"Studying what the different protein parts of an ion channel do is a huge challenge," Isacoff says. "Over the years, my group has developed techniques by which the domains of channel proteins can be labeled with site-specific fluorescent dyes. Structural rearrangements of the labeled sites in the channel can then be detected through changes in the fluorescence."
Isacoff and his group separated the C-terminal domain from the rest of the protein and tagged it with a green fluorescent protein (GFP) - a fluorescent protein from jellyfish commonly used to paint cells green for biological studies. Whereas the pore of the TREK1 ion channel is embedded in the plasma membrane of a neuron, the C-terminal is a short tail that protrudes out into the surrounding cytoplasm.
Using voltage clamps to measure electrical currents through the channel and fluorescence to monitor the disposition of the C-terminal domain, Isacoff and his group found that when the C-terminal tail is fully bound to the plasma membrane, the TREK1 potassium channel opens more; when the tail is unbound from the plasma membrane, the ion channel tends to close.
"We found that fluoxetine causes the isolated C-terminal domain to unbind from the membrane and also causes an inhibition of current from the full TREK1 channel," Isacoff says.
The next step will be to see how the C-terminal tail is affected by the pre
|Contact: Lynn Yarris|
DOE/Lawrence Berkeley National Laboratory