Abstract
The opening and closing of ion channels in the membrane of a neuron allows for
the flow of specific ions into and out of the cell. The flow of ions through membrane
channels creates current and this changes the voltage across the cell membrane. These
changes in voltage create the action potentials responsible for the propagation of neural
signals. If the number of ion channels changes or the kinetics of individual channels
change, the currents through the membrane will be altered and hence change the
excitability of the cell. Here I report changes in potassium conductance in the neurons of
Doriopsilla albopunctata after treatment and recovery from a thirty-minute heat shock at
30°C. By using a voltage clamp, currents of individual cells were characterized. The
cells were heat shocked and characterized again using the same stimulation regime.
Significant increases in potassium conductance were observed within many, although not
all, cells. In such cases, a novel current was found to be responsible for the change in
potassium conductance. I conclude that heat shock affects either the expression of novel
channels in the cell membrane or the kinetics of pre-existing channels, but only in some
neurons. What cellular mechanism is responsible for this change and what specifically it
changes, remains to be seen.
Introduction
The effect of heat shock has been shown to have lasting effects within the cells of
organisms across species. For poikilothermic organisms, this is of particular significance
as their body temperatures are dictated by daily vacillating ambient temperature. Most
research on heat shock has focused on the expression and distribution of heat shock
proteins and how that expression and distribution affects cellular function. Oddly
enough, the effect of heat shock in relationship to the nervous system has only recently
been explored (Ramierez et al 1999). In particular, the effect of heat shock on the
mechanisms of neural exitability is yet to be uncovered.
In light of this, I examined the effect of heat shock on Doriopsilla albopunctata, a
low intertidal poikilotherm. Due to its fluctuating temperature environment and large
head ganglia, this animal was ideal for studying the effect of heat shock on neurological
function.
To examine the effect of heat shock on the head ganglia of Doriopsilla, I created a
protocol that allowed for constant dual electrode voltage clamp recording pre and post
heat shock without ever removing the electrodes from the cell. Here I report an increase
in potassium conductance post heat shock due to the recruitment of a slow inactivating
channel. The expression of the channel increased with increasing time post heat shock.
This effect, however, was not observed in all cells.
Methods
Specimens were collected from Monterey Bay and kept in a tank at Hopkins
Marine Station. Each Doriopsilla was dissected and its head ganglia excised. The
ganglion was placed in a Silgard-plated dish with fresh seawater. The ganglion was then
pinned to the Silgard plate using cactus needles. Most of the seawater was removed from
the dish and 1 mg of non-specific protease (dispase) was applied to the sheath
surrounding the ganglion. A small piece of kimwipe was used to cover the ganglion.
The dish was then encapsulated in a glass container and kept at 4°C in a refrigerator. An
hour later, the kimwipe was removed and the ganglion was washed with freshwater
seawater. The dish was then refilled with fresh seawater and placed back into the
refrigerator. An hour later, the dish was removed from the refrigerator and the seawater
exchanged. The dish was placed onto a Peltier plate held at 12°C. Using micro forceps,
the sheath surrounding the ganglia was removed. Two glass microelectrodes were
prepared and filled with 3M KCl solution. Resistance of the electrodes was between 1-
2MS. A small amount of black ink was applied to the tip of the electrode for
visualization purposes. The two electrodes were then hooked up to a voltage clamp (as
described by Connor and Stevens with minimal modifications). A single cell within the
ganglia was penetrated by both electrodes and clamped at a holding potential near its
observed resting potential and recordings were made using the Clampex 8.0 computer
program while maintaining a 12°C temperature regime. Äfter stimulus was applied, the
Peltier was heated to 30°C with the electrodes remaining in the cell. A temperature of
30°C was maintained within the dish for 30 minutes. The dish was then cooled to 12°C.
The stimulation regime used prior to heat shock was applied again at fifteen-minute time
intervals up to 1.5 hours post heat shock.
Results
Single cell voltage clamp recordings using a holding potential of -80mV were
used to examine potassium current. 400ms, SmV voltage steps used to stimulate from
-40mV to -20mV revealed large increases in peak outward current amplitude as well as
steady state current amplitude post heat shock. This increase was correlated with time
post heat shock (Fig. 1). At 30 minutes post heat shock, peak amplitude had increased
22.3% (.435 nA to .535nA) and steady state amplitude increased 37.5% (.04 to .055) after
a 400ms depolarizing voltage step at -20mV was applied. At 1.5 hours post heat shock,
peak amplitude had increased 90.8% (from .435 nA to .83nA) and the steady state had
increased 1000% (.04nA to 4nA) when exposed to the same stimulus. This change is
reflected in the potassium IV curve in Fig. 2. A GV curve was constructed (Fig. 3) using
data from a second stimulation protocol that determined the instantaneous reversal
potential of potassium by holding the cell at -80mV, stepping to -10mV for 25 ms and
stepping down to-60mV in 10mV increments for 100ms. This result showed an increase
in conductance post heat shock. Similar changes in potassium conductance were
observed in three other cells (data not shown).
A control was run in order to qualify this result. A single cell was run through the
same heat shock protocol with a slightly augmented stimulation protocol (holding
potential of-80mV with voltage steps from -40mV to OmV with a delta of 10mV (Fig.
4)) and the same reversal potential protocol. The IV for the control is shown in Fig. 5.
There was no appreciable change in peak amplitude while a mild change in steady state
was observed. The GV for the control is shown in Fig. 6.
By looking more closely at the top current trace from the 1.5 hour stimulation
regime, it appears that the trace is comprised of two decaying logarithmic functions. This
is highlighted in Fig. 7. The linear approximation of the natural log of the current from
that trace is shown in Fig. 8.
In order to better analyze the data, I took the top current trace (-20mV
depolarizing step) from each time interval post heat shock (30min, lhour, and 1.5 hour)
and subtracted out the top current trace from the pre heat shock regime. The difference
of those traces is shown in Fig. 9. This difference in current shows the component that is
effected by the heat shock.
Discussion
These results show a large increase in potassium conductance post heat shock.
Moreover, conductance increases with time post heat shock. This implies a cellular
mechanism that not only responds to heat shock, but also responds on a very short time
scale (within one hour after the onset of heat shock). What mechanism is responsible for
this change is difficult to extrapolate from the given experiments. However, closer
analysis of the data gives insight into what may be happening.
As Fig. 7 shows, it appears that two exponentially decaying functions comprise
the current trace 1.5 hours post heat shock. Since inactivation can be described
exponentially, it appears that two different rates of inactivation are present. In order to
prove this, Fig. 8 shows the linear approximations of the log of current with respect to
time. There appears to be two separate straight lines that can describe the semi-log plot.
This demonstrates that there are two exponentially decaying functions that comprise the
post heat shock current trace. This implies that there exists a slow inactivating channel
that is recruited more strongly as time post heat shock increases. Fig. 9 shows the
increase in current with time. The subtraction of the initial current from the post heat
shock current appears to have its own set of kinetics of activation and inactivation. This
implies that there are two channels: one fast inactivating channel that does not change
with heat shock and one slow inactivating channel that is recruited more readily post heat
shock.
Two hypotheses can describe this occurrence: the number of slow-inactivating
channels in the cell membrane is increasing or the slow inactivating channels are pre-
existing in the membrane and are only getting recruited post heat shock. Both hypotheses
are certainly possible mechanisms to describe this occurrence.
When taking into consideration the first hypothesis, one can imagine that there are
intracellular vesicles that contain the slow inactivating channels. When heat shock is
induced, the vesicles travel along the microtubule array and deploy the silent channels
into the cell membrane. This mechanism would be similar to the one proposed by
Brismar and Gilly (1987) in their description of sodium channel synthesis in the squid
giant axon. Such a mechanism might account for the observed result within a one hour
time scale. Inhibition of the microtubule array with colchicine would be a good way to
test for such a mechanism.
Looking at the second hypothesis, it is possible that the channels are being
expressed the entire time in the cell membrane and only post heat shock are they
recruited. Thompson (1994) describes a calcium-dependent potassium current in
Doriopsilla. Such a current could be recruited if the internal calcium concentration
changes post heat shock. Since calcium is such a ubiquitous cellular agent and so many
proteins are responsible for changing the calcium concentration within a cell, it would be
difficult to isolate precisely what mechanism is changing to allow for such a vacillation in
calcium concentration. However, it would interesting to pursue this possibility if this
same experiment were run in the presence of fura-2 in order to ascertain accurately how
much the internal calcium concentration vacillates post heat shock.
The possibility of heat shock proteins playing a role in this mechanism or simply
novel protein translation should be considered. Although hsp's play a role in many
cellular processes, it seems unlikely that they would have any appreciable affect on ion
channels directly or any integral membrane protein for that matter. Hsp90 may have
some significance in the second hypothesis as it has been shown to have significance in
signal transduction (Bratt and Toft), although its function remains dubious at best. Also,
it is not known if Doriopsilla translates hsp’s in response to a thirty-minute 30°C heat
shock. Finally, novel protein synthesis does not seem realistic on the time scale
observed, although it would be interesting to inhibit protein synthesis and run the same
experiment.
Although there are many questions left unanswered, the observed result is
compelling. I show that a novel current is recruited post heat shock and increases in
amplitude with increasing time post heat shock. The observed effect is also cell specific
as some cells exhibit no appreciable change in current in response to heat shock. In order
to determine the cellular mechanism for this change, many more experiments must be run
and more rigorous stimulation regimes instigated. The data mandate experimentation to
characterize this novel change in neurological function in response to heat shock.
Acknowledgments
1 would like to thank Dr. William Gilly for providing appropriate literature, John Lee for
constructing the heat shock apparatus, Anna Kirby, Freya Sommer, and Melissa Coates
for collecting, Rob DeConde for computer assistance, Christian Reilly for constant
support, and Dr. Stuart Thompson without whom none of this would have been possible.
References
Brismar, Tom and Gilly, William F. (1987) Synthesis of sodium channels in the cell
bodies of squid giant axons. Proc. Natl. Acad. Sci. USA. 84: 1459-1463
Connor, J.A. and Stevens C.F. (1971) Inward and Delayed Outward Membrane Currents
in Isolated Neural Somata Under Voltage Clamp. J. Physiol. 213: 1-19
Pratt, WB. and Toft, DO. (1997) The role of hsp90-based chaperone system in signal
transduction by nuclear receptors and receptors signaling via MAP kinase. Endo Rev.
18: 1-55
Ramirez, J.M., Elsen, F.P., and Robertson, R.M. (1999) Long Term Effects of Prior Heat
Shock on Neural Potassium Currents Recorded in a Novel Insect Ganglion Slice
Preparation. American Physiological Society. 795-802
Thompson, Stuart H. (1994) Facilitation of calcium-dependent potassium current.
Journal of Neuroscience. 14: 7713-772.
Figure Legends
Figure 1: Four current traces taken pre heat shock, 30 minutes post heat shock, one hour
post heat shock, and 1.5 hours post heat shock. Current traces are in response to a -80mV
holding potential with 400ms voltage steps from —40mV to -20mV with a delta of 5mV.
Figure 2: Potassium IV curves taken from the current traces in Fig. 1
Figure 3: Potassium GV curves taken from current traces from the reversal potential
protocol.
Figure 4: Four current traces taken pre heat shock, 30 minutes post heat shock, one hour
post heat shock, and 1.5 hours post heat shock. Current traces are in response to a -80mV
holding potential with 400ms voltage steps between —40mV and OmV with a delta of
1OmV.
Figure 5: Potassium IV curves taken from the current traces in Fig. 3
Figure 6: Potassium GV curves taken from the current traces from the reversal potential
protocol
Figure 7: Current trace from 1.5 hours post heat shock in response to a -20mV 400ms
depolarizing step from a holding potential of -80mV.
Figure 8: Semi-log plot of current taken from Fig. 5 with respect to time with fitted linear
traces.
Figure 9: Novel current traces taken from subtracting the initial current from the current
traces 30 minutes, one hour, and 1.5 hours post heat shock.
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