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Voltage-gated sodium channels are essential in initiating
and propagating action potentials and are blocked by saxitoxin (STX)
and its analogs. Although all voltage-gated sodium channels are
blocked by STX, the toxin's potency depends on the specific channel
isoform. Zetekitoxin (ZTX) a recently purified and characterized
toxin from the Panamanian frog, Atelopus zeteki, was found to have
a structure similar to saxitoxin. The present study characterizes
ZTX's effect on voltage-gated sodium channels and compares the results
with STX. Sodium channel isoforms were expressed in Xenopus oocytes
and their activity was measured in the presence and absence of ZTX
by an oocyte two micro-electrode voltage clamp. Results showed ZTX
exhibiting similar isoform specificity as STX in native heart rat
and skeletal muscle sodium channel isoforms. ZTX though exhibited
an overall 10 to 100-fold higher potency for all isoforms compared
to STX making ZTX the most potent sodium channel toxin known.
Voltage-gated sodium channels can be found in excitable
cells including skeletal, cardiac and neuronal cells. The current
theory about the inactivation gate mechanism is the ball and chain
model. When the cell is at its resting potential, the gate remains
open, allowing ions to pass through. When the cell is held at a
voltage more positive than its resting potential, the gate slowly
closes, inactivating the channel. Therefore, the voltage-gated Na+
channel has an inactivated state in addition to the normal open
and closed states. This illustrates the normal bundling of the four
domains as well as the open Na+ channel and the open but inactivated
Na+ channel.The physiological significance of the Na+ channels involves
action potential propagation. A cell's resting potential is always
more negative than its surrounding environment, so when a cell depolarizes,
it becomes more positive, changing the polarity between the internal
and external environments. If a single spot on the membrane reaches
the threshold voltage, the sodium channels will open, allowing positive
sodium ions to rush into the cell, depolarizing the adjacent membrane.
This sends almost a wave-like effect of depolarization, which is
called an action potential. These action potentials are essential
to stimulus interpretations and responses. Because the exact structure
of sodium channels cannot be determined through crystallography,
a more indirect method must be used to develop a model. It is known
that there are multiple isoforms of the Na+ channel, located in
different cell types. There are three guanidinium toxins, tetrodotoxin
(TTX), saxitoxin (STX), and zetekitoxin (ZTX) that have been used
for this purpose. ZTX is the newest and least studied of these toxins.
The structure of ZTX was recently determined to be similar to STX,
suggesting ZTX will block sodium channels.
Step 1: Extract oocytes from Xenopus laevis. From
http://www.xenopus.com/products.htm
Step 2: Sort and collect oocytes in the 5th and 6th stage of development.
Step 3: Inject oocytes with 20 to 40 ng Na+ channel isoform cRNA.
Step 4: Let incubate at 17 degrees C for 48 hours. Step 5: Use two
micro-electrode oocyte clamp to measure Na+ channel current in the
presence and absence of ZTX.
ZTX Rapidly Blocks Na+ Channels, which is contrasted
by the slow current recovery reflecting slow toxin dissociation
from the channel. ZTX is More Potent than STX. Kd is the amount
of toxin at which 50% of the Na+ channel current is blocked. ZTX
is 10 to 100 times more potent than STX, making ZTX the most potent
Na+ channel toxin known to date. ZTX shows a similar preference
for muI skeletal muscle channels over hH1A human heart channels
when compared to STX. Nevertheless, ZTX responds differently to
mutations know to be responsible for the STX isoform differences
in Kd.
ZTX blocks Na+ channels. Similar to STX, ZTX more potently blocks
skeletal muscle Na+ channels (mu1) than heart Na+ channels (hH1a).
ZTX is 10 to 100 times more potent than STX. STX and ZTX responses
differ to mutations in their binding site.
Future Directions:
Apply the data to a computer model of the Na+ channel to refine
the structure of the outer pore of the channel. Use various voltage
protocols to assess state dependant binding of ZTX.
This research was funded by the Howard Hughes Medical Institute
Grant No. 52003727, the National Institutes of Health (NIH) grant
HL64828 (SCD), Department of Veterans Affairs merit grants (SCD),
an American Heart Association Established Investigator Award (SCD)
and American Heart Association Postdoctoral Research Award (AEP)
The authors offer their appreciation to Dr. Chris Hartzell for his
technical support of this project, and to Beth Boulden, Jon Allen,
Lisa Shang, Vijay Kasi, and Alice Huang for their guidance and help.
There is a newly identified toxin called zetekitoxin, which was
found to be similar to saxitoxin. Because they are similar in structure,
we hypothesized that they are also similar in function. Saxitoxin
blocks sodium channels, so we wanted to see if zetekitoxin blocked
sodium channels as well. To test this is a controlled setting, we
needed a system that would only contain sodium channels, not potassium
or calcium channels. To do this, we used Xenopus oocytes, which
are unfertilized eggs. We injected these eggs with sodium channel
RNA, which allowed the oocytes to produce the sodium channel protein.
We then tested these sodium channels in the presence and absence
of zetekitoxin to see if there were any differences. We found that
zetekitoxin does in fact block sodium channels like its relative
saxitoxin. It also shows a similar pattern of blocking in the different
sodium channel types. But the most exciting find of our project
was that zetekitoxin is 10 to 100 times more potent than saxitoxin,
which means it is the most potent sodium channel known to date.
Two micro-elecrtode oocyte clamping, DNA purification, RNA synthesis.
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