Molecular Mechanisms of Ion Channel Function 
Gary Yellen, Ph.D. — Assistant Investigator 
Dr. Yellen is also Assistant Professor of Neuroscience and Biophysics at the Johns Hopkins University 
School of Medicine. He received his undergraduate degree in biochemical sciences from Harvard College 
and his Ph.D. degree in physiology from Yale University, where he studied with Charles Stevens. Dr. Yellen 
did his postdoctoral research on ion channel physiology as a Life Sciences Research Foundation 
postdoctoral fellow at Brandeis University, where he worked with Christopher Miller. 
ALL electrical signaling in the nervous system 
is controlled by ion channels, a class of 
membrane proteins that form pores through the 
membrane. Charged ions such as sodium, potas- 
sium, and calcium pass through ion channels and 
carry an electrical current. The channels them- 
selves are regulated, so that the pores are only 
open when the proper chemical or electrical sig- 
nal is present, and only certain ions can pass 
through a particular kind of channel. By under- 
standing how channels open and close and how 
they are regulated, we define the repertoire of 
molecular changes used by neurons when they 
signal, sense, and learn. 
Ion channels, like other membrane proteins, 
have resisted standard biochemical and structural 
analysis. Their structure has only recently begun 
to be elucidated by a combination of protein 
chemistry and molecular biology. On the other 
hand, we have detailed knowledge of the func- 
tioning of ion channels. Because each ion chan- 
nel catalyzes the transport of millions of ions per 
second, we can measure electrically the current 
carried by just a single-channel protein molecule. 
This technique of single-channel recording has 
allowed us to make a detailed model for the con- 
formational changes between open and closed 
states induced by chemical ligands and changes 
in voltage, but we still have no knowledge of the 
protein structures that underlie these conforma- 
tional changes. 
My laboratory uses a combination of high- 
resolution functional analysis (by single-channel 
recording) and direct manipulation of the struc- 
ture of the channel protein. Site-directed muta- 
genesis allows us to modify any amino acid in a 
protein for which we have the cloned genetic ma- 
terial. Rather than modifying the protein directly, 
we change the DNA sequence and then inject the 
modified messenger RNA into immature frog eggs 
(oocytes) , which manufacture the modified pro- 
tein. This method allows us to test specific the- 
ories about which parts of the channel protein are 
important for specific functional features. 
We have applied this combination of strategies 
to voltage-activated potassium channels, which 
participate in electrical signaling. By systematic 
mutagenesis, we have identified the region of the 
potassium channel protein that lines the pore 
through which ions cross the membrane. We 
found specific amino acid residues in the protein 
sequence that control the sensitivity of the chan- 
nel to tetraethylammonium, an organic ion that 
can block current through the channel. Natural 
variation of one of these amino acids explains the 
differences in drug sensitivity between potassium 
channels in different organs or species. Amino 
acids in this region of the protein can also alFect 
the rate at which ions are conducted through the 
pore. These discoveries put us in a position to 
discover the basic mechanisms of ion selectivity 
and channel gating at the level of individual 
amino acids. 
We have also used recording from single potas- 
sium channels to demonstrate that one of the 
mechanisms by which these channels open and 
close is a simple occlusion of the pore by part of 
the channel protein. Earlier work established that 
the pore could be directly blocked or occluded 
by internal organic ions; we have established that 
the natural gating occurs by a very similar mecha- 
nism involving a tethered blocking particle. The 
most direct demonstration of this is that potas- 
sium ions passing through the pore from one side 
can clear the tethered blocking particle from the 
opposite side. 
Further work in progress to determine the 
structural basis for potassium channel gating in- 
cludes introducing chemically reactive cysteine 
residues at specific locations in the protein se- 
quence. Channel proteins with cysteines inserted 
at critical locations should show a specific 
change in function when treated with reagents 
that modify the cysteine side chain. The reactivity 
of these side chains will depend on both their 
location in the channel protein and the specific 
conformational state of the protein at the time of 
reaction. 
We have also used the site-directed strategy to 
study acetylcholine-activated cation channels, 
which convert neurochemical signals into elec- 
trical signals at synapses. We have changed amino 
465 
