Irwin Levitan's
laboratory is interested in the regulation of the electrical
activity of nerve cells. Such regulation of neuronal electrical
activity is critical for long term changes in behavior.
The specialized membrane proteins known as ion channels
are responsible for all electrical signaling in neurons,
and hence understanding how ion channel activity is modulated
is of fundamental importance. The ion channel modulatory
mechanism that has been most thoroughly studied and is
best understood is modulation by protein phosphorylation.
Ion channels, like many other proteins, are substrates
for protein kinases and phosphoprotein phosphatases, and
channel activity can be altered profoundly by phosphorylation.
Levitan began
his lecture with a summary of the effects of phosphorylation
on the properties of several different kinds of ion channels.
He emphasized that modulation by phosphorylation is not
confined to a particular class of ion channel, but is
widespread, and suggested that modulatability by phosphorylation
may be as intrinsic to ion channels as are such properties
as voltage dependence, conductance and selectivity. He
then moved on to the major theme of his lecture, that
ion channels do not exist alone in the plasma membrane,
but often are bound tightly to the modulatory enzymes
(such as protein kinases and phosphatases) that influence
channel activity.
This theme
was illustrated by two examples of work from Levitan's
laboratory. The first example concerned the modulation
of calcium-dependent potassium channels from rat brain,
reconstituted in artificial phospholipid bilayers. Under
these experimental conditions, proteins are effectively
at infinite dilution in the vast ocean of bilayer lipid.
Channel activity can be modulated in the bilayers by the
addition of ATP to their cytoplasmic sides, and a variety
of evidence demonstrates that this modulation results
from protein phosphorylation. Because no exogenous protein
kinase was added in these experiments, it could be inferred
that the modulation must be mediated by an endogenous
protein kinase activity that is tightly bound to the channel
and accompanies it in the bilayer. Similar experiments
demonstrated that a phosphoprotein phosphatase activity
is also part of the modulatory complex.
In another
set of experiments, biochemical methods were used to demonstrate
directly that another kind of potassium channel, a human
voltage-gated potassium channel, binds tightly to the
Src protein tyrosine kinase. Specific antibodies that
recognize the channel or the kinase were used in co-immunoprecipitation
experiments. These experiments demonstrated that when
channel is immunoprecipitated with its specific antibody,
the kinase can be detected in the immunoprecipitate, and
vice-versa. Other experiments defined the specific amino
acid sequences in both channel and kinase that are involved
in the binding interaction. Because these sequences occur
with high frequency in many other ion channels and signaling
proteins, it is likely that such ion channel/signaling
protein interactions are extremely common.
Levitan concluded
by emphasizing that the traditional picture of ion channels
as membrane loners is inappropriate. Their tight associations
with protein kinases, phosphoprotein phosphatases and
other signaling and scaffolding proteins has fundamental
implications for temporal features and specificity of
neuronal signaling.
