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Intermittent hypoxia (IH) is a physiological
stimulus that
arises in a number of situations, including the pathological settings
of sleep
apnea and neonatal recurrent apneas. In response to IH, changes occur
in
humans, rats, and cultured cells. I investigate IH-induced changes in
ion
channel density in cultured cells.
The main focus of my research is
the hERG potassium channel.
The hERG channel is a delayed rectifier channel that is important for
repolarization
in the heart, and helps to control resting membrane potential in many
neurons.
hERG channels are an appealing model for this type of study both
because of
their profound affect on cellular excitability and because their
trafficking
has been relatively well characterized for an ion channel. When lysates
from
stably transfected HEK293 cells are probed by immunoblot, two distinct
bands
can be seen, corresponding to immature, endoplasmic
reticulum(ER)-restricted
protein, and mature protein that may be trafficked to the membrane.
Thus
without extensive intervention, channel trafficking can be studied
based on the
pattern of mature and immature protein.
I have determined that hERG
channel density is reduced by
exposure to IH. This reduction can be seen both at the protein level by
Western
blot and in current density. Mature protein is reduced, while the
amount of
immature protein is increased. Further, immunofluorescence shows an
accumulation of immature protein in the ER of transfected cells. This
effect is
poorly reversible, in stark contrast to the effects of continuous
hypoxia, even
for much longer exposures. Both mature and immature protein density is
reduced
in SH-SY5Y neuroblastoma cells, which endogenously express hERG protein.
I have also investigated the
effects of hypoxia, both
continuous and intermittent, on the cardiac sodium channel, Nav1.5.
Neither
continuous nor intermittent hypoxia altered the current density of
Nav1.5 in a
stably expressing HEK293 cell line, even when the continuous hypoxia
was
extended over 72 hours, 3 times longer than necessary to achieve a
maximal
effect on hERG channels. Therefore, ion channels are subject to
differential
regulation by hypoxia, and are not simply reduced across the board.
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