Functional characterization of the voltage-gated bacterial sodium channel NaChBac using lanthanide-based resonance energy transfer (LRET)

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Voltage-gated ion channels are essential for Generation and propagation of action potentials in all excitable cells. They are made up of four homologous domains, each of which contains six transmebrane segments. Although various parts of channels have been shown to play a role in channel gating, seemingly undergoing some conformational changes in response to voltage, little is known about the nature of these changes. In our project, we intend to investigate the recently cloned six transmembrane segment voltage-dependent bacterial Na+-channel by the lanthanidebased resonance energy transfer (LRET) technique. We plan to study distances in reconstituted channel using an EF-hand in the protein. The EF-hand grabs the donor fluorophore Tb which emits owing to sensitization from an excited tryptophan residue close to the Tb. Voltage-dependent movements near the voltage-sensing regions can be effectively measured by determining intersubunit and intrasubunit distances as a function of voltage. Analysis of gating properties and permeation of the channel by the patch-clamp technique will provide us with an additional information about channel functioning. Combination of these two techniques is a powerful tool for biophysical examination of voltage-gated ion channels, because the conformational alterations occuring during channel gating can be visualized, establishing the link between changes in electrophysiological properties measured by patch-clamp and the source of these changes.