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'Blind' cells see the light; maybe someday humans will, too

o turn it into a remote-controlled switch that could be used to turn nerve cells on and off.

They decided to concentrate on the potassium channel, which opens when a voltage difference develops between the inside and outside of a nerve cell. The open channel lets positive potassium ions flow out of the cell, equalizing the voltage and turning the cell off.

Trauner, Kramer and their team designed a way to re-engineer the potassium channel to respond to light rather than voltage. To create this man-made channel and insert it into living cells, they took a two-step approach. First, they mutated the gene for the ion channel -- using as their starting material the potassium channel found in fruit flies -- so that, when expressed in a cell, the channel is broken and always stays open. They also added an extra molecule -- the amino acid cysteine -- to the channel protein so that, once the protein gets in place in the cell membrane, this molecule dangles off the outer surface of the cell like a fish hook.

They then inserted the mutated potassium channel gene into cells from the hippocampus of a rat -- cells that are found inside the brain and never see the light of day. To achieve this in their cell culture experiment, they flooded the culture with the mutated gene inside a circular piece of DNA called a plasmid, which cells readily take up. They checked to see how many of the hippocampal cells took up the gene by also washing the cells with a plasmid containing a gene for green fluorescent protein, which glows green when hit with UV light. Cells taking up one plasmid usually take up other plasmids, and nearly all the cells glowed green.

The second step was to wash the cells with a chemically synthesized switch that gloms onto the cysteine hook. The photoswitch an azobenzene compound was built like a drain plug on a rigid tether, so that when the end of the tether binds to cysteine, the plug fits snugly into the potassium channel.


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Contact: Robert Sanders
rsanders@berkeley.edu
510-643-6998
University of California - Berkeley
21-Nov-2004


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