedo electroplax. f Membr Biol 
124:199-206. 
Goldstein, S.A.N., and Miller, C. 1991. Site-specific 
mutations in a minimal voltage-dependent 
channel alter ion selectivity and open-channel 
block. Neuron 7:403-408. 
Goldstein, S.A.N., and Miller, C. 1992. A point 
mutation in a Shaker channel changes its cha- 
rybdotoxin receptor site from low to high affinity. 
Biophys f 62:5-7. 
Park, C.S., and Miller, C. 1992. Interaction of char- 
ybdotoxin with permeant ions inside the pore of a 
K"^ channel. Neuron 9:307-313. 
Stampe, P., Kolmakova-Partensky, L., and Miller, 
C. 1992. Mapping hydrophobic residues of the 
interaction surface of charybdotoxin. Biophys f 
62:8-9. 
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