Csi undergraduate conference on research, scholarship...

Research Poster Presentations
P O S T E R 3 7
Unraveling the Structural Basis
of a Snake Toxin/Potassium
Channel Interaction
Tony Jin
Faculty Mentor: Dr. Sebastien Poget
Department of Chemistry
Presentation Abstract: Potassium (K+) channels are
integral membrane proteins that, when activated,
allow highly specific K+ ion flux through the
plasma membrane of cells at near-diffusion rates.
They play central roles in such physiological
processes as neurotransmission, secretion of
insulin, and regulation of the heartbeat. KcsA is a
bacterial potassium channel known to share
homology with K+ channels of most other species,
including humans.
Certain varieties of toxins, poisonous biological
peptides, have long been known to target potassium
channels, collectively causing cardiac arrest and
respiratory failure. These toxins typically work by
binding to potassium channels to restrict K+ ion flow.
However, we isolated a toxin—coined Tx7335—from
the venom of the Eastern Green Mamba (Dendroapsis
angusticeps) and discovered via electrophysiological
assay that it increases the frequency of activation of as
well as the duration of openings, leading to an
increase of K+ ion flow through KcsA—a rare effect
among toxin-channel interactions.
This project aims at unraveling the structural basis
underlying KcsA’s increased frequency of activation
upon binding of Tx7335, via methods of 2D
solution-state protein NMR spectroscopy. Our
current approach involves 1) creating a solution of
KcsA that gives optimal spectral quality, 2)
assigning each peak in the spectrum to its
corresponding amino acid residue, and 3)
examining changes in peak position upon addition
of the toxin, which will reveal the location of the
Tx7335 binding site on KcsA. So far, we have found
a detergent species that allows us to reconstitute
KcsA and monitor toxin binding. We are currently
further optimizing NMR sample conditions.
Conclusive findings from this project will further
our understanding of the biophysical driving forces
in K+ channel activation and may aid the design of
novel and more effective K+ channel openers—
presently used to treat hypertension and hair loss—
as well as other drugs that act by modulating K+
channel activity.
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P O S T E R 3 8
Substrate-Dependent Regulation
of Cytochrome-2E1 (Cyp2E1)
Activity in Mouse Primary
Hepatocyte Culture
Kaitlin Kelly, Diana I. Aparicio-Bautista
Faculty Mentor: Dr. Jimmie E. Fata
Department of Biology
Primary hepatocyte cultures are widely used for in
vitro investigation and as a screening tool to
evaluate hepatic metabolism, cytochrome P450
(CYP) induction or inhibition, and hepatotoxic
potential of xenobiotics. However, the use of
cultured hepatocytes in these applications is
dependent on adequate and relative stable
expression of CYP enzymes over the culturing
period. The culture conditions, in particular the
configuration of cell-substrate interactions and the
formulation of culture medium are crucial for
maintaining the activity of CYP enzymes.
Cytochrome 2E1 (Cyp2E1), a member of the
cytochrome P450 family is involved in the
oxidative metabolism of different substrates such as
tobacco derived N-nitrosamines and benzene,
which are potential carcinogens in breast tissues of
human and rat respectively. In this study, we
evaluated the Cyp2E1 enzyme activity of mouse
primary hepatocyte, which were cultured 48 hrs on
three different substrates; plastic, collagen type I
and Matrigel (basement membrane). Our results
showed that the level of enzyme activity of Cyp2E1
increased significantly using Matrigel as substrate
when compared to collagen type I or plastic. These
results suggest that Matrigel is an effective way to
maintain the optimal cytochrome activity in mouse
primary hepatocyte cultures.