Voltage-gated ion channels from prokaryotic cells have been cloned recently. These.channels are of great interest
because they provide a unique opportunity to perform fine biochemical, biophysical and structural studies in the
same protein. Because it can be produced in large amounts in bacterial culture, the protein can be purified,
reconstituted and crystallized allowing detailed characterization of structure and function. In addition, the channels
can be expressed in mammalian cells allowing detailed studies of channel gating. One such channel is NaChBac, a
bacterial Na+ channel from B.halodurans that has interesting structural and functional characteristics. The present
project aims at determining the mechanism by which NaChBac gates and what parts of the protein are directly
involved in gating. For this, we will measure energy transfer between donor-acceptor pairs that will be positioned
by introducing at individual sites an EF-hand motif and a cysteine by site-directed mutagenesis. The modified
channel protein will be affinity purified and reconstituted into liposomes. Energy transfer between a donor (terbium
bound to the EF-hand motif) and an acceptor (a fluorophore attached to the introduced cysteine) will be determined
to measure the distance between donor and acceptor groups and the changes therein with changes in membrane
voltage. In parallel, all mutant channels will be functionally evaluated by whole-cell recording from channels
expressed in mammalian cells and by lipid bilayer recording of incorporated proteoliposomes containing
purified/reconstituted channel protein.